Anatoxin development

In our laboratory crude preparations of aphantoxins and anatoxin-a(s) are extracted similarly except at the final stages of purification (Fig. 2). A Bio-gel P-2 column (2.2 x 80 cm) is used for aphantoxins gel filtration and a Sephadex G-15 (2.6 x 42 cm) column for ana-toxin-(s). Both toxins are eluted with 0.1 M acetic acid at 1.5 ml/ min. Fractions of aphantoxins from Bio-gel P-2 run are spotted on thin-layer chromatography plates (Silica gel-60, EM reagents) and developed according to Buckley et al. (1976) (31). The Rf values for the aphantoxins, saxitoxin and neosaxitoxin standards (Table 1) indicates that two of the aphantoxins (i.e. I and II) are similar to saxitoxin and neosaxitoxin. 

An elegant synthesis of the neurotoxic alkaloid anatoxin has exploited the electrocyclic opening of the dibromobicyclo[5.1.0]octane followed by transannular cyclization (Scheme 13).238 Similarly, the thermal electrocyclic opening of the dichlorocyclopropane followed by intramolecular trapping of the developing allylic cation by a suitably positioned amine has been used in a homoaporphine synthesis.238... 

In a development involving racemic products, nonactivated /V-benzyl (3-lactam 117, is shown to be opened selectively with methyllithium to yield, after intramolecular oxirane opening, the methyl ketone 118, Scheme 39. This compound is transformed through several steps into the nicotinic acetylcholine receptor agonist anatoxin-a [106]. 

In order to guarantee the water and food quality and to preserve the human health, several detection and quantification methods, each one of them having advantages and disadvantages, have been developed for microcystins and anatoxin-a(s). 

Anatoxin-a(s) was first detected by the mouse bioassay. However, the technique most commonly used for its detection is HPLC, coupled to MS detection. The irreversible inhibitory power of this toxin towards acetylcholinesterase has been described [60] and the corresponding colorimetric inhibition assay has also been developed [61-63]. To date, no antibodies towards anatoxin-a(s) have been produced. 

Our research group is working on the development of electrochemical biosensors for the detection of microcystin and anatoxin-a(s), based on the inhibition of protein phosphatase and acetylcholinesterase, respectively. These enzyme biosensors represent useful bioanalytical tools, suitable to be used as screening techniques for the preliminary yes/no detection of the toxicity of a sample. Additionally, due to the versatility of the electrochemical approach, the strategy can be applied to the detection of other cyanobacterial toxins. 

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). 

Anatoxin-a (3) is a powerful neurotoxin (inhibitor of acetylcholine esterase) found in freshwater blue-green algae. The compound was required as an analytical standard and also for development of an immunoassay. It was synthesized by the 8-step sequence summarized in Scheme 29.1. The key intermediate 4 was resolved by separation of the dibenzoyl tartrates, and the remaining steps then gave both (+)- and (-)-anatoxin-a.2 The efficiency of the resolution was monitored by formation of the BocAla derivatives, which were distinguishable by NMR an ee of >98% was achieved even before recrystallization of the salts. It is also noteworthy that the published absolute configuration of the intermediate 53 was shown to be in error by X-ray crystallography. 

Subsequently, alternative methods were developed by Stjernlbf and co-workers for introducing the acyl moiety and different sidechains in the ketone intermediate 14 (Lindgren et al. 1987 Stjemlof et al. 1989 Ferguson et al. 1995), which was achieved using the Campbell synthesis (Campbell et al. 1979). The resolution of 14 was performed by traditional diastereomeric crystallisation of its (+)- and ( )-dibenzoyltartaric acid salts from ethanol (Stjemlof et al. 1989). This allowed the syntheses of both ( )- and (+)-anatoxin-a in 96% ee, respectively, as they could prove later in a different conversion of 14 into anatoxin-a (Ferguson et al. 1995). 

Somfai et al. demonstrated that it is also possible to use A-sulphonyl groups (Somfai and Ahman 1992 Ahman and Somfai 1992 Weinreb 1997) using the same concept for the cyclization step developed by Speckamp with the allylsilanes (Esch et al. 1987). In this case, the starting product was the L-pyroglutamic acid, inside the succinimide (Esch et al. 1987), allowing the enantioselective synthesis of the (+)-anatoxin-a. The key step consisted of an intramolecular cyclization of an 7V-tosyl iminium ion, catalysed by a Lewis acid, TiCl, to set up the desired bicyclic ring system (Somfai and Ahman 1992). 

Two other approaches using acyl iminium ion intermediates were developed by the Shono (Shono et al. 1987) and Skrinjar (Skrinjar et al. 1992) gronps in a synthesis of racemic and (+)-anatoxin-a,... 

An extensive work has been developed for the control of microcystins but not much effort has been made for the development of methods for the detection of other cyanobacterial toxins sneh as anatoxins or cylindrospermopsins despite their important acute toxicity as occurs in the case of the... 

Anatoxin-a(s) acts as an irreversible inhibitor of acetylcholinesterase, reducing blood pressure and heart rate in rats dramatically. " It has been identified as the active agent in a number of animal poisonings, including the deaths of numerous birds in 1993, which coincided with huge cyanobacterial blooms in freshwater lakes. "" As a result, a biosensor has recently been developed for the detection of 61 in environmental samples. " ... 

In addition, the synthesis of nor-anatoxin-or and -a from cocaine was published. As a new development, betalamic acid, the major building block of the betalaine pigments, was prepared as its dimethyl ester (19) from N-benzyl-norteloidinone (16) via the major intermediates (17) and (18), as shown in Scheme 1. 

Rotifers are part of freshwater zooplankton. Their metabolism is unresearched. It is unknown if they produce alkaloids. However, some planktonic rotifers are known to digest toxic cyanobacterium, producing the alkaloid neurotoxin, anatoxin which inhibits their development. ... 

The development of analytical methods for anatoxin-a has been facilitated by the fact that the structure of this toxin was already solved in the late 1970s and chemical synthesis yielded the pure... 

Wood SA, Holland FT, MacKcmzie L (2011) Development of solid phase adsmption toxin tracking (SPATT) for monitoring anatoxin-a and homoanatoxin-a in river wato. Otemosphere 82 888-894. doi 10.1016/j.chemosphere.2010.10.055... 

Azevedo J, Osswald J, GnUhermino L, Vasconcelos V (2011) Development and validation of an SPE-HPLC-FL method for the determination of anatoxin-a in water and trout (Oncorhincus mykiss). Anal Lett 44 1431-1441. doi 10.1080/00032719.2010.512682... 

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