Total Synthesis of Microcystin-LR,
Microcystin-LF, and Unnatural Derivatives thereof
Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.)
vorgelegt von Ivan Zemskov
an der
Mathematisch-Naturwissenschaftliche Sektion Fachbereich Chemie
Tag der mündlichen Prüfung: 15.09.2016 1. Referent: Prof. Dr. Valentin Wittmann 2. Referent: Prof. Dr. Daniel R. Dietrich 3. Referent: Prof. Dr. Tanja Gaich
Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-368120
Глаза боятся - руки делают (Русская пословица) Augen haben Angst, Hände sind fleißig (Russisches Sprichwort)
Dieser Arbeit entstand im Zeitraum von Dezember 2011 bis Januar 2016 an der Universität Konstanz im Fachbereich Chemie in der Arbeitsgruppe von Herrn Prof. Dr. Valentin Wittmann.
An erste Stelle danke ich Herrn Prof. Dr. Valentin Wittmann für die Überlassung des interessanten Themas, sein starkes Vertrauen und den vielen Freiraum um meine eigenen Ideen zu verwirklichen.
In diesen Jahren habe ich sehr viel von Herrn Wittmann gelernt.
Herrn Prof. Daniel R. Dietrich als Mitglied meines Promotionskomitees gilt mein Dank sowohl für seine Ideen bezüglich der toxikologischen Seite des Projektes, als auch für die Übernahme des Zweitgutachtens.
Bei Frau Prof. Dr. Tanja Gaich möchte ich mich für die Übernahme des Prüfungsvorsitzes und Ihre sehr motivierenden Naturstoffvorlesungen bedanken.
Für die kritische Durchsicht dieses Manuskripts möchte ich mich bei Verena Schart, Raphael Fahrner, Stefan Altaner, Oliver Baudendistel und Miriam Fontanillo Dolz bedanken.
Bei allen Mitgliedern der Arbeitsgruppe Wittmann möchte ich mich für eine hervorragende Arbeitsatmosphäre zu bedanken. Es war eine sehr schöne Zeit für mich hier in der AG. Mein besonderer Dank gilt Daniel „Manni“ Wieland, Martin Dauner, Oli Baudendistel und Jamsad „Jimmy“
Mannuthodikayil für viele spannende „Gangdiskussionen“ rundum die Chemie und einfach so. Bei Ellen Batroff und Daniel Wieland bedanke ich mich außerdem für ein kreatives gemeinsames HPLC (Auseinander)schrauben und (Kaputt)reparieren. Philipp Rohse gilt mein Dank für die musikalische Begleitung während den AG Fahrten.
Ein ganz besonderer Dank gilt meiner Laborkollegin Verena Schart für ihre Hilfsbereitschaft, Optimismus und Ihre ständigen Bemühungen unser Labor ordentlich und zu halten. Ich werde unser Labor sehr vermissen.
Meinen Bachelorstudenten Svenja Hutzler, Heike Kropp und Maximilian Häfner danke ich, für ihre engagierte Mitarbeit an den Teilen des Projektes. Max danke ich außerdem für seine sehr motivierte Mitarbeit während des Mitarbeiterpraktikums und HiWis, die bei der Verwirklichung dieses Projektes sehr weitergeholfen haben. Allen meinen Mitarbeiterpraktikanten danke ich für fleißiges Nachkochen der zahlreichen Bausteine.
Bei meinen Kooperationspartnern Stefan Altaner (AG Dietrich, Universität Konstanz) und Miriam Fontanillo Dolz (AG Köhn, EMBL) möchte ich mich für einen produktiven Austausch und Ihre Hilfe mit der Durchführung den Phosphatasenassays bedanken.
trockenen Lösungsmitteln. Bei Mikhail „Opa“ Kabdulov, Konstantin „Total“ Samarin und Magnus
„Badminton“ Pfaffenbach möchte ich mich für die Mitgestaltung der L8-Klimmzugstange und für ihre regelmäßige Teilnahme an „Pyramiden“ bedanken, die in der stressigen Abschlussphase meiner Doktorarbeit für einen sportlichen Ausgleich gesorgt haben.
Bei meine Frau Nadiia, Sohn Miron und meiner Eltern möchte ich mich ganz herzlich für Ihre Geduld und Ihren starken Rückhalt zu bedanken. Ohne Euch würde meine Arbeit kein Sinn machen. Спасибо вам за всё.
1. Intoduction and State of the Art 1
1.1 Introduction 1
1.2 Chemical structure of microcystins and nodularins 2
1.3 Toxicity of microcystins 3
1.3.1 General aspects 3
1.3.2 Interactions of microcystins with protein phosphatases 4
1.4 Analysis of microcystin-containing samples 6
1.5 Biosynthesis and role of microcystins in the cyanobacteria 8
1.6 Chemical modifications of natural microcystins 9
1.7 Synthesis of microcystins, nodularins and simplified analogs thereof 11 1.7.1 Total synthesis of microcystin-LA and its unnatural analogs 11
1.7.2 Towards the total synthesis of microcystin-LR 16
1.7.3 Synthesis of nodularin-V (motuporin) 19
1.7.4 Synthesis of Adda 20
1.7.5 Synthesis of simplified microcystin analogs 21
2. Aim of the thesis 27
3. Results and discussion 29
3.1 Solution synthesis of microcystins 29
3.1.1 General considerations 29
3.1.2 Synthesis of microcystin derivatives using Boc-/-COOMe strategy 29
3.1.3 Development of the novel synthetic route 32
3.1.4 Synthesis of the building blocks 34
3.1.4.1 Synthesis of Fmoc-D-MeAsp-Ot-Bu 34
3.1.4.2 Alternative access to the D-MeAsp building block 37
3.1.4.5 Synthesis of Boc-Adda-OH and Fmoc-Adda-OH building blocks 42
3.1.5 Assembly of the microcystin scaffold in solution 44
3.1.5.1 Synthesis of dipeptide fragments 45
3.1.5.2 Synthesis of tetrapeptide fragment 46
3.1.5.3 Assembly of the linear precursors 47
3.1.5.4 Macrocyclization and selenoxide elimination 49
3.2 Modifications of Adda 52
3.2.1 Synthesis of enantio-Adda derivatives 53
3.2.2 Design of simplified microcystin derivatives 56
3.2.2.1 Synthesis of Anda and Amba building blocks 58
3.2.2.2 Assembly of the simplified microcystin analogs 59 3.3 Synthesis of the microcystin derivatives by SPPS
3.3.1 Development of the SPPS approach 62
3.3.2 SPPS-based approach to [Amba5]-MC-LY(Prg) 63
3.3.3 SPPS-based approach to MC-LR 65
3.4. Protein phosphatase inhibition assays (PPIAs) 69
3.4.1 pNPP-based PPIAs with natural and synthetic microcystins 71 3.4.2 DIFMUP-based PPIAs with simplified microcystin analogs 73 3.5 Novel methods for the cleavage of microcystin-peptide thioether linkages 75
