CHEMICAL INVESTIGATIONS OF THE METABOLITES OF TWO STRAINS OF TOXIC CYANOBACTERIA Justin D. Isaacs A Thesis Submitted to the University of North Carolina Wilmington in Partial Fulfillment of the Requirements for the Degree of Master of Science Department of Chemistry and Biochemistry University of North Carolina Wilmington 2011 Approved by Advisory Committee Pamela J. Seaton Bongkeun Song Jeffrey L. C. Wright Chair Accepted by Dean, Graduate School
124
Embed
CHEMICAL INVESTIGATIONS OF THE METABOLITES OF TWO …
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
CHEMICAL INVESTIGATIONS OF THE METABOLITES OF TWO STRAINS OF TOXIC CYANOBACTERIA
Justin D. Isaacs
A Thesis Submitted to the University of North Carolina Wilmington in Partial Fulfillment
of the Requirements for the Degree of Master of Science
Department of Chemistry and Biochemistry
University of North Carolina Wilmington
2011
Approved by
Advisory Committee
Pamela J. Seaton Bongkeun Song Jeffrey L. C. Wright
Chair
Accepted by
Dean, Graduate School
ii
TABLE OF CONTENTS
ABSTRACT ................................................................................................................................... iii
ACKNOWLEDGEMENTS ........................................................................................................... iv
LIST OF TABLES .......................................................................................................................... v
LIST OF FIGURES ....................................................................................................................... vi
Cyanobacteria harmful algal blooms (CHABs) are an ever-increasing problem in
bodies of water that experience high levels of pollution or eutrophication, jeopardizing the health
of humans and animals who rely on these sources of water for drinking or recreation. A general
analytical method was employed to analyze interesting secondary metabolites produced by a
natural bloom of Microcystis aeruginosa in the Cape Fear river and a laboratory-cultured sample
of Planktothrix rubescens (UTCC 507). The Microcystis aeruginosa sample was found to
produce two known hepatotoxins, microcystin-LR and microcystin-RR, as well as two novel
Ahp-containing depsipeptides, micropeptin 1106 and micropeptin 1120. Planktothrix rubescens
(UTCC 507) was found to produce one previously known hepatotoxin, [D-Asp3, (E)-Dhb7]
microcystin-HtyR, and one novel Hph-containing microcystin, [D-Asp3, (E)-Dhb7] microcystin-
HphR. In addition Planktothrix rubescens (UTCC 507) also produced two novel cyclic peptides,
anabaenopeptin 872 and anabaenopeptin 856, that belong to the anabaenopeptin family. Both of
these anabaenopeptins contain versions of the extremely rare amino acids known as alternamic
acids, which previously had never been found in cyanobacterial metabolites. These isolates
demonstrate the diverse metabolic capacity of toxigenic cyanobacteria, which may as yet be
found to possess important biological activity.
iv
ACKNOWLEDGEMENTS
I would like to thank Dr. Jeffrey Wright for providing me with the opportunity to work in
his lab and learn natural product chemistry while developing the skills I need to successfully
accomplish future career goals. Another special thanks goes out to Dr. Ryan Van Wagoner,
Allison Drummond, and Jan Vincente for teaching me the ways of a natural product chemist and
offering advices to help me overcome obstacles in the lab. I would also like to thank Mrs. Eve
Wright for teaching me culturing techniques and growing the cyanobacteria cultures I used
throughout my study, as well as Dr. Mike Mallin and Matt McIver for providing the cell samples
of Microcysis from the Cape Fear River. I am very grateful for the wonderful experience I had
while working at the Center for Marine Science and that can be accredited to all the kind and
helpful people I met, Thank You.
