Chromatographic resolution of chiral intermediates in β-adrenergic blocker synthesis on chiral stationary phases

The enantiomeric purities of optically active intermediates for β-adrenergic blocking agents prepared via enzyme-assisted processes can be determined rapidly and with high accuracy using HPLC on commercially available columns with chiral supports [Chiralcel OD, OB; Chiralpak OT(+)]. The dependence of the resolution parameters on the substitution pattern of both hydroxy compounds and their esters is reported. © 1993 Wiley-Liss, Inc.     

Comparative HPLC enantioseparation of ferrocenylalcohols on two cellulose-based chiral stationary phases

The direct HPLC enantiomeric separation of several ferrocenylalcohols on the commercially available Chiralcel OD and Chiralcel OJ columns has been evaluated in normal-phase mode. Almost all the compounds were resolved on one or both chiral stationary phases (CSPs) with separation factor (alpha) ranging from 1.06 to 2.88 while the resolution (R(s)) varied from 0.63 to 12.70 In the separation of the alpha-ferrocenylalcohols 1a-e and the phenyl analogues 2a-e, which were all resolved except 1c, a similar trend in the retention behavior for the two series of alcohols was evidenced and the selectivity was roughly complementary on the two investigated CSP. For three ferrocenylacohols, chosen as model compounds, the influence of the mobile phase composition and temperature on the enantioseparation were investigated and additional information on the chiral recognition mechanism were deduced from the chromatographic behavior of their acetylderivatives.     

Separation of albendazole sulfoxide enantiomers by chiral supercritical-fluid chromatography

The enantioseparation of albendazole sulfoxide (ABZSO) by chiral supercritical-fluid chromatography (SFC) on two columns, based on the polysaccharide derivatives Chiralpak AD and Chiralcel OD, was studied. The effect of different modifiers, methanol, ethanol, 2-propanol, and acetonitrile, was examined. The results showed that ABZSO can be separated on both columns, using an alcohol-type modifier. Using the Chiralpak AD column, the best results were obtained with 2-propanol and, in the case of the Chiralcel OD, with methanol.     

Direct measurement of enantiomeric ratios of enzymatic resolution by chiral high-performance liquid chromatography

(R)- and (S)-Methyl 2-(phenoxy)propionate and their acids could be separated simultaneously by a Chiralcel OD or OK column, while (R)- and (S)-methyl 2-(4-chlorophenoxy)propionate and their acids were separated concurrently only by an OK column. This is a novel and facile way to measure the enantiomeric excesses of the remaining substrate and product in the reaction of enzymatic resolution; enantiomeric ratios could then be calculated.     

Chiral separation of novel iminonaringenin derivatives

A series of 4‐iminonaringenin derivatives 2 ‐ 6 have been prepared in good overall yields from a condensation reaction between naringenin and primary amines. The structures of all products were confirmed by ultraviolet, infrared, proton nuclear magnetic resonance, and carbon‐13 nuclear magnetic resonance spectroscopic techniques. These derivatives were analyzed by high‐performance liquid chromatography using polysaccharide‐based chiral stationary phases, namely, Chiralpak IB and Chiralcel OD, using various mobile phases. 2‐Propanol showed a high enantioselectivity for naringin and its derivatives using achiral column containing immobilized polysaccharides (Chiralpak IB).     

Chiral separation of neonicotinoid insecticides by polysaccharide‐type stationary phases using high‐performance liquid chromatography and supercritical fluid chromatography

The enantiomeric separations of three neonicotinoid insecticides (identified as compounds 1 , 2 , and 3 ) were performed on three polysaccharide‐type chiral columns, that is, Chiralcel OD‐H, Chiralpak AD‐H, and Chiralpak IB, by high‐performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC). Effects of the modifier percentage and column temperature on chiral recognitions of chiral stationary phases were also studied. Both 1 and 2 could be resolved on all three columns selected, with the highest R_s values obtained on Chiralpak AD‐H and Chiralcel OD‐H, respectively. However, satisfactory separation of the four stereoisomers of 3 was only achieved on Chiralcel OD‐H. Considering the effects of ethanol on the values of k, α, and R_s, we concluded that hydrogen bonding, π–π, and/or dipole–dipole interactions might be all responsible for the chiral separation. In comparison to HPLC, a shorter run time was achieved for 1 and 2 by SFC. However, 3 could not be stereoselectively resolved using SFC. On the basis of the calculated thermodynamic parameters, we found that the separation processes of enantiomers of 1 and 2 were entropy controlled and enthalpy controlled, respectively. Chirality, 2011. © 2010 Wiley‐Liss, Inc.     

