RESUMO
The immunosuppressant drug rapamycin is a complex polyene-containing natural product which undergoes autoxidation. The resulting product mixtures contained numerous monomeric and oligomeric compounds, which represented challenges for addressing mass balance in forced degradation studies and in analysis of aged developmental drug-eluting stents. A combination of SEC with ultraviolet and refractive index detection and RP-HPLC was used to account for drug loss and product formation. The mass balance methodology was subsequently validated for the determination of rapamycin and composite rapamycin autoxidation product material in developmental stent samples. This mass balance approach may find general applicability in other situations where drugs degrade to a plethora of products, which cannot be determined individually and summed.
Assuntos
Imunossupressores/química , Sirolimo/química , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Dimerização , Stents Farmacológicos , Imunossupressores/análise , Estrutura Molecular , Oxirredução , Refratometria/métodos , Sirolimo/análise , Espectrofotometria Ultravioleta/métodosRESUMO
Competitive interactions of ochratoxin A (OTA) and several other acidic compounds were utilized to gain insight into the localization of binding sites and the nature of binding interactions between anionic species and human serum albumin (HSA). Depolarization of OTA fluorescence in the presence of a competing anion was used to quantify ligand-protein interactions. The results obtained were rationalized in terms of OTA displacement from its major binding site. Based on their ability to displace OTA, two distinct groups of the anionic ligands were revealed. The first group contained structurally diverse compounds that shared a common binding site in subdomain IIA (Sudlow Site I). The second group consisted of three non-steroidal anti-inflammatory drugs, which showed much lower affinity to Site I than the OTA dianion. The major site for these drugs was located in domain III. Fluorescence spectroscopy measurements of OTA, warfarin (WAR) and naproxen (NAP) complexes with recombinant proteins corresponding to the domains of HSA (D1-D3) revealed binding to all domains but with different affinities. The binding constants for OTA and WAR decreased in the series D2z.Gt;D3>D1. In contrast, NAP showed the most favorable interaction with D3 and comparable affinities to the two remaining domains. The OTA binding constant for D2, 7.9 x 10(5) M(-1), was smaller than the largest constant for HSA by a factor of approximately 7. The binding constant for OTA with D3, 1.1 x 10(5) M(-1), was very close to that of the secondary binding site for HSA.
Assuntos
Ocratoxinas/metabolismo , Fragmentos de Peptídeos/metabolismo , Albumina Sérica/metabolismo , Ânions/metabolismo , Ligação Competitiva , Polarização de Fluorescência , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Naproxeno/metabolismo , Ocratoxinas/química , Fragmentos de Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Albumina Sérica/química , Varfarina/metabolismoRESUMO
The immunosuppressant drug rapamycin, also known as Sirolimus, underwent autoxidation under mild conditions to give numerous monomeric and oligomeric compounds, which were generally characterized by size-exclusion chromatography and NP-HPLC with UV and MS detection. Some of the more predominant products, epoxides and ketones, were isolated and identified. Two epoxides and 10S-epimer of rapamycin were described for the first time. Observed rapamycin isomers were also addressed. Computational chemistry was used to provide mechanistic insights. Formation of the majority of the rapamycin products could be rationalized with free radical-mediated autoxidation reactions involving alkene and alcohol sites. Methodological aspects of oxidative stress testing are discussed.
Assuntos
Compostos de Epóxi/isolamento & purificação , Imunossupressores/análise , Cetonas/isolamento & purificação , Sirolimo/análise , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Dimerização , Estabilidade de Medicamentos , Imunossupressores/química , Isomerismo , Espectrometria de Massas , Estrutura Molecular , Oxirredução , Sirolimo/químicaRESUMO
Effects of sterilization by electron beam (E-BEAM) on paclitaxel (1) mixed with poly(DL-lactide-co-glycolide) (PLG) in reservoirs of COSTAR Stents are examined by using liquid chromatography-mass spectrometry (LC-MS-MS) techniques with information-dependent acquisition (IDA). Numerous degradation products of 1 are formed in a ß-radiation dose-dependent manner to give plethora of low-level degradants. This behavior, together with multiple interferences from PLG-related compounds, creates considerable challenges for analysis of the drug/PLG mixtures. IDA methods with different survey scans are proven to be very efficient in elucidating degradation pathways and in identifying numerous products. Combined LC-MS-MS results from analysis of sterilized drug substance and stents indicate that water addition and oxidative processes together with the isomerization are largely responsible for degradation of 1 under E-BEAM sterilization conditions used.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Stents Farmacológicos , Paclitaxel/química , Esterilização/métodos , Espectrometria de Massas em Tandem/métodos , Estabilidade de Medicamentos , Elétrons , Oxirredução , Paclitaxel/administração & dosagem , Paclitaxel/efeitos da radiação , ÁguaRESUMO
Spin-unrestricted calculations and time-dependent DFT were used to characterize structure and reactivity of 1-methyl-8-nitronaphthalene (1) in the triplet state. Four hybrid models (B3LYP, PBE0, MPW1K, BHLYP) with significantly different amount of the exact exchange were employed. The triplet potential energy surface of 1 was mapped by using the UB3LYP and UMPW1K techniques. Both hybrid models provided qualitatively consistent pictures for the potential energy landscape. Thirty-one stationary points, of which 15 were minima, were found at the UB3LYP level of theory. Three minima corresponding to the nitro form of 1 were located on the triplet surface; just one was found for the singlet ground state. Two reaction paths leading from 1 either to a nitrite-type intermediate (2) or to the aci-form (3) were characterized. For both paths, reaction products were of diradical nature. The lower activation energy was obtained for the triplet-state tautomerization affording 3. The ground state of triplet multiplicity was predicted for two isomers of the aci-form. The triplet diradical 3 is expected to react through the thermal population of a close-lying singlet excited state. The results are discussed in relation to mechanisms of photoinduced rearrangements of peri-substituted nitronaphthalenes that can be used to develop novel photolabile protecting groups.
