Terpenes

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Introduction

The term terpene is derived from turpentine (lat. balsamum terebinthinae). Terpenes are very large class of most abundant natural hydrocarbons and commonly occur in higher plants as constituents of essential oils. Some insects can also produce terpenes. They are also major components of resin. Most of the terpenes have cyclic structures and their commercial applications include their use as fragrance in perfumery, as constituent of flavours for spicing food, etc [1]. The fundamental building block of terpenes is the isoprene unit (2-methyl-1,3-butadiene) linked in a head-to-tail fashion and is represented by general structural formula (C5H8)n where n is the number of linked isoprene units. Terpenes are classified into several classes depending on the number of isoprene units in the structure. The isoprene rule, developed by Ruzicka in 1921 played key role in structure determination [2].

Terpenoids are also naturally occurring compounds that contain oxygen functionality and show structural resemblance with terpenes. Two terms are often used interchangeably. In simple words, terpenoids are simply modified version of terpenes, in which methyl groups are removed and oxygen atoms are accomodated. They are also called isoprenoids.


Terpene is a natural product having various functions in the natural world [3]. They play pivotal role in plant physiology as well as important functions in all cellular membranes. Some of the terpenes are most potent drugs against life threatening disease such as cancer [4], malaria [5] and heart disease [6]. Some terpenes also show insecticidal properties [7].

Classification of Terpenes

Terpenes classification is based on number of isoprene units liked together in a head to tail fashion. They are classified [8] as monoterpenes, sesquiterpenes, diterpenes, triterpenes, tetraterpenes, polyterpenes etc. prefix represents the number of isoprene unit present in a molecule.

Monoterpenes

They consists of two isoprene units and has molecular formula C10H16. They are volatile natural products found in higher plants as essential oils and are widely used in perfumery and flavoring industries. For example geraniol is a main constituent of geranium oil (Pelargonium graveolens) and its isomer linalool is found in the oil of a garden herb, clary sage. Citral, a lemon oil component, is extracted from lemon grass oil (cymbopogon flexuousus). Menthol is isolated from Mentha arvensis. It has significant commercial values and widely used to flavor sweets, tobacco and toopaste. It is also used for local anaesthetic and refreshing effects.

Moreover, highly oxygenated monoterpenoids such as iridodial are found in ants and exist in equilibrium with its hemiacetal form [9].

The pine oil (turpentine) contains two monoterpenes viz. terpineol and α-pinene. Camphor was extracted from camphor tree, Cinnanomum camphora. It is used to protect cloths from moths. At present, α-pinene is a raw material for the commercial synthesis of camphor. Some of the other examples such as myrcene, limonene, citronellal, are also monoterpenes.

Sesquiterpenes

Sesquiterpens are also generally obtained from the essential oils but from higher boiling point fractions. It contains three isoprene units and has molecular formula C15H24. For example Caryophyllene from oil of cloves, humulene from oil of hops, cedrene from cedar wood oil and longifolane from Indian turpentine oil (Pinus ponderosa).

There are some other examples of sesquiterpenes such as farnesol, bisabolene, cadinene, selinene, vetivone, and antibiotic pentalenolactone etc.

Sesquiterpenoid lactones such as santonin from Artemisia maritima (warm wood) and artemisinin obtained from Artemisia annua are commonly used as medicine. Abscisic acid, a plant hormone is also an example of sesquiterpenoid. It stimulates leaf fall and dormancy in plants.

Diterpenes

Diterpenes are composed of four isoprene units having general molecular formula C20H32. Some of the diterpenes are wood resin product. For example, abietic acid (from Pinus and Abies species), podocarpic acid (from Podocarpus cupressinum) and neutral resin manoyl oxide.

Biologically active compounds such as phytol, vitamin A (Retinol) and casbene (phytoalexin) can also be considered as diterpenes.

Taxol or paclitaxcel is a diterpenoid, which was first isolated from the bark of the Pacific yew, Taxus brevifolia [10]. It is widely used in the treatment of breast and ovarian cancer.

Plant hormone, gibberellic acid is a diterpenoid which is synthesized as a phytotoxin by the fungus Gibberella fujikuroi. It is used in the malting step in beer manufacture to increase α-amylase production and also for growing of seedless grapes.

Sesterterpenes

It contains five isoprene units and it has molecular formula C25H40. Sesterterpenes are most abundant in marine sponges. Manoalide was first discovered in 1980 by Scheuer from the marine sponge Luffariella variabilis [11]. It showed antibacterial activity against Streptomyces pyogenes and Staphylococcus aureus.

Triterpenes

Triterpens contain six isoprene units having molecular formulaC30H48. Squalene is a simple linear triterpene which was first isolated from fish liver oil.

Subsequently, it is been reported from plant oils, and mammalian fats. Most of the members of this category contain tetracyclic structure.


The cyclopentaperhydrophenanthrene backbone is common skeleton of all steroids which belongs to the category of tetracyclic triterpens. Cholesterol is an important constituent of lipid membranes. Female steroid hormones such as progesterone, estradiol and male hormone testosterone also belongs to triterpenes.

