Eat me? Want to eat me again? Eat me again...

Food lovers must have a love-hate relationship with pufferfish. 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. Copyrighted image, unauthorized reproduction

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, but only achieved a yield of 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.

01

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.

Highly poisonous pufferfish. Copyrighted image, unauthorized reproduction

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 pufferfish toxicity 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) based on the name of the Tetraodontidae family to which pufferfish belong. 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 from the University of Tokyo, Hirata Yoshimasa from Nagoya University, and Woodward from 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."

02

When a chemist is overwhelmed

No functional group is innocent

We often look for 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 diagram. Image source: Wikipedia

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, it is densely packed with functional groups.

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

Image source: Wikipedia

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.

Image source: Wikipedia

There are many hydroxyl groups inside and outside this "cage", which also add a lot of complexity to the molecule. Among them, 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, and can be said to be "accomplices" in producing highly toxic substances.

Image source: Wikipedia

All in all, the entire molecule has 4 rings and 9 adjacent stereocenters.

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 reported the total synthesis of the racemate of tetrodotoxin for the first time 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 the synthetic strategy. However, the efficiency, yield and selectivity of the total synthesis of TTX have been unsatisfactory.

03

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 and 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.

Image source: Science

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

Image source: Science

The next question is how to get 2 in workshop 2. In workshop 2, nitromethane is a key player. 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 through intramolecular 1,3 cycloaddition to get 2. Therefore, the reactants and products in workshop 2 are 3 and 2 respectively.

Image source: Science

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.

Image source: Science

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!

Source: This article is produced by Science Popularization China, produced by Gu Miaofei (Science Pictorial), and supervised by China Science Popularization Expo

Submitted by: Computer Information Network Center, Chinese Academy of Sciences

The cover image and some images in this article are from the copyright gallery. The image content is not authorized for reprinting.