Isoprene?
When the small fragments of knowledge that were scattered here and there suddenly starts to connect each other and begin to make sense, we feel an exhilarating intellectual pleasure. As I was looking into the subject of lipids from the scratch from various angle, I inevitably came to delve into cholesterol, which is a hot potato for us. I was quite taken aback to learn that cholesterol does not have fatty acids as its basic components like other lipids, but rather originates from the unusual unit called isoprene. The term 'isoprene' was completely unfamiliar to me, and I had no prior knowledge of it. However, it turned out that this tiny molecular unit aggregates like Lego pieces to create an astonishing array of substances in nature! It exists in various forms very close to us. It is even released from our mouths when we breathe. The pure form of this small molecule is a colorless volatile liquid. Many plants and animals, including humans, synthesize isoprene within their bodies.
Terpene, isoprenoid, terpenoid
Isoprene consists of five carbon atoms and eight hydrogen atoms, and it is an unsaturated hydrocarbon compound with double bonds between the carbon atoms. To put it very simply, a compound formed by two isoprene units is called a terpene. Of course, starting from here, an enormous number or a variety of isoprene units can join together to create substances of different sizes and types, referred to as isoprenoids or terpenoids. There is a tendency to use these terms interchangeably. It's important to remember that terpenes, isoprenoids, and terpenoids all ultimately have isoprene as their most basic unit.
Isoprene-based substances found in our close surroundings
One familiar isoprene-based substance is carotenoids. These include well-known compounds such as carotene, which gives carrots their orange color, as well as β-carotene, a precursor to vitamin A, lutein, which is beneficial for vision, and lycopene. Carotenoids are composed of eight isoprene units, or in other words four terpenes, and are plant-based organic pigments found in photosynthetic organisms and some fungi. They exhibit various colors, including orange, yellow, and red. Carotenoids play important roles as pigments that absorb light in photosynthetic organs, as well as acting as photoprotectants, antioxidants, and color attractants that protect the photosynthetic apparatus from damage caused by strong light from singlet oxygen and other free radicals.
Representative secondary metabolites
Let’s take a closer look at isoprene-based natural substances. First, terpenes are synthesized as secondary metabolites in plants of the natural world. Secondary metabolites are not essential for the survival of the plant but are necessary for the plant to protect itself from the surrounding environment and to adapt to it. They are metabolic intermediates produced through a series of reactions when exposed to various stresses, functioning as a kind of defense molecule. These compounds are produced to manage both hostile relationships, where competition is necessary to avoid being eaten, and symbiotic relationships, such as those involved in pollination, where mutual assistance is required for reproduction.
Secondary metabolites, also known as specialized metabolites or natural products, include terpenoids, phenolic compounds (e.g., lignans, tannins, flavonoids), and alkaloids (e.g., nicotine, caffeine, cocaine). Among these, the most representative and diverse group of natural compounds is terpenes, or terpenoids. A chemical examination of these will be discussed further in the following sections.
If we think about the terpenoids of plants in this context, we can understand better the forms they actually take. They possesse not only delightful scents that please our noses—such as cinnamon, cloves, eucalyptus, ginger, lavender, citrus peels, and even coffee—but also odors, flavors, and colors that can repel pests or parasites.
Plants possess antimicrobial properties that can help build immunity against bacteria, fungi, and viruses. Stationary plants attract beneficial insects with pleasant fragrances while emitting unpleasant substances to deter harmful ones. Trees secrete rubber or resin to protect their surfaces from invasions by insects or bacteria. Isoprene can also protect trees from heat stress and stabilize plant cell membranes by integrating into them, helping to stabilize the membrane during significant temperature fluctuations in the leaves. During the hot, sunny days, plants release isoprene to resist singlet oxygen and reactive oxygen species (ROS) in the air. All of these factors are essential for plants to interact with their surrounding environment and protect themselves.
Phytoncides: another member of the isoprene family
Let’s look at another familiar example: phytoncides. You may have heard of them before. These are antimicrobial, volatile organic compounds primarily emitted by trees such as pine and conifers. They serve as a secret weapon that helps protect plants from pests and fight diseases. The suffix “-cide,” which means “to kill,” suggests their strong antimicrobial properties. Phytoncides are also based on isoprene. The reason phytoncides have received attention is that when humans inhale large amounts of them, such as through forest bathing, it increases the number and activity of white blood cells, like natural killer (NK) cells. This leads to enhanced anti-inflammatory and antimicrobial responses, as well as reduced levels of stress hormones. Furthermore, reports indicate that exposure to phytoncides can result in extended sleep duration, lowered blood pressure, suppressed sympathetic nervous activity in the kidneys, and a significant increase in NK cells. [1] This illustrates how natural substances produced by plants for their own defense and healing can be utilized for our health.