Most importantly, I would like to thank my mom, Donna Isaacs, and my grandfather,
Herman Williams, for telling me that I can accomplish anything I put my mind to, for telling me
I am great, and never letting me give up. Thank you for always being there for me, no matter
what, and supporting my every need. It is because of them that I shoot for the stars and never
look back; I only hope that I can give them as much as they have given me. I would also like to
thank my girlfriend, Claire Taylor, for being understanding and giving up cuddle time so that I
can accomplish my goals. It has been a sacrifice for us all.
v
LIST OF TABLES
Table Page
1. Commonly observed cyanotoxins in US fresh, estuarine and marine waters ..................... 3
2. Retention times for Marfey derivatives of amino acid standards ..................................... 23
3. Assignment of 1H and 13C NMR data for micropeptin 1106 in DMSO-d6 ....................... 32
4. Assignment of 1H and 13C NMR data for micropeptin 1120 in DMSO-d6 ....................... 34
5. Retention times for hydrolysate of micropeptin 1106 and 1120 ....................................... 37
6. Assignment of 1H and 13C NMR data for [D-Asp3, (E)-Dhb7] microcystin-HtyR in DMSO-d6 ...................................................................................................................... 46 7. Assignment of 1H and 13C NMR data for [D-Asp3, (E)-Dhb7] microcystin-HphR in DMSO-d6 ...................................................................................................................... 48 8. Retention times for hydrolysate of [D-Asp3, (E)-Dhb7] microcystin-HtyR and [D-Asp3, (E)-Dhb7] microcystin-HphR ............................................................................ 51 9. Assignment of 1H and 13C NMR data for anabaenopeptin 872 in DMSO-d6 ................... 60
10. Assignment of 1H and 13C NMR data for anabaenopeptin 856 in DMSO-d6 ................... 62
11. Retention times for hydrolysate of anabaenopeptin 856 and 872 ..................................... 65
12. Seven major topic areas and 23 subtopics identified by ISOC-HAB ............................... 68
vi
LIST OF FIGURES
Figure Page
1. Representatives of the common groups of cyanotoxins ..................................................... 4
2. Chemical structure of various microcystins produced by cyanobacteria ............................ 9
3. Representatives of anabaenopeptins/oscillamide peptides ............................................... 10
4. Representatives of Ahp-containing depsipeptides ............................................................ 11
5. Metabolite isolation method for Mycrocystis aeruginosa ................................................ 14
6. Purification process for Planktothrix rubescens (UTCC 507) metabolites ...................... 18
7. UV absorbance for microcystin-RR and microcystin-LR ................................................ 25
8. ESI-MS spectrum of microcystin-LR ............................................................................... 26
9. ESI-MS spectrum of microcystin-RR ............................................................................... 27
10. Structure of micropeptins 1106 and 1120 ......................................................................... 29
11. ESI-MS spectrum of micropeptin 1106 ............................................................................ 30
12. ESI-MS spectrum of micropeptin 1120 ............................................................................ 31
13. ROESY correlations for Ahp moiety ................................................................................ 38
14. Structure of microcystins from UTCC 507 ....................................................................... 41
15 UV absorbance for [D-Asp3, (E)-Dhb7] microcystin-HtyR .............................................. 42
16 ESI-MS spectra for [D-Asp3, (E)-Dhb7] microcystin-HtyR ............................................. 43
17. UV absorbance for [D-Asp3, (E)-Dhb7] microcystin-HphR ............................................. 44
18. ESI-MS spectra for [D-Asp3, (E)-Dhb7] microcystin-HphR ............................................ 45
19. Structure of new anabaenopeptins from UTCC 507 ......................................................... 55
20. UV absorbance for anabaenopeptin 856 ........................................................................... 56
21. ESI-MS spectra of anabaenopeptin 856 ............................................................................ 57
vii
22. UV absorbance for anabaenopeptin 872 ........................................................................... 58
23. ESI-MS spectra of anabaenopeptin 872 ............................................................................ 59
INTRODUCTION
Cyanobacteria, also known as blue-green algae, are ancient prokaryotic organisms that
have survived and flourished on the planet for over two billion years. Their name describes the
characteristic blue and green phycobilin pigments found within these photosynthetic organisms
which are believed to be responsible for producing the earth’s earliest oxygen, enabling aerobic
metabolism and all life forms that depend on it (Dismukes et al., 2001). Cyanobacteria are
prolific organisms, often the first plant life to colonize barren areas of rock and soil, where they
play an important role in the functional processes of ecosystems and the cycling of nutrients.
