Biochemistry Lipids As Co-Factors and Pigments

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Paper : 03 Structure and Function of Biomolecules -II Module : 25 Lipids as co-factor and pigments

Principal Investigator Dr. Sunil Kumar Khare,Professor Dept. of Chemistry, I.I.T. Delhi

Dr. Sunil Kumar Khare,Professor Paper Co-ordinator Dept. of Chemistry, I.I.T. Delhi

Dr. M.N.Gupta, Emeritus Professor Dept. of Biochemical Engg. and Biotechnology, I.I.T. Delhi

Dr. M.N.Gupta, Emeritus Professor Content Writer Dept. of Biochemical Engg. and Biotechnology, I.I.T. Delhi

Dr. Prashant Mishra, Professor Content Reviewer Dept. of Biochemical Engg. and Biotechnology, I.I.T. Delhi 1

Structure and Function of Biomolecules II Biochemistry Lipids as co-factors and pigments

Description of Module

Subject Name Biochemistry

Paper Name Structure and Function of Biomolecules II

Module Name/Title Lipids as co-factors and pigments

Dr. Vijaya Khader Dr. MC Varadaraj

Structure and Function of Biomolecules II Biochemistry Lipids as co-factors and pigments

1. Objectives

 To learn about diversity of molecules or part of molecules which originate in a C5 monomeric unit.  To learn about various carotenoids and terpenoids.

 To learn about the roles of carotenoids and terpenoids in biochemistry and their applications in many areas

2. Concept Map

Structure and Function of Biomolecules II Biochemistry Lipids as co-factors and pigments

3. Description

While talking of both steroids and lipid soluble vitamins, we have mentioned the importance of C5 unit as a precursor of these molecules or their side chains. We had specially mentioned Vit A being derived from carotenoids. In this chapter, we discuss carotenoids and terpenoids in detail.

Fats/oils are “saponified” by alkali to form soaps and glycerol. Large number of lipids do not undergo saponification. These are called nonsaponifiable lipids. We have seen steroids as one example. Carotenoids and terpenoids are other two important classes of nonsaponifiable lipids These fascinating molecules are responsible for colour and fragrance around us and in our daily life. Besides they have trememdous biochemical involvement.

Figure 1

Carotenoids are another group of unsaponifiable lipids of plants and animal origin. Many organisms like red and green algae, fungi and photosynthetic bacteria also have these pigments which are lipids. The name carotenoids has its origin in the observation that in 1831 the substance called carotene was isolated from carrots. It is necessary to point out that techniques like NMR, MS etc came much later. Even U.V. spectrophotometers were not commercially available to chemists.

Structure and Function of Biomolecules II Biochemistry Lipids as co-factors and pigments

Hence, the isolated and “characterization” of carotenoids could only profit from chromatography earlier invented by Tswett in 1906. Karrer, Kuhn, Lederer and Zechmeister pioneered their separation. Colored as they are, visualization did not need any additional staining/spraying step. Lycopene, the substance which makes tomato (and hence tomato sauce!) red, is perhaps the precursor of all naturally occurring carotenoids. Apart from tomato, lycopene is also found in many flowers and fruits and even in some micro organisms.

Lycopene (C40H56) has the extended conjugated double bond structure which of course, is responsible for the colour. Two C20H28 units, each consisting of 4 isoprene units CH2=C(CH3)-CH=CH2, are joined by a double bond at C15 (from either end!). So lycopene has symmetrical structure.

Figure 2

Structure and Function of Biomolecules II Biochemistry Lipids as co-factors and pigments

The other three important naturally occurring carotenoids also are C40H56 compounds and are called α, β, γ carotenoids. All higher plants have one or more of these three carotenoids and even in many unicellular organisms.