RESUMO
Irradiation of 2-nitrobenzyl alcohol (1, R = H) and 1-(2-nitrophenyl)ethanol (1, R = Me) in various solvents yields 2-nitroso benzaldehyde (4, R = H) and 2-nitroso acetophenone (4 R = Me), respectively, with quantum yields of about 60%. The mechanism of this reaction, known since 1918, was investigated using laser flash photolysis, time-resolved infrared spectroscopy (TRIR), and 18O-labeling experiments. The primary aci-nitro photoproducts 2 react by two competing paths. The balance between the two depends on the reaction medium. Reaction via hydrated nitroso compounds 3 formed by proton transfer prevails in aprotic solvents and in aqueous acid and base. In water, pH 3-8, the classical mechanism of cyclization to benzisoxazolidine intermediates 5, followed by ring opening to carbonyl hydrates 6, predominates. The transient intermediates 3 and 6 were identified by TRIR. Potential energy surfaces for these reactions were mapped by density functional calculations.
Assuntos
Álcoois Benzílicos/síntese química , Nitrobenzenos/química , Compostos Nitrosos/química , Concentração de Íons de Hidrogênio , Lasers , Estrutura Molecular , Nitrobenzenos/síntese química , Compostos Nitrosos/síntese química , Fotoquímica , Fotólise , Prótons , Teoria Quântica , Espectrofotometria Infravermelho/métodos , Raios UltravioletaRESUMO
The mechanism of methanol photorelease from 2-nitrobenzyl methyl ether (1) and 1-(2-nitrophenyl)ethyl methyl ether (2), and of ATP release from adenosine-5'-triphosphate-[P(3)-(1-(2-nitrophenyl)ethyl)] ester ('caged ATP', 3) was studied in various solvents by laser flash photolysis with UV-vis and IR detection. In addition to the well-known primary aci-nitro transients (A, lambda(max) approximately 400 nm), two further intermediates preceding the release of methanol, namely the corresponding 1,3-dihydrobenz[c]isoxazol-1-ol derivatives (B) and 2-nitrosobenzyl hemiacetals (C), were identified. The dependencies of the reaction rates of A-C on pH and buffer concentrations in aqueous solution were studied in detail. Substantial revision of previously proposed reaction mechanisms for substrate release from 2-nitrobenzyl protecting groups is required: (a) A novel reaction pathway of the aci-tautomers A prevailing in buffered aqueous solutions, e.g., phosphate buffer with pH 7, was found. (b) The cyclic intermediates B were identified for the first time as the products formed by the decay of the aci-tautomers A in solution. A recently proposed reaction pathway bypassing intermediates B (Corrie et al. J. Am. Chem. Soc., 2003, 125, 8546-8554) is shown not to be operative. (c) Hemiacetals C limit the release rate of both 1 (pH < 8) and 2 (pH < 10). This observation is in contrast to a recent claim for related 2-nitrobenzyl methyl ethers (Corrie et al.). Our findings are important for potential applications of the 2-nitrobenzyl protecting group in the determination of physiological response times to bioagents ('caged compounds').
Assuntos
Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Metanol/química , Éteres Metílicos/química , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Conformação Molecular , Fotólise , Espectrofotometria Ultravioleta , Água/químicaRESUMO
Proton-transfer reactions in aqueous solutions of poly(methacrylic acid) (PMA) were studied using a fluorescent probe and Fourier transform infrared (FTIR) spectroscopy. Protolytic photodissociation of 1-hydroxypyrene (HP) in water was found to be very slow. The PMA polyanion appeared to be very inefficient as a proton acceptor in the excited-state reaction with HP. However, a drastic increase in the deprotonation efficiency was observed in PMA solutions with the same pH values close to neutral when dodecyltrimethylammonium chloride (DTAC) was added. The protonated form of HP, as well as its anion, was shown to be solubilized in polyion-covered micelles. Time-resolved fluorescence data suggested at least two localization sites with different reactivities toward PMA. FTIR spectroscopy was used to quantify the degree of ionization of PMA in PMA-DTAC mixtures. The IR data indicated that protolytic dissociation of PMA could be well described by the Henderson-Hasselbach equation with an apparent pK of 6.6. In contrast, the fluorescent data revealed cooperative protonation of the PMA groups interacting with HP localized within surfactant assemblies. This selective protonation at a pH close to neutral may be associated with a conformational transition in the polymer-surfactant complex.
RESUMO
Ochratoxins are a class of naturally occurring compounds produced by several fungi. The most toxic is ochratoxin A (OTA), and occurrence of some human nephropathies and tumors correlate with enhanced OTA exposure. In this Account, the following areas are examined: molecular details of the binding of OTA to human serum albumin (HSA), the influences of binding to HSA on the trans-port of OTA across epithelial cell membranes by organic anion transport proteins, the oxidative activation of OTA, and the formation of OTA adducts with biological molecules. These studies are beginning to provide a detailed chemical model for the trans-port, accumulation, and genotoxic and carcinogenic effects of OTA.