They also flourish in salty, brackish or fresh water, in cold and hot springs, and other extreme
environments where no other microalgae can exist (Mur et al., 1999). The tremendous
adaptability of these microorganisms has allowed them to inhabit a diverse range of aquatic and
terrestrial environments covering all corners of the earth (Carmichael et al., 1993; Mur et al.,
1999). Awareness of the presence and effects of cyanobacteria has been documented throughout
history. For example, this description by a medieval clergyman and chronicler of his time,
Gerald of Wales, describing the presence of buoyant cyanobacteria in 1188: “The lake has many
miraculous properties --- it sometimes turns bright green, and in our days it has been known to
become scarlet, not all over, but as if blood were flowing along certain currents and eddies”
(Codd et al., 2005a). Such blooms of cyanobacteria can produce toxins and the earliest
documented reports of cyanobacteria causing death were published during the 19th century, when
livestock and wildlife in Australia, Denmark and Poland died by ingesting waters containing
toxic cyanobacteria blooms (Codd et al., 2005a).
2
If the conditions of light, turbidity, and nutrients in a body of water are favorable,
cyanobacteria often form dense blooms, defined as visible colorations of a body of water due to
the presence of suspended cells, filaments and/or colonies and, in some cases, subsequent surface
scums (Fristachi et al., 2008). Blooms are especially prevalent in areas that experience high
levels of pollution or eutrophication, the excessive richness of nutrients, usually phosphorous and
nitrogen, in a lake or other body of water that increases biological production of plant life while
simultaneously killing animal life due to the lack of oxygen. While eutrophication happens
naturally in some bodies of water, many times it is caused by anthropogenic activity such as
municipal wastewater discharge or run-off from fertilizers and manure spread on agricultural
areas (Bartram et al., 1999). This ability to thrive in eutrophic or polluted bodies of water make
cyanobacteria blooms common in rivers, lakes, and reservoirs that are in close proximity to
developed areas or farmlands where they are more likely to make an impact. Cyanobacteria have
several vectors by which they create problems for people and animals; diminishing water clarity,
reducing oxygen levels, producing bad odors and tastes, and toxin production (Chorus et al.,
1999).
Many bloom-forming genera of cyanobacteria contain toxic members, referred to as
cyanobacteria harmful algal blooms (CHAB’s). Some examples include Anabaena,
Anabaenopsis, Aphanizomenon, Cylindrospermopsis, Raphidiopsis, Microcystis, Nodularia and
Planktothrix (Carmichael at al., 1992; Codd et al., 2005a, 2005b; Gademann et al., 2008). These
toxigenic genera are responsible for producing an impressive and chemically diverse array of
toxic secondary metabolites that can be grouped according to their chemical structures and
biological effects (see Table 1 and Figure 1 for representative examples). Table 1 also highlights
the variety and range of toxicological properties of these compounds.
3
Toxin Type and (# congeners) Mode of Action Hepatotoxins
Microcystins Cyclic heptapeptides (>70) Protein phosphatase inhibitor, tumor promoter
Nodularins Cyclic pentapeptides (9) Protein phosphatase inhibitor, tumor promoter, carcinogenic
Cylindrospermopsins Guanidine alkaloid (3) Protein phosphatase inhibitor, genotoxic and necrotic injury to liver and other organelles
Neurotoxins
Anatoxin-a (including homoanatoxin-a)
Azobicyclic alkaloid (5) Postsynaptic neuromuscular blocking agent and Acetylcholinesterase agonist
Anatoxin-a (S) Guanidine methyl phosphate ester (1)
Ecosystem Effects • Aquatic Vertebrates • Trophic Status & Ecological
Conditions Risk Assessment • Economic Impact
• Toxic Microbes & Mixtures • Human & Ecological Integration
Table 12. Seven major topic areas and 23 subtopics identified by ISOC-HAB.
