HPLC Elution Order Research Articles

Synthesis and HPLC-ECD Study of Cytostatic Condensed O,N-Heterocycles Obtained from 3-Aminoflavanones.

Racemic chiral O,N-heterocycles containing 2-arylchroman or 2-aryl-2H-chromene subunit condensed with morpholine, thiazole, or pyrrole moieties at the C-3-C-4 bond were synthesized with various substitution patterns of the aryl group by the cyclization of cis- or trans-3-aminoflavanone analogues. The 3-aminoflavanone precursors were obtained in a Neber rearrangement of oxime tosylates of flavanones, which provided the trans diastereomer as the major product and enabled the isolation of both the cis- and trans-diastereomers. The cis- and trans-aminoflavanones were utilized to prepare three diastereomers of 5-aryl-chromeno[4,3-b][1,4]oxazines. Antiproliferative activity of the condensed heterocycles and precursors was evaluated against A2780 and WM35 cancer cell lines. For a 3-(N-chloroacetylamino)-flavan-4-ol derivative, showing structural analogy with acyclic acid ceramidase inhibitors, 0.15 μM, 3.50 μM, and 6.06 μM IC50 values were measured against A2780, WM35, and HaCat cell lines, and apoptotic mechanism was confirmed. Low micromolar IC50 values down to 2.14 μM were identified for the thiazole- and pyrrole-condensed 2H-chromene derivatives. Enantiomers of the condensed heterocycles were separated by HPLC using chiral stationary phase, HPLC-ECD spectra were recorded and TDDFT-ECD calculations were performed to determine the absolute configuration and solution conformation. Characteristic ECD transitions of the separated enantiomers were correlated with the absolute configuration and effect of substitution pattern on the HPLC elution order was determined.

High-performance liquid chromatographic and mass spectrometric analysis of fluorescent carbon nanodots

Amino/hydroxyl-functionalized fluorescent carbon nanodots (C-NanoD) are conveniently synthesized based on hydrothermal carbonization of chitosan at 180°C. Dialysis membranes with small cut-off masses (500–1000Da) were found useful for removing the side-products and low molecular mass species to purify the C-NanoD product. Herein, reversed-phase high-performance liquid chromatography (RP-HPLC) has been successfully applied to fractionate the C-NanoD product. The elution order of the C-NanoD species present in the sample follows approximately their core sizes from small to large. The separated C-NanoD fractions are collected and characterized by UV absorption spectroscopy, photoluminescence (PL) spectroscopy, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and transmission electron microscopy (TEM). All the C-NanoD fractions display a distinctive absorption band at 300nm, attributing to the n→π⁎ transition of CO bond. The PL spectra of the fractions display emission peaks at 400–415nm which are slightly red-shifted with their increase in relative molecular masses. The C-NanoD fractions are fully anatomized by MALDI-TOF MS, displaying their fragmentation mass ion features. The core sizes of some selected C-NanoD are determined as 1.6, 1.8, 2.5, and 3.1nm by TEM which are in consistent with their HPLC elution order. The findings highlight the virtues of RP-HPLC to fractionate and reveal the unique characteristics of individual C-NanoD species present in an as-synthesized C-NanoD product which may have potential applications in the fields of bioanalysis, bioimaging, catalysis, chemosensing, energy storage, and optoelectronics device.

Absolute configurations and stability of cyclic guanosine mono-adducts with glyoxal and methylglyoxal

Glyoxal and methylglyoxal are two endogenous and mutagenic 1,2-dicarbonyl compounds, which can readily form adducts with guanosine. The molecular structures of cyclic guanosine-glyoxal (G-g) and guanosine-methylglyoxal (G-mg) mono-adducts have been extensively studied before. However, diastereoisomers of these adducts have not yet been studied in detail. In this work, one pair of G-g and two pairs of G-mg diastereoisomers were baseline separated by reverse phase HPLC, whose structures were identified as the previously reported cyclic forms, and their absolute configurations were determined by circular dichroism, the octant rule, and molecular modeling. According to the HPLC elution order, configurations of two G-g (as well as trans G-mg) were (6R,7R) and (6S,7S), respectively. Meanwhile, the stability of each isomer in neutral solution was also investigated, which revealed the stability order G-g > cis G-mg > trans G-mg and also indicated distinct transformation processes for different G-mg configurations. Trans G-mg only racemized between each other, while cis G-mg transformed to both cis and trans forms. Different intermediates in the racemization processes were proposed to explain the observations. These results may shed light on further understanding the roles of these two small molecules in mutagenesis.

