Science: Resiniferatoxin, Chemistry

Resiniferatoxin was first isolated from the resin spurge, Euphorbia resinifera (Euphorbiaceae).

The most ‘spicy’ substance known to science, resiniferatoxin (RTX), is a vanilloid diterpene of the daphnane skeleton. This may sound alien to you, but it’s exactly what we are dealing with today. RTX may not be as deadly as other plant toxins, but it sure as hell will make you suffer! It is one thousand times more potent than pure capsaicin, and 10,000 times more spicy than world’s spiciest chilli, the Carolina reaper. As far as I know, no one has tested pure RTX on his or her tongue, but that’s probably a wise thing. However, believe it or not, RTX is a molecule that inspired  tremendous research in pain science, to fight the very pain it induces above all else.

Figure 1: Chemical structure of resiniferatoxin.

Figure 2: Heterocyclic ether fragment of resiniferatoxin.

Look carefully at the chemical structure of RTX (Figure 1). It’s a fairly complex molecule with 37 carbon, 40 hydrogen and 9 oxygen atoms. At the left hand side of its structure, you can see three carbon rings labelled as A, B and C, which are numbered in red. This is called a daphnane diterpene (top left, Figure 1). Daphanane is so named because the first instance of its structure was isolated from plants in the Daphne genus (Thymelaceae). Here, we see it being elaborated by the resin spurge (Euphorbia resinifera, Euphorbiaceae). If you recall my previous article on carboxyatractyloside, we have actually encountered this type of plants metabolites called the diterpene. They all comprise a backbone with  20-carbon atoms, daphnane being no exception. Count the red numbers on the RTX structure, starting from C-1 to C-4 in ring A, followed by C-5 to C-10 in ring B, and C-11 to C-14 in ring C, with extra six carbons attached to C-13, C-11, C-2 and C-6, respectively. They should sum up perfectly to twenty. Besides, at ring C of the daphnane skeleton, RTX is incorporated with three additional heterocyclic rings containing two oxygen atoms , which are merged back to back, forming a sort of a molecular cage (bottom of  Figure 2). They comprise of a seven membered dioxepane (C-1’’–O–C-9–C-11–C-12–C-13–O-C-1’’), a six membered dioxane (C-1’’–O–C-14–C-8–C-9–O–C-1’’), and a five membered dioxolane  (C-1’’–O–C-14–C-13–O–C-1’’) rings (top right, Figure 2). The three heterocyclic rings all converge at carbon 1’’, which is connected to an aromatic  benzyl group (PhCH₂)  labelled as ring D in Figure 1. Take note that RTX  does not contain any nitrogen atom, so it is not classified as an alkaloid. Instead, at carbon-20 of RTX, we have this entire fragment that I’ve highlighted in blue, which is called a vanilloid (containing ring E, Figure 1). Vanilloids are all derived from the fragrant compound vanillin (isolated from vanilla orchid), but we will only examine in part II, which focuses on the structure activity relationship of RTX. In 3D space, there are nine stereocenters in RTX, while ring D and E are planar. The rests all adopt shapes that best relax the RTX molecule by avoiding clashes with other atoms, hence the heterocyclic rings tend to sit at the opposite side of rings A, B and C, which rings D and E point away from others. I honestly can’t depict a 3D structure via a 2D diagram, so if you really want to visualise how RTX, you can try it on 3D molecular simulators online. Anyhow, so much so for a vanilloid-daphnane-diterpene, I hope it is not that hard? Let me know if you think my articles are getting more and more challenging. Should you find it too hard, I can always downgrade the technical level in future.

Figure 3: Proposed biosynthesis pathway of daphnane (diterpene).

Now to make spice things up, we will look at how Euphorbia resinifera synthesise RTX from elements of nature. Since the entire biosynthesis pathway of RTX and even daphnane has not been fully resolved, we will only look at the proposed biosynthesis of a daphnane skeleton. Similar to all diterpene, the building blocks of daphnane are made up of individual five carbon units called isoprene. To make 20 carbons, we will need to condense four isoprene units (4 x 5 = 20) into an upstream precursor called geranylgeranyl pyrophosphate (GGPPi). Examine Figure 3 carefully. From the GGPPi molecule, we can fold up the backbone of a daphnane, but without the crucial bonds that make up rings A, B and C. This first diterpene precursor is called casbene, and it was isolated from the castor oil plant (also from the family Euphorbiaceae). Look carefully at the numbering on the casbene molecule in Figure 3, and compare it with the daphnane ring on RTX. Can you see the scaffold? Hence, by connecting C-4 and C-10, C-13 and C-14, as well as C-8 and C-9 of the casbene skeleton, we will end up with a prototypical diterpene molecule of the Euphorbiaceae family called phorbol, which has similar rings A, B and C as daphnane. Follow the red arrows and red bonds that indicate sequential bond formations by crucial atoms. Finally, to convert phorbol into daphnane, all we need to do is to disconnect the covalent bond between C-14 and C-13. This then produces the characteristic C-15–C-16–C-17 prong that’s attached to C-13 in RTX.  There are more complicated steps that would later introduce oxygen atoms into the daphnane skeleton of RTX, but those are not well elucidated by science as yet. Last but not least, the most crucial biosynthesis step of RTX would be the incorporation of a vanilloid fragment at C-20, but that will be my subsequent article.

That’s it for today, and I’ll leave you some questions to think about. RTX only comprises about 0.002% of the latex of Euphorbia resinifera. There are many more toxins contained in this plant. Is it possible that there are compounds like monoterpenes (10C), or triterpenes (30C), or tetraterpenes (40C), or something in between, what would those be? If that’s the case, how can chemists like me isolate and purify something as minute as 0.002% from the latex of Euphorbia resinifera?