Ylindrospermopsin Alkaloids - Research Paper Example

? CHEMISTRY PAPER HYPOTHESES: The incidence of cylindrospcermopsin is very common in many countries worldwide and exclusively in fresh water bodies. The reason why researchers are so interested in this phenomenon is because of its biological aspect which includes a wide array of disruption and toxic levels of metabolic pathways. Another aspect of this compound which is of measurable interest to our research group is its isolation, as possibility for the alteration of the natural product in order to increase accessibility towards finding a more precise and even therapeutic activity is of a great degree. AIMS AND OBJECTIVES: The continuing research under the discipline of synthetic chemistry on the cylindrospermopsin alkaloids has attracted great interest from researchers and the community, alike. As a direct result of this increasing interest, the development and culturing of blue-green algae’s (cyanobacteria) naturally occuring species which create these alkaloids has also become an area of curiosity. Listed below are the three aims of this project: I) The execution of of bioreactor growth technology in cooperation with Merlin Biodevelopment Ltd. at the School of Chemistry in Bangor. II) Thorough implementation of this technology to cylindrospermopsin.which is produced by cyanobacteria III) Using preparative HPLC, ELISA antibody testing and affinity chronography to separate and recognize natural products in large numbers. ABSTRACT: The purpose of this report is to carry out a purposeful evaluation and analysis of the current information that is available on the accumalation of on cylindrospermopsin. It also focuses on making use of the information available on on the worldwide network of cylindrospermopsin producers as well as providing a summary of its human and ecological effects. In order to carry out this task in the most effective manner the report has been created by making use of research conducted on the bioaccumulation of cylindrospermopsin and systematically assessing the information in the said reports and research. The paper also contains a discussion related to the factors that influence both bioaccumalation rates as well as potential. The initial part of this research paper will determine production of cylindrospermopsin in one type of cyanobacteria, that is known as Anabaena. CHAPTER 1: INTRODUCTION The description of cylindrospermopsin alkaloids is such that they consist of three soluble water toxins that comprise of cylindrospermopsin (CYN) (1), 7-deoxy-cylindrospermopsin (7-deoxy-CYN) (2) and also 7-epi-cylindrospermopsin (7-epi-CYN) (3). Researchers have signaled towards certain problems related to the toxicological characteristics of these natural compounds, their existence in water and which was followed by their removal. The structural features of these alkaloids are in fact extraordinary comprising of a sulfonated tricyclic guanidine linked to a uracil ring. The beginning of this analysis centers on the isolation of these innate products followed by the investigation into the environmental impact of cylindrospermopsin alkaloids contamination. (1) Figure 1. The cylindrospermopsin alkaloids of cylindrospermopsin (1),7-deoxy-cylindrospermopsin (2) and 7-epi-cylindrospermopsin (3). 1.1 ISOLATION AND CHARACTERIZATION Credited to Moore and his team using a combination of NMR and mass spetroscopy, the extraction of Cylindrospermopsin (1) took place in 1992 from the cyanobacterium Cylindrospermopsis raciborskii. While 7-deoxy-cylindrospermopsin (2) was discovered in 1999, owing to the purification of cylindrospermopsin which was performed on a regular basis. It was also concluded that the derivate of cylindrospermopsin could also exist in the form of a pair of tautomers because of the fact that the uracil ring’s vinylic proton was not identified during the application of the H NMR technique as shown in figure 2. However, as every sample of 7-deoxy-cylindrospermopsin (2) was observed the incidence of the uracil group (4) was confirmed by examining the absorbance maximum (?max). Even though, the measurement of approximate quantity was not possible, it was evident that as a mixture of compounds the natural material indeed existed. Fig 2 Proposed tautomeric forms of 7-deoxy-cylindrospermopsin The discovery of the 7-epi-cylindrospermopsin (3) took place in 2002 and it was originally established that it was synthesized with Aphanizomenon ovalisporum(5). The approximation of cylindrospermopsin’s relative stereochemistry was made by considering the unusualtautomer enol presence of the uracil D ring. To explain the concept, uracil D as described in structure 6 is a intramolecularly hydrogen linked to a nitrogen terminus of the guanidine group. The NMR evidence was made to be the basis of the product correlation which understandably led the research participants to conclude that structure 7 was the most appropriate and relevant description of cylindrospermopsin (1). Deduction of the alkaloids as uracil tautomers aided in the establishment of the respective structures of cylindrospermopsin (1) and 7-epi-cylindrospermopsin (2). Figure 3. Projected structures of cylindrospermopsin (1) and 7-epi-cylindrospermopsin(3) CHAPTER 2: EXPERIMENTAL 2.1.1 Isolation of CYN using charcoal filtration method: According to numerous studies that have been conducted pertaining to the CYN, research has suggested that its isolation can be accomplished by certain absorption techniques using a number of substrates supplies. It has also been found that Powdered Activated Charcoal or PAC is of use in eradicating CYN from the water with results suggesting that it can lower the levels of CYN concentration in water to 1 microgram per liter from values as substantial as 25-50 microgram per liter. However, the factors that determine the level of concentration are the source of the water along with the amount of Dissolved Organic Carbon at hand. Further experiments also suggest that by making use of activated charcoal, CYN can also be isolated from PAC, if a CYN: PAC ratio of 1:100 is established for the process. Reseachers have discovered certain limitations of the use of activated charcoal however, of which the most important drawback is that it can lead to the formation of biofilm in water treatment plants if used for isolation of CYN. The formation of the biofilm is known to intervene greatly with the isolation process by hampering the ability of CYN to absorb. Furthermore, as biofilm is not biodegradable that can also pose as a major problem. 