69
Microcystis aeruginosa
Applying this analytical method to a naturally occurring sample of M. aeruginosa has
yielded two common microcystins and two novel micropeptins. The production of hepatotoxic
microcystins by this strain is of great concern considering the bloom was found in the drinking
water supply of New Hanover County, NC. Microcystin-LR (1) is commonly considered to be
the most toxic of the microcystins and has been commonly isolated from many strains of
toxigenic cyanobacteria. Microcystin-RR (2) is another commonly isolated microcystins, and
has a much higher LD50 than MC-LR, 235.4 µg/kg vs 43.0 µg/kg (Gupta et al., 2003). The
production of these toxins in drinking water supplies is a serious concern since not all water
treatment processes are effective at removing the toxins.
From the same organism, two new micropeptins (3 and 4) isolated from M. aeruginosa
are similar to a previously known group of micropeptins, micropeptin 88-A through 88-F (Ishida
et al., 2008). The micropeptins in this group all contain the same non-polar side chain, butyric
acid, linked to tyrosine then glutamic acid. The difference between these known micropeptins
and the newly isolated ones is the incorporation of arginine in the fifth position from the carboxy
terminus. The only difference between compounds 3 and 4 is the extra O-methyl functional
group attached to the glutamic acid moiety of 4, which is also found in micropeptin 88-F. The
Ishida group that isolated micropeptins 88-A through 88-F found all but micropeptin 88-B
displayed potent chymotrypsin inhibition ranging from IC50 of 0.4-10.0 µg/mL (Ishida et al.,
1998). The activity of these previously known micropeptins can be attributed to the hydrophobic
amino acids, Tyr and Leu, in the fifth position from the carboxy terminus. However, 3 and 4
contain the basic amino acid Arg in this position, which should deem them trypsin inhibitors
instead (Gademann et al., 2008).
70
Planktothrix rubescens
Applying the general method described to extracts of Planktothrix rubescens
(UTCC507), four interesting cyclic peptides (5-8) were identified and unambiguously
characterized by NMR and confirmed by Marfey’s method.
The two microcystins isolated from this strain (5 and 6) were initially thought to be MC-
YR and MC-FR based on the primary LC/UV/MS data, however, the 1D and 2D NMR data
established 5 and 6 to actually be [D-Asp3, (E)-Dhb7] microcystin-HtyR and [D-Asp3, (E)-Dhb7]
microcystin-HphR, respectively. This observation underscores the need for full structural
characterization of metabolites using all the analytical techniques necessary including NMR,
rather than data based on preliminary LC/UV/MS analysis alone. There is speculation that this
may be a common occurrence and mis-identification of cyanotoxins pose a significant problem
when trying to assess the toxicity of a particular bloom or when attempting to identify a
causative agent within a toxic sample. It is accepted that an integral relationship exists between
the three-dimensional structure of a compound and the biological activity is may possess, though
the exact mode of action may be unknown. The actual structures of compounds 5 and 6 have
several variations from the structures of MC-YR and MC-FR. These include Dhb7 instead of
Mdha7, D-Asp3 instead of D-MeAsp3, and L-Hty/L-Hph instead of L-Tyr/L-Phe. Some of these
changes are more subtle than others and probably affect bioactivity accordingly.
In this regard, [D-Asp3, (E)-Dhb7] microcystin-HtyR (5) was originally isolated by Sano
and Kaya (1998) from a strain of Oscillatoria agardhii. Two forms of Dhb exist, Z and E, that
can easily be distinguished by the 1H shift of the olefinic quartet, 6.49 ppm vs. 5.73 ppm,
respectively (Sano et al., 1998), and the double bond configuration determined by ROESY and
NOESY data. Several of these Dhb containing microcystins have also been tested for bioactivity
71
by an intraperitoneal mouse bioassay where they showed an acute toxicity, sharing similar
characteristics to Mdha containing microcystin toxicity (Beattie et al., 1992).