4. Conclusions and outlook 81
5. Zusammenfassung 86
6. Experimental 90
6.1 General Methods 90
6.2.2 Peptide synthesis in solution 93
6.3 Synthesized compounds 96
6.4 Cleavage of MC-LF-glutathione adduct 158
6.5 Protein phosphatase inhibition assay 159
7. References 160
8. Appendix 166
8.1 NMR spectra of synthesized compounds 166
8.2 Selected LC-MS and HPLC chromatograms 216
8.2.1 LC-MS Chromatograms 216
8.2.2 Selected HPLC chromatograms 226
8.2.2.1 Cleavage studies with [enantio-Adda5]-microcystin dimethyl esters 226
8.2.2.2 Macrocyclization using Pfp-esters 228
8.2.2.3 Macrocyclization using HATU 229
8.2.2.4 Selenoxide Elimination 230
Abbreviations
Ac acetyl
Adda (2S,3S,4E,6E,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid enantio-Adda (2R,3R,4E,6E,8R,9R)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid ADMAdda (2S,3S,4E,6E,8S,9S)-3-amino-9-acetyloxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid
AU absorption units
aq. aquoeus
Bn benzyl
Boc tert-butyloxycarbonyl
Bt benzotriazolyl
t-Bu tert-butyl
calcd calculated conc. concentrated
CuAAC Cu-catalyzed azide-alkyne cycloaddition
chemical shift
DCM dichloromethane
DCC N,N’-dicyclohexylcarbodiimide dH2O MilliQ-grade water
Dhb 2-amino-2-dehydrobutyric acid (2-aminoacrylic acid) DIBAL-H diisobutylaluminium hydride
DIFMUP 6,8-difluoro-4-methylumbelliferyl phosphate DIPEA N-ethyldiisopropylamine
DMF N,N-dimethylformamide DMSO dimethylsulfoxide
DVB divinylbenzene
EC50 half maximal effective concentration ELISA enzyme-linked immunosorbent assay equiv. equivalents
ESI electrospray ionization
Et ethyl
FA formic acid
5,6-FAM 5(6)-carboxyfluorescein FC flash column chromatography
--D-Glu- D-glutamic acid incorporated into a peptide via -carboxy group
h hour(s)
Har homoarginine
HATU 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate
HBTU 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate HPLC high-pressure liquid chromatography
HOBt hydroxybenzotriazole
HRMS high-resolution mass spectrometry HWE reaction Horner-Wadsworth-Emmons reaction
Hz Hertz
i.p. intraperitoneal
IC50 half maximal inhibitory concentration
J coupling constant
LC50 median lethal dose
LC-MS liquid chromatography-mass spectrometry LHMDS lithium bis(trimethylsilyl)amide
m/z mass-to-charge ratio
M molar
MC microcystin
Me methyl
D-MeAsp (2R,3S)-2-amino-3-methylsuccinic acid ((3S)-3-methyl-D-aspartic acid)
--D-MeAsp- (3S)-3-methyl-D-aspartic acid incorporated into a peptide via the -carboxy group Mdha N-Methyldehydroalanine
Mdhb 2-(methylamino)-2-dehydrobutyric acid (2-(methylamino)acrylic acid)
min minute(s)
mL milliliter (cm3) m.p. melting point
MPLC medium-pressure liquid chromatography Ms mesyl (methanesulfonyl)
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide NEM N-ethylmorpholine
NMeAla N-methyl-alanine NMeGly N-methyl-glycine
NMeSecPh N-methyl-Se-phenyl-L-selenocysteine NMR nuclear magnetic resonance
OATP organic anion-transporting polypeptide
o/n over night
Orn ornithine
Oxyma ethyl 2-cyano-2-(hydroxyimino) acetate
Pac phenacyl
pdb RSCB protein databank (http://www.rcsb.org/pdb/home/home.do) Pfp pentafluorophenyl
Ph phenyl
Phth phthaloyl
PhFl 9-phenylfluorenyl
Pip piperidine
pNPP para-nitrophenyl phosphate PL parameter logistic
PP protein phosphatase
PP1 protein phosphatase 1 PP2A protein phosphatase 2A PP2B protein phosphatase 2B
PPIA protein phosphatase inhibition assay
Prg propargyl
Pr propyl
i-Pr iso-propyl
PS polystyrene (resin)
RP reversed phase
Rf retention factor
Rt retention time
rt room temperature
Sar sarcosine
sat. saturated (solution)
Sec selenocystein
Su succinimidyl
TAMRA 5(6)-carboxytetramethylrhodamine Tce trichloroethyl
TBS tert-butyldimethylsilyl
Tf triflyl (trifluoromethanesulfonyl) TFA trifluoroacetic acid
TFE trifluoroethanol
THF tetrahydrofurane
TIC total ion chromatogram TIPS triisopropylsilyl
TIS triisoprpylsilane
TLC thin-layer chromatography TMS trimethylsilyl
TOF time-of-flight
Ts tosyl (para-toluenesulfonyl) U units (enzyme activity)
UV ultraviolet
Xaa variable amino acid
Z carboxybenzyl
Amino acid nomenclature
IUPAC-recommended nomenclature and symbolism were used for the amino acids and peptides (Pure & Appl.
Chem., 1984, 56, 595 – 624).
1 1. Introduction and state of the art
1.1 Introduction
Cyanobacteria are among the oldest living and most diverse groups of organisms on Earth.[1] It is hypothesized, that 2.2 - 2.4 billion years ago during the so called Great Oxidation Event oxygen- producing cyanobacteria have contributed to significant changes in the Earth atmosphere transforming it from a reducing to an oxidizing one.[2-3] Nowadays cyanobacteria can still be found in the vast majority of fresh, brackish and salty waters. Global warming along with eutrophication of water basins caused by anthropogenic factors such as sewage disposal and fertilizer run-off, pave the way for so called algal blooms - rapid growth of cyanobacterial populations. Algal blooms are often accompanied by the release of cyanobacterial metabolites like microcystin-LR 1, saxitoxin 2, cylindrospermopsin 3, anatoxin-a 4 or anatoxin-a(S) 5 (Figure 1.1). These can be toxic for aquatic organisms as well as for water consuming land animals and humans. The toxins found in the majority of harmful algal blooms (HABs) all over the globe are microcystins.[4-5] Recent microcystin related HABs in densely populated areas around the Great Lakes in the USA and Canada and lake Taihu in China resulted in the shut-down of the public water supply for millions of people, making microcystin related HABs a global problem.[6-8]
Figure 1.1. Structures of the most abundant cyanobacterial toxins.
