RESUMO
Predicting whether a chiral column will be effective is a daily task for many analysts. Moreover, finding the best chiral column for separating a particular racemic compound is mostly a matter of trial and error that may take up to a week in some cases. In this study we have developed a novel prediction approach based on combining a random forest classifier and an optimized discretization method for dealing with enantioselectivity as a continuous variable. Using the optimization results, models were trained on data sets divided into four enantioselectivity classes. The best model performances were achieved by over-sampling the minority classes (α ≤ 1.10 and α ≥ 2.00), down-sampling the majority class (1.2 ≤ α < 2.0), and aggregating multicategory predictions into binary classifications. We tested our method on 41 chiral stationary phases using layered fingerprints as descriptors. Experimental results show that this learning methodology was successful in terms of average area under the Receiver Operating Characteristic curve, Kappa indices and F-measure for structure-based prediction of the enantioselective behavior of 34 chiral columns.
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The use of a coulometric array detector in tandem with HPLC-UV was evaluated for the absolute quantitation of pharmaceutical compounds without standards, an important capability gap in contemporary pharmaceutical research and development. The high-efficiency LC flow-through electrochemical detector system allows for the rapid evaluation of up to 16 different potentials, aiding in the identification and quantitation of electrochemically reactive species. By quantifying the number of electrons added or removed from an analyte during its passage through the detector, the number of moles of the analyte can be established. Herein we demonstrate that molecules containing common electroactive functional groups (e.g. anilines, phenols, parabens and tertiary alkyl amines) can in some cases be reliably quantified in HPLC-EC-UV without the need for authentic standards. Furthermore, the multichannel nature of the CoulArray detector makes it well suited for optimizing the conditions for electrochemical reaction, allowing the impact of changes in potential, flow rate, temperature and pH to be conveniently studied. The electrochemical oxidation of albacivir, zomepirac, diclofenac, rosiglitazone and several other marketed drugs resulted in large linear ranges, predictable recoveries and excellent quantitation using the total moles of electrons and back-calculating using Faraday's law. Importantly, we observed several instances where subtle structural changes within a given class of molecules (e.g. aromatic ring isomers) led to unanticipated changes in electrochemical behavior. Consequently, some care should be taken when applying the technique to the routine quantitation of compound libraries where standards are not available.
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An extract of Eudistoma sp. provided eudistidine C (1), a heterocyclic alkaloid with a novel molecular framework. Eudistidine C (1) is a racemic natural product composed of a tetracyclic core structure further elaborated with a p-methoxyphenyl group and a phenol-substituted aminoimidazole moiety. This compound presented significant structure elucidation challenges due to the large number of heteroatoms and fully substituted carbons. These issues were mitigated by application of a new NMR pulse sequence (LR-HSQMBC) optimized to detect four- and five-bond heteronuclear correlations and the use of computer-assisted structure elucidation software. Synthesis of eudistidine C (1) was accomplished in high yield by treating eudistidine A (2) with 4(2-amino-1H-imidazol-5-yl)phenol (4) in DMSO. Synthesis of eudistidine C (1) confirmed the proposed structure and provided material for further biological characterization. Treatment of 2 with various nitrogen heterocycles and electron-rich arenes provided a series of analogues (5-10) of eudistidine C. Chiral-phase HPLC resolution of epimeric eudistidine C provided (+)-(R)-eudistidine C (1a) and (-)-(S)-eudistidine C (1b). The absolute configuration of these enantiomers was assigned by ECD analysis. (-)-(S)-Eudistidine C (1b) modestly inhibited interaction between the protein binding domains of HIF-1α and p300. Compounds 1, 2, and 6-10 exhibited significant antimalarial activity against Plasmodium falciparum.
Assuntos
Alcaloides/síntese química , Compostos Heterocíclicos de 4 ou mais Anéis/síntese química , Biologia Marinha , Urocordados/química , Alcaloides/química , Animais , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Cromatografia Líquida de Alta Pressão , Compostos Heterocíclicos de 4 ou mais Anéis/química , Estrutura Molecular , Espectroscopia de Prótons por Ressonância Magnética , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Multiple-injection techniques have been shown to be a simple way to perform high-throughput analysis where the entire experiment resides in a single chromatogram, simplifying the data analysis and interpretation. In this study, multiple-injection techniques are applied to gas chromatography with flame ionization detection and mass detection to significantly increase sample throughput. The unique issues of implementing a traditional "Fast" injection mode of multiple-injection techniques with gas chromatography and mass spectrometry are discussed. Stacked injections are also discussed as means to increase the throughput of longer methods where mass detection is unable to distinguish between analytes of the same mass and longer retentions are required to resolve components of interest. Multiple-injection techniques are shown to increase instrument throughput by up to 70% and to simplify data analysis, allowing hits in multiple parallel experiments to be identified easily.
