Parallel Chromatography in Natural Products Chemistry: Isolation of New Secondary Metabolites from Streptomyces sp

Integration of parallel chromatography on both, gel permeation and silica gel chromatography by making use of the CombiFlashTM si1000s system (ISCO, Lincoln, USA) into purification process to speed up the isolation of secodary metabolites from microorganisms. As an example, we applied this approach to Streptomyces sp. (GT 061089) which led to the isolation and structural characterization of six 2-3-disubstituted butanoids (1 to 6), four 2,4disubtituted butanoids (7 to 10), a monoterpenoid (11), two indol compounds (12, 13), a furan-3-carboxylic acid (14), as well as two already known isocoumarins (15, 16). The isolated pure compounds were characterized by spectroscopic methods and chemical transformations. The results of biological tests showed that both 15 and 16 possess medium cytotoxic activity and strong inhibiting activity on horse radish peroxidase. 15 also exhibits antiviral activity as well as a distinct inhibiting activity on 3a-hydroxysteroid dehydrogenase (3a-HSD).


Introduction
The search for new pharmacologically active agents obtained from natural sources has led to the discovery of many clinically useful drugs that play a major role in the treatment of human diseases. Numerous examples impressively demonstrated the innovative potential of natural products and their impact on the progress in the drug discovery and development [1,2,3] . However, natural products research as a part of drug discovery effort faces increasing challenges: how to improve diversity and quality of sample sources and reduce incidence of false positive and interfering material in biological screening attempts, how to accelerate dereplication; and automatic sample preparation and isolation. A new technology based on solid-phase-extraction (SPE) and the automation concept of the CyBi TM -Xtract (CyBio AG, Jena, Germany) focused on the preparation of high-quality samples from natural origin which fulfilled quality and quantity of the high throughput screening (HTS) [4] . The next challenge is to speed up the subsequent isolation and structure characterization procedure of striking compounds from the crude extracts. In consequence, we used parallel chromatography approach for purification of several natural products simultaneously.

Isolation and Parallel Chromatography Approach
After harvesting, the culture filtrate was passed through a Amberlite-XAD 16 column and eluted with water/methanol (gradient from 20% to 70% methanol, then 100% methanol) to yield three fractions. Figure 1 and Figure 2 showed the process of separation and purification of the first two fractions. After first chromatography of this two fractions on silica gel columns yielded three and two enriched fractions, respectively. This five fractions were then separated by parallel gel permeation chromatography on Sephadex LH-20 (five columns: 2.5 × 50 cm, Methanol, 0.5 ml/min) using CombiFlash TM si1000s system. The combined fractions were further purified by parallel chromatography on silica gel (five columns: 1.1 × 30 cm, n-hexane/EtOAc, gradient from 4:1 to 2:1) or/and RP-C18 HPLC (2.5 × 25 cm, 7 mm, MeOH/H2O) (Figure 1 and 2) to obtain 0.37 mg/l of 1, 0.15 mg/l of 2, 0.36 mg/l of 3, 0.19 mg/l of 4, 0.03 mg/l of 5, 0.05 mg/l of 6a and 6b, 0.04 mg/l of 7, 0.13 mg/l of 8, 0.06 mg/l of 9, 0.10 mg/l of 10, 0.30 mg/l of 11, 0.15 mg/l of 12, 0.08 mg/l of 13, 0.05 mg/l of 14, 0.30 mg/l of 15, 0.10 mg/l of 16. 1.0 g of 15 (1.0 mg/l) was obtain from the third fraction after extraction with EtOAc and re-crystallized in methanol.  The isolated pure compounds were characterized spectroscopically. The molecular formulae were determined by mass spectrometry and the structures were elucidated by both, detailed analysis of the 1 H-, 13 C-, 1 H-1 H-, and 1 H-13 C-shift correlation NMR-spectra, and chemical transformations.
The relative stereochemistry of 2 and 3 was assigned by analysis of the NOESY NMR data. An NOE effect observable between H-3 (d 2.74) and H-6 (d 4.12) in the NOESY spectrum of 2 indicates a syn-facial position of 2-OH and 3-hydroxymethyl group. The syn-substituted pattern in the tetrahydrofurane ring in 2 was deduced from the NOE effect between H-6 (d 4.12) and H-9 (d 3.96).
The correlation signal between H-3 (d 2.65) and H-7 (d 1.95) in the NOESY spectrum of 3 indicates the syn-facial position of 2-OH and 3-hydroxymethyl group, which is identical to that in 2. However, the NOE effect observed between H-6 (d 4.21) and the methyl group (d 1.11) points to the anti-substituted pattern in the tetrahydrofurane ring, which is instead of the syn-substituted pattern in 2 and agreement with the optical rotation values {2: [a] D = + 7.4 (c = 3.51, methanol); 3: Table 3. 13 C-NMR data of the compounds 1 to 5, 7 to 10 and 17.
Proton -proton connections arose from both, a comparison of coupling constants, and a 1 H-1 H COSY spectrum. It reveals two segments: Carbon and proton signal assignments resulted from a 2D 1 H-13 C correlation spectrum. The connections of the proton attached carbon atoms with the quaternary carbon atoms are demonstrated by appropriate correlation in the HMBC spectrum data and lead to the constitution of 4.