2
Microcystins are a family of over 100 cyclic heptapeptides which are produced by at least 10 different cyanobacterial genera[9-10] and are well known for their hepatotoxicity.[11-12] Pentapeptides, which are structurally related to microcystins are called nodularins.[13] Even though microcystin and nodularin-related poisonings of domestic animals are long known,[14-16] the structure of these toxins was first determined in 1980s.[17-20]
The scientific interest in microcystins is not limited to studying the toxic algal blooms alone. The cytostatic properties of these toxins as well as their selective uptake by certain types of tumors make microcystins suitable lead structures for the development of novel anti-cancer drugs.[21-22]
1.2 Chemical structure of microcystins and nodularins
Up to date over 100 different microcystin congeners have been isolated and identified.[12] All the microcystins contain a 25-membered macrocycle, which is formed by 7 amino acid residues (Figure 1.2A). Structurally microcystins consist of three D-amino acids D-Ala1, -D-MeAsp3, and -D-Glu6 at positions 1, 3 and 6, two L-amino acids (positions 2 and 4), the -amino acid Adda ((2S,3S,4E,6E, 8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid, position 5) and a Michael acceptor containing Mdha (N-methyldehydroalanine) residue (position 7). The --D-Glu6- and --D- MeAsp3- residues, which are incorporated in the macrocycle via their side chains, as well as Adda5 and Mdha7, sometimes also Mdhb7 (2-(methylamino)-2-dehydrobutyric acid) or Dhb7 (2-amino-2- dehydrobutyric acid), are highly conserved structural elements for majority of the congeners, with only minor varieties observed. By contrast, the L-amino acids at positions 2 and 4 are frequently varied, which make up the major differences between single congeners.
Figure 1.2. Microcystin-LF 6a (A) and nodularin-V 7 (B) (black) as well as frequent natural microcystin and nodularin variations (grey).
For utility reasons, a two letter abbreviation is often used for the notation of single congeners, whereas the first letter corresponds to the L-amino acid at position 2 and the second letter to the L-
3 amino acid at position 4. Microcystin-LF 6a with Leu2 and Phe4 is abbreviated as MC-LF (Figure 1.2A).
The deviations in all other residues, if present, are mentioned explicitly.
The most common microcystin congeners are: MC-LR 1, MC-LF 6a, MC-RR, MC-LA 8, MC-LW, MC-YR and MC-LY,[5] however the exact composition of produced toxins is frequently complex and highly variable from one algal bloom to another.[4] The most studied congener is the MC-LR 1, which was also the first commercially available microcystin.[4] Most of the published toxicological studies have been performed using this congener.[23] Another frequently found microcystin is more hydrophobic MC-LF 6a, which is considered to be among the most toxic ones.[24]
Nodularins (Figure 1.2B) are related pentapeptides, which structurally resemble the western part of the microcystin macrocycle. Currently 9 different nodularins are known.[13] In contrast to microcystins, nodularins have only one variable L-amino acid at position 2 and standardly have Mdhb4 instead of Mdha (Figure 1.2B). The most frequent nodularin-V 7 is also called motuporin.[25-26]
Interestingly some unusual structural variants of the highly conserved amino acid Adda have been identified in nodularins, including inversed configuration of stereocenter 2 and the lack of the substituent at position 9 of Adda.[27]
1.3 Toxicity of microcystins 1.3.1 General aspects
The most frequent way how organisms are exposed to microcystins is the ingestion of the toxins with water. Two different aspects regarding microcystin toxicity should be considered separately. First, acute poisoning caused by a one-time ingestion of contaminated water and second, a long-term cancerogenic effect due to the chronic exposure to toxins in low concentrations.[28-29]
The most severe and best documented case of acute microcystin poisoning occurred in 1996 in Caruaru, Brazil.[30] 131 renal dialysis patients have been treated with water, which was contaminated with microcystins. As a result, most of the patients developed severe neuro- and gastroenterological symptoms as well as liver failure. 71 persons died in consequence of this accident. [30] The correlation of chronic exposure to small microcystin concentrations with higher level of primary liver cancer in certain regions of China[29] and Serbia[28] is discussed. Another possible way for microcystin exposure is the transfer of the toxin between the different trophic levels of the food chains.[31] An example of such a transfer is the bioaccumulation of microcystins by fresh water or marine invertebrates, in lakes, rivers and coastal areas, where the toxin concentration can make up to 107 fold excess compared to the ambient water[32-33] Consumption of such microcystin containing shellfish by marine animals or humans could lead to severe liver damages. Such cases were observed e.g. for the sea otters which are listed as threatened species.[33]
4
Different aspects of microcystin toxicity like hepato-,[34-35] nephro-, [36] and neurotoxicity[37-38] as well as gastric toxicity[5, 39] have been investigated so far. The typical i.p. LD50 values for different microcystin congeners in a mouse model lay between i.p. LD50 = 36 - 50 g·kg-1 (MC-LR, one of the most potent congeners)[40-41] and i.p. LD50 = 111 - 600 g·kg-1 (MC-RR, one of the least potent congeners)[41-44] dependent on the study.
