Fungi are the second largest group of organisms in the world after the insects. It is estimated that there are one and a half million types of fungi in existence. However, with just 5% of this total having being described , a huge, still unknown and untapped microbial pool remains, which promise the discovery of novel, useful and economically profitable compounds. Organisms such as fungi, which generally living in highly competitive environments, are considered as major producers of secondary metabolites. Fungal secondary metabolites are characterized, not only by their structural diversity , but also by diversity of biological activity.
Endophytic microorganisms are to be found in virtually every plant on earth . The most widely accepted definition of an endophyte is given by Bacon
2. New methodology for rapid isolation and identification of known bioactive polyesters
Polyester is a widely used and useful material. Its usefulness extends into many fields including the medical field, industry, textiles and as bioactive natural and synthetic compounds.
Dereplication is a process for the rapid identification of already known natural products. This is strategically important for scientists when screening crude extracts from natural sources for novel bioactive compounds. The continued demand to get new drugs to the market more quickly and more cheaply, requires that the analytical technologies that support this work keep up with, for example, the rate at which new chemical entities (NCEs) are synthesized for high-throughput screening programmes .There are numerous approaches to dereplication based on hyphenated techniques, and each has its own advantages, be they sensitivity, resolution, or scale (mg vs μg). The most common approaches are LC-UV, LC-MS, LC-MS/MS and LC-NMR, or combinations thereof, and the increasing use of capillary and cryo-NMR probes [8-16]. Of course, any technique involving mass spectrometry will always potentially suffer from problems associated with ionization (or lack thereof) of the compounds being studied. Despite this, in the pharmaceutical industry these hyphenated techniques are very powerful for the monitoring, characterization and identification of impurities . Take LC-NMR as an example. There are three main coupling technologies, on flow, stopped-flow and loop-storage. However, they all have disadvantages. On flow results in poor signal-to-noise (S/N) ratio for the NMR spectra unless a reduced flow rate is used. However, reduced flow can then reduce the effectiveness of the chromatographic separation which makes this method only suitable for the more intense signals arising from the major constituents. Stopped-flow has the advantage that a number of chromatographic peaks can be studied, but the frequent stops then necessary for data acquisition can disturb the quality of separation, and concentrated samples from the major components can contaminate the NMR detection cell. Therefore, this approach is most suitable for mixtures having only a small number of constituents. In the loop-storage mode, the chromatographic run is not interrupted; instead each analyte is stored in a separate capillary loop in order for NMR acquisition to be carried out at a later stage. A prerequisite for this technique however, is that the analyte must be stable during the long NMR analysis time .Also, it is very reliant on the sensitivity of the NMR instrument. For assisting with the sensitivity problem, the use of a cryoprobe has been a recent advancement in LC-NMR. In NMR cryoprobes, the electronic components are cryogenically cooled to ~20K while the sample remains at ambient temperature which reduces the electronic noise thus gaining a better S/N. Cryoprobes provide quite significant sensitivity gains.
2.2. Advantages of dereplication with CapNMR
In the past, without CapNMR, the dereplication process was based mainly on data from the mass and UV spectra. However, both techniques have disadvantages and are not totally reliable. The mass spectrum can contain impurities which makes it difficult to assign the correct molecular mass. Furthermore, the spectrum may be dominated by the preferential ionization of a minor component, again making it difficult to assign a correct molecular mass to the major component. The UV spectrum only gives definitive structure information for those compounds with strong chromophores. The UV library of the Marine Group within the Dionex analytical HPLC system only contains the known compounds which the group has worked on, and to date this is not yet a comprehensive collection. Furthermore, many entries in the AntiMarin database do not have reported UV data for the compound listed. However, even typical UV profiles may also give wrong answers since similar chromophore may give the same UV spectra. It is for these reasons the NMR data is so important because it can deliver definitive structural information. The AntiMarin database includes information on, for example, the number and type of methyl groups, that can be recognized from 1H NMR spectra. With the introduction of CapNMR 1H NMR spectra can be obtained from a single HPLC-MT plate collection derived from only 200-500 μg of crude extract. The 1H NMR spectrum can then provide specific structural information for searching the AntiMarin database. The most readily interpreted information available is that for methyl groups which can be described as singlets, doublets or triplets depending on their environment. Other features, such as the type of substituted benzene rings, are also easily recognized. From the HPLC separation of 200-500 μg of crude extract, a master MT plate can be prepared for the CapNMR experiments and daughter plates made for P388 bioactivity tests to locate the bioactive components and ESMS (Electrospray mass spectra)measurements. The UV profiles are obtained while collecting the MT plate. Therefore, it is now possible to determine from ~ 500 μg if a crude extract contains bioactive new compounds, or known compounds. This task can be easily completed in one or two days. Small volume NMR flow probes were first constructed in the laboratories of Sweedler and Albert in the 1990s [20-22]. These were developed for coupling to various chromatographic methods, resulting in so-called “hyphenated” techniques . They were designed especially for coupling to capillary electrophoresis (CE) and capillary HPLC (CapLC) for the detection of the small volumes associated with these techniques.
A diagrammatic view and photograph of the Protasis capillary probe NMR (CapNMR) system is presented in
2.3. Isolation of known bioactive polyesters using dereplication strategies
The natural products chemistry group at the University of Canterbury has been focused on natural products of fungal and marine origin. As well as searching for new bioactive natural products, the group has also focused on the development of new methodologies for dereplication.
Fungal extract 9PR2 which was isolated from the internal root tissue of
Endophytic extract was diluted at 1 mgL-1with methanol. 30 µL of the diluted extract was injected into HPLC. 10 % standard gradient programme was chosen with 80 % of 0.5 % formic acid plus distilled water and 20 % acetonitrile of HPLC grade.