[D-Asp3, (E)-Dhb7] Microcystin-HphR is a novel microcystin that contains the very
unusual amino acid, homophenylalanine. [Dha7] Microcystins-HphR is the only other
microcystin described to date that contains this rare amino acid and was simultaneously isolated
with a homotyrosine (Hty) congener mirroring the isolation of both congeners from UTCC 507
(Namikoshi et al., 1992). When Namikoshi tested these two MC’s in an intraperitoneal mouse
bioassay, death occurred within 1-3 hours. Considering MC’s containing Dhb, Hty and Hph
have all proven to be toxic individually, it is assumed that 5 and 6 isolated in this experiment are
also hepatotoxic.
The two novel anabaenopeptins (7 and 8) isolated from UTCC 507 are very similar to the
previously reported anabaenopeptin B (Figure 3). However, these new anabaenopeptins contain
a couple of very rare amino acids in place of the more common ureido linked arginine. These
two unusual amino acids were identified as 2-amino-5-phenyl-pentanoic acid and 2-amino-5-
hydroxyphenyl-pentanoic acid, found in 7 and 8, respectively. This is the first known example
of cyanobacteria using these unusual amino acids. In fact, the only compounds previously
known to contain these two amino acids are the alternarolides, isolated from the fungus
Alternaria mali (Okuno et al., 1975) and given the name alternamic acids. The alternarolides are
host specific phytotoxins that cause leaf-spot disease in various varieties of apples. There are
three congeners of alternarolides and each contains a different congener of alternamic acid.
72
CONCLUSION
In addition to the known toxins they produce, many unrelated families of cyclic peptides
are produced by toxigenic genera of cyanobacteria and may pose a risk to humans and animals
that come in contact with contaminated water. In this study, strains of M. aeruginosa and P.
rubescens were analyzed for the production of unusual peptides using a general procedure that
was developed in this laboratory. The M. aeruginosa strain was gathered from a natural bloom
that occurred in the Cape Fear river of North Carolina and found to produce two common
microcystins, MC-LR and MC-RR, as well as two novel micropeptins, characterized as
micropeptin 1106 and 1120. The P. rubescens strain (UTCC 507) was originally gathered
beneath the ice of a lake in Ontario and cultured in the lab under controlled environmental
conditions. From this strain, two unique microcystins were isolated, [D-Asp3, (E)-Dhb7]
microcystin-HtyR and [D-Asp3, (E)-Dhb7] microcystin-HphR, along with two novel
anabaenopeptins, named anabaenopeptin 856 and 872. These P. rubescens products were found
two contain several unique amino acids, namely 2-amino-5-phenyl-pentanoic acid, 2-amino-5-
hydroxyphenyl-pentanoic acid, homotyrosine and homophenylalanine. Detailed spectroscopic
analysis established the structure and connectivity of the amino acid components in all the
isolated peptides and the absolute stereochemistry of the peptides was confirmed by Marfey’s
method.
73
LITERATURE CITED Barco, M.; Flores, C.; et al. Determination of Microcystin Variants and Related Peptides Present
in a Water Bloom of Planktothrix rubescens in a Spanish Drinking Water Reservoir by LC/ESI-MS. Toxicon, 2004, 44, 881-886.
Bartram, J.; Carmichael, W. W.; Chorus, I.; Jones, G.; Skulberg, O. M. Introduction. In Toxic
Cyanobacteria in Water: A Guide to Their Public Health Consequences, Monitoring and Management; Chorus, I., Bartram, J., Eds.; World Health Organization: London, England, 1999; 1-14.
Beattie, K.; Kaya K.; Sano, T.; Codd, G. A. Three Dehydrobutyrine-Containing Microcystins
From Nostoc. Phytochemistry, 1998, 47 (7), 1289-1292. Blom, J.F.; Robinson, J.A.; Jüttner, F. High Grazer Toxicity of [D-Asp3,(E)-Dhb7]Microcystin-
RR of Planktothrix rubescens as Compared to Different Microcystins. Toxicon, 2001, 39, 1923-1932.