The toxic effect of microcystins is mediated by their uptake into cells. It is known that no passive diffusion of microcystins through cellular membranes can occur. The toxins are rather taken up by the cells via an active transport.[34, 45] It was shown, that organic anion transporting polypetides (OATPs) are responsible for the microcystin transport.[22, 34] Up to date there are over 300 different types of OATPs identified in over 40 organisms, which can be found both ubiquitously or in specific organs.[46-47] In human organism OATPs can be found in hepatocytes (OATP1B1, OATP1B3, OATP1A2), kidney cells (OATP1A2) and the blood-brain barrier (OATP1A2).[34]
Interestingly OATP1B3, but not OATP1B1 can be found in many types of cancer cells making it an interesting therapeutic target.[22] Recently it was shown, that the presence of Arg-residue the position 2 of the microcystin scaffold favours the selective uptake by OATP1B3 over OATP1B1 by factor of 30.[22] The observed selectivity makes microcystin a suitable lead structure for the devolpment of selectively transported cytostatic derivatives, which is relevant for further development of anti-cancer drugs. [21-22]
The taken up toxin inhibits the protein phosphatases, which leads to disruption of the cellular signaling pathways, apoptosis and generation of reactive oxygen species.[45] It was also shown that certain amount of intracellular microcystin forms a thioether linkage between the Mdha-residue of microcystins and glutathione or cysteine residues of other proteins.[45, 48] The formation of covalent microcystin-glutathione adducts is believed to be a part of the cellular detoxification mechanism.[45,
48]
1.3.2 Interactions of microcystins with protein phosphatases
Reversible phosphorylation is a ubiquitous post-translational modification which is fundamental for cellular signal transduction.[49-51] Protein phosphatases 1 and 2A (PP1 and PP2A) are responsible for more than 90% activity of all serine/threonine phosphatases within the cell.[52-53] Selective inhibition of these enzymes is important for the investigation of the intracellular signal transduction cascades, as well as for drug development.[49, 54] However their high structural similarity, makes the selective inhibition of these enzymes challenging.[54] Although fostriecin (IC50 (PP2A) = 3.2 nM; IC50 (PP1) = 131 M),[53] is a known selective PP2A inhibitor, a highly selective PP1 inhibitor would be of importance for the development of novel potential HIV, type II diabetes and anti-cancer drugs.[49]
5 The protein phosphatases PP1 and PP2A are the best studied intracellular interaction partners of microcystins.[45] There is little data about the binding of microcystin to other phosphatases.[55] However it could be shown, that microcystins are potent inhibitors of PP4-PP6,[56-57] they do not inhibit the tyrosine phosphatases, PP2B or PP2C.[57-58] Typical IC50 valuesfor the PP1 and PP2A inhibition with microcystins lay between 0.15 and 6 nM, depending on the microcystin congener and type of assay used.[59] The high affinity makes microcystins high-potential lead structures for the development of novel selective synthetic phosphatase inhibitors.[60] Up to date there are several X-ray structures of the covalently linked microcystin-phosphatase complexes, which are published for MC-LR 1 and PP1 (pdb code: 1FJM[61], Figure 1.3.2), MC-LR 1 and PP2A (pdb codes: 2IE3,[62] 2IAE,[63] 3DW8,[64] and 3FGA[65]) as well as for the non- covalent complex of dihydro-MC-LA and PP1 (pdb: 2BDX).[66] Additionally NMR structures of MC-LR 1 (pdb: 1EVA[67] and 1LCM[68]), [D-Leu1]-MC-LR[69] and MC-RR[70] in solution have been published. Certain structure-activity relationships between different positions within the microcystin backbone and regions on the enzyme surface can be established based on the crystal structures of the PP1- and PP2A- microcystin adducts and the toxicological data.[43, 71]
Figure 1.3.2. X-ray structure of MC-LR 1 bound to PP1-enzyme (pdb: 1FJM).[61] The image was created using the PyMOL Molecular Graphics System, Version 1.3 Schrödinger, LLC.
Interaction of microcystin with PP1 occurs in a two-step mechanism. Within the first, fast step, microcystin coordinates to the enzyme surface.[59, 72-73]
The toxin binds to the phosphatase by the interacting with the active site and the hydrophobic groove adjacent to it.[61, 72] The two carboxy groups of
-D-Glu6 and -D-MeAsp3 interact indirectly via two water molecules with the same PP1 Adda5
Arg4
-D-Glu6
-D-MeAsp6
Leu2
D-Ala1
Thioether linkage
6
residue, Arg-96.[61],[51, 72] Additionally the carboxy group of -D-Glu6 accepts the hydrogen bonds from a water molecule that bridges a metal cation in the active site of the enzyme.[51, 61] Besides Arg-96, the free carboxy moiety of -D-MeAsp3 of the toxin interacts with and Tyr-134 of an enzyme, thus disturbing the interactions of the phosphatase with other substrates and blocking the active site.[61, 72] The carbonyl groups of Adda5 and Arg4 of MC-LR 1 form hydrogen bonds to Arg-221 of the enzyme.[51, 61] These interactions make carboxy groups of -D-Glu6- and -D-MeAsp3-residues crucial for binding. For these reasons, natural microcystins like [-D-Glu(OMe)6]MC-LR (LD50 > 1000 g·kg-1) and [-D- Glu(OCH2CH(OH)CH3)6]MC-LR (LD50 = 1000 g·kg-1) which contain ester moieties instead of carboxylic acids show greatly reduced or no activity.[43, 71]
Position 2 of the microcystin scaffold is occupied by a frequently varied L-amino acid. During the binding to PP1 the unpolar residues in this position can undergo hydrophobic interactions with the aromatic ring of the Tyr-272 of the enzyme which is located in close proximity.[72, 74]
As it can be seen from the X-ray structure of the phosphatase-enzyme complex the Adda5-residue is tightly packed within the hydrophobic groove of PP1. It undergoes lipophilic interactions with the residues within this part of enzyme, including Trp-206, which seems to be essential for the binding.[61, 72] The configuration of the double bonds within the Adda5 side chain seems to be important for these interactions. The rare natural [6Z-Adda5]MC-LR[75] and [6Z-Adda5]MC-RR[75] were shown to be non-toxic in doses up to 1000 g·kg-1. As it can clearly be seen from both crystal structure and toxicological data, the only part within the microcystin backbone, which is not involved in any interaction with the phosphatases, is the position 4. [61, 71] The residue in this position is directed away from the binding sites (Figure 1.4.2), which makes it ideal for introduction of a label into the toxin scaffold.[59, 72]
After the toxin is bound to the enzyme surface, a thioether bond involving Cys-273 of PP1 and Mdha7 is slowly formed (kinetics in order of hours).[59] The formation of this covalent linkage leads to irreversible enzyme inhibition.[61] The tendency to form such covalent enzyme-toxin adducts makes the analysis of microcystin-containing tissue samples by standard immunological methods uncertain.[76] Interestingly it was shown that the reduction of the Mdha with sodium borohydride to a racemic N-MeAla-derivative, which makes impossible the formation of the covalent thioether bond between, does not influence the microcystin-phosphatase binding affinity.[77]
1.4 Analysis of microcystin-containing samples
The high toxicity of microcystins even in very small concentrations as well as the discussed transfer of these toxins between different levels of the food chain[78] makes it essential to have a highly sensitive and reliable methodology for the determination of microcystin content, both in water and in biological samples. However the tendency to form covalent adducts with cysteine residues makes
7 the determination of the overall microcystin content in the biological samples (total microcystin burden) complicated.[79] The main analytical methods for microcystin detection and quantification include LC-MS-MS,[80-84] protein phosphatase inhibition assays and ELISA,[85-86] which is based on the anti-Adda antibody.[79, 87] The LC-MS-MS methodology is the only analytical technique, which enables both quantification of the toxin as well as the identification of single congeners.[55, 88] Currently the main limitation of this method is lack of certified analytical materials as internal standards.[88]
According to the analytical method issued by the US EPA,[55] an internal standard (surrogate analyte) should chemically be as similar as possible to the natural toxins but should be absent in any natural sample. The surrogate analyte is added to the sample prior to the extraction in order to be able to evaluate the toxin loss during the extraction procedures.[55] The ideal standard would thus be a microcystin labeled with stable isotopes (e.g. deuterium). Its physicochemical properties are identical to those of natural toxins; however it can clearly be distinguished from the natural toxins due to the m/z-shift caused by the incorporation of the stable isotopes.
