Of -anatoxin

HPLC analysis of anatoxin-a was first carried out by Astrachan and Archer, " who extracted the toxin from Anabaenaflos-aquae using chloroform followed by hydrochloric acid. The HPLC analysis was carried out on an ODS column using hypochlorate-methanol. Other systems used since include acetic acid extraction and analysis on a reversed-phase C g column using methanol-water mobile phase, and extraction in water after ultrasonication and analysis on reversed-phase... 

The ability to identify and quantify cyanobacterial toxins in animal and human clinical material following (suspected) intoxications or illnesses associated with contact with toxic cyanobacteria is an increasing requirement. The recoveries of anatoxin-a from animal stomach material and of microcystins from sheep rumen contents are relatively straightforward. However, the recovery of microcystin from liver and tissue samples cannot be expected to be complete without the application of proteolytic digestion and extraction procedures. This is likely because microcystins bind covalently to a cysteine residue in protein phosphatase. Unless an effective procedure is applied for the extraction of covalently bound microcystins (and nodiilarins), then a negative result in analysis cannot be taken to indicate the absence of toxins in clinical specimens. Furthermore, any positive result may be an underestimate of the true amount of microcystin in the material and would only represent free toxin, not bound to the protein phosphatases. Optimized procedures for the extraction of bound microcystins and nodiilarins from organ and tissue samples are needed. 

The debut of the selective AChR agonist (+)-anatoxin-a has provided a new tool for AChR physiology and pharmacology. (+)-Anatoxin not only has high affinity for the nicotinic AChR but it also has high selectivity for nicotinic over muscarinic receptors in the mammalian CNS. Recently, the use of (+)-anatoxin-a was essential to the identification of nicotinic receptors on cultured neurons (4), We are studying the features which allow it to bind with high affinity to the peripheral and central nicotinic receptors and the kinetic effects on receptor conformational... 

Using synthetic enantiomers, we found that anatoxin-a is highly stereospecific with the (+) isomer having 150-fold greater potency than the (-) isomer (Figure 2) 19). The semi-rigid nature of anatoxin-a undoubtedly facilitates its stereospecificity. 

Figure 1. Structure of (+)-anatoxin-a and two analogs with agonist and noncompetitive antagonist activity, respectively.

Figure 2. Potency of anatoxin-a analogs to induce contracture in frog rectus abdominis muscle. The data from two experiments are combined in this figure. In one, anatoxinmethylester was found to be equipo-tent with carbamylcholine. In the other, the anatoxin isomers were assayed against ACh [after cholinesterse inhibition by diisopropylfluoro-phosphate (DFP) followed by washing of the preparation] (19). Maximal contracture was measured by depolarization with KCl at the end of each experiment.

Onodera H, Oshima Y, Henriksen P, Yasumoto T (1997) Confirmation of anatoxin-a(s) in the cyanobacterium Anabaena lemmermanni as the cause of bird kills in Danish lakes. Toxicon 35 1645-1648... 

The HPLC elution pattern of anatoxin-s(s) is shown in Fig. 5 and is the last peak in the profile. Determination of LD gave an approximate value of 40 yg/kg body weight, which is five times more potent than anatoxin-a. 

Figure 1. Structure of anatoxin-a from Anabaena flos-aquae NRC-44-1, saxitoxin and neosaxitoxin. Saxitoxin and neosaxitoxin is produced by certain species of marine algae and by the freshwater cyanobacteria Aphanizomenon flos-aquae NH-5.

Figure 2. Flow diagram for the extraction of Anatoxin-a(s) and Aphantoxins.

Figure 5. HPLC profile of Anatoxin-a(s) toxic peak (far-left ...

Harada, K. Nagai, H. Kimura, Y. Suzuki, M. Park, H.-D. et al. (1993) Liquid chromatography/mass spectrometiy detection of anatoxin-a, a neurotoxin from cyanobacteria. Tetrahedron, 49, 9251-60. 

Total synthesis of anatoxin-a was achieved by Martin and Mori through the same strategy (Scheme 38). 

The key step is the construction of a bicyclo[4.2.1] system. To this end, pyrrolidine derivative 95 having fif-substituents is synthesized from (+)-pyroglutamic acid and subjected to enyne metathesis using Ig to result in formation of 96 in 84% yield. From 96, synthesis of (+)-anatoxin-a is straightforward and successfully achieved. 

The Pd-catalyzed intramolecular aminocarbonyiation has also been applied to the formal total synthesis of Anatoxin-A 219, an acetylcholine mimic (Equation (16)). Thus, the reaction of 5-(methoxycarbonylamino)-cyclooctene 216a in the presence of a catalytic amount of PdCl2 and cupric chloride (CUCI2) (3 equiv.) in methanol under ambient pressure of CO gave the desired azabicyclo[4.2.1]nonane 217 as the predominant product. The regioselectivity of this reaction is highly dependent on the nature of the A -substituent. Thus, the... 

Reproduced from Oh, C.-Y. Kim, K.-S. Ham, W.-H. A fonnal total synthesis of (+)-anatoxin-A by an intramolecular Pd-catalyzed aminocarbonylation reaction. Tetrahedron Lett. 1998, 39, 2133-2136, with pennission from Elsevier. 

