Pufferfish: We don’t produce toxins, we are just the “carriers” of toxins

When it comes to pufferfish, gourmets must have a love-hate relationship with it. It is delicious, but its toxicity is also terrifying. It is more than 1,000 times more toxic than potassium cyanide, and it takes effect very quickly, killing people within minutes to hours.

Pufferfish in the ocean

(Photo source: veer photo gallery)

This love-hate relationship with pufferfish is not only shared by gourmets, but also by chemists. What they love is that pufferfish toxin has a specific inhibitory effect on nerve excitation, and can be used as an excellent nerve blocker, which is capable of playing a major role in medical fields such as analgesia and anesthesia; what they hate is that it is too difficult to synthesize! In 1972, when the pufferfish toxin molecule was first synthesized in the laboratory, it took a full 67 chemical reaction steps to finally achieve a yield of only 1%. Such tedious steps and such low output make this synthetic route almost unusable in practice.

So, are chemists helpless? Of course not. They have been working hard to reduce the number of steps and improve the yield. In July this year, a study published in Science shortened the total synthesis of tetrodotoxin molecules to 22 steps , with a yield of 11% . This means that the synthesis of tetrodotoxin can be used in industry, and the development of new drugs based on tetrodotoxin will also become possible.

One man's poison is another man's honey

Why do pufferfish carry such a strong toxin? Although tetrodotoxin is found in pufferfish, the real source is not pufferfish. The toxins of pufferfish mainly come from the microorganisms they eat (also from symbiotic and infected bacteria). In other words, a lot of the toxins of pufferfish are also eaten. Tetrodotoxin also appears in other animals that also feed on these microorganisms, such as starfish, conchs, toads, etc. However, these animals have a complete "anti-toxin" mechanism in their bodies, so even if the toxins enter through the mouth, they will be fine.

Poisonous pufferfish

(Photo source: veer photo gallery)

If humans eat tetrodotoxin, they are not so lucky. After entering the human body, this toxin will quickly act on nerve endings and nerve centers, blocking the sodium ion channels on the nerve cell membrane, hindering nerve conduction, and thus causing nerve paralysis and death.

Its toxicity is so strong that it seems mysterious. People can't help but wonder: What does such a highly toxic molecule look like?

In the early days when the toxicity of pufferfish was known, the molecular structure of tetrodotoxin remained a mystery due to the imperfect analytical methods. As early as 1909, Japanese scholars described the toxic components of pufferfish eggs and named them Tetrodotoxin (TTX) after the Latin name of pufferfish, Tetraodontidae. In 1938, scientists extracted relatively pure toxic components from pufferfish for the first time. In the following decades, people only knew the name of tetrodotoxin but not its structure. It was not until the 1950s that the monomer crystals of tetrodotoxin were separated. More than a decade later, in 1964, at an international conference in Kyoto, three research teams, Tsuda Kyosuke of the University of Tokyo, Hirata Yoshimasa of Nagoya University, and Woodward of Harvard University, reported the correct structure of tetrodotoxin at the same time, and the true face of tetrodotoxin finally surfaced.

The chemical formula of the tetrodotoxin molecule is C11H17O8N3, and the molecular weight is 319.27, which is not a very large molecule. Now, chemists and biologists are even more interested: this little thing, although small in size, has great capabilities! Is it worth studying carefully?

As the saying goes, "one man's arsenic is another man's honey." On the surface, tetrodotoxin appears to be a deadly poison, but when used in the right place it can have the miraculous effect of "fighting poison with poison."

Since tetrodotoxin can selectively bind to the sodium ion channel receptors on the surface of nerve cell membranes, thereby blocking action potentials and inhibiting the conduction of nerve excitation, people can use it to synthesize a series of drugs that control the action mechanism of nerve cell membranes, regulate the "silence" and "excitement" of nerve cells, and play analgesic, anesthetized, and sedative roles. In addition, tetrodotoxin can also be used as a drug addiction treatment drug. In 1998, a Canadian company successfully developed a new drug addiction treatment drug called tetrodin using tetrodotoxin, which can be described as a major initiative of "fighting poison with poison."

When chemists are troubled, no functional group is innocent

We often look for some natural substances in the biological world that can replace synthetic chemicals, because they often have exquisite structures and specific functions that are naturally created, and we can use these characteristics to achieve the purpose of pleasing others. For example, biological enzymes can be used as a clever catalyst. They are precise and efficient, and their catalytic activity and selectivity crush a large number of catalyst products that have been painstakingly synthesized in the laboratory; for example, mRNA technology can use the regulatory mechanism of RNA on proteins to produce the desired protein molecules, eliminating the trouble of step-by-step production in the workshop. The idea of ​​these works is to replace "artificially synthesized things" with "natural things", and the synthesis of TTX is actually a bit "opposite" to this conventional idea - it is to use "artificial" methods to replicate TTX, a "natural" neurotoxin. What's more, the difficulty of synthesizing TTX is really not small.