RESUMO
The market of protein therapeutics is exploding, and characterization methods for proteins are being further developed to understand and explore conformational structures with regards to function and activity. There are several spectroscopic techniques that allow for analyzing protein secondary structure in solution. However, a majority of these techniques need to use purified protein, concentrated enough in the solution to produce a relevant spectrum. In this study, we describe a novel approach which uses ultrahigh pressure liquid chromatography (UHPLC) coupled with mass-spectrometry (MS) to explore compressibility of the secondary structure of proteins under increasing pressure detected by hydrogen-deuterium exchange (HDX). Several model proteins were used for these studies. The studies were conducted with UHPLC in isocratic mode at constant flow rate and temperature. The pressure was modified by a backpressure regulator up to about 1200 bar. It was found that the increase of retention factors upon pressure increase, at constant flow rate and temperature, was based on reduction of the proteins' molecular molar volume. The change in the proteins' molecular molar volume was caused by changes in protein folding, as was revealed by differential deuterium exchange. The degree of protein folding under certain UHPLC conditions can be controlled by pressure, at constant temperature and flow rate. By modifying pressure during UHPLC separation, it was possible to achieve changes in protein folding, which were manifested as changes in the number of labile protons exchanged to deuterons, or vice versa. Moreover, it was demonstrated with bovine insulin that a small difference in the number of protons exchanged to deuterons (based on protein folding under pressure) could be observed between batches obtained from different sources. The use of HDX during UHPLC separation allowed one to examine protein folding by pressure at constant flow rate and temperature in a mixture of sample solution with minimal amounts of sample used for analysis.
Assuntos
Cromatografia de Fase Reversa , Medição da Troca de Deutério , Pressão , Conformação Proteica , Animais , Bradicinina/química , Bovinos , Citocromos c/química , Citocromos c/metabolismo , Cavalos , Humanos , Insulina/química , Espectrometria de Massas , Mioglobina/química , Dobramento de Proteína , Temperatura , Uracila/químicaRESUMO
In recent years, the use of halogen-containing molecules has proliferated in the pharmaceutical industry, where the incorporation of halogens, especially fluorine, has become vitally important for blocking metabolism and enhancing the biological activity of pharmaceuticals. The chromatographic separation of halogen-containing pharmaceuticals from associated isomers or dehalogenation impurities can sometimes be quite difficult. In an attempt to identify the best current tools available for addressing this important problem, a survey of the suitability of four chromatographic method development platforms (ultra high-performance liquid chromatography (UHPLC), core shell HPLC, achiral supercritical fluid chromatography (SFC) and chiral SFC) for separating closely related mixtures of halogen-containing pharmaceuticals and their dehalogenated isosteres is described. Of the 132 column and mobile phase combinations examined for each mixture, a small subset of conditions were found to afford the best overall performance, with a single UHPLC method (2.1 × 50 mm, 1.9 µm Hypersil Gold PFP, acetonitrile/methanol based aqueous eluents containing either phosphoric or perchloric acid with 150 mM sodium perchlorate) affording excellent separation for all samples. Similarly, a survey of several families of closely related halogen-containing small molecules representing the diversity of impurities that can sometimes be found in purchased starting materials for synthesis revealed chiral SFC (Chiralcel OJ-3 and Chiralpak IB, isopropanol or ethanol with 25 mM isobutylamine/carbon dioxide) as well as the UHPLC (2.1 × 50 mm, 1.8 µm ZORBAX RRHD Eclipse Plus C18 and the Gold PFP, acetonitrile/methanol based aqueous eluents containing phosphoric acid) as preferred methods.