Figure 3

Ring I (LHS) of all these three carotenoids is same and also similar to ring II (RHS) of β-Carotene. This ring is β-ionine residue. Ring II (RHS) of α-Carotene is α-ionone residue. Either of these two kinds of ionone rings are present in all carotenoids. Table 1: Some naturally occurring carotenoids

Structure and Function of Biomolecules II Biochemistry Lipids as co-factors and pigments

Majority of carotenoids are oxygenated compounds called xanthophylls. These can be considered as related to α- ,β-, γ- carotenes. Thus a derivative of lycopene is called Lycoxanthin. Apart from tomato, another good source for this is rhodospirillum. Other lycopene derivatives are lycophyll which is present in berries of Solanum dulcamera. Lutein or xanthophylls is another well known carotenoid. The parent compound for lutein is α-carotene and it occurs in green leaves, flowers and fruits. Thus Lutein is wide spread in plant kingdom. The carotenoids related to β-carotene cover a wide range and include cryptoxanthin [occurs in fruits, berries and zea mays (yellow corn)], zeaxanthin (also present in yellow corn), violaxanthin which also occurs in some green leaves and flowers. Eichinenone is 4-keto-β-carotene and occurs in marine invertebrates. Canthaxanthin occurs in both mushroom and cornybacterium, its structure is 4, 4-diketo-β-carotene. Astacin occurs in lobster shells and is 3, 4, 4’, 3’- tetraketo-β-carotene. Astaxanthin from green algae is 3,3’- dioxy-4,4’-diketo-β-carotene. The derivative of γ-carotene Rubixanthin occurs in some flowers and green sulphur bacteria. It is 3-oxy-γ-carotene. 7

Structure and Function of Biomolecules II Biochemistry Lipids as co-factors and pigments

Figure 4 Oxidative cleavage of the carotenoids gives few carotenoids containing carboxyl groups, naturally occurring and obviously have fewer than 40 C atoms. Notable among these are Bixin (pods of bixa orellana), crocetin (the carotenoid responsible for the colour of saffron, crocus sativus) and tolularhodrin (found in red yeast Torula rubra). The carotenoids in both vertebrate and invertebrate animals are present in fat droplets of ovaries and eggs in many cases and milk. Eye tissues and epidermal outgrowths (feather’s, shells and wings) of birds, crustaceans and butterflies are coloured because of the presence of carotenoids. Many marine invertebrates and fishes have pigmented tissues due to the presence of carotenoids. The only other common source of colours in birds/animals which has such widespread presence are melanins and their derivatives. Formed from o-quinones, these are polymeric materials. Apart from o-quinones, p-quinones are also part of biologically important lipids. Vitamin K is discussed Many carotenoids are converted to Vit A inside our bodies. At one time, when only animal assays were available to assay vitamins, carotenoids were evaluated/graded in terms of relative Vit A activity. Table 2: Relative vitamin A activity of some naturally occurring pigments

Structure and Function of Biomolecules II Biochemistry Lipids as co-factors and pigments

The carotenoids with β-ionone ring are vitamin A precursors. Xanthophyll ring because of the presence of OH groups do not have any vit A activity. Lycopene does not have closed rings. γ-carotene has only one closed β-ionone ring. Lycopene, thus has no vit A activity. γ-carotene has significant vit A activity. We also have to consider presence of stereoisomers due to cis-trans isomerism. Kemmerer and Fraps found that in rats, some neo-β-carotene can be converted into β-carotene and this results in neo-β-carotene showing some vit A activity. Thus, relative vit A activity of various isolated preparations of carotenes may differ. The relative vit A activity may also differ depending upon which animal is being used for this assay.

Structure and Function of Biomolecules II Biochemistry Lipids as co-factors and pigments

Figure 5 10

Structure and Function of Biomolecules II Biochemistry Lipids as co-factors and pigments

β-carotene, after oxidation of the central double bond, cleaves symmetrically and forms two molecules of vitA1 (retinol) which after oxidation of the alcoholic group forms 11-cis-retinal. 11-cis-retinal, as we discuss elsewhere is the visual pigment involved in animal vision. We should also recall

that retinol 2 or vit A2 which occurs in fresh water fish, though it contains an additional bond in the ionone ring is just active as vit. A1. 1U of vit A is equivalent to 0.6 μg of pure β-carotene. As conjugated double bonds are very vulnerable to oxidation, antioxidants present in food alongwith carotenoids serve the useful action of preventing this.

