Bacteria and Fungi as source for Biologically active Compounds

Transcription

1 Natural Product The organic compounds isolated from the living organism i.e plants, animals and micro organism are generally known as Natural products. These includes carbohydrates, protein, aminoacids, alkaloides, terpenes, antibiotics etc The term natural product is applied to material derived from plants microorganisms and invertebrates, which are fine biochemical s factories for synthesis of both primary and secondary metabolites

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4 Bacteria and Fungi as source for Biologically active Compounds

5 Classical and Advance methods

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9 Structure of Morphine HO- Group is needed for activity HO O HO N H H CH 3 Morphine (Astramorph) HO- Group not important to activity

10 Tinkering with the structure of morphine produced heroin HO- Group is needed for activity Easily enzymatically hydrolyzed to AcOH and HO-Ar HO O HO O H H N Morphine (Astramorph) HO- Group not important to activity CH 3 AcO AcO H H N CH 3 Heroin (Diamorphine) (2X as potent as morphine) (Conversion of two -OH groups to -OAc facilitates crossing of the BBB)

11 The heart beat may be too fast or too slow

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25 Terpenoids

26 INTRODUCTION Terpenoids are the secondary metabolites synthesized by plants, marine organisms and fungi by head to tail joining of isoprene units. They are also found to occur in rocks, fossils and animal kingdom. Isoprene (short for isotepene), or 2-methyl-1,3-butadiene, is a common organic compound with the formula CH2=C(CH3)CH=CH2. Under standered conditions it is a colorless liquid. However, this compound is highly volatile because of its low boiling point. Isoprene Isoprene

27 Terpenes Terpenes are natural products that are structurally related to isoprene. CH 3 H 2 C C CH CH 2 or Isoprene (2-methyl-1,3-butadiene)

28 Isoprene

29 Head Tail Isoprene Head Tail

30 Head Tail Isoprene Head Tail

31 Terpenes and terpenoids posses a carbon fram work consisting of five carbon units known as isoprene unit. It is represented by symbol C 5 H 8 In oldest days the term terpene was used for those compounds containing 10 carbon atoms. This is still used in Modern classification of Terpenes. Terpenes are classified in to the following groups. Hemeterpens 1 x C 5 H 8 = C 5 H 8 Monoterpens 2 x C 5 H 8 = C 10 H 16 sesquiterpens 3 x C 5 H 8 = C 15 H 24 Diterpens 4 x C 5 H 8 = C 20 H 32 sesterpens 5 x C 5 H 8 = C 25 H 40 triterpens 6 x C 5 H 8 = C 30 H 48 Tetraterpenes 7 x C 5 H 8 = C 35 H 58 Polyterpenes n x C 5 H 8 = C 5 H 8 )n Rubber n= 100 or above

32 CALASSFICATION TYPE OF NUMBER OF ISOPRENE TERPENOIDS CARBON ATOMS UNITS hemiterpene monoterpenoid sesquiterpenoid diterpenoid triterpenoid tetraterpenoid C 5 C 10 C 15 C 20 C 30 C 40 one two three four sesterterpenoid C 25 five six eight NOTE : hemi = half di = two sesqui = one and a half tri = three tetra = four

33 Mnonoterpenoids Monoterpenes are a class of terpenes that consist of two isoprene units and have the molecular formula C 10 H 16. Monoterpenes may be linear (acyclic) or contain rings. Biochemical modifications such as oxidation or rearrange-ment produce the related monoterpenoids.

34 Representative Monoterpenes OH O H a-phellandrene (eucalyptus) Menthol (peppermint) Citral (lemon grass)

35 Representative Monoterpenes OH O H a-phellandrene (eucalyptus) Menthol (peppermint) Citral (lemon grass)

36 Representative Monoterpenes a-phellandrene (eucalyptus) Menthol (peppermint) Citral (lemon grass)

37 Mnonoterpenoids Acyclic monoterpenoid: Bicyclic monoterpenoid CHO CH 2 OH nerol geranial Monocyclic monoterpenodi Â-myrcene Á-pinene OH OH O O d-borneol l-menthol menthone cineole

38 Acyclic monoterpenoid Biosynthetically, isopentenyl pyrophosphate and dimethylallyl pyrophosphate are combined to form geranyl pyrophosphate Geranyl pyrophosphate

39 Acyclic monoterpenoid Elimination of the pyrophosphate group leads to the formation of acyclic monoterpenes such as ocimene and the myrcenes. Myrcene

40 Acyclic monoterpenoid Hydrolysis of the phosphate groups leads to the prototypical acyclic monoterpenoid geraniol. geraniol

41 Acyclic monoterpenoid Additional rearrangements and oxidations provide compounds such as citral, citronellol, and many others. citral

42 Acyclic monoterpenoid Many monoterpenes found in marine organisms are halogenated, such as halomon. Halomon is a polyhalogenated monote rpene first isolated from the marine red algae Portieria hornemannii. Halomon It has attracted research interest becau se of its promising profile of selective cy totoxicity that suggests its potential use as an antitumor agent.

