RESUMEN
PNU-159682 is a highly potent secondary metabolite of nemorubicin belonging to the anthracycline class of natural products. Due to its extremely high potency and only partially understood mechanism of action, it was deemed an interesting starting point for the development of a new suite of linker drugs for antibody drug conjugates (ADCs). Structure activity relationships were explored on the small molecule which led to six linker drugs being developed for conjugation to antibodies. Herein we describe the synthesis of novel PNU-159682 derivatives and the subsequent linker drugs as well as the corresponding biological evaluations of the small molecules and ADCs.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Doxorrubicina/análogos & derivados , Inmunoconjugados/química , Inmunoconjugados/farmacología , Animales , Antineoplásicos/síntesis química , Línea Celular Tumoral , Doxorrubicina/síntesis química , Doxorrubicina/química , Doxorrubicina/farmacología , Humanos , Ratones Endogámicos NOD , Ratones SCID , Neoplasias/tratamiento farmacológicoRESUMEN
RATIONALE: The icosahedral carborane anion HCB11 H11-1 is well known for its exceptional stability in solution and the solid state, but the gas-phase properties of this molecule have not previously been explored. METHODS: Electrospray ionization ion trap mass spectrometry, collisional activation, and isotopic labelling were used to examine the formation and reaction pathways of covalent and noncovalent carborane clusters. RESULTS: Covalent attachment of an amino group to the C-vertex of HCB11 H11-1 dramatically alters its stability in the gas phase. Interestingly, covalently bound carborane dimers are produced during electrospray ionization. Subsequent collisional activation of these dimers leads to facile hydrogen losses (7 H2 molecules). The loss of H2 does not follow a stochastic pattern, but occurs preferentially by loss of 3 H2 , followed by loss of 2 H2 and then another 2 H2 molecules. CONCLUSIONS: This study provides insight into new possible modes of B-H activation and functionalization in carborane cages. Copyright © 2016 John Wiley & Sons, Ltd.
RESUMEN
Maintaining redox homeostasis, or the balance of oxidant and antioxidant forces, is essential for proper cellular functioning in biology. Although the antioxidant nature of many small molecules such as vitamin c and glutathione have been thoroughly investigated, contributions to redox homeostasis from larger biomolecules have received less attention. Evidence has shown that some proteins are antioxidant (in a non-catalytic sense), but large scale examination of this property for a diverse set of proteins has proven difficult. Herein, radical-directed dissociation mass spectrometry (RDD-MS) is used to examine the antioxidant capacity of a series of proteins with diverse biological roles, persistence intervals, and localizations. Digestion of these proteins reveals that all contain antioxidant peptide regions. Examination of the amino acid content of the antioxidant peptides does not reveal significant differences relative to normal peptides, suggesting that sequence may be more important than residue content. Sequence homology analysis across organisms reveals that antioxidant regions are frequently conserved, although many of these regions are also known to have other functions which may have influenced evolutionary pressure. Regardless of the origin, it is clear that many proteins may play secondary roles as sacrificial antioxidants within the cellular milieu.
Asunto(s)
Aminoácidos/análisis , Antioxidantes/química , Proteínas/química , Secuencia de Aminoácidos , Animales , Gatos , Bovinos , Pollos , Secuencia Conservada , Perros , Caballos , Humanos , Espectrometría de Masas , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Ratas , Homología de Secuencia de Aminoácido , PorcinosRESUMEN
An anionic self-folding deep cavitand is capable of immobilizing unmodified proteins and enzymes at a supported lipid bilayer interface, providing a simple, soft bioreactive surface that allows enzymatic function under mild conditions. The adhesion is based on complementary charge interactions, and the hosts are capable of binding enzymes such as trypsin at the bilayer interface: the catalytic activity is retained upon adhesion, allowing selective reactions to be performed at the membrane surface.