While vit A is discussed along with vitamins, its synthesis and metabolism is best covered alongwith the discussion on carotenoids. It should also be emphasized here that while any discussion on vit A focuses its role in vision, it obviously impacts other metabolic process. Vit A deficiency affects almost all the organs! That in turn points towards the nutritional value of carotenoids. A general advice is to eat vegetables of different colours! Carotenoids being largely responsible for colours in vegetables underpins the science behind this advice. In mammals, ingestion of β-Carotene results in sharp rise in the level of retinol in liver.Persons with severe liver disease fail to show this result. Hence, it was thought that β-carotene is converted to Vit A in the liver. However, later studies indicated that this conversion takes place in intestinal mucosa. Hence why patients of liver disease fail to show rise in levels of hepatic vitamin A after β-carotene administration is not clear. Dioxygenase from rat intestinal mucosa formed part of the early picture of the enzymology of carotene conversion to vit A. It is a soluble enzyme and is NADH-dependent. The dioxygenase is not very different from other dioxygenase. It shows maximal activity in presence detergents. Free –SH group (s) is essential for the activity as –SH reacting reagents inactivate the enzyme. In the intestines, the ester form of retinol (present in liver and fish oils) is first hydrolysed to the alcoholic form and then reesterified to retinyl palmitate. This forms chylomicron particles and reaches liver to be stored in hepatic kupffer cells. It is interesting to note that many tissues contain specific retinyl esterases. That also indicates that some form of vit A activity is involved in many metabolic process other than vision. There are reports that diabetic patients show impaired conversion of carotene to vit A. Sobel showed that alloxan induced diabetes in rats led them to have diminished capacity for this conversion.

Structure and Function of Biomolecules II Biochemistry Lipids as co-factors and pigments

Similarly, involvement of thyroid in vit A production and its metabolism is suspected. Hypothyroid animals show poor conversion of carotenoids to vit A. Again, use of thiouracil or thiourea administration to decrease thyroid function also led to lower conversion to vit A. Apart from their roles in vit A production in animals, carotenoids have also attracted attention in the context of metabolic roles in plants and microorganisms.

Figure 6 In chlorophyll, the phytol side chain is derived from carotenes. Apart from that carotene them selves form part of photosynthetic pigments in many photosynthetic organism. Quite often three important carotenoids, α-, β-, γ-carotenes occur together in natural sources. In carrots, their % are about 15, 85 and 0.1 respectively. Bixin is a monomethyl ester and its second –COOH group can be esterified to give methyl bixin. Bixin occurs in nature in cis form but is readily converted into trans form which is more stable. Crocetin occurs in saffron as a digentiobioside which is called crocin. Crocetin also has four carboxylic acid groups. Carotenoids and steroids have something in common. Both classes of compounds results from joining of the C5 unit isoprene. In nature, the building block is isopentenyl pyrophosphate.

Structure and Function of Biomolecules II Biochemistry Lipids as co-factors and pigments

Figure 7: The formation of isopentenyl pyrophosphate from HMG-CoA

We have mentioned elsewhere as HMG CoA being a key intermediate in cholesterol biosynthesis. This is typical of compounds at branch points being the site of metabolic regulation. HMG-CoA can form isopentenyl pyrophosphate (IPP). The reactions involve enzymes HMGCoA reductase to form mavalonate which is phosphorylated by Mevalonate-5-phosphotransferase. A kinase action followed by catalysis by pyrophosphomevalonate decarboxylase yields IPP. In fact, not just steroids and carotenoids, a variety of compounds are formed from IPP. These compounds play important roles in diverse biochemical phenomena.