Compared to water analysis, determination of the microcystin content in biological samples is more challenging. A common practice is the extraction of microcystins from tissue or blood samples with a suitable solvent (most frequently methanol) prior to the quantification of the toxins.[89] This treatment does not cleave thioether linkages between microcystins and intracellular proteins. Thus the covalently-bound microcystins, which are still bioavailable and may represent up to 90% of the total toxin amount[78] are frequently not registered.[89] The lack of the bound toxin fraction leads to an underestimation of the toxin content in the biological samples.[78-79, 87, 89-90]
Furthermore, the larger covalent microcystin-protein conjugates were shown to be digested to smaller microcystin- containing fragments.[78] These fragments retained at least partial inhibitory potency towards protein phosphatases, thus suggesting the further transfer of the microcystins in the food web and making the covalently bound toxins a potential hidden danger.[78]
8
Scheme 1.4.1. Possible approaches for the cleavage of microcystin-protein conjugates.[89]
The only technique with which both protein-bound as well as free microcystins can be detected, is the so called Lemieux-oxidation (Scheme 1.4.1).[76, 89-93]
As a result of treatment of the Adda side- chains with KMnO4/KIO4, the 2-methyl-3-methoxy-4-phenylbutyric acid 9 is formed, which can be quantified either by GC-MS or by LC-MS.[89, 91] However, the main limitation of this approach is its inability to evaluate the amount of the single congeners as well as its poor reproducibility.[79, 89] This methodology can also lead to overestimation of the microcystin content in case of algal probes, which are reported to also contain non-toxic Adda-containing intermediates.[87, 89, 94]
Therefore, a new method which would enable the estimation of bound and unbound microcystin concentration and at the same time identification of single congeners is highly demanded.[89]
1.5 Biosynthesis and role of microcystins in the cyanobacteria
The production of microcystins is an energetically demanding process for the cyanobacteria, nevertheless the amount of the produced toxin can compose up to 2% of dry mass of the cell.[95] The toxins are produced by non-ribosomal peptide synthetases (NRPS), polyketide synthetases and further tailoring enzymes.[96-97] The corresponding microcystin synthetase gene cluster is probably one of the most intensively investigated NRPS gene clusters considering its evolution and diversification.[96] Despite intensive investigation, the function of the microcystin within the cyanobacterial cell is not yet completely understood.[97] Extracellular roles of the toxins as a defense against grazers,[98] parasites[99] or as infochemicals[97, 100-101]
are discussed.[102] The assumed advantages of the microcystin producing strains also involve defense against oxidative stress upon high light conditions[103] and improved growth under low carbon conditions.[102-103] Recently it was shown that covalent binding of microcystins by the formation of a thioether linkage between Mdha7
9 and Cys-residues to the cyanobacterial proteins such as RbcL, (the large subunit of RubisCO) could protect the modified proteins against degradation and could be seen as part of a response mechanism at high light stress conditions.[103] In order to shed light on the intracellular functions of microcystins, the identification of further microcystin binding proteins is of interest. However the number of suitable analytical methods is limited. The application of an anti-Adda antibody in the cyanobacterial samples is questionable, because of the probable presence of non-toxic Adda- containing metabolites.[94] An alternative method for the detailed investigation of the role of microcystins in cyanobacteria could be the use of microcystin derivatives with biorthogonal tags.
After incubation e.g. fluorescent dyes or other reporter molecules can be attached using bioorthogonal ligation reactions. This methodology can be used for the identification of further microcystin binding proteins, or for monitoring the effects of adding the labeled microcystins to cyanobacterial populations. In this context, the use of labeled toxins would enable the differentiation between endogenous microcystins produced by cyanobacteria and added labeled standards.
1.6 Chemical modifications of natural microcystins
In order to investigate the toxicological properties of microcystins, several modified derivatives have been synthesized starting from natural MC-LR 1. The guanidinium moiety of its Arg-side chain was used to introduce various modifications into the microcystin scaffold (Figure 1.6.1). For example, it was used for the reaction with 1,3-diketones leading to the formation of a N-(2-pyrimidyl)ornithyl residue.[59] The main drawback of the Arg-side chain labeling methodology is that it is not bioorthogonal. Thus the labeled microcystin derivative should be prepared separately, before the addition of the toxin to the biological sample.
Using this type of side chain modification, a TAMRA-MC-LR-derivative 10 was synthesized and evaluated with a protein phosphatase inhibition assay (PPIA).[59] The fluorescently labeled compound 10 was an approx. 10-fold less potent phosphatase inhibitor compared to natural MC-LR 1, but still showed affinity in a low nanomolar range (IC50 (PP1) = 4 nM, compared to IC50 (PP1) = 0.3 nM for 1 within the same assay).[59] The derivative 10 was further used as an affinity based probe, for the identification of protein phosphateses PP1 and PP2A in complex mixtures.[59] Another example is a series of compounds including biotin- and diazirine-derivatives 11 and 12, as well as MC-LR-fluorescent dye conjugates MC-LR-(Alexa- 430) 13, MC-LR-(Alexa-488), MC-LR-(5,6-FAM) 14, MC-LR-(TexasRed)). Those were synthesized by the conjugation of the guanidine moiety of MC-LR 1 with an excess of carboxy-preactivated precursor and 2- tert-butyl-1,1,3,3-tetramethylguanidine (Barton’s base).[104-105] The fluorescently labeled derivatives 13 and 14 were ca. 30 times less potent PP2A inhibitors (IC50 = 1.5 nM and 1.6 nM respectively) compared to unmodified MC-LR 1 (IC50 = 0.05 nM).[104] In contrast to these data the MTT assay performed with 13 (EC50 = 16.3 M for 13, compared to EC50 = 4.4 M for MC-LR 1), as well as toxicity studies with 13,14 and