Carmichael, W. W. Cyanobacteria Secondary Metabolites- the Cyanotoxins. Journal of Applied
Bacteriology, 1992, 72, 445-459. Carmichael, W. W.; Falconer, I. R. Diseases Related to Freshwater Blue-green Algal Toxins, and
Control Measures. In Algal Toxins in Seafood and Drinking Water; Falconer, I. R., Ed.; Academic Press: San Diego, CA, 1993, 187-210.
Codd, G. A.; Lindsay, J.; Youg, F. M.; Morrison, L. F.; Metcalf, J. S. Harmful Cyanobacteria:
From Mass Mortalities to Management Measures. In Harmful Cyanobacteria; Huisman, J., Matthijs, H. C. P., Visser, P. M., Eds.; Springer: Dordrecht, The Netherlands, 2005a; 1-24.
Codd, G. A.; Morrison, L. F.; Metcalf, J. S. Cyanobacterial Toxins: Risk Management for Health
Protection. Toxicology and Applied Pharmacology, 2005b, 203, 264-272. Dismukes, G. C.; Klimov, V. V.; Barnov, S. V.; Kozlov, Y. N.; Dasgupta, J.; Tyryshkin, A. The
Origin of Atmospheric Oxygen on Earth: The Innovation of Oxygenic Photosynthesis. Porc. Natl. Acad. Sci., 2001, 98, 2170-2175.
Falconer, I.; Bartram, J.; Chorus, I.; Kuiper-Goodman, T.; Utkilen, H.; Burch, M.; Codd, G. A.
Safe Levels and Safe Practices. In Toxic Cyanobacteria in Water: A Guide to Their Public Health Consequences, Monitoring and Management; Chorus, I., Bartram, J., Eds.; World Health Organization: London, England, 1999; 155-178.
Fenical, W., Jenson, P. Developing a New Resource for Drug Discovery: Marine Actinomycete
Bacteria. Natural Chemical Biology, 2006, 2, 666-673.
74
Fristachi, A.; Sinclair, J. L. Occurrence of Cyanobacterial Harmful Algal Blooms Workgroup Report. In Cyanobacterial harmful Algal Blooms: State of the Science and Research Neeeds; Hudnell, H. K., Ed.; Springer Science + Business Media: New York, 2008; 45-104
Gademann, K.; Portmann, C. Secondary Metabolites From Cyanobacteria: Complex Structures
and Powerful Bioactivities. Current Organic Chemistry, 2008, 12, 326-341. Grach-Pogrebinsky, O.; Sedmak, B.; Carmeli, S. Seco[D-Asp3] Microcysin-RR and [D-Asp, D-
Glu(OMe)6] Microcystin-RR, Two New Microcystins From a Toxic Water Bloom of the Cyanobacterium Planktothrix rubescens. J. Nar. Prod., 2008, 67, 337-342.
of Cyanobacterial Cyclic Peptide Toxin Microcystin Variants (LR, RR, YR) in Mice. Toxicology, 2003, 188, 285-296.
Hoeger S. J.; Schmid, D.; Blom, J. F.; Ernst, B.; Dietrich, D. R. Analytical and Functional
Characterization of Microcystins [Asp3] MC-RR and [Asp3, Dhb7] MC-RR: Consequences for Risk Assessment. Environ. Sci. Technol., 2007, 41, 2609-2616.
Hudnell, H. K.; Dortch, Q.; Zenick, H. An Overview of the Interagency, International
Symposium on Cyanobacterial Harmful Algal Blooms (ISOC-HAB): Advancing the Scientific Understanding of Freshwater Harmful Algal Blooms. In Cyanobacterial harmful Algal Blooms: State of the Science and Research Neeeds; Hudnell, H. K., Ed.; Springer Science + Business Media: New York, 2008; 45-104.
Ishida, K.; Matsuda, H.; Murakami, M. Micropeptins 88-A to 88-F, Chymotrypsin Inhibitors
from the Cyanobacterium Microcystis aeruginosa. Tetrahedron, 1998, 54, 5545-5556. Llano, D. G.; Herraiz, T.; Polo, M. C. Peptides. In Handbook of Food Analysis: Physical
Characterization and Nutrient Analysis; Nollet, L. M. L., Ed.; Marcel Dekker: Basel, Switzerland, 2004; 125-167.