Simultaneous monitoring of the enantiomeric purities of both substrate and product in the enzymatic resolution of (R,S)-2-acetoxy-3-bromopropyl para-toluenesulfonate by HPLC chiral column chromatography

The enantiomeric purities of both substrate, 2-acetoxy-3-bromopropyl para-toluenesulfonate (I), and product, 2-hydroxy-3-bromopropyl p-toluenesulfonate (II) were examined in one analysis. The enzymatic resolution was conducted by Amano lipase AK and the enantiomeric excess as well as the conversion rate were monitored by HPLC analysis utilizing a Chiralcel OD column. After 7 h of reaction, HPLC results indicated that the enantiomeric purities of both substrate (I) and product (II) were greater than 95% and the conversion rate was around 55%. © 1995 Wiley-Liss, Inc.     

Semipreparative enantioseparation of Tröger base derivatives by HPLC

We describe the enantioseparation of functionalized derivatives of the Tr?ger base by HPLC on commercially available chiral stationary phases. Cellulose-derived Chiralcel OJ and brush-type Whelk O1 are demonstrated to be complementary to each other in their scope. On the basis of the results obtained, the separation of selected compounds was successfully transferred onto semipreparative columns. We believe that the availability of enantiopure functionalized derivatives of the Tr?ger base will stimulate the further use of this interesting molecular scaffold in molecular recognition, asymmetric catalysis, and related areas of research and technology.     

High-Performance liquid chromatography (HPLC) chiral separations of guaifenesin,methocarbamol, and racemorphan

HPLC chiral separations on silica gel coated with derivatized cellulose stationary phases are described. Most examples make use of the Chiralcel OD column from Daicel, Inc. With a judiciously chosen mobile phase, baseline separations of the enantiomers can be achieved. If those separations are used as a base for enantiomeric purity determination, detectable limits of 0.1% of the minor enantiomer are routinely accessible. Examples are given concerning separations of guaifenesin, methocarbamol, and racemorphan. © 1993 Wiley-Liss, Inc.     

Two-dimensional HPLC on-line analysis of phosphopeptides using titania and monolithic columns

Conventional and comprehensive two-dimensional (2D) HPLC systems using the combination of titania and monolithic columns were established for the on-line analysis of phosphopeptides. Compared with immobilized metal affinity chromatography of a general method for the analysis of phosphopeptides, the use of titania columns in the analysis permits the specific isolation of phosphopeptides in a higher yield. Using the current 2D HPLC systems, phosphopeptides were specifically isolated from nonphosphorylated peptides by the first-dimension titania column, followed by the high-speed separation of the phosphopeptides by the second-dimension monolithic column. Proteolytic digests of beta-casein were analyzed within 30 min using the comprehensive 2D HPLC system; all phosphopeptides from beta-casein could be efficiently isolated and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The comprehensive 2D HPLC system coupled with mass spectrometry will be useful for high-throughput and on-line phosphoproteome analyses.     