10
freshwater crustacean T. platyurus (LC50 = 15.7 M for 13, LC50 = 5.7 M for 14 compared to LC50 = 10.8
M[106] for MC-LR 1) revealed a comparable toxicity of the labeled derivatives compared to unmodified MC-LR 1.[104]
Figure 1.6.1. Modifications of the Arg-side chain of MC-LR 1 leading to N-(2-pyrimidyl)ornithyl derivative 10,[59] MC-LR-biotin 11,[105] MC-LR-diazirin 12,[105] and MC-LR-(Alexa-430) 13[104]and MC-LR-(5,6-FAM) 14.[104]
Another residue within the microcystin scaffold which can be easily modified, is the Michael acceptor of Mdha7. This methodology was inspired by the observed Michael addition of the Cys residue to the microcystins which occurs under physiological conditions.[107-110] The modification of MC-LR 1 with a fluorescein-derivatized cystein led to the formation of a fluorescent non-covalent phosphatase inhibitor 15 which was used for the development of a biosensor (Figure 1.6.2 A).[107] The reaction of Mdha7 with ethanthiol derivatives leading to the formation of thioethers 16 was also used to faciliate the mass- spectrometric detection of microcystins (Figure 1.6.2 B).[108, 110] The differences in the reactivity of Michael acceptor moieties towards the thiols in case of Mdha7, Mdhb7 and Dhb7 caused by the presence or
11 abscence of a -methyl group were used to differentiate between microcystins, which contain these different types of Michael acceptors.[109]
In order to be able to investigate the distribution of microcystins in the toxin-exposed organism, 125I- and
3H-labeled microcystin derivatives were prepared.[111-113] For the preparation of the 125I- iodinated microcystins, MC-YM was treated with 125I-iodine, NaI and lactoperoxidase.[111] 3H- labeled MC-LR was prepared using an exchange procedure treating MC-LR 1 with 3H2O in presence of acetic acid and pyridine. The 3H-label was shown to be incorporated into -D-MeAsp3 and -D-Glu6 (ratio 1.5:1) and the obtained 3H-microcystin derivatives were shown to be stable in blood (59% remaining compound after 7 days, based on radioactivity).[112]
Figure 1.6.2. The derivatives of MC-LR 1, which are modified at position 7. (A) MC-LR-fluorescein derivative 15[107] (B) thioether derivatives 16.[108-109]
1.7 Synthesis of microcystins, nodularins and simplified analogs thereof
1.7.1 Total synthesis of microcystin-LA and its unnatural analogs
The increasing abundance of toxic algal blooms, high biological activity and structural complexity of microcystins and related nodularins have stimulated the efforts towards the total synthesis of these natural products during the last two decades. Surprisingly, up to date only one synthesis of a single microcystin congener MC-LA 8 has been published.[114] Several unnatural derivatives of 8 were synthesized later using the same approach with the aim to target the selectivity between PP1 and PP2A.[60] Generally, for all the amino-groups and the carboxy-moieties of -D-Glu6- and -D-MeAsp3, the Boc-/-COOMe protecting group strategy was used. The C-terminal L-Ala residue was protected with trichloroethyl (Tce) or –COOt-Bu protecting groups.[60, 114] For macrocyclization, the position between Ala4 and Adda5 was used.[60, 114] This position also corresponds to the cyclization site
12
exploited during the biosynthesis of microcystins.[115] In order to facilitate the synthesis, the MC-LA scaffold was assembled in a convergent way from three fragments: tetrapeptide 17, dipeptide 18 and Boc-Adda-OH 19, which were synthesized independently (Scheme 1.7.1.1).[114]
Scheme 1.7.1.1. Retrosynthetic analysis of the MC-LA 8 according to the Chamberlin approach.[114]
The key residue within fragment 17 is Mdha7 which was generated by the HWE-reaction of a phosphonylsarcosine derivative with formaldehyde early in the synthesis.[114] Peptide 17 was assembled from Boc-D-Glu-OMe 20 and tripeptide 26 which contained D,L-phosphonylsarcosine. The peptide 26 was prepared in 4 steps starting from methylglyoxalate hemiacetal 22 (Scheme 1.7.1.2).
Compound 22 was first converted to Z--hydroxysarcosine derivative 24 by an equilibrium reaction with Z-methylamine 23. Derivative 24 was treated with MsCl under basic conditions and the corresponding mesylate was in situ transformed to dimethylphosphonylsarcosine derivative 25 using trimethylphosphite and sodium iodide. The obtained amino acid 25 was then coupled to the N- terminally unprotected dipeptide H-D-Ala-Leu-Ot-Bu to give the desired tripeptide 26.
13 Scheme 1.7.1.2. Synthesis of phosphonylsarcosine containing tripeptide 26.[114]
Dipeptide 18 was assembled from the trichloroethyl alanine derivative 27 and Boc--D-MeAsp-OH 28.
Scheme 1.7.1.3. Synthesis of -D-MeAsp3-building block 28.[114, 116]
The erythro--D-MeAsp3 residue is specific for the microcystins and nodularins and was synthetically one of the most challenging ones. The Boc--D-MeAsp-OH 28 building block was synthesized in 6 steps with an overall yield of 20% starting from H-D-Asp-OH 29 (Scheme 1.7.1.3). First both carboxy groups of 29 were protected and dimethyl ester 30 was subsequently benzylated by reductive amination to give 31. The benzylamine derivative 31 was treated with a phenylfluorenyl bromide to give the fully protected derivative 32. Next, compound 32 was methylated using LHMDS as base and MeI as methylating agent which led to an regio- and stereoselective formation of the erythro-
14
product 33. The observed regioselectivity of the -methylation versus -methylation of 32 can be explained by the sterical hindrance caused by the phenylfluorenyl protecting group, thus shielding the -position of 32.[116-117] The stereoselective formation of -erythro-product 33 can be attributed to the formation of the (Z)-lithium enolate, which is attacked from the direction opposite to the bulky phenylfluorenyl group.[116] The subsequent saponification of the methyl ester 33 with LiOH led to the formation of diastereomers 34a and 34b which were separated by column chromatography. Milder cleavage conditions could not be used because of the low reactivity of the sterically hindered diester 33. The erythro-configured amino acid 34a was hydrogenated and protected using Boc2O to give building block 28.