Scheme 39 Synthesis of anatoxin-a precursor 118 via MeLi mediated [3-lactam ring opening...

Fig. 16.2. Chemical structure of anatoxin-a(s). This image is licensed under the http //www.gnu.org/copyleft/fdl.html GNU Free Documentation License. It uses material from the http //en.wikipedia.org/wiki/Cyanotoxin Wikipedia article.

The developed biosensor was applied to the analysis of cyanobacterial bloom samples from freshwater lakes of Spain, Greece, France, Scotland and Denmark. Two samples from Scotland and one from Denmark irreversibly inhibit the acetylcholinesterase. The estimated concentrations were between 1.5 and 30nmol/g of dry weight, values extremely high when compared to the intraperitoneal 50% lethal dose of anatoxin-a(s) in mice (121 nmol/kg). 

C. Edwards, K.A. Beattie, C.M. Scrimgeour and G.A. Codd, Identification of anatoxin-a in benthic cyanobacteria (blue-green algae) and in associated dog poisonings at Loch Insh, Scotland, Toxicon, 30 (1992) 1165-1175. 

P. Henriksen, W.W. Carmichael, J. An and O. Moestrup, Detection of anatoxin-a(s)-like anticholinesterase in natural blooms and cultures of cyanobacteria/blue-green algae from Danish lakes and in stomach contents of poisoned birds, Toxicon, 25 (1997) 901-903. 

E. Devic, D. Li, A. Dauta, P. Henriksen, G.A. Codd, J.-L. Marty and D. Fournier, Detection of anatoxin-a(s) in environmental samples of cyanobacteria by using a biosensor with engineered acetylcholinesterases, Appl. Environ. Microbiol., 68 (2002) 4102 4106. 

The first partial synthesis7,8 of anatoxin-a (12) (a neurotoxin from algae) has been reported, starting with (-)-cocaine. 

Hemscheidt, T., Rapala, J., Sivonen, K., and Skulberg, O. M., Biosynthesis of anatoxin-A inAnabaena flos-aquae and homoanatoxin-A in Oscillatoriaformosa, J. Chem. Soc. Chem. Commun., 1361, 1995. 

The unique structure of anatoxin-a, and its potential as a pharmacological tool, has inspired many different chemical syntheses of anatoxin-a and analogs. The earliest synthesis, in 1977, used (—)-cocaine as starting material [23]. Subsequently, there... 

The SARs of anatoxin-a and analogs have been extensively studied [33-36], and only a few important findings dealing with chirality, conformation, and other structural features of anatoxin-a and homologs vfill be summarized here. A recent review that addresses the chemistry and pharmacology of anatoxin-a and analogs provides a more detailed treatment of this topic [37]. 

The naturally occurring enantiomer, (+)-anatoxin-a, is much more potent than (—)-anatoxin-a [38]. For example, using conditions that preferentially labeled rat brain a4p2 nACh receptors with [ H]nicotine, the natural enantiomer was found to be 1000-fold more potent than (—[-anatoxin [39]. This enantiospecificity of anatoxin-a has provided an excellent tool for probing the stereospedfidty of the ACh binding site on the nicotinic receptor. 

As compared to the natural ligand ACh, the conformation of anatoxin is relatively rigid, with only one rotatable bond as compared to four in ACh. This simplifies the ability to correlate the structural conformation of the ligand to efficacy, and, consequently, attention has been directed toward determining the active conformation through the use of sterically constrained analogs of the s-ds and s-trans conformers of anatoxin-a [19,40-44]). Although current evidence leans toward s-trans as the active conformation, condusive evidence, such as a crystal structure of (+)-anatoxin-a in complex with an nACh receptor, is still needed. 

Homoanatoxin-a, obtained from various freshwater cyanobacteria, is a relatively rare natural analog of anatoxin-a for which the C-11 side chain is extended by one methylene unit (Figure 6.4). It was originally isolated from Planktothrix sp. (formerly Oscillatoria) in 1992 [51]. It has recently been isolated from Raphidiopsis mediterranea Skuja from Japan [52] and Planktothrix (formerly Oscillatoria) formosa blooms in Ireland [53]. 

The total synthesis of homoanatoxin-a, as an analog of anatoxin-a, was also accomplished in 1992 [33]. It is a potent nicotinic agonist active at the postsynaptic nicotinic ACh receptor channel complex [54]. Although not as potent as anatoxin-a, homoanatoxin-a has provided valuable insight for SAR studies of anatoxin-a and its homologs. 

Anatoxin-a(s) is produced in both Anabaena flos-aquae [56] and Anahaena lemmer-mannii [57]. The symptoms of anatoxin-a(s) intoxication are similar to those of anatoxin-a but cause increased salivation in vertebrates hence, the similarity in the names of these compounds with the (s) added to indicate salivation [18]. The structures, however, are quite different, as are the mechanisms of action. 

Anatoxin-a(s) inhibits acetylcholinesterase by acting as an irreversible active-site-directed inhibitor [61]. This prevents degradation of ACh and leads to over-stimulation of the muscle cells (Figure 6.1) [56,62]. Thus, although the mechanism of action of anatoxin-a(s) is quite different from that of anatoxin-a, the observed toxicity is similar. In addition, it was the first irreversible acetylcholinesterase inhibitor to be found in a cyanobacterium. 

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