低浓度哇巴因对负鼠肾小管上皮细胞增殖的影响

目的:用低血清培养液来模拟肾脏供血不足的营养不良状态,研究低浓度哇巴因对低血清培养下OK细胞(负鼠肾小管上皮细胞)增殖的影响。方法:用低浓度哇巴因(1-30n M)处理0.2%血清培养下OK细胞,MTT实验和Brdu掺入法检测哇巴因对OK细胞增殖的影响;Western blot检测Akt和ERK1/2的磷酸化水平;用LY294002和PD98059分别抑制PI3K/Akt和ERK1/2蛋白激酶活性,观察抑制PI3K/Akt和ERK1/2对哇巴因促进OK细胞增殖的影响。结果:低浓度哇巴因(1-30n M)促进OK细胞的增值,上调OK细胞中Akt和ERK1/2磷酸化水平。用LY294002和PD98059特异抑制Akt和ERK1/2的活化能够抑制哇巴因的促增殖作用。结论:低浓度哇巴因(1-10n M)能够促进OK细胞的增值,PI3K/Akt和ERK1/2信号通路参与哇巴因对OK细胞促增殖作用的调节。     

High-performance liquid chromatographic enantioseparation of 1-(aminoalkyl)-2-naphthol analogs on polysaccharide-based chiral stationary phases

The enantiomers of various 1-(alpha-aminobenzyl)-2-naphthol and 1-(aminoalkyl)-2-naphthol analogs were separated on cellulose-tris-3,5-dimethylphenyl carbamate-based chiral stationary phases (Chiralcel OD-H and Chiralcel OD-RH), using n-hexane/2-propanol/diethylamine or phosphate buffer/organic modifier mobile phases. The 3,5-dimethylphenyl carbamoylated cellulose columns were effective in both normal and rev ersed-phase modes. The effects of the mobile phase composition, the pH, the buffer concentration, and the structures of the substituents on the 2-naphthol on the enantioseparations were studied. The absolute configuration and elution sequence were determined for 1-(1-amino-2-methylpropyl)-2-naphthol: the elution sequence was S < R.     

Enhancement of rat hepatic macrophages by treatment with interleukin-2 and streptococcal preparation OK432, with reference to antitumor activity,soluble factor production and Ia expression

Summary The effect of biological response modifiers, such as interleukin-2 (IL-2) and streptococcal preparation OK432, on the functions of hepatic macrophages was investigated. The macrophages, even with no exogenous stimulation, produced superoxide anion (O ₂ ^- ) and tumor necrosis factor (TNF), displayed cytotoxicity against K562 cells and cytostasis against P815 cells and expressed immune-region-associated antigen (Ia). IL-2 administered in vitro or in vivo enhanced O ₂ ^- production by hepatic macrophages and the intravenous injection of OK432 also enhanced O ₂ ^- production. Furthermore, IL-2 added to the culture medium of hepatic macrophages isolated from OK432-injected rats augmented O ₂ ^- production even more. The TNF production and Ia expression of the macrophages were also increased by the intravenous injection of OK432. As with O ₂ ^- production, the cytotoxicity of the cells was enhanced by OK432 injection or by IL-2 added to the culture medium and the combination of OK432 and IL-2 augmented their cytotoxicity even more. Thus, the present study suggested that IL-2 and OK432 induce the augmentation of the antitumor activity of hepatic macrophages, partly as a result of the increase in production of O ₂ ^- and TNF and Ia expression.     

Enantioselective chromatography and absolute configuration of N,N-dimethyl-3-(naphthalen-2-yl)-butan-1-amines: potential sigma1 ligands

We describe the preparation of racemic N,N-dimethyl-3-(naphthalen-2-yl)-butan-1-amines, potential sigma1 ligands, and their resolution via chiral HPLC. In order to obtain enantiopure compounds, direct chromatographic methods of separation using chiral stationary phases were investigated. Different methods suitable for both analytical and semipreparative purposes are proposed. The best resolutions were achieved using cellulose tris (3,5-dimethylphenyl carbamate) (Chiralcel OD and OD-H) and amylose tris (3,5-dimethylphenyl carbamate) (Chiralpak AD). On the basis of the preliminary chromatographic results, the resolution of compound 1 was transferred onto a Chiralcel OD semipreparative column. The enantiomers were obtained in high enantiomeric excess. The configurational assignment was performed by circular dichroism. Computational analysis was used to explore the enantioselective recognition process of compound 1 with the Chiralcel OD stationary phase.     