Preparation of Single-Enantiomer Biofunctional Molecules with (S)-2-Methoxy-2-(1-naphthyl)propanoic Acid

(RS)-beta-Ionol and (RS)-2-methyl-4-octanol were resolved by using (S)-2-methoxy-2-(1-naphthyl)propanoic acid [(S)-MalphaNP acid]. The specific stereochemistry of each MalphaNP ester was elucidated by 2D NMR analyses, and shielding by the 1-naphthyl group was observed in both the 1H- and 13C-NMR spectra. Solvolysis of the individual (S)-MalphaNP esters gave four single-enantiomer alcohols. The normal-phase HPLC elution order of each MalphaNP ester is also discussed.

Retinoid quantification by HPLC/MSn

Retinoic acid (RA) mediates most of the biological effects of vitamin A that are essential for vertebrate survival. It acts through binding to receptors that belong to the nuclear receptor transcription factor superfamily (Mangelsdorf et al. 1994). It is also a highly potent vertebrate teratogen. To determine the function and effects of endogenous and exogenous RA, it is important to have a highly specific, sensitive, accurate, and precise analytical procedure. Current analyses of RA and other retinoids are labor intensive, of poor sensitivity, have limited specificity, or require compatibility with RA reporter cell lines (Chen et al. 1995. Biochem. Pharmacol. 50: 1257–1264; Creech Kraft et al. 1994. Biochem. J. 301: 111–119; Lanvers et al. 1996. J. Chromatogr. B Biomed. Appl. 685: 233–240; Maden et al. 1998. Development. 125: 4133–4144; Wagner et al. 1992. Development. 116: 55–66). This paper describes an HPLC/mass spectrometry/mass spectrometry product ion scan (HPLC/MSn) procedure for the analysis of retinoids that employs atmospheric pressure chemical ionization MS. The retinoids are separated by normal-phase column chromatography with a linear hexane-isopropanol-dioxane gradient. Each retinoid is detected by a unique series of MSn functions set at optimal collision-induced dissociation energy (30% to 32%) for all MSn steps. The scan events are divided into three segments, based on HPLC elution order, to maximize the mass spectrometer duty cycle. The all-trans, 9-cis, and 13-cis RA isomers are separated, if desired, by an isocratic hexane-dioxane-isopropanol mobile phase.This paper describes an HPLC/MSn procedure possessing high sensitivity and specificity for retinoids.

Absolute configuration in 4-alkyl- and 4-aryl-3,4-dihydro-2(1H)-pyrimidones: a combined theoretical and experimental investigation.

Structural features (orientation of the carboxyl group, ring puckering), electronic absorption, and circular dichroism spectra of 4-alkyl- and 4-aryl-dihydropyrimidones 1-5 are calculated by semiempirical (AM1, INDO/S), ab initio (HF/6-31G, CIS/6-31G, RPA/6-31G), and density functional theory (B3LYP/6-31G) methods. These calculations allow an assignment of the absolute configuration by comparison of simulated and experimental CD spectra. Although the ab initio methods greatly overestimate electronic transition energies, the general appearance of the experimental CD spectra is quite nicely reproduced by these calculations. Thus, comparison of experimental with calculated CD spectra is a reliable tool for the assignment of the absolute configuration. For 4-methyl derivatives 1, the first enantiopure DHPM examples with no additional aromatic substituent, the stereochemistry at C4 provided by the theoretical results is confirmed by X-ray structure determination of the diastereomeric salt 6. Additional support is the consistent HPLC elution order found for all investigated DHPMs on a cellulose-derived chiral stationary phase.

(S)-homo-AMPA, a specific agonist at the mGlu6 subtype of metabotropic glutamic acid receptors.