Asunto(s)
Éteres Cíclicos/química , Membrana Dobles de Lípidos/química , Proteínas/química , Resorcinoles/química , Secuencia de Aminoácidos , Aniones/química , Datos de Secuencia Molecular , Unión Proteica , Proteínas/metabolismo , Electricidad EstáticaRESUMEN
The fragmentation behavior of a set of model peptides containing proline, its four-membered ring analog azetidine-2-carboxylic acid (Aze), its six-membered ring analog pipecolic acid (Pip), an acyclic secondary amine residue N-methyl-alanine (NMeA), and the D stereoisomers of Pro and Pip has been determined using collision-induced dissociation in ESI-tandem mass spectrometers. Experimental results for AAXAA, AVXLG, AAAXA, AGXGA, and AXPAA peptides are presented, where X represents Pro, Aze, Pip, or NMeA. Aze- and Pro-containing peptides fragment according to the well-established "proline effect" through selective cleavage of the amide bond N-terminal to the Aze/Pro residue to give yn (+) ions. In contrast, Pip- and NMA-fragment through a different mechanism, the "pipecolic acid effect," selectively at the amide bond C-terminal to the Pip/NMA residue to give bn (+) ions. Calculations of the relative basicities of various sites in model peptide molecules containing Aze, Pro, Pip, or NMeA indicate that whereas the "proline effect' can in part be rationalized by the increased basicity of the prolyl-amide site, the "pipecolic acid effect" cannot be justified through the basicity of the residue. Rather, the increased flexibility of the Pip and NMeA residues allow for conformations of the peptide for which transfer of the mobile proton to the amide site C-terminal to the Pip/NMeA becomes energetically favorable. This argument is supported by the differing results obtained for AAPAA versus AA(D-Pro)AA, a result that can best be explained by steric effects. Fragmentation of pentapeptides containing both Pro and Pip indicate that the "pipecolic acid effect" is stronger than the "proline effect."
Asunto(s)
Fragmentos de Péptidos/química , Ácidos Pipecólicos/química , Prolina/química , Espectrometría de Masas en Tándem/métodos , Iones/químicaRESUMEN
Antioxidant peptides such as glutathione play critically important roles within cells by opposing the action of oxidative species. Similarly all proteins may, as a secondary function, potentially contribute to the antioxidant capacity of the cellular milieu, though this possibility has not been thoroughly explored previously. Herein it is demonstrated that, in addition to radical quenching solution-phase behavior, antioxidant peptides display an astonishing ability to sequester radicals in the gas phase. Compared to other peptides of similar sequence and size, radical antioxidant peptides exhibit very little radical-directed dissociation when subjected to collisional activation in the gas phase. Importantly, this property can be leveraged in highly sensitive and rapid mass spectrometry based experiments to identify antioxidant peptides. Examination of peptides derived from human serum albumin (HSA), which is a protein known to behave as an antioxidant, revealed three previously unknown peptide regions that exhibit antioxidant capacity. One of these peptides, VAHRFK, shows antioxidant capacity comparable to that of glutathione. It is likely that these peptide regions contribute to the overall antioxidant capacity of HSA. In comparison with previous methods, the present technique is significantly more sensitive and less time-consuming, which should enable more wide-scale examination of antioxidant peptides that are relevant to redox homeostasis, food chemistry, and disease.
Asunto(s)
Antioxidantes/metabolismo , Espectrometría de Masas/métodos , Proteínas/metabolismo , Cromatografía LiquidaRESUMEN
The connection between charge state distributions, protein structure, and mechanistic details of electrospray are discussed in relation to the emerging field of gas phase structural biology. Comparisons are drawn with the established area of enzymatic catalysis in organic solvents, which shares many similar challenges. Charge solvation emerges as a dominant force in both systems that must be dealt with to enable kinetic trapping of native structures in foreign environments. Potential methods for mediating unfavorable charge solvation effects are discussed and, ironically, do not include partial solvation by water. The importance of timescale in relation to the evolution of protein structure during the process of electrospray ionization is discussed. Finally several prospects for future endeavors are highlighted.