Assuntos
Química Farmacêutica/métodos , Contaminação de Medicamentos , Halogenação , Halogênios/análise , Cromatografia Líquida de Alta Pressão/métodos , Halogênios/química , IsomerismoRESUMO
In this study we describe the evaluation of a recently developed supercritical fluid chromatography (SFC) instrument for automated chiral SFC method development. The greatly improved gradient dwell volume and liquid flow control of the new instrument in combination with the use of shorter columns containing smaller stationary phase particles affords chiral SFC method development that is faster and more universal than previous systems.
Assuntos
Cromatografia/métodos , Estereoisomerismo , Estilbenos/químicaRESUMO
Bombesin receptor subtype 3 (BRS-3) is an orphan G-protein coupled receptor expressed primarily in the hypothalamus which plays a role in the onset of both diabetes and obesity. We report herein our progress made towards identifying a potent, selective bombesin receptor subtype-3 (BRS-3) agonist related to the previously described MK-7725(1) Chobanian et al. (2012) that would prevent atropisomerization through the increase of steric bulk at the C-2 position. This would thereby make clinical development of this class of compounds more cost effective by inhibiting racemization which can occur over long periods of time at room/elevated temperature.
Assuntos
Benzodiazepinas/química , Desenho de Fármacos , Receptores da Bombesina/agonistas , Sulfonamidas/química , Sulfonamidas/síntese química , Animais , Humanos , Camundongos , Ligação Proteica , Ratos , Receptores da Bombesina/metabolismo , Estereoisomerismo , Sulfonamidas/farmacocinética , TemperaturaRESUMO
A simple approach to the automated screening of four different columns on a single gas chromatography (GC) instrument is used for rapid chiral GC method development. Configuration of a conventional GC instrument with a second autosampler and several inexpensive Y-splitters enables simultaneous evaluation of two different columns, allowing a total of four different columns to be evaluated in two automated back to back runs. The resulting system affords a simple and effective approach to chiral GC method development that speeds analysis while eliminating the need for slow and tedious manual interchange of columns. An example of developing a rapid isothermal GC method from the screening results obtained by the instrument is also shown.
Assuntos
Cromatografia Gasosa/métodos , Cromatografia Gasosa/instrumentação , EstereoisomerismoRESUMO
The emergence of new therapeutic modalities requires complementary tools for their efficient syntheses. Availability of methodologies for site-selective modification of biomolecules remains a long-standing challenge, given the inherent complexity and the presence of repeating residues that bear functional groups with similar reactivity profiles. We describe a bioconjugation strategy for modification of native peptides relying on high site selectivity conveyed by enzymes. We engineered penicillin G acylases to distinguish among free amino moieties of insulin (two at amino termini and an internal lysine) and manipulate cleavable phenylacetamide groups in a programmable manner to form protected insulin derivatives. This enables selective and specific chemical ligation to synthesize homogeneous bioconjugates, improving yield and purity compared to the existing methods, and generally opens avenues in the functionalization of native proteins to access biological probes or drugs.
Assuntos
Insulina , Penicilina Amidase , Peptídeos , Engenharia de Proteínas , Sequência de Aminoácidos , Humanos , Insulina/análogos & derivados , Insulina/biossíntese , Lisina/química , Penicilina Amidase/química , Penicilina Amidase/genética , Peptídeos/química , Peptídeos/genética , Engenharia de Proteínas/métodosRESUMO
Calculation of chromatographic enantioselectivity (α) is critically important in enantioselective chromatographic method development studies. The fact that α can only be calculated from isocratic elution conditions, whereas gradient elution conditions are predominantly used in method development screening, presents some problems for the use of α as a scoring indicator for automated, intelligent enantioselective chromatography method development systems. In this study, an empirical algorithm was developed to estimate α at isocratic conditions based upon information collected from a gradient elution. The algorithm was validated for SFC applications and has been shown to accurately predict enantioselectivity for a wide variety of racemic test analytes eluted on different chiral column and mobile phase conditions.
