1,312 research outputs found
Exploiting MS-based techniques to unveil elusive reaction intermediates of bioinorganic relevance
No full textThe periodic table for a medicinal chemist or a biochemist is usually restricted to very few elements. More than 95% of the mass of living systems is indeed composed by carbon, hydrogen, nitrogen and oxygen. Besides, elements present only in trace amount can have irreplaceable roles in the chemistry of life. Even more surprisingly, transition metals completely unrelated to living systems have found their way in therapy and, nowadays, antineoplastic drugs containing for example platinum are widespread. However, many techniques routinely exploited for analyzing chemical reactions in solution fail to characterize the properties of metal complexes, in particular in their interaction with biological molecules. The aim of this thesis is to show how electrospray ionization (ESI) mass spectrometry (MS) may excel in capturing elusive species from solution. One can thus shed light on reaction mechanisms of biological relevance and gain insight about coordination sites of biomolecules in binding metals. This work is focusessed on platinum complexes moving from the PtII-containing anticancer drug cisplatin to novel PtIV complexes, which are promising candidates to be at the forefront of future platinum-based therapies. To obtain structural insights about the species of interest, several MS-based techniques have been exploited. IR multiple photon dissociation (IRMPD) spectroscopy was used to obtain the vibrational features of mass selected species. IRMPD spectroscopy combined with calculations at the DFT level, to interpret the experimental vibrational features, allowed us to tackle a variety of issues. Among them, we could unveil the nature of the encounter complex lying on the reaction coordinate of ligand exchange of cisplatin with model biological targets. IRMPD spectroscopy was also employed to characterize the primary intermediates formed by cisplatin reacting with histidine and methionine, major platination targets in proteins. Eventually, IRMPD kinetics on isomer- and conformer-specific vibrational modes were also used to obtain semi-quantitative information about the conformational landscape of cisplatin derived complexes. Collision induced dissociation (CID) was instead the MS/MS technique of choice to gain information about the gas-phase reactivity of platinum(IV) complexes. Using high-resolution mass spectrometry a complete fragmentation pattern was achieved by assigning an unambiguous molecular formula and so characterizing exotic species generated by reduction of PtIV
Extracellular ATP Induces a Distorted Maturation of Dendritic Cells and Inhibits Their Capacity to Initiate Th1 Responses
No full textDendritic cells (DCs) express functional purinergic receptors, but the effects of purine nucleotides on DC functions have been marginally investigated. In this study, we report on the ability of micromolar concentrations of ATP to affect the maturation and Ag-presenting function of monocyte-derived DCs in vitro. Chronic stimulation (24 h) of DCs with low, noncytotoxic ATP doses increased membrane expression of CD54, CD80, CD86, and CD83, slightly reduced the endocytic activity of DCs, and augmented their capacity to promote proliferation of allogeneic naive T lymphocytes. Moreover, ATP enhanced LPS- and soluble CD40 ligand-induced CD54, CD86, and CD83 expression. On the other hand, ATP markedly and dose-dependently inhibited LPS- and soluble CD40 ligand-dependent production of IL-1alpha, IL-1beta, TNF-alpha, IL-6, and IL-12, whereas IL-1 receptor antagonist and IL-10 production was not affected. As a result, T cell lines generated from allogeneic naive CD45RA(+) T cells primed with DCs matured in the presence of ATP produced lower amounts of IFN-gamma and higher levels of IL-4, IL-5, and IL-10 compared with T cell lines obtained with LPS-stimulated DCs. ATP inhibition of TNF-alpha and IL-12 production by mature DCs was not mediated by PGs or elevation of intracellular cAMP and did not require ATP degradation. The inability of UTP and the similar potency of ADP to reproduce ATP effects indicated that ATP could function through the P2X receptor family. These results suggest that extracellular ATP may serve as an important regulatory signal to dampen IL-12 production by DCs and thus prevent exaggerated and harmful immune responses
Photoionization mass spectrometry of ω-phenylalkylamines: Role of radical cation-π interaction
No full textLinear ω-phenylalkylamines of increasing alkyl chain length have been investigated employing synchrotron radiation in the photon energy range from 7 to 15 eV. These molecules have received considerable interest because they bear the skeleton of biologically relevant compounds including neurotransmitters and because of the possible interaction between the amino moiety and the phenyl ring. Recently, the contribution of this interaction has been assayed in both neutral and protonated species, pointing to a role of the polymethylene chain length. In this work, the ionization energy (IE) values of benzylamine (BA), 2-phenylethylamine (2-PEA), 3-phenylpropylamine (3-PPA), and 4-phenylbutylamine (4-PBA) were investigated in order to ascertain the impact of the different alkyl chain lengths and to verify an amino radical cation-π interaction. The IEs obtained experimentally, 8.54, 8.37, 8.29, and 8.31 eV for BA, 2-PEA, 3-PPA and 4-PBA, respectively, show a decreasing trend that is discussed employing calculations at the CBS-QB3 level. Moreover, the appearance energy values for major fragments produced by the photofragmentation process are reported