Third building block Boc-Adda-OH 19 was assembled from two the fragments 35a and 35b using Suzuki coupling (Scheme 1.7.1.1 and Section 1.7.4).[114]
Scheme 1.7.1.4. Macrocyclization and final deprotection steps.[114]
15 The fragments 17, 18 and 19 were coupled to give the linear heptapeptide 36, which was then C- terminally deprotected, activated using Pfp-OH/DCC, and macrocyclized by adding highly diluted peptide to a CHCl3/phosphate buffer mixture (Scheme 1.7.1.4).[114] No isomerization related to the macrocyclization was mentioned.[114] The obtained macrocyclic diester 37 was treated with LiOH in THF/H2O to give a mixture of MC-LA 8 and unidentified isomers. The isomers were separated by HPLC and compared to an authentic MC-LA sample, to identify MC-LA 8.[114]
Four unnatural MC-LA analogs 38-41 were synthesized, using a slightly modified synthetic route (Figure 1.7.1.5).[60] In case of these derivatives one of the most challenging steps was the saponification of methyl esters.[60] In analogy to the synthesis of MC-LA 8 the formation of 2-3 isomers was reported for compounds 38-41 during this final basic deprotection step.[60] The attempts to avoid the harsh basic conditions (LiOH in H2O/THF) using for example (CH3)3SiOK, CeCl3, CoCl2 in H2O/THF orlipases from Candida cylindracea or Pseudomonas species led either to no improvement or no reaction.[60]
Figure 1.7.1.5. Unnatural MC-LA derivatives 38-41.[60]
In contrast to the synthesis of natural MC-LA 8 where the isomeric mixture was compared to the natural toxin to identify the desired product, there was no standard available for the derivatives 38- 41.[60] To identify the desired unnatural derivatives, the obatined isomers of 38-41 were re-esterified using TMS-diazomethane and the resulting diesters were compared with the original ones by TLC- analysis.[60] To evaluate the binding affinity and possible selectivity towards PP1 and PP2A, a series of
16
PPIA was performed with the unnatural microcystins 38-41. The peptides 38-41 showed a binding affinity in a nanomolar range,[60] with the highest selectivity (7:1 PP1 vs PP2A) observed for compound 40.[60]
To sum up, the Boc-/-COOMe approach was a landmark achievement which for a first time enabled the synthesis of natural MC-LA 8[114] and its unnatural derivatives.[60] Even though the synthetic microcystin derivatives are highly demanded,[88] there are several serious challenges which have been limiting the further applications of this approach for over 15 years. Most important of them is the isomerization during the final synthetic step which leads to formation of several isomers and cannot be avoided. This makes the isolation and identification of the final product complicated and considerably lowers its overall yield. Furthermore the Michael-acceptor containing Mdha7 residue is generated early in the synthesis. This limits the possibilities to change the protecting group strategy by introducing the moieties which are cleaved under nucleophilic conditions. Therefore a novel approach to the microcystin scaffold is desired.
1.7.2 Towards the total synthesis of microcystin-LR
Microcystin-LR 1 is the most abundant and well-studied natural microcystin. Compared to MC-LA 8, the synthesis of MC-LR 1 is more challenging due to the presence of the guanidine moiety in the Arg- side chain, which requires significant changes in the protecting group strategy. An attempt to synthesize MC-LR 1 by a solution-based approach was developed as a PhD-project in a Rinehart group.[118]
17 Scheme 1.7.2.1. Retrosynthetic analysis of the MC-LR-scaffold.[118]
In analogy to the synthetic route developed for the MC-LA 8,[114] the MC-LR scaffold was split into three fragments: tetrapeptide 42, dipeptide 43 and Boc-Adda-OH 19 (Scheme 1.7.2.1). Similarly, Boc-/-COOMe protecting group strategy was used for all the amino groups and the carboxy groups of
-D-MeAsp3 and -D-Glu6. Howeverthe presence of an arginine side chain required introduction of additional protecting groups.[118] Therefore, instead of the direct incorporation of Arg-residue, H- Orn(Fmoc)-OPac 44 was first introduced in the dipeptide fragment 43 and later converted into arginine derivative. To avoid possible addition of nucleophiles to the Michael acceptor of Mdha7 during the numerous backbone transformation steps, the phenylselenocysteine[119-120] containing tripeptide 45 was synthesized and coupled to Boc-D-Glu-OMe 17 to give the tetrapeptide 42.[118] The phenylselenocysteine moiety was converted to the Mdha during the final step of the synthesis.[118]
Boc--D-MeAsp-OMe 28 was synthesized analogously to the synthesis of MC-LA.[114] Boc-Adda-OH 19 was synthesized in 13 steps starting from phenylacetaldehyde 46 according to the linear synthetic approach published earlier by the same group.[121]
18
Scheme 1.7.2.2. Macrocylization and deprotection steps.
In contrast to the former synthetic approach,[114] the amide bond between Leu2 and -D-MeAsp3 was used as a macrocyclization site for the MC-LR scaffold. In order to synthesize the corresponding linear precursor, first tetrapeptide 42 and Boc-Adda-OH 19 were coupled to obtain a pentapeptide which was then attached to the dipeptide 43, giving heptapeptide 47 (Scheme 1.7.2.2). The subsequent macrocyclization was performed using highly diluted linear precursor 47 and coupling reagent PyAOP in DMF.[118] The resulting macrocyclic derivative was treated with piperidine to remove the Fmoc-protecting group and the ornithine residue was transformed into the di-Boc protected arginine via coupling with an activated guanidine derivative 48 to give the cyclic peptide 49.[118] In analogy to the synthesis of MC-LA 8, the main challenges in case of the described MC-LR synthesis were the final deprotection steps. Treatment of the macrocycle 49 with hydrogen peroxide in order to transform the phenylselenocystein moiety into Mdha7, subsequent basic saponification of -COOMe moieties with LiOH in THF/H2O and final treatment with TFA to remove Boc-protecting groups, led to 200 g of a complex mixture, which could not be separated by HPLC.[118] Unfortunately
19 no evidences confirming the formation of MC-LR 1 could be found in the analytical data provided[118]
for the obtained inseparable mixture.
This synthesis featured several innovative ideas used to overcome the limitations arising from the former synthetic approach (Section 1.7.1), like early incorporation of Mdha residue. However the same protecting group pattern used for the carboxy moieties of -D-Glu6 and -D-MeAsp3 residues led to same challenges which made impossible the obtaining and/or purification of the desired MC- LR 1.
1.7.3 Synthesis of nodularin-V (motuporin)
Nodularins are toxic cyclic pentapeptides, which are structurally related to microcystins. They can be found in the cyanobacteria Nodularia spumigena and the marine sponge Theonella swinhoei.[27, 122] In contrast to microcystins, which can be found in both fresh and brackish waters,[4] nodularins occur exclusively in brackish waters.[122]
Scheme 1.7.3.1. Retrosynthetic analysis of the nodularin-V scaffold (motuporin) 4.[26, 123-126]
Up to date there are several synthetic approaches published, but only for a single nodularin-V (motuporin) 4 (Scheme 1.7.3.1).[26, 123-126]
These approaches differ by the syntheses of single building blocks and cyclization strategies, but all of them share the same Boc-/-COOMe protecting group pattern previously discussed for MC-LA 8 and MC-LR 1 syntheses. In contrast to the published synthesis of microcystins[60, 114, 118]
no isomerization during the final methyl ester saponification using aq. Ba(OH)2[26, 123-124, 126]
or aq. LiOH[125] have been reported in case of motuporin 4. However, there is a footnote reporting the isomerization in amounts comparable to those observed during the total synthesis of MC-LA (see footnote 65 in the published total synthesis of MC-LA).[114] Similarly to the synthesis of microcystins, the macrocyclization was another crucial step during the synthesis of motuporin 4. Several different cyclization positions including -D-MeAsp1 and Val2,[124, 126]
Val2 and Adda3,[26, 125] as well as Mdhb5 and -D-Glu4 [123] have been exploited using HATU,[26, 123-124]
FDPP,[125] or
20
Pfp-Ester[126] as activation reagents. The best cyclization yield of 79% could be achieved by using HATU/NEM and the amide bond between -D-MeAsp1 and Val2 as a cyclization site.[124]
1.7.4 Synthesis of Adda
The -amino acid Adda5 ((2S,3S,4E,6E,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6- decadienoic acid) is a building block which is specific for microcystins and nodularins. The presence of Adda was shown to be crucial for the biological activity of these cyanotoxins.[72-73, 75, 127]
Given the structural complexity of this residue, which contains four stereocenters (positions 2, 3, 8, and 9) and two E-configured double bonds, the reliable and high-yielding synthesis of Adda is one of the most essential prerequisites for the successful total synthesis of natural microcystins and nodularins as well as the unnatural analogs thereof. Therefore, there have been numerous syntheses of Adda,[121,
128-137] as well as enantio-Adda ((2R,3R,4E,6E,8R,9R)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl- 4,6-decadienoic acid),[138-139] published. Most of these synthetic strategies were optimized to deliver Boc-Adda-OH 19 used with the Boc-/-COOMe strategy. A synthesis, which delivers Fmoc-protected Adda derivatives needed for the Fmoc/-COOt-Bu based SPPS has not yet been published.
