Preparative isolation and purification of anti‐tumor agent ansamitocin P‐3 from fermentation broth of Actinosynnema pretiosum using high‐performance counter‐current chromatography

Ansamitocin P‐3 is a potent anti‐tumor maytansinoid found in Actinosynnema pretiosum. However, due to the complexity of the fermentation broth of Actinomycete, how to effectively separate ansamitocin P‐3 is still a challenge. In this study, both analytical and preparative high‐performance counter‐current chromatography were successfully used to separate and purify ansamitocin P‐3 from fermentation broth. A total of 28.8 mg ansamitocin P‐3 with purity of 98.4% was separated from 160 mg crude sample of fermentation broth in less than 80 min with the two‐phase solvent system of hexane–ethyl acetate–methanol–water (0.6:1:0.6:1, v/v/v/v). The purity and structural identification were determined by HPLC, ¹H NMR, ¹³C NMR and mass spectroscopy.     

Efficient isolation and purification of five products from microbial biotransformation of cinobufagin by high‐speed counter‐current chromatography

An efficient separation method of using high‐speed counter‐current chromatography was successfully established to directly purify cytotoxic transformed products of cinobufagin by Cordyceps militaris. The two‐phase solvent system composed of n‐hexane–ethyl acetate–methanol–water (4:6:3:4, v/v) was used in high‐speed counter‐current chromatography. A total of 9 mg of 4β,12α‐dihydroxyl‐cinobufagin ( 1 ), 15 mg of 12β‐hydroxyl‐cinobufagin ( 2 ), 8 mg of 5β‐hydroxyl‐cinobufagin ( 3 ), 12 mg of deacetylcinobufagin ( 4 ) and 6 mg of 3‐keto‐cinobufagin ( 5 ) were obtained in a one‐step separation from 400 mg of the crude extract with purity of 98.7, 97.2, 90.6, 99.1 and 99.4%, respectively, as determined by HPLC. Their chemical structures were identified on the basis of ¹H‐NMR and ¹³C‐NMR technology. All products ( 1 – 5 ) showed the potent activities against human carcinoma cervicis (Hela) and malignant melanoma (A375) cells in vitro.     

Synthesis,characterization, and crystallization of thermoplastic aromatic semi‐crystalline poly(ester‐imide)s based on bis(trimellitic acid anhydride) phenyl ester

A high molecular weight aromatic homopoly(ester‐imide) (homoPEI) was synthesized from homopoly(ester‐amic acids) (homoPEAA), which was obtained from the reaction of bis(trimellitic acid anhydride) phenyl ester (BTAH) with 4‐4'‐oxydianiline ether (ODA). This homoPEI was melt‐processable semi‐crystalline polymer and displayed dual endothermic transitions which were attributed to the different levels of crystal perfection and size in the crystal structures. Four high molecular weight aromatic copoly(ester‐imide)s (coPEIs) were synthesized via copoly(ester‐amic acids)s (coPEAA) from the reaction of 4, 4'‐oxydiphthalic anhydride (ODPA) and BTAH with ODA. The molar percentage of BTAH varied from 10 to 40%. When the molar percentages of BTAH were 30 and 40%, the resulting two coPEIs were crystallinable and their melting temperatures were 361°C and 356/371°C, respectively. Differential scanning calorimetry (DSC) and wide angle X‐ray diffraction (WAXD) results indicated that the crystal structures included short segments of BTAH/ODA and ODPA/ODA. The initial crystallization of the two coPEIs took place during imidization process. Thermogravimetric (TA) and mechanical analysis confirmed that both homoPEI and coPEIs showed almost no weight loss until 400°C in N₂ and good mechanical properties. Copyright © 2009 John Wiley & Sons, Ltd.     

Phytoquinoids and Secoprezizaane‐Type Sesquiterpenes from Illicium arborescens

A phytochemical investigation of the MeOH extract of Illicium arborescens yielded the two new phytoquinoid epimers, 2,3‐didehydro‐5‐O‐methyl‐11‐epiillifunone E ( 1 ) and 2,3‐didehydro‐5‐O‐methylillifunone E ( 2 ), as well as five new sesquiterpene lactones (8,9‐secoprezizaane‐type sesquiterpenes). Two of them, i.e., 3 and 4 , were minwanensin‐type sesquiterpenes, the other two, i.e., 5 and 6 , had the anisatin‐type (or floridanolide type) skeleton, and the fifth, i.e., 7 , was a dunnianin‐type sesquiterpene. Their structures were established by analyses of 1D‐ and 2D‐NMR, HR‐MS, and chemical evidence. The in vitro cytotoxic activity of compounds 1 – 7 was tested against four human tumor cell lines, including HeLa (cervical epitheloid), WiDr (colon), Daoy (medulloblastoma), and Hep2 (liver carcinoma) human‐tumor cells.     