TXX molecular structure

TTX is a molecule that looks quite confusing. In fact, the carbon skeleton of the molecule is not complicated, just a cyclohexane with C1 and C2 side chains, but in sharp contrast to it are the densely packed functional groups on it.

First, the rightmost part with nitrogen atoms in the above picture is called guanidine. Guanidine is the "culprit" of TTX's toxicity because it will be positively charged at physiological pH and interact with the negatively charged groups on the sodium channel receptor protein;

If you follow the guanidine group toward the center, you will see a cage-like structure (that is, a part composed of two six-membered rings interlaced). This is a dioxacycloadamantane, which is also the core structure of TTX.

There are many hydroxyl groups inside and outside this "cage", which also add a lot of complexity to the molecule. The hydroxyl groups near the guanidine group are not good things either. They will bind to the receptors of the sodium ion channel in the form of hydrogen bonds, which can be said to be "accomplices" in producing highly toxic substances. All in all, the whole molecule has 4 rings and 9 adjacent stereo centers.

When chemists are troubled, no functional group is innocent. The density of functional groups and the high stereospecificity make the synthesis of TTX very difficult. Therefore, TTX has a high status in the field of synthetic chemistry and has always been regarded by chemists as a very challenging research target.

The first to succeed in the challenge were Kishi and Fukuyama of Nagoya University in Japan, who first reported the total synthesis of the racemate of tetrodotoxin in 1972, a milestone achievement in organic synthesis that was not surpassed for more than 30 years . After more than 30 years of stagnation, the total synthesis of TTX has experienced rapid development since 2003, with multiple research teams providing a variety of synthetic routes and continuously optimizing synthetic strategies. However, the efficiency, yield, and selectivity of the total synthesis of TTX have been unsatisfactory.

Let’s take a look at this simple and efficient new route

Until July this year, a joint team of scientists from Germany, the United States, and Japan published this new TTX total synthesis route in Science. They used a glucose derivative as the starting material and only needed 22 steps to get TTX - first of all, it won in terms of simplicity. The other side of simplicity is practicality and economy, which means that the wonderful uses of tetrodotoxin that we have imagined, such as using it as an anesthetic, a "magic drug" for drug addiction treatment, etc., will all become a reality.

Like many classic total synthesis designs, this route also has amazing ingenuity and design-filled transformations. Of course, although the reaction steps have been "significantly" reduced to "only" 22 steps, it is still a bit confusing to laymen. Fortunately, the research team summarized these 22 steps into 4 major steps in the paper and explained their synthesis strategy in a result-oriented, reverse-deduction manner.

If we compare this TTX synthesis route to the four workshops on a production line in a factory, then the product of the last workshop should be TTX. Conversely, the reactant entering the fourth workshop is alkynyl isoxazolidine (represented by 1), which undergoes an oxidation reaction in the fourth workshop - of course, 1, as a reactant in the fourth workshop, is also the product of the third workshop.

Now we work backwards to workshop 3. To obtain 1 at the end of workshop 3, we can allow bicyclic isoxazoline (represented by 2) to enter the workshop as a reactant to undergo alkynyl nucleophilic addition reaction.

The next question is how to get 2 in workshop 2. In workshop 2, nitromethane plays a key role. We can understand that it has been in workshop 3 for a long time, just waiting for 3 to come in, and then it can react with it in an intramolecular 1,3 ring addition reaction to obtain 2. Therefore, the reactant and product in workshop 2 are 3 and 2 respectively.

Although 3 seems to be the starting point of a complete synthetic route, the research team found a more suitable starting material than 3 - a glucose derivative (represented by 4). If 4 is used as the starting material to complete this route, all carbons and two stereo centers will be retained throughout the process, so the workload of the subsequent workshops will be slightly smaller and less difficult. So, in the first workshop, what happened was the conversion from 4 to 3.

At this point, this new TTX total synthesis route has been completed. With 22 steps and an 11% yield, it has set a record for the shortest route and the highest efficiency in history . So, what can it be used for?

First of all, its high efficiency makes it valuable for industrial application, and it can lay the foundation for the development of new drugs based on tetrodotoxin. In addition, tetrodotoxin actually has a series of analogs, and this route can be slightly modified to synthesize other difficult-to-obtain tetrodotoxin analogs. Besides, there are still many things worth exploring about tetrodotoxin in many fields such as biology, ecology, toxicology, and neuroscience . This work may provide inspiration for research in other fields!

Produced by: Science Popularization China

Author: Gu Miaofei (Science Pictorial)

Producer: China Science Expo