3′-Fluorine-Substitution in 3-Fluorocarbocyclic Oxetanocin A Augments the Drug's Inhibition of HHV-6B Propagation in Chronically Infected Cultures

An infection of TaY cells, which originated from an adult T-cell leukemia, with an HHV-6B OK isolate resulted in a chronically infected culture, termed TaY(OK). Cell cloning analysis revealed that the TaY(OK) culture consisted of a mixture of cells permissive and refractory to the infection, and that the permissive cells were continuously produced from the refractory cell population. Since the chronically infected culture has been maintained for over 2 years without the addition of uninfected TaY cells, we used it for an evaluation of the antiviral potency of nucleoside analogs, especially carbocyclic oxetanocins (COXTs). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assays showed a lack of toxicity of ganciclovir (GCV), COXTs, and their derivatives, to TaY(OK) cells at 1 μm . Therefore we compared the antiviral potencies of these drugs at 1 μm by monitoring the viral loads produced during a 1-day period during the course of the drug treatment. Among the drugs tested, 3′-fluorocarbocyclic oxetanocin A (3′-C.OXT-A) was the most effective for inhibiting the virus production, and at concentrations ranging from 0.5 μm to 10 μm , the inhibition of the viral production was dose-dependent. A comparison of the chemical structures of the derivatives with that of C.OXT-A, which is the parental molecule, suggested that the 3′-fluorine-modification might account for the higher anti-HHV-6 activity and lower cytotoxicity.     

Characteristics of transport of selenoamino acids by epithelial amino acid transporters

Selenoamino acids are the main form of organic selenium derived from the diet. They are efficiently absorbed in the intestine and reabsorbed in kidney, but the transporter proteins that mediate their cellular uptake have not yet been identified. We here describe the transport pathways of selenoamino acids and derivatives, including selenomethionine, methylselenocysteine, selenocystine, selenobetaine and selenocystamine. Transport studies employed the Xenopus laevis oocyte system expressing the amino acid transporters SIT1, b^0,+rBAT, B⁰ or PAT1 and intestinal Caco-2 and renal OK cell lines that possess a multitude of amino acid transporters. Our results suggest that the major route for the uptake of selenomethionine is the system b^0,+ rBAT in Caco-2 cells and B⁰ in OK cells. Affinity of selenomethionine or methionine for these transporters did not differ, but for SIT1 selenomethionine shows a higher affinity than methionine. Methylselenocysteine displayed a higher affinity than cysteine for all transporters tested and in both OK and Caco-2 cells, system B⁰ seems to be the primary uptake route. Selenocystine is taken up well by the b^0,+ rBAT system, while selenobetaine is a low-affinity substrate only for SIT1 and PAT1. Selenocystamine was not transported by any of the transport systems investigated. When cells were exposed to selenoamino acids, intracellular selenium levels in OK cells considerably exceeded those in Caco-2 cells, indicating effective renal reabsorption capacity. In conclusion, selenoamino acids but not the seleno-derivatives selenobetaine and selenocystamine, are effectively transported by various intestinal and renal amino acid transporters and are thus available for selenium metabolism and therapeutic approaches.     

Direct HPLC separation of enantiomers of pantoprazole and other benzimidazole sulfoxides using cellulose-based chiral stationary phases in reversed-phase mode

A direct, isocratic, and simple reversed-phase HPLC method was described for the separation of enantiomers of the proton pump inhibitor, rac-pantoprazole (PAN) using cellulose-based chiral stationary phases (Chiralcel OD-R and Chiralcel OJ-R). Some structurally related chiral benzimidazole sulfoxides, rac-omeprazole (OME) and raclansoprazole (LAN), were also studied. Chiralcel OJ-R was successful in the resolution of enantiomers of rac-PAN and rac-OME, while Chiralcel OD-R was most suitable for resolving the enantiomers of rac-LAN. Highest enantioselectivity to rac-PAN and rac-OME was achieved on Chiralcel OJ-R by using acetonitrile as an organic modifier, whereas methanol afforded better resolution of rac-LAN on Chiralcel OD-R than acetonitrile. Increases in buffer concentration and column temperature decreased retention and did not improve the resolution of the enantiomers on both columns. Using a mixture of 50 mM sodium perchlorate solution and acetonitrile as a mobile phase at a flow rate of 0.5 ml/min, maximum separation factors of 1.26 and 1.13 were obtained for the enantiomers of rac-PAN and rac-OME using a Chiralcel OJ-R column, while maximum separation factor of 1.16 was obtained for the enantiomers of rac-LAN using a Chiralcel OD-R column. © 1995 Wiley-Liss, Inc.     