Monoterpenoids from the Fruit of Gardenia jasminoides

Eight new monoterpenoids, jasminoside J ( 1 ), jasminoside K ( 2 ), 6′‐O‐trans‐sinapoyljasminoside B ( 3 ), 6′‐O‐trans‐sinapoyljasminoside L ( 4 ), jasminosides M–P ( 5 – 8 ), together with three known analogues, jasminoside C ( 9 ), jasminol E ( 10 ), and sacranoside B ( 11 ), were isolated from the fruit of Gardenia jasminoides Ellis (Rubiaceae). Their structures were elucidated by spectral and chemical methods.     

Four New Terpenoids from Xylaria sp. 101

One new diterpenoid, xylarenolide ( 1 ), and three new sesquiterpenoids, xylaranol A ( 2 ), xylaranol B ( 3 ), and xylaranic acid ( 4 ), were obtained from the fungal strain Xylaria sp. 101, which was isolated from the fruiting body of Xylaria sp. collected in Gaoligong Mountain, Yunnan Province. Their structures were elucidated by spectroscopic analyses, including 1D‐ and 2D‐NMR experiments, and by HR‐Q‐TOF mass spectrometry. Their antimicrobial activities were evaluated.     

An automated capillary electrophoresis system for high-speed separation of DNA fragments based on a short capillary

A high-speed DNA fragment separation system was developed based on a short capillary and a slotted-vial array automated sample introduction system. The injection process of DNA sample in a short capillary was investigated systematically with three injection techniques including constant-field-strength, low-field-strength and translational spontaneous injections. Under the optimized conditions, picoliter-scale sample plugs (corresponding to ca. 20-μm plug length) were obtained, which ensure the high-speed and high-efficiency separation for DNA fragments with a short effective separation length. Other separation conditions including the sieving matrix concentration, separation field strength and effective separation length were also optimized. The present system was applied in the separation of ΦX174-Hae III digest DNA marker. With an effective separation length of 2.5 cm, the separation could be achieved in <100 s with plate heights ranging from 0.21 to 0.74 μm (corresponding to plate numbers from 4.86 × 10(6) to 1.36 × 10(6)/m). The repeatabilities for the migration time of the eleven fragments were between 0.4 and 1.1% RSD (n=8). By using the automated continuous injection method, the separation for four different DNA samples could be achieved within 250 s. The present system was further applied in the fast sizing of real DNA samples of PCR products.     

High-performance affinity monolith chromatography for chiral separation and determination of enzyme kinetic constants

A new kind of immobilized human serum albumin (HSA) column was developed by using the sub-micron skeletal polymer monolith based on poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) [poly(GMA-EDMA)] as the support of high-performance affinity chromatography. Using the epoxide functional groups presented in GMA, the HSA immobilization procedure was performed by two different means. The affinity columns were successfully adopted for the chiral separation of d,l-amino acids (AAs). Then this method was shown to be applicable to the quantitative analysis of d-tryptophan, with a linear range between 12.0 μM and 979.0 μM, and a correlation coefficient above 0.99. Furthermore, it was used for the analysis of urine sample. This assay is demonstrated to be facile and relatively rapid. So it allows us to measure the enzyme catalytic activity in the incubation of d,l-AAs with d-AA oxidase and to study the kinetics of the enzyme reaction. It implied that the affinity monolithic columns can be a useful tool for studying DAAO enzyme reaction and investigating the potential enzyme mechanism requirement among chiral conversion.     

Identification of methylated regions with peak search based on Poisson model from massively parallel methylated DNA immunoprecipitation-sequencing data

DNA methylation is one of the most important epigenetic modification types, which plays a critical role in gene expression. High efficient surveying of whole genome DNA methylation has been aims of many researchers for long. Recently, the rapidly developed massively parallel DNA‐sequencing technologies open the floodgates to vast volumes of sequence data, enabling a paradigm shift in profiling the whole genome methylation. Here, we describe a strategy, combining methylated DNA immunoprecipitation sequencing with peak search to identify methylated regions on a whole‐genome scale. Massively parallel methylated DNA immunoprecipitation sequencing combined with methylation DNA immunoprecipitation was adopted to obtain methylated DNA sequence data from human leukemia cell line K562, and the methylated regions were identified by peak search based on Poison model. From our result, 140 958 non‐overlapping methylated regions have been identified in the whole genome. Also, the credibility of result has been proved by its strong correlation with bisulfite‐sequencing data (Pearson R²=0.92). It suggests that this method provides a reliable and high‐throughput strategy for whole genome methylation identification.