Scheme 1.7.4.1. Fragment based approach for the synthesis of Adda-building blocks.[128-129, 135]
The vast majority of published Adda-syntheses feature the convergent approach to the synthesis of this building block (Scheme 1.7.4.1). According to this strategy Adda is assembled from two separately synthesized fragments: A, which corresponds to the positions 8 and 9 as well as the phenyl ring of Adda, and B, which includes stereocenters corresponding to the positions 2 and 3 of Adda. The connection of these two fragments is performed using either a cross-coupling or by a double bond-forming reaction. The published syntheses suggest attachment of the fragments A and
21 B using Wittig or HWE reactions (fragments 50a and 50b,[125-126, 128-130, 134-135]
or 50a and 51b[133]), coupling of a Zn-organic derivative with the corresponding vinyliodide (fragments 52a and 52b),[124,
136] Suzuki coupling (fragments 35a and 35b),[114] or Stille coupling (fragments 53a and 53b).[132] In case of the of enantio-Adda derivative, a metathesis reaction was used to connect the corresponding fragments.[138-139] Alternatively, Boc-Adda-OH 19 was synthesized via a linear approach starting from phenylacetaldehyde 46 (Scheme 1.6.4.2).[121, 131] In this case, the first aldol reaction with corresponding (R)-Evans or (R)-Crimmins auxiliaries (R)-54 or (R)-55 determined the configuration of the stereocenters at positions 2 and 3. The second aldol reaction with enantiomeric auxiliaries (S)-54 or (S)-55 was responsible for the configuration of stereocenters 8 and 9. The E-configured double bonds were synthesized via two consecutive Wittig reactions. The linear synthetic approach, which is based on the Evans aldol reaction is the highest yielding Adda synthesis (40% over 13 steps) published.[121] For further details considering this approach see section 3.1.4.5.
Scheme 1.7.4.2. Linear approach to Boc-Adda-OH 19 based on Evans[121] or Crimmins[131] aldol reactions.
1.7.5 Synthesis of simplified microcystin analogs
The microcystins are perspective lead structures for the development of protein phosphatase inhibitors and cytotoxic agents.[45] However, these compounds consist of synthetically demanding building blocks, which makes their synthesis challenging as well as time consuming and cost expensive. Thus the amount of available natural microcystin derivatives is limited, series of simplified microcystin and nodularin analogs were designed, synthesized and evaluated.[140-147]
22
Figure 1.7.5.1. Adda-containing linear peptides 67-73.[148]
Inspired by the observation, that linear Adda-containing degradation products of MC-LR (e.g. peptide 67), showed phosphatase inhibition in the nanomolar range,[149-150] two series of linear phosphatase inhibitors have been synthesized by the Chamberlin group.[147-148] The longer linear peptides 67-73 (Figure 1.7.5.1), including the microcystin degradation product 67, resemble the substructure of natural microcystins comprising residues 5-6-7-1-2 of the natural microcystin scaffold.[148] The N- terminus of the linear analogs was unprotected (67 and 70) or acetylated (68, 69, 71, 72, and 73). In the position corresponding to position 7 of the microcystin scaffold, either Mdha (67, 69, 72) or NMeGly (70, 71, 73) residues were incorporated.[148] However, it was impossible to reproduce the reported nanomolar[149-150] affinity of peptide 67, which along with the compounds 68-73 showed activity in a 16-400 M range.[148]
Figure 1.6.5.2. Adda-containing linear microcystin analogs 74-80.[147]
23 Another series of shorter Adda-based inhibitors have been synthesized by the same group (Figure 1.7.5.2).[147] In this case, the N-acetylated Adda was extended with a single amino acid residue (74- 76) or with substituted proline derivatives (77-80). The dipeptides 74 and 75 have shown inhibitory potency in the nanomolar range.[147] Interestingly, the IC50 values for compounds 75-77 were demonstrated to be dependent on the type of assay used. The phosphorylase-based assay led to an over 40-fold lower IC50 values compared to the para-nitrophenyl phosphate (pNPP)-based assay.[147]
The substituted proline derivatives 77-80 have shown an inhibitory potency in the M range with the pNPP-based assay.[147]
Scheme 1.7.5.3. Adda-based PP1-activating derivative 81.[57]
As it can be seen from the structure of the small Adda-based phosphatase inhibitors, the Adda-side chain is capable to undergo strong binding to the phosphatases PP1 and PP2A. By combining the Adda-moiety with the structural motive RVXF, which is responsible for the activation of the protein phosphatase PP1, a compound 81 was designed by the Chamberlin group, which was positively modulating the phosphatase activity towards the substrate by 200 %.[57] Derivative 81 was assembled starting from the Adda fragment 50a, by coupling it to aldehyde 82, which resembles the valine side chain, by a Wittig reaction, subsequent attachment of the arginine residue followed by deprotection and acetylation.[57]
Based on the microcystin and nodularin structures, series of simplified cyclic phosphatase inhibitors were synthesized using SPPS by the group of Gani.[143, 146]
The binding affinities of compounds 83-87 were evaluated in an inhibitory assay with PP1 (Figure 1.7.5.4).[151] Peptides 83-87 share a similar macrocyclic structure, including the free carboxy-groups of -D-Asp3 and -D-Glu6 which are known to be crucial for the binding in case of natural microcystins.[143, 146]
The macrocyclic phosphatase inhibitors 83-87 differ by having either proline (84 and 86) or sarcosine (compounds 83, 85, and 86) in the eastern part of macrocycle (Figure 1.7.5.4, blue). Additional differences are the Adda