2.1.1 Purification of Cylindrospermopsin: CYN requires an effective extraction procedure to acquire its purest form as it most commonly exists in dissolved fraction. It has been observed that more dependable results of CYN extraction and purification have been found by making use of carbon as in, in SPE cartridges and graphitized carbon. Another process that has been suggested is that of extraction using freeze dried cells. The process involves the usage of 10 grams of freeze dried cells that are dissolved in methanol solution of 500 millilitres. Once, extracted the separation of methanol from the cells occurs by putting the solution in centrifuge. Then flash evaporation allows the methanol to be separated from cells followed by the dilution of the cells in 100 milliliteres of acetic acid of 0.1 M concentration. After this the solution is processed through a diatomaceous earth bed of 150 mililiter and for elution the solution undergoes a carbograph column using a 5% formic acid solution that contains methanol. Once eluted, a HPLC purification on a C18 column allows the pure form of the CYN compound to be obtained. In recent times, Norris et al has formulated a procedure to make the graphitized carbon based SPE more efficient by focusing more on the aspects of how the sample is prepared and the process involving solvent selection. This procedure in its optimized form had the objective of achieving large quantities of CYN. The method revolved around the separation of the cells through a centrifugation process which then allowed the supernatant and the concentrate to be used in a range of experiments. This extraction of the CYN cells is attained with 5% formic acid with 0.9% saline solution and an ultrasonication step involved in at two minute intervals in 70% duty cycles. The CYN present in the supernatant solution is then purified via filtration with the procedure of recovery conducted by using carbograph cartridges in triples. The final elution in a particular solvent succeeds the process of passing the sample through a step involving a washing which is 5 milliliter per minute and finally another round of the same process but in distilled water. It has been discovered that washing process works most appropriately with a 10 milliliter volume. The drying out of the eluent in a vacuum occurs once it has been purified after which it is analyzed by utilizing HPLC or ELISA. 2.1.2 Analysis of CYN: The regular method used for cylindrospermopsin quantification and identification is based upon LC-MS. LC-MS however is not cost effective and inaccessible for countries experiencing toxic blooms. For this particular reason, when LC-MS may not be applicable or preferred characterization with nuclear magnetic resonance (NMR) and High Performance Liquid Chromatography (HPLC) may come up as trustworthy options. HPLC is highly regarded for such reasons that can permit in quantification, identification and purification of an individual compound. Individual compounds are isolated by pressure which is utilized to fill the compounds in a separate chamber where the process takes place under the control of liquid solvents. On the contrary, NMR is a technique that employs magnetic nuclei which soak up before re-emitting the electromagnetic radiations at a precise resonance frequency. 2.1.4.1 Detection and analysis of cylindrospermopsin using HPLC Compared to microcystins and saxitoxins, Cylindrospermopsins do not have many ways through which they can be detected, because of its signature UV spectra (?max at 262 nm), High-performance liquid chromatography is one method which has been proven to be superior in its identification of cylindrospermopsins, however, it has the limitation which makes sample purification mandatory as it is usually co-eluted with a range contaminants. HP-20 resin is used for the purification of cylindrospermopsin as it helps in the removal of certain ionic components. Cylindrospermopsin can be detected by HPLC once it has been extracted. Ultrasonication in 75% methanol enables the water sample to extracted and filtered on glass fibre filters. Extraction procedure The procedure involves the addition of 1.2 ml of methanol combined with a bath ultrasonicator for a total of 15 minutes to the air dried frozen samples after putting them borosilicate glass tubes and thawing them two times. Further ultrasonication of the samples should be performed for one minute, while extracted aliquots are centrifuged at 10,000 ? g for 10 min. This step precedes the transfer of 500 µl of the supernatants into borosilicate vials which at 40°C under argon are evaporated till dry. The reconstitution of the dried extracts is conducted as 100 µl of 75% methanol then centrifuged at 10,000 ? g for 10 min in vials with another option entailing their filtration using the HPLC grade filter. It must be made sure that before the HPLC system is run it should be according to the manufacturer’s directions and the samples of chromatogram should be in accordance with HPLC gradients with the usage of 10 µl injections. The analysis of chromatogram should then be conducted so that the