Our previous publication (J. Med. Chem. 1996, 39, 3188-3194) described (RS)-2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid (Homo-AMPA) as a highly selective agonist at the mGlu6 subtype of metabotropic excitatory amino acid (EAA) receptors. Homo-AMPA has already become a standard agonist for the pharmacological characterization of mGlu6 (Trends Pharmacol. Sci. Suppl. 1997, 37-39), and we here report the resolution, configurational assignment, and pharmacology of (S)- (6) and (R)- (7) Homo-AMPA. Using the "Ugi four-component condensation", 3-(3-ethoxy-5-methylisoxazol-4-yl)propanal (10) was converted into the separable diastereomeric derivatives of 6 and 7, compounds 12 and 11, respectively. Deprotection of 12, in one or two steps, gave extensively racemized 6, which was converted in low yield into 6 (99.0% ee) through several crystallizations. 6 (99.7% ee) and 7 (99.9% ee) were finally obtained by preparative chiral HPLC. The configurational assignments of 6 and 7 were based on 1H NMR spectroscopic studies on 12 and 11, respectively, and circular dichroism studies on 6 and 7. Values of optical rotations using different solvents and the chiral HPLC elution order of 6 and 7 supported the results of the spectroscopic configurational assignments. The activities of 6 and 7 at ionotropic EAA (iGlu) receptors and at mGlu1-7 were studied. (S)-Homo-AMPA (6) was shown to be a specific agonist at mGlu6 (EC50 = 58 +/- 11 microM) comparable in potency with the endogenous mGlu agonist (S)-glutamic acid (EC50 = 20 +/- 3 microM). Although Homo-AMPA did not show significant effects at iGlu receptors, (R)-Homo-AMPA (7), which was inactive at mGlu1-7, turned out to be a weak N-methyl-D-aspartic acid (NMDA) receptor antagonist (IC50 = 131 +/- 18 microM).

Identification of Isomeric Naphthyl-Anthracenes Generated from the Pyrolysis of an Anthracene/Naphthalene Mixture

Abstract A 1:1 mole-ratio mixture of anthracene and naphthalene was pyrolyzed in a drop-tube furnace. the product mixture was analyzed by high performance liquid chromatography with diode-array, UV-absorption spectrometric detection (HPLC/DAD) and by gas chromatography/mass spectrometry (GCMS). In addition to previously characterized bianthracene and binaphthalene isomers, 6 isomeric naphthyl-anthracenes were identified. Because of the unavailability of standard reference compounds, structural elucidation was achieved by comparing UV spectral features, mass spectral fragmentation patterns, and GCMS and HPLC elution order with those for bianthracenes, binaphthalenes and other structurally similar molecules. In addition, steric hindrance to rotation at the naphthalene-anthracene bond, a factor that governs the degree of non-planarity of the isomers and thus influences HPLC elution order and MS fragmentation patterns, was estimated using a semi-empirical molecular orbital model.

HPLC-based method for determination of absolute configuration of alpha-chiral amines.

We introduce a novel, HPLC-based method for facile determination of the absolute configuration of alpha-chiral amines. Our method is easily applied to a variety of compounds, including amino acid derivatives. The method involves initial derivatization of the chiral amine analyte with the chiral derivatizing reagent, N-succinimidyl alpha-methoxyphenylacetate (SMPA), to produce the corresponding diastereomeric adducts. Inspection of a particular rotomer of the SMPA adduct and application of simple rules correlates absolute configuration and HPLC elution order. A key aspect of our method is that it can be used to determine absolute configuration without using enantiomeric standards of the amine analytes. Furthermore, it is of utmost significance that our method can also be used to determine absolute configuration even when only one analyte enantiomer of unknown absolute configuration is present, as is often the case for enzymatic products, naturally derived compounds, or enantiomerically enriched compounds prepared via chiral syntheses. We have observed strict adherence between predicted and observed absolute configuration for a wide variety of alpha-chiral amines. The chromatographic method we present in this paper is very practical and has several important advantages over NMR-based approaches which have been previously developed. For example, microgram quantities of an analyte in a complex enzymatic mixture can be directly analyzed by our HPLC-based method while the impurities often preclude definitive proton assignments in the NMR approach.