5-(1-Hydroxyethyl)-4´-hydroxymethyl-tetrahydro-[2,3´]bifuranyl-2´-one (5):
The molecular formula, C 11 H 16 O 5 , was determined from the HREI-MS spectrum of 5 (m/z = 228.1020, calcd. 228.0998) and supported by its ESI-MS spectrum. The IR spectrum shows the presence of hydroxyl group (3325 cm -1 ) and a,b-unsaturated carbonyl group (1721, 1659cm -1 ). The 1 H-and 13 C -NMR (300 MHz, CD 3 OD) spectra show signals of 14 protons and 11 carbons, respectively ( Table 2 and Table 3). A comparison of NMR data of 2 and 5 shows the closely structural similarities. The difference is the presence of a double bond between C -2 (d = 95.0) and C -6 (d = 173.7) in 5. This causes the downfield-shift of 7-H 2 (from d H = 2.27/2.00 in 2 shifting to d H = 3.20/2.98 in 5). It seems that 2 lose the 2-OH and the 6-H to form an a,b-unsaturated ester and yielded the dehydrated product 5. Two dimensional correlation [COSY, HSQC, HMBC] allowed assignments of all proton and carbon resonance and fully confirmed this hypothesis. Thus,    (Table 4 and Table 3) indicate their identically partial structure expect an isopropyl group in 8 instead of an isobutyl group in 7. This is conformed by detailed interpretation of 2D NMR ( 1 H-1 H COSY, HSQC and HMBC) data of 8. Identical coupling constants of H-2, H-3, H-4 and H-5 in 7 to those in 8 indicate the same relative stereochemistry, trans-2,4-disubstituted, in the g-lactone moiety.

2-(1,4-Dihydroxy -4-methyl-pentyl)-4-hydroxymethyl-butanolide (9):
Combination of the results from HREI-MS and ESI-MS spectra of 9 revealed the molecular formula C 11 H 20 O 5 , possessing one more oxygen atom in comparison to 8. The IR, 1 H-NMR, and 13 C -NMR spectra of 8 and 9 (Table 4 and Table 3 In comparison to 8, the analysis of the 2D-NMR spectra led to the constitution of 9 as depicted in Scheme 2. The trans relative orientation of the substituent groups at C -2 and C -3 in the g-lactone ring was proposed due to the comparable coupling constants of 8 and 9.

2-(1,4-Dihydroxy -4-methyl-hexyl)-4-hydroxymethyl-butanolide (10):
Compound 10 possesses a molecular formulae C 12 H 22 O 5 which was determined by HREI-MS and ESI-MS and shows one more oxygen atom in comparison to 7. A comparison of IR, 13 C -and 1 H-NMR spectra (Table 4 and Table 3) of 10 and 7 led to the structure of 10 as depicted in Scheme 2. This is conformed by the analysis of 2D-NMR data of 10. From the biosynthesis view, 10 was suggested to possess a trans-2,4-disubstituted pattern in the g-lactone ring, which is supported by the comparable coupling constants of 7 and 10.

Biological Activities
Ten compounds (1 to 4, 8, 10 to 12, 15 and 16) were tested in a number of biological tests [7] (antibacterial, antifungal, antiviral, cytotoxic and enzyme assays) and was found to be inactive except that both 15 and 16 showed medium cytotoxic activity and strong inhibiting activity on horse radish peroxidase. 15 also exhibited antiviral activity as well as a distinct inhibiting activity on 3a-hydroxysteroid dehydrogenase (3a-HSD). Three 2,3-disubstituted butanolides (1, 2 and 4) and two 2,4-disubstituted butanolides (8 and 9) were also tested in an A-factor assay [11] , but have been found inactive.

Discussion
Parallel chromatography allows to separate several samples simultaneously and rapidly shortens the time of isolation procedure. It will play vital role in High-Throughput-Isolation of natural products and improve competitiveness of natural products with synthetic and combinatorial libraries in drug discovery process. Integration of parallel chromatography on both, gel permeation and silica gel chromatography by making use of the CombiFlash TM si1000s system (ISCO, Lincoln, USA) into purification procedure allowed us rapidly to isolate sixteen secondary metabolites belong to different classes compounds from Streptomyces sp. (GT 061089). It shows that parallel chromatography is efficient approach for speeding up the process of purification natural products.
However, there are still some problems which has to be solved. Parallel chromatography generates hulk fractions which have to be examined by TLC, because on-line detection system is not possible to integrate into parallel chromatography at present. Automatic TLC spot system will partially solve this problem. The CombiFlash TM si1000s system (ISCO) requires the isolated samples possessing similar polarity because of one solvent system for all columns. A new parallel system called Biotage Quad3 TM (Biotage UK Limited, Hertford, UK) can run up to 12 pre-parked cartridges, in parallel. The advantage of the system is that each cartridge has its own pump head, which allows to use different solvent system for different sample. The number of fractions of each column per run of both parallel chromatography systems are limited, typically 20 to 40 fractions per column. This might make parallel chromatography to be not suitable for the first isolation step because the extracts of microorganisms or plants are usually complex mixtures.

Experimental Section
General method. See ref. [6,7] . Parallel chromatography: CombiFlash -10 (ISCO). Fermentation: A 1 cm 2 slant of agar from 7 d old cultures of GT 061089 grown on medium A was used to inoculate a 300-ml Erlenmeyer flask containing 100 ml of medium B. The flask was cultivated for 6 d at fermentor containing medium B (duration of fermentation: 5 d, at 28 ° C, 500 rpm, aeration 10 l/min).
The third fraction (elution of second part from 100% methanol, 10 l) was dried (30 g) and was extracted with EtOAc (5 l) to give 10 g of viscous material after evaporated the solvent. To this material 200 ml of EtOAc was added and filtered. The residue was re-crystallized in methanol to yield 1.0 g of 15.