retention times and spectra is compared to the set standards for the eluents that consist of water, acetonitrile, methanol and their mixtures. Preparative chromatography is also made use of to obtain great quantities of cylindrospermopsins. Henceforth, chromatography can be utilized for the extract’s preparation as well as purification. The Reversed phase HPLC is utilized for this purpose which is a method that enables the time for elution of polar molecules to be increased as the elution of less polar compounds occurs in a quicker manner. In summation, HPLC-PDA is a highly reliable technique for the analysis of cylindrospermopsins, in order to carry out the identification of these toxins as the HPLC spectrum has an adequate retention time. But its limitation is such that the detection limit for the technique is particularly small for such samples of the environment that are low on cylindrospermopsins, moreover, if this toxin is not cleaned up and separated from its analogues otherwise it the chromatogram can be affected as in fig 8: Figure 8. HPLC-PDA chromatogram (?=262 nm) of a cyanobacterial bloom sample dominated 2.1.4.2 Analysis of CYN using Nuclear Magnetic Resonance (NMR): The Nuclear magnetic resonance (NMR) has been widely employed as a successful and highly regarded method to test purity of cylindrospermopsins by dealing with nuclei such as hydrogen’s magnetic properties. NMR functions by through the fundamental that in an applied constant magnetic field the polarization of the magnetic nuclear rotates, resulting in the shakeup of the nuclear rotations by a pulse which is electromagnetic and most likely in the form of radio frequency. The NMR technique involves the selection of two perpendicular fields that helps in the maximization of the signal strength. Observation suggests that that the attachment of the external magnetic field needed in assisting hydrogen to resonance decreases if attached to a greater electronegative element. Hence, electronegativity of the atoms is sensitive to even small changes which explains why the hydrogen atom needs the integration of a different magnetic field to attain resonance. Resonance for any particular compound, obtained in a magnetic field, as a matter of principle is directly related or proportional to the magnetic field’s strength. In the NMR continuum, the environments of the hydrogen which has been linked with different compounds will determine the quantity of peaks. In this scenario, sizes of the peaks are more important and of more relevance than their heights as the sizes of the peaks assign how many hydrogen atoms the environment contains. CHAPTER 3: RESULTS AND DISCUSSION As per the reports of Norris(3) on the procedure of Cylindrospermopsis raciborski’s growth into cylindrospermopsin by employing a method of culture growth succeeded by extraction through the means absorption on activated charcoal columns. It was the aim of the paper to imitate this methodology so that two species of Oscillatoria sp.and Aphanizomenon flos-aquae could be obtained from CCAP. The supply of the first species experienced a 3 month delay while the second species was rendered incapable of being generated by the CCAP. Therefore, it was decided that the growth of non-toxic algae Chlorophylls should be probed instead. Following the methods outlined in section (2.1.1), a sample of Chlorophylls was placed in the growth medium being shown in an active sample in the figure (10) shown below. It was established that this method was indeed successful in creating a culture of the Chlorophylls, the growth took 5 weeks in an incubator with daily agitation levels recorded at 22-25 ?C. Figure 10. Culture of Chlorophyllsgrown in the incubator 200 ml of the sample was transferred to the bio-reactor from the incubator along with 2 L of sterilized and 1 ml/L of Joborski growth medium and also 1ml/L of a vitamin solution. The Chlorophylls experienced an immense level of growth owing to 3 weeks of aeration, which was evaluated to be a single species. Once the Oscillatoria sp sample arrived, 150 ml of sterilized water, 10 ml of Oscillatoria sp and 1m of vitamin solution along with 1ml of Jaworski’s Medium was used to grow a stock solution in the incubator, which showed evident emergence of Oscillatoria sp within 5 weeks. The stock solution was placed with 2L of sterilized water, 1ml/L of vitamin solution and 1ml/L quantity of the growth medium in the bioreactor but showed no results for 4-5 days. However, XXg/mL of sodium bicarbonate was then added along with a continued agitation lasting 1 week which also showed no change. Other ineffective changes also led to no success which leads to the conclusion that a bio-reactor cannot act as a suitable container for growth of this type of cyanobacterium. Therefore, it can be concluded that the Chlorophylls algae which is non-toxic has the capacity to grow in both incubator and bio-reactor environments on the contrary, Oscillatoria sp only responds to successful growth in an incubator but not in a bio-reactor. Reasons for this premise remain unclear but may be related to factors such as poor lighting, lack of nourishment from suitable nutrients or over oxygenation. HPCL ANALYSIS: The HPCL analysis of these samples, as conducted quite commendably in the paper of Norris for the separation of cylindrospermopsin as well as 7-deoxy-cylindrospermopsin could not be conducted, however, it still took place on reverse phase HPCL and produced admirable results in terms of purity and separation for mixtures as shown below. Figure 9. HPLCUV trace for cylindrospermopsin (A) and 7-deoxy-cylindrospermopsin.(B) from Cylindrospermopsis raciborskiigrowth media Upon investigation and analysis it can be concluded that this is an effective and highly recommended method for supervising and probing metabolites production. Read More