The fragrances which are such pleasing part of the plant kingdom, in many cases is due to volatile C10-C15 compounds called terpenes. 13

Structure and Function of Biomolecules II Biochemistry Lipids as co-factors and pigments

Figure 8 The bay leaves fragrance is due to myrcene which has just two isoprene units. Limonene, the well known

fragrance compound from lemon oil also is C10 compound. The fragrance compound from lemon oil also is C10 compound. The fragrance compound Zingiberina from zinger oil is bigger and has the molecular formula of

C15H24.

The C40 carotenoids are synthesized by IPP forming a C20 intermediate called geranyl geranyl pyrophosphate. Two molecules of this condense tail to tail.

Structure and Function of Biomolecules II Biochemistry Lipids as co-factors and pigments

Figure 9 The side chains of vit K, ubiquinone and phytol side chains of chlorophylls are also made from condensation of isoprenoid units. The creation of diverse structures and functions is a part of divergent evolution at the molecular level. The basic biological design is not to start formation of structures at any level ab initio. Create module (s) and put them together in different ways. Terpenes, the largest number of them belong to the plant kingdom. Essential oils are used in cosmetics and in aromatherapy. Their main constituents are mono- and sesquiterpenes. Essential oils are volatile oils obtained from sap and tissues of plant/trees. There are di- and tri-terpenes which are not steam volatile and occur in plants and from tree gums and resins. 15

Structure and Function of Biomolecules II Biochemistry Lipids as co-factors and pigments

The essential oils should not be confused with oil/fats which are triglycerides. Nor they should be confused with mineral oils which are petroleum products. The common word oil refers more to their “oily” consistency.

Natural terpenes are mostly hydrocarbons with the molecular formula of (C5H8)n which essentially underlines the fact that these formed from condensation of C5 isoprene units. The value of n forms the basis of classifying terpenes. Natural rubber is an industrially important polyterpene. Carotenoids, which we have been discussing can be viewed as tetraterpenes! Many of the terpenes also form alcohol, aldehyde or ketone derivatives. Some people prefer the term terpene to

be used only for C10H16 (monoterpenes) and refer to other classes of terpene as Terpenoids. Thermal decomposition of many terpenes gives isoprene as the common product. That is how the early chemists realised that terpenes are formed from condensation of isoprene units. This observation was called isoprene rule and made first by Wallach in 1887. Ingold, one of the leading chemists of his times, proposed the so called special isoprene rule according to which natural terpenes are formed from head to tail condensation of isoprene units. We have already seen that carotenoids do not follow Ingold’s rule and are formed from tail to tail condensation. Lavandulol, consisting of just two isoprenes also has tail to tail condensation.

Figure 10: Lavandulol Terpenes which are open chain and follow Ingold’s rule have the following kind of C-chain. Monocyclic terpenes on the other hand contain a six member ring. All natural monocyclic terpenes can be considered a derivative of p-cymene.

Structure and Function of Biomolecules II Biochemistry Lipids as co-factors and pigments

Figure 11: p-cymene

Myrcene C10H16 is an important example of acyclic monoterpene. Its well known sources are oils in verbena and bay. Ocimene, is also a C10H16 compound and another acyclic monoterpene .

However the most important acyclic monoterpene is Citral. Widely distributed, its important source is lemon Grass oil in which it is present in the range of 60-80%. A liquid, it smells of lemons. Both geometrical isomers, the cis-form called citral-b or neral has a boiling point of 117-118 ºC. The trans form is also called citral-a or geranial also has a boiling point quite close to neral at 118-119 ºC as reported values.

Structure and Function of Biomolecules II Biochemistry Lipids as co-factors and pigments

Geraniol, a constituent of rose oil, is also found in many other essential oils. The cis-isomer nerol also occurs naturally, notably in oil of neroli and bergamot. Some other well known terpenoids present in essential oils are citronellal, rhodinol, limonene, menthol, piperitone (eucalyptus oil), Borneols and the fennel oil constituent Fenchone.