An integrated approach to study novel properties of a MALDI matrix (4-maleicanhydridoproton sponge) for MS imaging analyses
No full textThe chemical properties accounting for the operation of a valuable matrix used in matrix assisted laser desorption ionization (MALDI) to perform mass spectrometry imaging (MSI), namely 3-(4,5-bis(dimethylamino)napthalen-1-yl)furan-2,5-dione (4-maleicanhydridoproton sponge, MAPS), have been elucidated also by comparison with the parent molecule 1,8-Bis(dimethylamino)naphthalene (so-called Proton Sponge, PS). Both compounds present the bis(dimethylamino) groups, apt to efficiently trap a proton imparting positive charge. Only MAPS, though, owns the maleicanhydrido function acting as electrophile and yielding covalently bound adducts with a variety of analytes. In this way MAPS performs as ‘carrier’ for the analyte (A) of interest, at the same time minimizing the presence of useless, background ions. The covalent character of the adducts, [MAPS+H+A]+, is testified by their collision induced dissociation pattern, quite distinct from the one displayed by [PS+H]+, while PS does not form any [PS+H+A]+, thus confirming the key role of the maleicanhydrido functionality of MAPS. Vibrational spectroscopy of [MAPS+H+A]+ adducts (A = H2O, NH3) provided further structural evidence. The presence of a mobile proton on A was found to be a requisite for adduct formation by electrospray ionization of acetonitrile solutions, pointing to a possible role of MAPS in discriminating competing analytes based on molecular features. The performance of MAPS has been verified in MALDI-MSI of Atropa belladonna berries, exploiting MAPS binding to atropine
Adenosine triphosphate (ATP) induces a distorted maturation of dendritic cells and inhibits their capacity to initiate TH1 responses.
No full textDendritic cells (DCs) express functional purinergic receptors, but the effects of purine nucleotides on DCs functions have been only marginally investigated. Here we report the ability of ATP to affect the maturation and antigen presenting function of monocyte-derived DCs in vitro. Chronic stimulation of DCs with low, non cytotoxic ATP doses, but not with UTP, increased membrane expression of CD54, CD80, CD86 and CD83 as well as their capacity of inducing T lymphocyte proliferation in the primary mixed leukocyte reaction assay. Moreover, ATP enhanced LPS- or sCD40L-induced DC membrane maturation. On the other hand, ATP markedly and dose-dependently inhibited the production of TNF-alpha, IL-6 and IL-12 by LPS-stimulated DCs. In contrast, IL-10 production was only slightly affected. These effects did not require ATP metabolism as they were shared by the poorly hydrolyzable ATP analog ATP-gamma-S and were not prevented by specific inhibitors of the adenosine receptors. In addition, ATP activity was not blocked by treating DCs with indomethacin, suggesting that it was not mediated by prostaglandins. More interesting, T cell lines generated from allogeneic naive CD45RA+ T cells with DCs matured in the presence of ATP, produced lower amounts of IFN-gamma and higher levels of IL-4, IL-5 and IL-10. Addition of IL-12 and/or anti-IL-10 mAb restored IFN-gamma production, and reduced IL-4, IL-5 and IL-10 release. The results indicate that chronic stimulation with ATP, although induces phenotypic maturation, can potently inhibit the ability of maturing DCs to initiate Th1 responses
Cisplatin binding to biological ligands revealed at the encounter complex level by IR action spectroscopy
No full textCisplatin [cis-diamminedichloroplatinum(II)] was the first platinum-based antineoplastic agent and is still a cornerstone for the treatment of various solid tumors. Reactive events responsible for cisplatin activity are unveiled here at the molecular level. Simple ligands (L) representing ubiquitous functional groups in the biological environment likely to be encountered by administered cisplatin have been allowed to react with cis-[PtCl(NH3)(2)(H2O)](+), the primary intermediate from cisplatin hydrolysis. The substitution reactions have been examined by a combined experimental and computational approach and the structural features of the substitution product, cis-[PtCl(NH3)(2)(L)](+), have been probed by IR multiple-photon dissociation (IRMPD) spectroscopy. Furthermore, IRMPD spectroscopy has been exploited to elucidate the structure of [PtCl(NH3)(2)(L)(H2O)](+) clusters, also obtained by electrospray ionization (ESI) from the aqueous solution and representing the major focus of this investigation. These ions conform to the encounter complex of cis-[PtCl(NH3)(2)(H2O)](+) with the incoming ligand and represent the first direct evidence of a prototypical Eigen-Wilkins encounter complex in solution, lying on the reaction coordinate for ligand substitution and extracted by ESI for mass spectrometric analysis. Activated [PtCl(NH3)(2)(L)(H2O)](+) ions dissociate by the loss of either H2O or L, the former process implying a ligand substitution event. IRMPD spectroscopy has thus revealed both structural details and reaction dynamics at the level of the isolated encounter complex
