Tetrodotoxin
Sashimi is one of the most famous Japanese cuisine which is made from raw fish. One of this delicacy and the most expensive one is made from a species of pufferfish which is known as fugu in Japan, the most notable one is torafugu or tiger pufferfish (Takifugu rubipes). One thing that make this type of sashimi is so expensive is due to labourous preparation and the adequate preparation is necessary since this fish is so toxic. This high toxicity is due to a compound called tetrodotoxin (TTX).
As mentioned earlier, fugu is the Japanese word for pufferfish which literally means "river pig" and this fish contains lethal amount of toxin which mainly in its organ such as liver, the eyes and the ovaries. This highly toxic fish requires laborious and careful preparation to make it edible and only certified chefs are allowed to prepare this fish. Furthermore, the Japanese ministry of health, labour and welfare has a list of all the species of fugu and their parts that are edible.
The toxin is called tetrodotoxin and it is 1200 more potent than cyanide, and its name is derived from the order Tetraodontiformes which includes pufferfish, porcupinefish, ocean sunfish and triggerfish.
TTX is not produced by fugu itself but it is produced by certain bacteria such as Pseudoalteromonas tetradonis and other bacteria from genus of Pseudomonas and Vibrio which reside in the body of the fish. TTX is also found in several other animals such as blue-ringed octopus, rough-skinned newt, and Nactidae or sea snails.
Besides that, TTX is a potent neurotoxin and it inhibits the firing action potentials in nerves by binding to the voltage-gated sodium channel in nerve cell membranes and blocking the passage of sodium ions into the nerve cell. Since TTX is neurotoxin, it has similar symptons with nerve agents Sarin and VX.
This mode of action can paralyse the diapraghm which lead to the respiratory failure. Most of the death by TTX is due to asphyxation.
The first ever recorded case of TTX poisoning was the logs of Captain James Cook from 7 September 1774 where all the crews fed on tropical fish and the remaining was given to pigs kept on board. Then, the crew experienced numbness and shorthness of breath, while all the pigs were found dead in the next morning.
The biochemistry of TTX and its lethal properties lead organic chemists to attempt synthesising TTX for further study on its properties. Besides that, the unique structure of TTX which based on dioxa-adamantane skeleton with modified hydroxy groups, and an orthoester group which has acidic character (pKa = 8.7). In acidic condition, TTX forms a mixture othoester and lactone forms and as also its analogue.
The latest development in the synthesis of TTX was done by Isobe's group from Japan and it can be divided into 4 different stages which are:
(a) synthesis of the common intermediate,
(b) synthesis of the fully functionalised cyclohexane ring,
(c) synthesis of the lactone structure, and
(d) installation of the guanidine and completion of the total synthesis.
Isobe's group used Diels-Alder approach to synthesise the common intermediate 2 with starting material of levoglucosenone 1. This step consists of 8 steps and the common intermediate 2 was formed by using Overman rearrangement.
In this stage, there are two main problems encountered which was the low regioselectivity of Diels-Alder approach, while the stereoselectivity was perfect due to steric hindrance of 1,6 anhydro structure. The way to overcome this problem wass to inculde BF3.OEt2 as Lewis acid and MeCN which gave higher yield and better stereoselectivity. The inclusion of MeCN as solvent is crucial to quench the Lewis acid to inhibit oligomerisation. The second problem was lower yield of Overman rearrangemenet at higher scale production of intermediate 2. The inclusion K2CO3 improved the yield and it is suggested that K2CO3 might quench the acid.
Since simple cyclohexane 2 was chosen to be the intermediate, the important of regioselective and stereoselective of hydroxylation to produce 3 became the most challenging issues in the second stage.
The first hydroxy group attached on 10 was prepapred by forming dibromide then treated with DBU in DMF to form oxazoline intermediate via SN2'. The oxazoline intermediate is treated with TsOH to give the first hydroxy group. The other hydroxy groups were attached using epoxydation which the stereocontrol rellied on a conformational fixed by two sterically-hindered groups, not the hydroxy group.
The most difficult challenge in this stage was the stereoselective reduction of 1,2 diketone formed from the oxidation with IBX. The most effective reduction to give 15 was done by two steps, LiAlH(OtBu)3 followed Luche reduction and this gave the desired stereochemistry. The intermediate 3 was completed by protecting hydroxy group with TES (triethylsilyl) followed by allylic oxidation by SeO2. to form unsaturated aldehyde. Then, it finished off with Luche reduction, protecting hydroxy group and epoxydation using mCPBA.
The third stage is the formation of the lactone structure which is essential for the total synthesis of TTX. The desired configuration of 18 was achieved by the adduct with MgBr. The formation of the lactone structure was achieved via regioselective epoxide ring-opening by carboxylate group. The carboxylate group was formed via the cleavage of acetylinic group 19 in two steps reaction which generated the carboxylate group. The second step using hydrogen peroxide promoted the ring opening and finished by reprotection using TES to form 4.
The remaining problems left for this total synthesis from 4 is the installation of guanidine and the cleavage of the protected diol. This transformation is challenging because the nature of highly functionalised intermediate. The othoester was synthesised by deprotection with TBAF was followed by reprotection using acetylation. The 1,2-diol was cleaved with HIO4 to give dimethylacetal. Selective deprotection with aqueous ammonia and reprotection of orhtoester with TBS were followed. The protection using TBS gave regioselectivity for the reduction using DIBAL was possible. The installation of guanidine as the final step in this total synthesis was done with 27 to form the protected 28 and interesting enough all the protected group on 28 are acid sensitive which global deprotection using TFA can be used to finally give TTX.