Azadirachtin from Neem tree is highly oxidized tetranortriterpenenoid which shows strong insect anti-feedant and growth inhibitor activity [12].

Moreover, cortical steroids (e.g. cortisone) are also triterpenes which have an immunosuppressive activity and reduce inflammation. They are used in the treatment of asthma, rheumatoid arthritis etc. Vitamin D helps in the absorption of calcium and phosphate from gastrointestinal.

Lanosterol, a tetracyclic triperpene is present in wool fat and its ester are present in lanolin cream while the α- and β-amyrins are commonly found in wood resins and the bark of many trees.

Tetraterpenes

It is composed of eight isoprene units having molecular formula C40 H56. Some of the biologically active compounds like lycopene, monocyclic γ-carotene and bicyclic α and β-carotenes are common examples of tetraterpenes. Thus, the red colour of carrot is due to the presence of β-carotene while the deep-red pigment of tomato is due to the presence of lycopene. The carotenoids possess anti-oxidants properties. Moreover, carotenoids are precursors of vitamin A, which has vital role in vision process.

Polyterpenes

Polyterpenes are polymer in which several isoprene units are joined through head-to-tail fashion. Natural rubber(Heva brasilensis) is best known example of this series.

Terpenoid Biosynthesis

The “isoprene rule” developed by Ruzicka in 1920 was very useful to established structure of various terpenes. However, the biosynthetic origin of the isoprene unit (C5) was recently established. There are two most acceptable pathways; i) mevalonic acid pathways, and ii) 1-Deoxyxylulose pathways.


In general, terpenoid biosynthesis can be divided into four parts.


i) First step is synthesis of the isoprene unit, isopentenyl pyrophosphate ,

ii) Second step involves assembly of isopentynyl pyrophosphate into (C5)n isoprenoid backbone.

iii) Third step, involves cyclization of (C5)n isoprenoid backbone into the carbon skeltons

iv) Last step is formation of individual terpenoids


Mevalonic Acid Pathway

The first step is formation of (S)-3-hydroxyl-3-methylglutaryl co-enzyme A (HMG-Co A) by the condensation of acetyl co-enzyme A with acetoacetyl co-enzyme CoA. The next irreversible step involves the enzymatic reduction of (S)-3-hydroxyl-3-methylglutaryl co-enzyme A (HMG-Co A) with hydrogen from nicotinamide adenine dinucleotide phosphate (2 × NADPH) to give (R)-mevalonic acid. Mevaloic acid undergoes two successive phosphorylation by adenosine triphosphate (2 × ATP) to give the 5-pyrophosphate. This undergoes a trans-elimination of the tertiary hydroxyl group and the carboxyl group to form 3-methylbut-3-enyl pyrophosphate (isopentenyl pyrophosphate, IPP). This is in equilibrium with dimethylallyl pyrophosphate (DMAPP).

1-Deoxyxylulose pathway

In this pathway [9], pyruvic acid (2-oxopropanoic acid) and glyceraldehydehde monophosphate undergo condensation reaction to form 1-deoxyxylulose. The pinacol-type rearrangement of 1-deoxyD-xylulose 5P, followed by reduction with NADPH yields di-alcohol; 2-C-methyl-D-erythritol 4-P; which further undergoes series of reactions to produce 3-methylbut-2-enyl pyrophosphate (isopentenyl pyrophosphate, IPP). Next step is a stereospecific and reversible isomerization of the double bond of IPP that produces 3-methylbut-2-enyl pyrophosphate (dimethylallyl pyrophosphate, DMAPP). This step is very important because it generates reactive allylic pyrophosphate which helps in joining of two isoprene units to form the C10 geranyl pyrophosphate.

In both pathways, equilibrium favours DMAPP over IPP.

General Approach for the Biosynthesis of Terpenes

IPP and DMAPP are universal building blocks for the synthesis of various classes of terpenes. The enzyme, isopentenyl pyrophosphate isomerase catalyses reaction between IPP and DMAPP in presence of metal ion to produce geranylpyrophospahte (GPP), farnesyl pyrophosphate (FPP), geranylgeranylpyrophosphate (GGPP), squalene from IPP and DMAPP; a precursor for mono-, sesqui-, di-, tri-, tetra-, and poly-terpenes.The simple schematic representation [9] is shown here.

The mechanism for the synthesis of GPP, FPP, GGPP and squalene is very simple [9]. In the presence of enzyme, pyrophosphate group is activated and acts as leaving group to generate an allylic-tertiary carbocation. The enzyme needs M+2 for activity. The carbocation acts as electrophile and is attacked by the double bond of IPP, a second substrate to generate new carbocation which on stereospecific loss of proton produces various precursors for the synthesis of terpenes. This enzymatic reaction involves removal of the pro-R H-atom and formation of new C=C double bond with the E configuration. The overall mechanism is shown here.

Mechanism:

Thus, mono-, sesqui-, di-, tri-, and tetra-terpenes are biosynthesized using the following substrates.

The next step is cyclization of GPP, FPP, or GGPP to mono-, sesqui-, or di-terpenoids respectively. This is achieved by an enzyme known as terpene cyclases, a large family of enzymes that use GPP, FPP or GGPP as substrate for the formation of mono-, sesqui-, di-terpenoid products. The enzyme, terpene cyclases uses Mg+2 or Mn+2 as co-factor during catalysis. For example cyclization of geranyl pyrophosphate to menthol is shown below.