In defense of platinum: amino acidic polydentate ligands as a shield against sulphur-donor nucleophilic attacks
No full textThe interactions of sulfur-containing peptides and proteins with platinum-based anticancer agents are of significant interest in the field of bioinorganic chemistry. We previously modified the lead compound potassium {trichlorido[eta 2-(but-3-en-1-yl)-2-acetoxybenzoate]platinate(II)} (Pt-butene-ASA) by exchanging two chlorido ligands for an amino acid chelating ligand to improve its stability in aqueous solutions. In the current study, we demonstrate how this amino acid ligand strengthens the inertness of this class of complexes against sulfurcontaining compounds. We incubated four complexes, each bearing an amino acid chelating ligand, with various sulfur-containing compounds of biological interest, such as DMSO, methionine, glutathione, and substance P. The reactions were monitored using NMR spectroscopy and mass spectrometry, allowing the identification by combination with IR ion spectroscopy of exotic pentacoordinate platinum(II) complexes as reaction intermediates. The results indicated that the reactivity of complexes 1-4 ((ethene,N-trans)(L-alaninato-N,O) chlorido(eta 2-ethene)platinate(II), (ethene,O-trans)(beta-alaninato-N,O)chlorido(eta 2-ethene)platinate(II), (L-histidinato-N,N)chlorido(eta 2-ethene)platinate(II), and (olefin,N-trans)(L-alaninato-N,O)chlorido(eta 2-(but-3-en-1-yl)-2acetoxy benzoate)platinate(II), respectively) is determined by the inner coordination sphere, and the addition of an amino acid ligand significantly reduces the rate of nucleophilic substitutions by sulfur-containing nucleophiles. In particular, the N,ethylene-trans isomers reacted only to a low extent with the free thiol in glutathione, while the O,ethylene-trans isomer was completely consumed within 2 h. Complex 4, the new lead compound, showed low reactivity towards S-nucleophiles in general, which confirms the concept of using amino acids as biocompatible bidentate ligands to adjust the reactivity and stability of this kind of metallodrugs
Danger signals in the immune system: extracellular nucleotides trigger dendritic cell activation
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IRMPD spectroscopy of deprotonated selenocysteine. The 21st proteinogenic amino acid
No full textThe effect of deprotonation on the structure and stability of the 21st proteinogenic amino acid selenocysteine, generated as bare deprotonated species by electrospray ionization, has been investigated by infrared multiple photon dissociation (IRMPD) spectroscopy over an extended frequency range (700-3600 cm- 1 ), encompassing both the fingerprint and X-H (X = C, N, O) stretching ranges. IRMPD spectra, interpreted by anharmonic DFT calculations, provide evidence of a thermally averaged ion population of two types of low-lying canonical conformers deprotonated at the selenol (Se-H) group and involved in different H-bonding motifs. The broadened and diffuse band structures observed in the H-stretching range are well interpreted by Born- Oppenheimer molecular dynamics computations that provide a valuable description of the flexible backbone arrangements of Sec and of the proton sharing dynamics in the Se-HO and Se-HN moieties. Other prototropic isomers, deprotonated at the carboxylic group or with a zwitterionic structure, should not be significantly populated, according to their higher free energy and calculated IR spectra inconsistent with experimental evidence
Initial protein unfolding events in Ubiquitin, Cytochrome c and Myoglobin are revealed with the use of 213 nm UVPD coupled to IM-MS
No full textThe initial stages of protein unfolding may reflect the stability of the entire fold, and can also reveal which parts of a protein can be perturbed, without restructuring the rest. In this work we couple UVPD with activated ion mobility mass spectrometry to measure how three model proteins start to unfold. Ubiquitin, cytochrome c and myoglobin ions produced via nESI from salty solutions are subjected to UV irradiation pre-mobility separation, experiments are conducted with a range of source conditions which alter the conformation of the precursor ion as shown by the drift time profiles. For all three proteins the compact structures result in less fragmentation than more extended structures which emerge following progressive in-source activation. Cleavage sites are found to differ between conformational ensembles, for example, for the dominant charge state of cytochrome c [M+7H]7+, cleavage at Phe10, Thr19 and Val20 was only observed in activating conditions while cleavage at Ala43 is dramatically enhanced. Mapping the photo-cleaved fragments onto crystallographic structures provides insight into the local structural changes that occur as protein unfolding progresses, which is coupled to global restructuring observed in the drift time profiles
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