Next time, you use a cosmetic, deodorant, after shave etc, look for its constituent. You are likely to locate a terpenoid there now which is responsible for that nice smell! Some are also used in food to impart a nice smell to what you eat. The usefulness of terpenoids however is not restricted to just smell. Let us briefly look at these other important properties of terpeneoids. With renewed focus on “natural” “alternative system of remedies” and “neutraceuticals” you are likely to find this class of compounds more often. Epidemiological studies indicate that D-limonene and perilyl alcohol may have a chemopreventive property for some cancers in human. The suspected mechanism of action is that these may inhibit binding of carcinogens to DNA and cancer cell development and migration. Gram positive bacteria are more vulnerable to terpenoids as compared to gram negative bacteria. Some terpenoids which have shown antibacterial activity are linalool, farnesol, nerolidol and menthol. Carvone was found to be against listeria moncytogens and E-coli. Ferruginol, ursolic acid, carvone, pinene and linalool are examples of terpenoids which are reported to possess antifungal activity. Isoborneol has given some positive results as an antiviral substance. With the number of diabetic patients increasing at an alarming rate, the market for artificial sweetners is also growing exponentially. In India, saccharine has been around for a long time. More recent entrants in the market has been aspartame and sucralose. With the approval of FDA (USA), Stevia is now perceived as “safe” by larger number of people. Stevia contains a large amount of organic compounds. An important constituent is a diterpene steviol glycoside extracted from leaves of Stevia rebandiana. This terpenoid is reported to have insulinotropic, glucagonostatic and antihyperglycemic effects. It is known to lead to increase in glycolysis and gluconeogenesis. Besides steviol glycoside, others like rebandioside A-F, steviol bioside and dulcoside A also contribute to the sweet taste of stevia.

Structure and Function of Biomolecules II Biochemistry Lipids as co-factors and pigments

Figure 12: Structures of selected terpenes with anti-inflammatory activity Linalool, 1,8-cineole, Artemisolide etc have been shown to possess anti inflammatory properties. Betulin and

cucurbitacins inhibit phospholipase A2 and thereby show anti-inflammatory properties. Espintanol, piguerol A and menthol derivatives show antiparasitic properties. Linalool, carvone and thymol enhance permeability of drugs through skin and mucosal membranes. D-limonene is approved by regulatory agencies as an active enhancing agent for steroids.

Structure and Function of Biomolecules II Biochemistry Lipids as co-factors and pigments

Pigments of photosynthesis

Figure 13 The main photoreceptor in chloroplasts of green plants is chlorophyll. A magnesium porphyrin, it has a phytol side chain. With conjugated double bonds, both chlorophyll a and b absorb light with molar extinction coefficient (at λmax) > 105 cm-1M-1. This places these among the organic compounds with the highest molar extinction coefficient.

Structure and Function of Biomolecules II Biochemistry Lipids as co-factors and pigments

Figure 14 Blue-green algae (cyanobacteria) and red algae, the marine organisms with habitats at few meters below water surface do not get any red or blue light. Their photoreceptor phycocyanobilin and phycoerythrobilin are prosthetic groups of phycobiliprotein subunits.

Structure and Function of Biomolecules II Biochemistry Lipids as co-factors and pigments

Figure 15: the absorption spectra of various photosynthetic pigments

The various photosynthetic pigments ensure that depending upon the various biological niche, some part of solar energy is available to various organisms which have the capability of carrying out photosynthesis. Some other parts of the redox chain in photosynthesis also contain lipids like plastoquinones. So does the respiratory chain: ubiquinone and prosthetic groups of cytochromes.

Food, flavours, fragrances and neutraceuticals! Carotenoids and terpenoids encompass these various domains of applications. β-Carotene as precursor of Vit A and photosynthetic pigments show the usefulness of extended conjugation among chemical compounds.

Structure and Function of Biomolecules II Biochemistry Lipids as co-factors and pigments

Summary  Structures of various carotenoids and terpenoids.  Properties and applications of carotenoids and terpenoids.  Biochmical importance of carotene and terpenoids.

Structure and Function of Biomolecules II Biochemistry Lipids as co-factors and pigments