Aromatherapy seems to fall into the same context. It primarily uses essential oils extracted through distillation from plants such as flowers and herbs, as well as trees. The largest component of these essential oils is isoprene. Aromatherapy is an auxiliary therapy that aims to utilize the anti-inflammatory, antimicrobial, and antioxidant effects of essential oils, such as those derived from phytoncides, lavender, and tea tree oil, to treat physical or mental health issues.
Isoprene in the animal kingdom: cholesterol
Next, if we examine isoprene found in the animal kingdom, cholesterol might be the most well known example. The functions and roles of cholesterol are indeed significant. In the cell membranes of over 30 trillion cells that make up the human body, cholesterol plays a crucial role in ensuring that the membranes remain fluid and elastic rather than becoming rigid and inflexible, thus maintaining permeability. While there is an enormous variety of components that make up cell membranes, cholesterol accounts for about 40% of them, nestled within the phospholipid bilayer.[2] Considering that approximately 3.8 million cells are replaced every second, a substantial amount of cholesterol must be maintained. Furthermore, cholesterol serves as a precursor for various hormones, including sex hormones in both men and women and adrenal hormones. We obtain about 30% of the cholesterol we need through food, while a remarkable 70% is synthesized within the body.[3] Cholesterol is a substance that the body synthesizes very carefully and strictly through more than 30 steps, reflecting how important it is. Therefore, it is reasonable to view cholesterol and some other sterols as primary metabolites involved in growth, development, reproduction, and essential for normal physiological functions.
At first glance, isoprene may seem quite unfamiliar, but we have seen that it is actually the fundamental unit of many substances that are very close to us. Let’s take a closer look at isoprene in more detail.
Chemical composition of isoprene
Isoprene (C₅H₈) has the formal chemical name 2-methyl-1,3-butadiene. I has double bonds at carbon number 1 and 3, with a methyl group attached to the carbon number 2. A term often associated with isoprene is terpene (C₅H₈)ₙ, which refers to large polymers formed by the combination of two or more isoprene molecules. While isoprene and terpenes are simple hydrocarbon structures, chemical modifications can occur, such as the addition of oxygen (oxygenated compounds) or rearrangement/removal of oxidized methyl groups (CH₃) at various positions. These modified compounds are referred to as terpenoids or isoprenoids. However, in practice, these terms are often used interchangeably. In simple terms, isoprene units, which are five-carbon unsaturated hydrocarbons, act as building blocks that can be repeatedly linked together like Lego blocks to form terpenoids (or isoprenoids).
It seems that the terminology related to isoprene can get somewhat mixed up and complicated. Let’s try to clarify the names that appears in this context. The smallest unit is isoprene (C₅), and 2 of isoprenes come together to form a terpene. From there, additional isoprene or terpene units can combine to create larger substances, but it seems that the naming conventions are primarily based on terpenes. Specifically, a single isoprene unit is referred to as a hemiterpene (C₅), which means "half" of a terpene. When two isoprene units combine, it is considered one terpene, thus called a monoterpene (C₁₀). When three units combine, it is termed a sesquiterpene (C₁₅), with "sesqui" in Latin meaning one and a half. Continuing this pattern, four units combine to form a diterpene (C₂₀), six units create a triterpene (C₃₀), and eight units result in a tetraterpene (C₄₀). All of these names reflect the increase in the number of isoprene units, yet they incorporate the term "terpene." Perhaps it is simpler to use a base of ten for naming conventions.
Terpenes with one to three isoprene units, meaning those with a carbon count of around 15 (hemiterpene ~ sesquiterpene), are mostly highly volatile and are emitted by plants primarily to attract or repel nearby living organisms. In contrast, larger terpenes with higher molecular weights, are generally non-volatile and perform biological functions. For example, carotenoids, which we examined earlier, are tetraterpenes synthesized from eight isoprene units (four terpenes).
Starting Point of Isoprenoids: IPP and DMAPP
However, when we look at the process by which isoprene synthesizes various isoprenoid substances in nature, we find that isoprene itself is relatively unreactive. Therefore, it first combines with two phosphate molecules. The hydrophobic isoprene binds with the polar phosphates, resulting in the compound isopentenyl pyrophosphate (IPP). With the help of enzymes, this compound is converted into its isomer, dimethylallyl pyrophosphate (DMAPP). As mentioned in previous discussions about phosphates, when two phosphates are bonded together, it is referred to as pyrophosphate (or diphosphate). An isomer is a substance that consists of the same elements but differs in the arrangement of atoms, resulting in different chemical properties despite having the same molecular formula. Strictly speaking, IPP and DMAPP are the starting points for all isoprenoids. In other words, all terpenes are derived from these two molecules.