This total synthesis showed the importance controlling regio- and stereoselectivity by employing protecting group and fixed conformation to give the desired configuration. This general method is very common in the field of total synthesis of natural products which have specific configuration or conformation. This method of synthesis give the way to synthesise TTX analogues via common intermediate 2. Furthermore, the syntesised TTX can give a way to study its biological activity, including its activity as sodium channel inhibitor.
Reference
T. Nishikawa and M. Isobe, Chem. Rec., 2013, 13, 286.
As mentioned earlier, fugu is the Japanese word for pufferfish which literally means "river pig" and this fish contains lethal amount of toxin which mainly in its organ such as liver, the eyes and the ovaries. This highly toxic fish requires laborious and careful preparation to make it edible and only certified chefs are allowed to prepare this fish. Furthermore, the Japanese ministry of health, labour and welfare has a list of all the species of fugu and their parts that are edible.
 |
| The member of Tetraodontiformes |
TTX is not produced by fugu itself but it is produced by certain bacteria such as Pseudoalteromonas tetradonis and other bacteria from genus of Pseudomonas and Vibrio which reside in the body of the fish. TTX is also found in several other animals such as blue-ringed octopus, rough-skinned newt, and Nactidae or sea snails.
Besides that, TTX is a potent neurotoxin and it inhibits the firing action potentials in nerves by binding to the voltage-gated sodium channel in nerve cell membranes and blocking the passage of sodium ions into the nerve cell. Since TTX is neurotoxin, it has similar symptons with nerve agents Sarin and VX.
The first ever recorded case of TTX poisoning was the logs of Captain James Cook from 7 September 1774 where all the crews fed on tropical fish and the remaining was given to pigs kept on board. Then, the crew experienced numbness and shorthness of breath, while all the pigs were found dead in the next morning.
 |
| TTX in acidic condition |
The biochemistry of TTX and its lethal properties lead organic chemists to attempt synthesising TTX for further study on its properties. Besides that, the unique structure of TTX which based on dioxa-adamantane skeleton with modified hydroxy groups, and an orthoester group which has acidic character (pKa = 8.7). In acidic condition, TTX forms a mixture othoester and lactone forms and as also its analogue.
 |
| General scheme of the total synthesis of TTX |
The latest development in the synthesis of TTX was done by Isobe's group from Japan and it can be divided into 4 different stages which are:
(a) synthesis of the common intermediate,
(b) synthesis of the fully functionalised cyclohexane ring,
(c) synthesis of the lactone structure, and
(d) installation of the guanidine and completion of the total synthesis.
Isobe's group used Diels-Alder approach to synthesise the common intermediate 2 with starting material of levoglucosenone 1. This step consists of 8 steps and the common intermediate 2 was formed by using Overman rearrangement.
 |
| Step 1 - The synthesis of common intermediate 2 |
In this stage, there are two main problems encountered which was the low regioselectivity of Diels-Alder approach, while the stereoselectivity was perfect due to steric hindrance of 1,6 anhydro structure. The way to overcome this problem wass to inculde BF3.OEt2 as Lewis acid and MeCN which gave higher yield and better stereoselectivity. The inclusion of MeCN as solvent is crucial to quench the Lewis acid to inhibit oligomerisation. The second problem was lower yield of Overman rearrangemenet at higher scale production of intermediate 2. The inclusion K2CO3 improved the yield and it is suggested that K2CO3 might quench the acid.
Since simple cyclohexane 2 was chosen to be the intermediate, the important of regioselective and stereoselective of hydroxylation to produce 3 became the most challenging issues in the second stage.
 |
| Step 2 - The synthesis of the fully functionalised cyclohexane ring |
The first hydroxy group attached on 10 was prepapred by forming dibromide then treated with DBU in DMF to form oxazoline intermediate via SN2'. The oxazoline intermediate is treated with TsOH to give the first hydroxy group. The other hydroxy groups were attached using epoxydation which the stereocontrol rellied on a conformational fixed by two sterically-hindered groups, not the hydroxy group.
 |
| Stereocontrol of epoxydation |
 |
| Step 3 - The synthesis of the lactone structure |
The third stage is the formation of the lactone structure which is essential for the total synthesis of TTX. The desired configuration of 18 was achieved by the adduct with MgBr. The formation of the lactone structure was achieved via regioselective epoxide ring-opening by carboxylate group. The carboxylate group was formed via the cleavage of acetylinic group 19 in two steps reaction which generated the carboxylate group. The second step using hydrogen peroxide promoted the ring opening and finished by reprotection using TES to form 4.
 |
| Step 4 - The installation of the guanidine and completion of the total synthesis |
The remaining problems left for this total synthesis from 4 is the installation of guanidine and the cleavage of the protected diol. This transformation is challenging because the nature of highly functionalised intermediate. The othoester was synthesised by deprotection with TBAF was followed by reprotection using acetylation. The 1,2-diol was cleaved with HIO4 to give dimethylacetal. Selective deprotection with aqueous ammonia and reprotection of orhtoester with TBS were followed. The protection using TBS gave regioselectivity for the reduction using DIBAL was possible. The installation of guanidine as the final step in this total synthesis was done with 27 to form the protected 28 and interesting enough all the protected group on 28 are acid sensitive which global deprotection using TFA can be used to finally give TTX.
This total synthesis showed the importance controlling regio- and stereoselectivity by employing protecting group and fixed conformation to give the desired configuration. This general method is very common in the field of total synthesis of natural products which have specific configuration or conformation. This method of synthesis give the way to synthesise TTX analogues via common intermediate 2. Furthermore, the syntesised TTX can give a way to study its biological activity, including its activity as sodium channel inhibitor.
Reference
T. Nishikawa and M. Isobe, Chem. Rec., 2013, 13, 286.
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