High resolution screening of plant natural product extracts for estrogen receptor alpha and beta binding activity using an online HPLC-MS biochemical detection system.

A new screening technology that combines biochemical analysis with the resolution power of high-performance liquid chromatography (HPLC), referred to here as high-resolution screening (HRS) technique, is described. The capability of the HRS technology to analyze biologically active compounds in complex mixtures is demonstrated by screening a plant natural product extract library for estrogen receptor (ER) alpha and beta binding activity. The simultaneous structure elucidation of biologically active components in crude extracts was achieved by operating the HRS system in combination with mass spectrometry (MS). In contrast to conventional microtiter-type bioassays, the interactions of the extracts with the ER and the employed label, coumestrol, proceeded at high speed in a closed, continuous-flow reaction detection system, which was coupled directly to the outlet of a HPLC separation column. The reaction products of this homogeneous fluorescence enhancement-type assay were detected online using a flowthrough fluorescence detector. Primary screening of the extract library was performed in the fast-flow injection analysis mode (FlowScreening) wherein the chromatographic separation system was bypassed. The library was screened at high speed, using two assay lines in parallel. A total of 98% of the identified hits were confirmed in a traditional 96-well microplate-based fluorescence polarization assay, indicating the reliability of the FlowScreening process. Active extracts were reassayed in a transcriptional activation assay in order to assess the functional activity of the bioactive extracts. Only functional active extracts were processed in the more time-consuming HRS mode, which was operated in combination with MS. Information on the number of active compounds, their retention times, the molecular masses, and the MS/MS-fingerprints as a function of their biological activity was obtained from 50% of the functional active extracts in real time. This dramatically enhances the speed of biologically active compound characterization in natural product extracts compared to traditional fractionation approaches.

Application of liquid chromatography-nuclear magnetic resonance spectroscopy to the identification of natural products.

LC-NMR combines the separation power of high-performance liquid chromatography (HPLC) with the superior structural information content of nuclear magnetic resonance (NMR). These two techniques traditionally have been the primary tools used by natural products chemists to isolate and determine the structures of molecules of interest. Recent advances in NMR technology have allowed the practical application of LC-NMR, thus providing natural products chemists with a hyphenated technique which combines the two most important tools in their field. A brief review of the literature describing how LC-NMR has been applied to natural products research is followed by a specific example illustrating how this technique was used to identify the marine alkaloid aaptamine (1). Aaptamine was identified as the active component in the crude dichloromethane extract of the sponge Aaptos sp. which had been determined to possess inhibitory activity against the enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT) by a high throughput screening (HTS) effort. Isolated aaptamine (1) exhibited an IC50 = 120 microM against this enzyme. The experience gained from the identification of aaptamine was used to define a strategy for the use of LC-NMR in a natural products HTS program.

LC-NMR: a new tool to expedite the dereplication and identification of natural products.

The rapid identification of known or undesirable compounds from natural products extracts - "dereplication" - is an important step in an efficiently run natural products discovery program. Dereplication strategies use analytical techniques and database searching to determine the identity of an active compound at the earliest possible stage in the discovery process. In the past few years, advances in technology have allowed the development of tandem analytical techniques such as liquid chromatography mass spectrometry (LC-MS), LC-MS-MS, liquid chromatography nuclear magnetic resonance (LC-NMR), and LC-NMR-MS. LC-NMR, despite its lower sensitivity as compared to LC-MS, provides a powerful tool for rapid identification of known compounds and identification of structure classes of novel compounds. LC-NMR is especially useful in instances where the data from LC-MS are incomplete or do not allow confident identification of the active component of a sample. LC-NMR has been used to identify the marine alkaloid aaptamine as the active component in an extract of the sponge Aaptos sp. This extract had been identified as an enzyme inhibitor by a high throughput screening (HTS) effort. Isolated aaptamine exhibited an IC(50)=120 µM against this enzyme. Strategies for the identification of aaptamine and for the use of LC-NMR in a natural products HTS program are discussed.

Chemistry and chemical ecology of the Bahamian spongeAplysilla glacialis.

Chemical investigation of the secondary metabolites of the marine spongeAplysilla glacialis collected at French Wells, Crooked Island, Bahamas, resulted in the isolation and characterization of four diterpenes, two sterol endoperoxides, and two methylated primary metabolites. Some of these compounds, along with crude extracts of the sponge, were investigated for their ability to deter fish predation, affect the fouling of surfaces, and inhibit the growth of marine microorganisms. The diterpene manoöl (3), cholesterol endoperoxide (4), and the crude nonpolar extract ofA. glacialis were shown to deter feeding by a natural assemblage of fish predators in an in situ assay conducted at French Wells. Pure secondary metabolites and crude extracts ofA. glacialis also were tested in a laboratory fish feeding assay employing the wrasseThalassoma lunare. A mixture of sterol endoperoxides was isolated from the mucus that coats the surface ofA. glacialis and is exuded in large quantities when the sponge is disturbed. These compounds are thereby distributed in a manner in which they can best serve a defensive role for the sponge. An in situ assay was designed to determine the effect that pure secondary metabolites and crude extracts have on the fouling of surfaces. Manoöl (3) and cholesterol endoperoxide (4) were determined to increase the rate of fouling when compared to control surfaces. 1-Methyladenine (5) was identified as an antimicrobial constituent ofA. glacialis that inhibited the growth of four marine bacteria isolated from seawater samples collected at French Wells.

Diterpenes from the Pohnpeian marine sponge Chelonaplysilla sp.

Nine diterpenes have been isolated from the marine sponge Chelonaplysilla sp. collected in Pohnpei, Federated States of Micronesia. These include the known diterpenes 1-bromo-8-ketoambliol A acetate, dendrillolide A, dendrillolide D, norrisolide, 12-desacetoxyshahamin C, and aplyviolene. Three novel rearranged spongian diterpenes, chelonaplysins A, B, and C, were identified by interpretation of spectral data and chemical correlation with known compounds. Aplyviolene, chelonaplysin B, and chelonaplysin C exhibited antimicrobial activity against the bacterium Bacillus subtilis. The secondary metabolite composition of this collection of Chelonaplysilla sp. is compared to the composition previously reported for samples from Palau.

Identification of okadaic acid from a Caribbean dinoflagellate, Prorocentrum concavum.

Lipid-soluble toxins were isolated from a Caribbean strain of the epiphytic dinoflagellate Prorocentrum concavum Fukuyo. The major lipid-soluble toxin (LD50 = 210 +/- 15 micrograms/kg i.p. in mice) was purified by normal and reversed-phase column chromatography and characterized by 1H NMR and mass spectrometry. The toxin was identified as okadaic acid by interpretation of the spectral data. Okadaic acid was previously identified as a toxic component of the related species P. lima (Ehrenberg) Dodge. The finding of okadaic acid production in P. concavum and P. lima, abundant primary producers in the ciguatera-endemic Caribbean, suggests that the role of this toxin in the etiology of ciguatera may be more significant than previously thought.