Mur, L. C.; Skulberg, O. M.; Utkilen, H. Cyanobacteria in the Environment. In Toxic
Cyanobacteria in Water: A Guide to Their Public Health Consequences, Monitoring and Management; Chorus, I., Bartram, J., Eds.; World Health Organization: London, England, 1999; 15-40.
Namikoshi, M.; Rinehart, K.; Sakai, R. Identification of 12 Hepatotoxins from a Homer Lake
Bloom of the Cyanobacteria Microcystis aeruginosa, Microcystis viridis, and Microcystis wesenbergii: Nine New Microcystins. J. Org. Chem., 1992a, 57 (3), 866-870.
Namikoshi, M.; Sivonen, K.; Evans, W. R.; Carmichael, W. W.; Rouhiainen, L.; Luukkainen, R.;
Rinehardt, K. Structures of Three New Homotyrosine-Containing Microcystins and a New Homophenylalanine Variant from Anabaena sp. Strain 66. Chem. Res. Toxicol., 1992b, 5, 661-666.
75
Nishiwaki-Matsushima, R.; Nishiwaki, S.; Ohta, T.; Yoshizawa, S.; Suganuma, M.; Harada, K.; Watanabe, M. F.; Fujiki, H. Structure-Function Relationships of Microcysitns, Liver Tumor Promoters, in Interaction with Protein Phosphatase, Jpn. J. Cancer Res., 1991, 82, 993-996.
Okumura, H. S.; Philmus, B.; Portmann, C.; Hemscheidt, T. K. Homotyrosine-Containing
Cyanopeptolins 880 and 960 and Anabaenopeptins 908 and 915 from Planktothrix agardhii CYA 126/8. J. Nat. Prod., 2009, 72, 172-176.
Okuno, T.; Ishita, A.; Sugawara, Y.; Sawai, M. K.; Matsumoto, T. Structure of the Biological
Active Cyclopeptides Produced by Alternaria mali Roberts. Tetrahedron Letters, 1975, 5, 335-336.
Rogers, E. D.; Henry, T. B.; Twiner, M. J.; Gouffon, J. S.; McPherson, J. T.; Boyer, G. L.;
Saylor, G. S.; Wilhelm, S. W. Global Gene Expression Profiling in Larval Zebrafish Exposed to Microcystin-LR and Microcystis Reveals Endocrine Disrupting Effects of Cyanobacteria. Environmental Science and Technology, 2011, 45 (5), 1962-1969.
Sano, T.; Beattie, K. A.; Codd, G. A.; Kaya, K. Two (Z)-Dehydrobutyrine-Containing
Microcystins from a Hepatotoxic Bloom of Oscillatoria agardhii from Soulseat Loch, Scotland. J. Nat. Prod., 1998a, 61, 851-853.
Sano, T.; kaya, K. Two New (E)-2-Amino-2-Butenoic Acid (Dhb)-Containing Microcystins
Isolated from Oscillatoria agardhii. Tetrahedron, 1998b, 54 (3-4), 463-470. Sano, T.; Usui, T.; Ueda, K.; Osada, H,; Kaya, K. Isolation of New Protein Phosphatase
Inhibitors From Two Cyanobacteria Species, Plantkothrix spp. Journal of Natural Products, 2001, 64, 1052-1055.
Sedmak, B.; Elersěk, T.; Grach-Pogrebinsky, Olga.,; Carmeli, S.; Sever, N.; Lah, T. T.
Ecotoxicologically relevant cyclic peptides from cyanobacterial bloom (Planktothrix rubescens) – A threat to human environmental health. Radiol. Oncol., 2008, 42 (2), 102-113.
Ueno, T.; Nakashima, T.; Hayashi, Y.; Fukami, H. Isolation and structure of AM-toxin III, A
host specific phytotoxic metabolite produced by Alternaria mali. Agr. Biol. Chem., 1975a, 39 (10), 2081-2082.