Assuntos
Algoritmos , Celulose/análogos & derivados , Técnicas de Química Analítica/métodos , Cromatografia Líquida/métodos , Cromatografia com Fluido Supercrítico/métodos , Bibliotecas de Moléculas Pequenas/química , EstereoisomerismoRESUMO
The physical properties of active pharmaceutical ingredients (API) are critical to both drug substance (DS) isolation and drying operations, as well as streamlined drug product (DP) processing and the quality of final dosage units. High aspect ratio, low bulk density, API 'needles' in particular are a hindrance to efficient processing, with a low probability that conventional crystallization routes can modify the challenging morphology. The compound evaluated in this manuscript demonstrated this non-ideal morphology, with the added complexity of shear sensitivity. Modest shear exposure resulted in conversion of the thermodynamically stable crystalline phase to the amorphous phase, with the amorphous phase then undergoing accelerated chemical degradation. Slow filtration during DS isolation resulted in uncontrolled and elevated amorphous levels, while subsequent DP operations including blending, densification and compression increased amorphous content still further. A chemically stable final dosage unit would ideally involve a high bulk density, free flowing API that did not require densification in order to be commercialized as an oral dosage form with direct encapsulation of a single dosage unit. Despite every effort to modify the crystallization process, the physical properties of the API could not be improved. Here, an innovative isolation strategy using a thin film evaporation (TFE) process in the presence of a water soluble polymer alleviated filtration and drying risks and consistently achieved a high bulk density, free flowing co-processed API amenable to direct encapsulation. Characterization of the engineered materials suggested the lower amorphous levels and reduced shear sensitivity were achieved by coating surfaces of the API at relatively low polymer loads. This particle engineering route blurred conventional DS/DP boundaries that not only achieved improved chemical stability but also resulted in a optimized material, with simplified and more robust processing operations for both drug substance and drug product.
Assuntos
Química Farmacêutica , Preparações Farmacêuticas , Cristalização , Composição de Medicamentos , Estabilidade de Medicamentos , Polímeros , ÁguaRESUMO
A novel family of atropisomers based on a conformationally constrained seven membered ring system is investigated using a combination of preparative chiral chromatography, circular dichroism, and other analytical techniques. The influence of structure on the rate of atropisomer interconversion was explored with a series of analogs showing a range of interconversion rates ranging from very fast (undetectable on the HPLC timescale) to very slow (half life of many days).
Assuntos
Antipsicóticos/química , Antipsicóticos/classificação , Sulfonamidas/química , Sulfonamidas/classificação , Cromatografia Líquida de Alta Pressão/métodos , Dicroísmo Circular/métodos , Meia-Vida , Conformação Molecular , Estrutura Molecular , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The small column size (0.3 mm i.d. x 15 cm) used in microscale HPLC contains only a small fraction (<1%) of the chromatographic packing material of a typical analytical HPLC column. Consequently, chromatographic stationary phases that are prohibitively expensive in conventional HPLC, owing either to synthetic complexity or costly starting materials, may become commercially viable in the microscale format. To illustrate this point, a previously described, synthetically complex, crown ether chiral stationary phase was prepared and evaluated in the microscale format, showing excellent separation of the enantiomers of underivatized amine analytes.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Aminas/química , Aminas/isolamento & purificação , Técnicas de Química Analítica/métodos , Éteres de Coroa/síntese química , Éteres de Coroa/química , Indicadores e Reagentes , Microquímica/métodos , EstereoisomerismoRESUMO
The suitability of the Eksigent Express 800 microfluidic eight-channel HPLC instrument for multiparallel normal-phase chiral analysis in support of high-throughput pharmaceutical process research was investigated. Analysis of test mixtures containing the two enantiomers of benzoin and the closely related (R,S)-dihydrobenzoin, was carried out in a 96-well microplate, affording rapid (<2 h) and accurate assessment of enantiopurity. In a second example, use of the instrument to support high-throughput catalyst screening of the asymmetric hydrogenation of a prochiral unsaturated ester is presented, in which method development (gradient screening of four columns and two eluents, followed by optimization to afford a fast analytical method) and analysis of a 96-well microplate was carried out within a single working day. This represents a considerable improvement over conventional analysis techniques that usually take several days to complete.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Microfluídica/métodos , Benzoína/química , Hidrogenação , Microfluídica/instrumentação , Estrutura Molecular , Reprodutibilidade dos Testes , EstereoisomerismoRESUMO
Recent developments in fast chromatographic enantioseparations now make high throughput analysis of enantiopurity on the order of a few seconds achievable. Nevertheless, routine chromatographic determinations of enantiopurity to support stereochemical investigations in pharmaceutical research and development, synthetic chemistry and bioanalysis are still typically performed on the 5-20 min timescale, with many practitioners believing that sub-minute enantioseparations are not representative of the molecules encountered in day to day research. In this study we develop ultrafast chromatographic enantioseparations for a variety of pharmaceutically-related drugs and intermediates, showing that sub-minute resolutions are now possible in the vast majority of cases by both supercritical fluid chromatography (SFC) and reversed phase liquid chromatography (RP-LC). Examples are provided illustrating how such methods can be routinely developed and used for ultrafast high throughput analysis to support enantioselective synthesis investigations.