Biosynthesis of Squalene

Squalene is precursor of tri-terpenes and all steroids. Biosynthesis of squalene involves assembly of two molecules of farnnesyl pyrophosphate joined together in a head-to-tail fashion. In the presence of enzyme, pyrophosphate of one of the two fanesyl pyrophosphate is activated, resulting in the formation of allylic carbocation [9]. This carbocation acts as electrophile and is attacked by double of second farnesyl pyrophosphate molecule. This reaction involves formation of cyclopropyl intermediate, pre-squalene pyrophosphate as a result of an alkylation of the double bond of one farnysyl unit by the pyrophosphate of the second molecule. During reaction, NADPH acts as H donar and replaces one hydrogen stereospecifically. Rearrangement and reduction of intermediate gives squalene terpene.

Conversion of Squalene to Lanosterol

Squalene is tetracyclic tri-terpenoid. It is precursor for synthesis of lanosetrol, a steroid commonly found in wood, fat and yeast. Biosynthesis of lanosterol involves epoxidation of squalene to squaene-2,3-epoxide by enzyme squalene epoxidase that uses moleulcar oxygen and NADPH and FAD. In the next step, squalene-2,3- epoxide is converted to a protosterol cation by enzyme protosterol synthase and finally to lanosterol by a stereospecific cyclizations and rearrangements (hydride shifts) through series of intermediates.

Conversion of Lanosterol to Cholestoreol

Lanosterol is a precursor for the biosynthesis of cholesterol which in turn become precursor for steroid hormones, lipoproteins, bile acids, vitamin D. Cholesterol is an important molecule as it maintains membrane fluidity, structure and permeability. It is also involves in number of disesaes such as atherosclerosis, cholestasis (broad and variable impairment of bile secretion), hypercholesterolemia, cholesterol gallstone disease, and Nieman–Pick type C disease. The conversion of lanosterol into cholesterol is very complex process and involves nineteen steps. It requires total nine different enzymes from the endoplasmic reticulum. Out of nine, two enzymes catalyse multiple steps while three are used only two times in this transformation. Lanosterol has three methyl groups viz. at C30, C31, and C32 which are oxidized and removed as formic acid and two carbon dioxide molecule. The conversion of lanosterol to cholesterol [13] is shown in figure


Gibberellin biosynthesis

Gibberllins, tetracyclic diterpene acids are group of plant growth hormones. They have significant influence on many physiological processes in higher plants. Eiichi Kurosawa discovered Gibberellin in 1926 while working on the cause of bakanae, the foolish seeding disease in rice that is main cause of low yield of rice crops in Japan. Later on, it was first isolated by Teijiro Yabuta in 1935 from fungal strains, Gibberella fujikuroi. All gibberellins contain kaurene atom skelton. Gerenylgeranyl pyrophosphate (GGPP) is precursor of Gibberellins. Tetracyclic diterpenoid hydrocarbon, ent-kaurene is synthesized from GGPP by two step process. Oxidation of the hydrocarbon at C-19 and C-7 gives ent-7α-hydroxykaurenoic acid. This is substrate for unique oxidative ring contraction to form gibberellins A12 7-aldehyde. The key feature in all pathways is loss of C-20 and the formation of the γ-lactone such as gibberelic acid. Biosynthesis of gibberellin [14] is shown below.

References

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2. L. Ruzika, The isoprene rule and the biogenesis of terpenic compounds, Experientia, 1953, 9(10), 357 – 367.

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4. S. S. Ebada, W.H. Lin and P. Proksch, Bioactive Sesterterpenes and Triterpenes from Marine Sponges: Occurrence and Pharmacological Significance, Marine Drugs 2010, 8, 313-346.

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8. S. P. Bhutani, Chemistry of Biomolecules, Ane Book Pvt. Ltd. 2009.

9. P. M. Dewick, Medicinal Natural Products, A Biosynthetic Approach, Wiley, 2009.

10. D. M. Gibson, R. E. B. Ketchum, N. C. Vance, and A. A. Christen, Initiation and growth of cell lines of Taxus brevifolia (Pacific yew), Plant Cell Reports, 1993, 12, 479-482.

11. E. Dillp de Sllval and Paul J, Scheuer, Manoalide, an antibiotic sesterpenoid from the marine sponge Luffariella Variabilis (Polejaeff) Tertrahedron Letters, 1980, 21, 1611-1614.

12. S. V. Ley, Synthesis and chemistry of the insect antifeedant azadirachtin, pure and applied chemistry, 1994, 66, 2099-2102.

13. J. M. Risley, Cholesterol Biosynthesis: Lanosterol to Cholesterol, Journal of Chemical Education, 2002, 79, 377 – 384.

14. P. Hedden and Y. Kamiya, GIBBERELLIN BIOSYNTHESIS: Enzymes, Genes and Their Regulation, Annual Review of Plant Physiology and Plant Molecular Biology, 1997, 48, 431 – 460.

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