Terpenes in Metabolism and Synthesis
Terpenes are synthesized in all living organisms and perform antioxidant and various functions in major metabolism. The terpene synthesis pathway in vivo starts from the combination of just two molecules of acetyl coA. Acetyl coA (acetyl-CoA) is produced from pyruvate from glycolysis or from lipolysis (fatty acid beta oxidation). This synthesis pathway is also the very first step in cholesterol synthesis in the human body.
Starting from IPP (five carbons) and DMAPP (five carbons), let's examine the process through which triterpenes (thirty carbons) are produced.[4] Reports indicate that over 20,000 different triterpene structures have been found in natural products, each exhibiting more than 100 skeletal variations.[5] This suggests that triterpenes may represent the largest category among terpenoids. Among them, squalene, known as a precursor to cholesterol, is particularly important. In the process of condensation, where each polymer combines to form larger products, the thirty carbon atoms typically acquire a fused ring structure due to the action of cyclization enzymes.
Lanosterol: Precursor of sterols
In nature, most triterpenes (30 carbons) have four-ring and five-ring structures, but there are also examples that are non-cyclic or have one, two, three, or six-membered rings. The four-ring structures can be further classified into numerous subcategories, but here we focus on lanosterol, which is a precursor in the biosynthesis of sterols.
Lanosterol is synthesized into cholesterol in animals, cycloartenol in plants, and ergosterol in fungi. It is important to remember that the human body must go through an additional 19 complex steps to synthesize cholesterol from lanosterol. About 70% of cholesterol in the body is synthesized in this manner. To briefly look at how the linear structure of squalene is converted into the cyclic structure of lanosterol, this process involves several intricate steps.
As squalene undergoes cyclization, it generates over 150 polycyclic structures, which include up to five interconnected carbon rings and various lengths of side chains. The illustration above is an example of one such structure. Depending on the biological context—whether in animals, plants, or fungi—numerous downstream triterpenoid structures are derived from these carbon ring skeletons. These can lead to compounds such as steroids like cholesterol, as well as terpenoid-derived natural substances that exhibit color, fragrance, toxicity, medicinal properties, and consist of materials like rubber and wax.
The term "sterol" refers to an alcohol with a steroid backbone that has an OH group attached (steroid + alcohol). Consequently, cholesterol also has an OH group at the carbon-3 position.
Use of cholesterol in the human body
Cholesterol plays a vital role in the human body, as it is involved in the synthesis of bile acids, vitamin D, and steroid hormones. Steroid hormones include adrenal cortex hormones such as aldosterone and cortisol secreted by the adrenal glands, and sex hormones such as testosterone, progesterone, and estrogen secreted by the ovaries, testes, and placenta. Unlike progesterone, which is associated with pregnancy, testosterone is produced in both males and females, although the amount secreted is different. If we look at the skeletal structures of these hormones, it is evident that they all fundamentally share the same steroid backbone.
As we have seen, terpenes are biosynthesized from repeating units of isoprene, which is an unsaturated hydrocarbon consisting of five carbon atoms. The type of terpene is determined by the number of these repeating units, which can range from a minimum of 2 to as many as 10,000. This indicates the vast diversity of compounds based on isoprene that exist in nature, giving us a sense of the extensive variety of isoprene-derived substances found in the natural world.
Isoprene and lipids
Lastly, let's highlight the important characteristics of isoprene in relation to its original starting point, lipids. Carotenoids serve as precursors to fat-soluble vitamins, and isoprene-based essential oils are literally oils. Cholesterol, too, is classified as a lipid. Both isoprene and terpenes are non-polar, meaning they do not dissolve in water and exhibit strong hydrophobic properties.
[References}
[1] Transfer of Environmental Health and Preventive Medicine
https://environhealthprevmed.biomedcentral.com/articles/10.1007/s12199-009-0091
[2] Cholesterol makes cell membranes flexible and robust
https://www.fau.eu/2023/12/14/news/research/cholesterol-makes-cell-membranes-flexible-and-robust/
[3] The Role of Dietary Cholesterol in Lipoprotein Metabolism and Related Metabolic Abnormalities: A Mini-review
https://pubmed.ncbi.nlm.nih.gov/26055276/
[4] Terpenes, hormones and life: isoprene rule revisited.
https://joe.bioscientifica.com/view/journals/joe/242/2/JOE-19-0084.xml
[5] Biosynthesis of Structurally Diverse Triterpenes in Plants: the Role of Oxidosqualene Cyclases
https://insa.nic.in/writereaddata/UpLoadedFiles/PINSA/2016_Art96.pdf