Uptake of leptin and albumin via separate pathways in proximal tubule cells

The adipokine leptin and oncotic protein albumin are endocytosed in the proximal tubule via the scavenger receptor megalin. Leptin reduces megalin expression and activates cell signalling pathways that upregulate fibrotic protein expression. The aim of this study was to investigate if leptin uptake in proximal tubule cells was via the albumin-megalin endocytic complex. In immortalised proximal tubule Opossum kidney cells (OK) fluorescent leptin and albumin co-localised following 5 min exposure, however there was no co-localisation at 10, 20 and 30 min exposure. In OK cells, acute exposure to leptin for 2 h did not alter NHE3, ClC-5, NHERF1 and NHERF2 mRNA. However, acute leptin exposure increased NHERF2 protein expression in proximal tubule cells. In OK cells, immunoprecipitation experimentation indicated leptin did not bind to ClC-5. Leptin uptake in OK cells was enhanced by bafilomycin and ammonium chloride treatment, demonstrating that uptake was not dependent on lysosomal pH. Thus, it is likely that two pools of megalin exist in proximal tubule cells to facilitate separate uptake of leptin and albumin by endocytosis.     

Congenic diabetes-prone BB.Sa and BB.Xs rats differ from their progenitor strain BB/OK in frequency and severity of insulin-dependent diabetes mellitus.

Two newly established congenic diabetes-prone BB rat strains designated BB.Sa and BB.Xs carrying a region of chromosome 1 (Sa-Lsn-Secr-Igf2-Tnt, 16 cM) and a region of chromosome X (DXMgh3-Mycs/Pfkb1-Ar, 36 cM) of the SHR rats, respectively, were studied to determine whether the transferred chromosomal regions influence diabetes frequency, age at onset, and clinical picture. Therefore, 4 complete litters of BB/OK (n = 43), BB.Sa (n = 45), and BB.Xs (n = 41) were observed for diabetes occurrence up to the age of 30 weeks. From these litters 6 diabetic males of each strain manifesting in an interval of 1 week were chosen to study body weight, blood glucose, insulin requirement to survive, and several diabetes-related serum constituents at onset of diabetes and after a diabetes duration of 150 days. The diabetes frequency was significantly lower in BB.Xs than in rats of the parental strain BB/OK, whereas comparable frequencies were found between BB/OK and BB.Sa rats. Obvious differences were observed 150 days after diabetes onset between BB/OK and both BB.Sa and BB.Xs rats. BB/OK rats were significantly heavier and needed significantly more insulin/100 g body weight than BB.Sa and BB.Xs rats. Comparisons of the serum constituents as lipids, proteins, and minerals revealed significant differences between diabetic BB/OK rats and their diabetic congenic derivatives in several traits studied at onset and after 150 days of insulin treatment. These results not only show the power of congenic lines in diabetes research, but indicate for the first time that there are genetic factors on chromosomes 1 and X influencing frequency and severity of diabetes in the BB/OK rat.     

Direct high-performance liquid chromatography (HPLC) separation of etodolac enantiomers using chiral stationary phases

The enantiomers of the antiinflammatory drug Etodolac were separated without derivatization on Chiralcel OD and Pirkle (R)-DNBPG columns. Enantiomeric purity can be determined in less than 10 min. Optimization of separation was evaluated using various concentrations of 2-propanol (doped with TFA) in hexane as the mobile phase. © 1993 Wiley-Liss, Inc.