RESUMO
The use of NMR chiral solvating agents (CSAs) for the analysis of enantiopurity has been known for decades, but has been supplanted in recent years by chromatographic enantioseparation technology. While chromatographic methods for the analysis of enantiopurity are now commonplace and easy to implement, there are still individual compounds and entire classes of analytes where enantioseparation can prove extremely difficult, notably, compounds that are chiral by virtue of very subtle differences such as isotopic substitution or small differences in alkyl chain length. NMR analysis using CSAs can often be useful for such problems, but the traditional approach to selection of an appropriate CSA and the development of an NMR-based analysis method often involves a trial-and-error approach that can be relatively slow and tedious. In this study we describe a high-throughput experimentation approach to the selection of NMR CSAs that employs automation-enabled screening of prepared libraries of CSAs in a systematic fashion. This approach affords excellent results for a standard set of enantioenriched compounds, providing a valuable comparative data set for the effectiveness of CSAs for different classes of compounds. In addition, the technique has been successfully applied to challenging pharmaceutical development problems that are not amenable to chromatographic solutions. Overall, this methodology provides a rapid and powerful approach for investigating enantiopurity that compliments and augments conventional chromatographic approaches.
RESUMO
ChirBase, a database for the chromatographic separation of enantiomers containing more than 200,000 records compiled from the literature, was used to develop quantitative structure activity models for the prediction of which chiral stationary phase will work for the separation of a given molecule. Constructuion of QSAR models for the enantioseparation of nineteen chiral stationary phases was attempted using only analyte structural information, leading to the producton of self-consistent models in four cases. These models were tested by predicting which in-house racemic compounds would and would not be resolved on the different columns. Some degree of success was observed, but the sparseness of data within ChirBase, which contains enantioseparations for only a subset of molecules on a subset of columns under a variety of conditions may limit the creation of effective models. Augmented data sets gleaned from automated chromatographic method development systems deployed in academic and industrial research laboratories or the use of models that take other factors such as solvent composition, temperature, etc. into account could potentially be useful for the development of more robust models.
Assuntos
Técnicas de Química Analítica/métodos , Cromatografia , Modelos Químicos , Bases de Dados Factuais , Solventes , Estereoisomerismo , Relação Estrutura-Atividade , TemperaturaRESUMO
Averaging of chromatograms can lead to enhancement of signal to noise ratio (S/N) in proportion to the square root of the number of measurements. Although the general principle has been known for decades, chromatogram averaging is almost never used in current pharmaceutical research. In this study we explore the utility of this approach, showing it to be a simple and easily accessible method for boosting sensitivity for quantification of minor components and trace impurities, where current techniques deliver insufficient S/N.
Assuntos
Cromatografia Líquida de Alta Pressão , Alanina/química , Alanina/isolamento & purificação , Humanos , Razão Sinal-Ruído , EstereoisomerismoRESUMO
Commercially available ultra high pressure liquid chromatography (UHPLC) equipment offers the ability to explore the influence of backpressure on chromatographic separations. However, the influence of pressure on the chromatographic separation of enantiomers on chiral stationary phases remains largely unexplored. In this investigation we surveyed the effects of pressure on the separation of enantiomers using several reversed-phase chiral stationary phases. The experiments were conducted at constant flow rate and column temperature, using isocratic conditions. The only variable parameter was pressure, which was adjusted using a post-column backpressure regulator. For the separation of enantiomers on chiral stationary phases, an increase in pressure from approximately 2,000 psi (138 bar) to approximately 8,000 psi (552 bar) at constant flow rate and temperature led to an increase of retention factors for some analytes and a decrease for others. Achiral separations on a C-18 stationary phase always led only to an increase of retention factor. Interestingly, changes in pressure led to small changes in enantioselectivity during reversed-phase chiral separation of enantiomers, suggesting that such studies may be of value for better understanding the mechanisms underlying chromatographic enantioseparation.