Ueno, T.; Nakashima, T.; Hayashi, Y.; Fukami, H. Structures of AM-toxin I and II, Host Specific
Phytotoxic Metabolites Produced by Alternaria mali. Agr. Biol. Chem., 1975b, 39 (5), 1115-1122.
Welker, M.; Brunke, M.; Preussel, K.; Lippert, I.; Von Döhren, H. Diversity and Distribution of
Microcystis (Cyanobacteria) Oligopeptide Chemotypes from Natural Communities Studied by Single-Colony Mass Spectrometry. Microbiology, 2004, 150, 1785-1796.
76
Welker, M.; Von Döhren, H. Cyanobacterial Peptides – Nature’s Own Combinatorial Biosynthesis. FEMS Microbiol. Rev., 2006, 30, 530-563.
Wiegand, C.; Pflugmacher, S. Ecotoxicological Effects of Selected Cyanobacterial Secondary
Metabolites a Short Review. Toxicology and Applied Pharmacology, 2005, 203, 201-218. World Health Organization. Guidelines for Drinking-water Quality, 2nd ed.; Geneva,
Switzerland, 1993. Yamaki, H.; Sitachitta, N.; Sano, T.; Kaya, K. Two New Chymotrypsin Inhibitors Isolated From
the Cyanobacterium Microcystis aeruginosa NIES-88. J. Nat. Prod., 2005, 68, 14-18. Zafrir-Ilan, E.; Carmeli, S. Eight Novel Serine Proteases Inhibitors From a Water Bloom of the
Cyanobacterium Microcystis sp. Tetrahedron, 2010a, 66, 9194-9202. Zafrir-Ilan, E.; Carmeli, S. Micropeptins From an Israeli Fishpond Water Bloom of the
Cyanobacterium Microcystis sp. J. Nat. Prod, 2010b, 73, 352-358.
77
APPENDIX Appendix A. Solvent suppliers (all purchased through VWR).
Stock Formula Stock Soln mL/L Sodium Nitrate NaNO3 75g/500mL 10mL Potassium Phosphate Dibasic K2HPO4�3H2O 8.0g/200mL 1 mL Magnesium Sulfate MgSO4�7H2O 15.0g/200mL 1 mL Calcium Chloride CaCl2�2H2O 7.2g/200mL 1 mL Citric Acid with 1.2g/200mL 0.5 mL Ferric Ammonium Citrate 1.2g/200mL 0.5 mL EDTA 0.20g/200mL 1 mL Sodium Carbonate Na2.CO3 4.0g/200mL 1 mL Trace Metal Soln See below 1 mL Trace Metal Solution From Rippka Makes 1 Liter Boric Acid H2BO3 2.86g Manganous Chloride MnCl2�4H20 1.81g Zinc Sulfate ZnSO4�7H2O 0.22g Sodium Molybdate Dihydro Na2MoO4�2H2O 0.39g Cupric Sulfate CuSO4�5H2O 0.08g Cobalt Nitrate Co(NO3)2�6H2O 0.05g
Note: Dissolve each of the above substances separately prior to adding the next on the list. Reference: Rippka, R.; Deruelles, J.; Waterbury, J.; Herdman, M.; Stonier, R. Generic Assignments, Strain Histories and Properties of Pure Cultures of Cyanobacteria. J. Gen. Microbiol., 1979, 111, 1-61.
79
Appendix C. 1H NMR spectrum of microcystin-LR in DMSO-d6.
80
Appendix D. TOCSY NMR spectrum of microcystin-LR in DMSO-d6.
81
Appendix E. HSQC NMR spectrum of microcystin-LR in DMSO-d6.
82
Appendix F. HMBC NMR spectrum of microcystin-LR in DMSO-d6.
83
Appendix G. 1H NMR spectrum of microcystin-RR in DMSO-d6.
84
Appendix H. TOCSY NMR spectrum of microcystin-RR in DMSO-d6.
85
Appendix I. HSQC NMR spectrum of microcystin-RR in DMSO-d6.
86
Appendix J. HMBC NMR spectrum of microcystin-RR in DMSO-d6.