The HPLC analysis revealed that the extract contained seven compounds. From the similarity of their UV profiles (Fig 2) the seven compounds were related. Based on the HPLC-UV profiles the assumption was made that the compounds contained a highly conjugated or aromatic system. These seven compounds were isolated from the appropriate wells in the MT plate (Fig 3) and each examined by CapNMR to obtain their 1H NMR spectra. Compound
These features were entered into the AntiMarin database, together with the supposed mass of 384 Da Two matches were found with the same structure, a polyester named 15G256V . The literature NMR data for this compound also matched those obtained for compound
The next compound examined, had signals for a doublet methyl group and for a 1,2,3,5-tetrasubstituted benzene ring in its 1H NMR spectrum. These features, together with the supposed mass 194 Da, were used in an AntiMarin search. The NMR data for a (+)-6-hydroxymellein  matched with the data for Compound
The 1H spectrum of
The next compounds
2.4. Cytotoxicity test
Fungal code 9PR2 showed excellent cytotoxicity in the P388 assay with an IC50 value 42.8 μg/mL . Extract 9PR2 was tested for cytotoxic effect against a murine leukaemic cell line P388 and incubated for 72 h after which the MTT [3-(4, 5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] assay was carried out as described in the literature , but with minor modifications. The cytotoxic activity was expressed as the mean concentration of extract required to kill 50% of the cell population (IC50).
2.5. Antifungal activity
3. Novel macrocyclic polyesters from fungus menisporopsis theobromae
In a study to uncover the chemical diversity of plant associated microorganisms, the influence of culture conditions on metabolite production of the fungal strain
The fungus was collected from Khao Yai National Park, Thailand and was fermented in peptone-yeast extract-glucose medium (PYGM) under static condition. Chemical investigation led to the isolation of a novel macrocyclic polylatone menisporopsin A (
A recent study showed that fructose instead of glucose in PYGM is more suitable as carbon source. Shaking not only enhance the production of menisporopsin but also reduced the incubation time . On the basis of these two factors fermentation of BCC 4162 was further studied which led to the isolation of new analogue, menisporopsin B (
4. Macrosphelides A and B, novel inhibitors of cell-cell adhesion molecule from microsphaeropsis sp. FO-5050
Cell adhesion molecules play important roles in various types of pathological conditions such as tumor, allergy and inflammatory diseases in the course of a screening program aimed at cell adhesion inhibitors obtained from microorganisms; macrosphelide was discovered in the fermentation broth of
Macrosphelide is a newly discovered anti-cell adhesion substance, which is, a low molecular weight, unique 16-membered macrolide antibiotics possessing three ester bonds.
4.1. Biological activities
Anti-adherent activity:Mcrosphelides were assayed in an adhesion assay system using HL-60 cells and HUVEC cells both Macrosphelide
Antimicrobial activity: The antimicrobial activity of Macrosphelides was determined by the agar dilution method using paper disks. Macrosphelides
5. Macrosphelides C and D, novel inhibitors of cell adhesion
For further research the fermentation of strain
The molecular formula of
5.1. Biological activity
Biological activities of Macrosphelides
6. Macrosphelides J (14) and K (15)
To clarify the structure-activity relationship of the Macrosphelides and to apply the results to the design of more potent inhibitors of cell-cell adhesion, more attention was devoted to the purification of other Macrosphelide derivatives from the broth of strain FO-5050 which resulted in the isolation of two new members of Macrosphelides J (
The molecular formula of J (
The molecular formula (C18H26O9) of
Biological Activities : These compounds were evaluated in an adhesion assay system using human leukemia cells (HL-60 cells) and HUVECS (human umbilical vein endothelial cells). The IC50 values of all compounds tested were greater than 100 µg/ml and did not indicate any effects on the cell growth of HL-60 and B16/BL6 melanoma when tested at 50 µg/ml.
SAR study of Macrosphelides: Macrosphelides J and K are compared to other neither showed inhibitory activity against HL-60 cells and HUVEC cells, the primary structural differences between the Macrosphelides J and K and Macrosphelides B was a double bond at C-12.
On the other hand, Macrosphelides J and K were not artifacts of Macrosphelide B, because of their stability in MeOH or EtOH solution. These results show the double bond at C-12 to be important for the inhibitory activity and the analysis can help in the development of an inhibitor of the cell-adhesion molecule.
The Marine Group’s dereplication method is a fast and reliable method for obtaining the information on compounds. An effort that would previously have taken possibly months of work, often with the unfortunate outcome of finding a known compound. This example using the 9PR2 extract illustrates just how quickly and efficiently the use of LC-UV-MS-NMR, through the intermediary of a microtitre plate and in combination with appropriate databases, can be usedto identifythe presence of known compounds starting from a very small amount of extract. The dereplication method developed by the Marine Group greatly saves the time taken to determine whether the compounds are known or unknown. Firstly, the sample collection amounts required are reduced from grams or kilograms down to milligrams only, which can provide a reduction in time from months to less than a week for working on the extracts. Furthermore, in the purification step, only several μg of compounds are required instead of the mg of compounds previously required, again reducing the time from weeks of effort to only one or two days using the MT plate HPLC method. The compounds are isolated from HPLC then can be analyzed by CapNMR and mass spectrometry to provide useful information for subsequent AntiMarin database searching to determine the novelty or otherwise of the compounds.
The author gratefully acknowledge her collaborators Lin SUN, Cole A.L.J., Blunt J.W., Munro M.H.G (Marine group) atUniversity of Canterbury, Christchurch, New Zealand, for identification and characterization of known polyesters (