Isolation and characterization of LS1924A, a new analog of emycins

Abstract

In the course of our screening program for new bioactive compounds from our natural product library,1 a number of ‘talented’ strains were discovered based on their bioactivity spectrum or novel peaks on liquid chromatography-diode array detection-mass spectrometry (LC-DAD-MS) analysis. A Streptomyces sp. LS1924 strain was found to have the ability to produce bioactive metabolites and was selected to be studied further leading to the discovery of LS1924A (1), a new analog of emycins, together with three known compounds of angucyclinone family. In this paper, we report the fermentation, isolation, chemical characterization of these compounds and the bioactivity of the new compound. The producing strain Streptomyces sp. LS1924 was isolated from a soil sample collected from Yaoli Virgin Forest (elevation 850 m) of Jiangxi Province, China. Strain LS1924 was deposited at the China General Microbiological Culture Collection Center (CGMCC) with the accession number CGMCC No.5240. The strain was grown and maintained on a GT-medium agar slant consisting of soluble starch 2.0%, L-asparagine 0.05%, KNO3 0.1%, K2HPO4 !H2O 0.05%, NaCl 0.05% and MgSO4 ! 7H2O 0.05% (pH 7.5) at 28 1C. The stock culture was transferred into 250-ml Erlenmeyer flasks containing 40 ml of the seed medium with the same components as the agar slant medium. The culture was incubated on a rotary shaker (220 r.p.m.) at 28 1C for 4 days. Ten milliliters of the seed culture was transferred to 1000-ml Erlenmeyer flasks containing 250 ml of the producing medium, which contains glucose 1.0%, millet meal 2.0%, cottonseed protein powder 2.0% and MOPS 2.0% (pH 7.0). The fermentation broth was incubated at 28 1C for 8 days on a rotary shaker at 220 r.p.m. The fermentation broth (10 l) was fractionated by centrifugation. The mycelium was extracted with methanol and the supernatant was applied on a Diaion HP-20 resin (Mitsubishi Chem. Ind., Co., Ltd., Tokyo, Japan) column (1 l), which was then washed with deionized water (3 l), and the bioactive components were subsequently eluted with ethanol. The organic phases from the mycelium and the HP-20 resins were combined and then evaporated to give a general extract (15 g). The general extract was then chromatographed on a column of silica gel (300 ml, Qingdao Silica Manufactory, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) first with petroleum ether/ acetone gradient from 90:10 to 50:50 and later with chloroform/ methanol gradient from 90:10 to 50:50 and two bioactive fractions (petroleum ether/acetone 80:20 and chloroform/methanol 90:10) were obtained. Fraction 1 was chromatographed on a silica gel column (300 ml, Qingdao Silica Manufactory) again with petroleum ether/acetone gradient elution from 98:2 to 80:20, resulting in three sub-fractions. The purification of sub-fraction 1 on a column of sephadex LH-20 (1.5" 100 cm, Pharmacia. Uppsala, Sweden) with chloroform/methanol (1:1) elution afforded pure 2 (11.5 mg). Subfraction 2 was chromatographed on a column of sephadex LH-20 (1.5" 100 cm, Pharmacia) with chloroform/methanol (1:1), followed by preparative high-performance liquid chromatography on a SB-C18 column (9.4" 250 mm, 5mm, Agilent, Santa Clara, CA, USA) with methanol/water (95:5) as mobile phase at 2 ml min#1 to give pure 3 (6.0 mg). Subfraction 3 was chromatographed on a column of LH-20 (1.5" 100 cm, Pharmacia), developed with chloroform/methanol (1:1) and the active fraction was finally purified by preparative high-performance liquid chromatography on a SB-C18 column (9.4" 250 mm, 5mm, Agilent) with methanol/water (65:35) as mobile phase at 2 ml min#1 to yield pure 1 (3.2 mg). Fraction 2 was applied on a silica gel column with petroleum ether/acetone gradient elution from 90:10 to 50:50 and a subsequent purification for the bioactive fraction by preparative high-performance liquid chromatography on a SB-C18 column (9.4" 250 mm, 5mm, Agilent) with methanol/water (30:70) at 2 ml min#1 to give pure 4 (6.7 mg).