43 Monocyclic monoterpenoid In addition to linear attachments, the isoprene units can make connections to form rings. The most common ring size in monoterpenes is a six-membered ring. A classic example is the cyclization of geranyl pyrophosphate to form limonene.

44 Bicyclic monoterpenoid Geranyl pyrophosphate can also undergo two sequential cyclization reactions to form bicyclic monoterpenes, such as pinene which is the primary constituent of pine resin.

45 Bicyclic monoterpenoid Other bicyclic monoterpenes include carene and camphene. Camphor, borneol and eucalyptol are examples of bicyclic monoterpenoids containing ketone, alcohol, and ether functional groups, respectively. carene camphor borneol

46 Sesquiterpenoids Sesquiterpenes are a class of terpenes that consist of three isoprene units and have the molecular formula C 15 H 24. Like monoterpenes, sesquiterpenes may be acyclic or contain rings, including many unique combinations. Biochemical modifications such as oxidation or rearrangement produce the related sesquiterpenoids.

47 Representative Sesquiterpenes H a-selinene (celery)

48 Representative Sesquiterpenes H a-selinene (celery)

49 Representative Sesquiterpenes a-selinene (celery)

50 Sesquiterpenoids Acyclic sesquiterpenoids Monocyclic sesquiterpenoids O Á-farnesene Â-farnesene Bicyclic sesquiterpenoids H Á-eudesmol OH H cadinene guaiazulene

51 Acyclic Sesquiterpenoids When geranyl pyrophosphate reacts with isopentenyl pyro- phosphate, the result is the 15- carbon farnesyl pyrophosphate, which is an intermediate in the biosynthesis of sesquiterpenes such as farnesene. Oxidation can then provide sesquiterpenoids such as farnesol. Sesquiterpenes are found naturally in plants as defensive agents. farnesyl pyrophosphate farnesene farnesol

52 Monocyclic Sesquiterpenoids With the increased chain length and additional double bond, the number of possible ways that cyclization can occur is also increased, and there exists a wide variety of cyclic sesquiterpenes. In addition to common six-membered ring systems such as is found in zingiberene, a consitituent of the oil from ginger, cyclization of one end of the chain to the other end can lead to macrocyclic, rings such as humulene. 姜稀 zingiberene

53 Bicyclic Sesquiterpenoids In addition to common six-membered rings such as in the cadinenes, one classic bicyclic sesquiterpene is caryophyllene, from the oil of cloves which has a nine-membered ring and cyclobutane ring. Additional unsaturation provides aromatic bicyclic sesquiter- penoids such as guaiazulene. H H cadinene caryophyllene guaiazulene

54 Tricyclic Sesquiterpenoids With the addition of a third ring, the possible structures become increasingly varied. Examples include longifolene, copaene and the alcohol patchoulol. longifolene copaene isomers of patchoulol

55 Diterpenoids

56 Diterpenoids Diterpenes are composed for four isoprene units and have the molecular formula C 20 H 32. They derive from geranylgeranyl pyrophosphate. Examples of diterpenes are cafestol, kahweol, cembrene and taxadiene (precursor of taxol). geranylgeranyl pyrophosphate cafestol cembrene

57 Representative Diterpenes OH Vitamin A

58 Representative Diterpenes OH Vitamin A

59 Representative Diterpenes Vitamin A

60 Diterpenoids Diterpenes also form the basis for biologically important compounds such as retinol, retinal, and phytol. They are known to be antimicrobial and antiinflammatory. The herb Sideritis contains diterpenes. retinol retinal

61 Structure ----Diterpenoids CH 2 OH CH 2 OH phytol vitamin A Acyclic diterpenoids Monocyclic diterpenoids O HO O H H HO CH 2 OH andrographolide COOH pimaric acid H kaurene Bicyclic diterpenoids Tricyclic diterpenoids Tetracyclic diterpenoids

62 Triterpenoids

63 Triterpenoids Triterpenes consist of six isoprene units and have the molecular formula C 30 H 48. The linear triterpene squalene, the major constituent of shark liver oil, is derived from t he reductive coupling of two molecules of farnesyl pyrophosphate. Squalene is then processed biosynthetically to generate either lanosterol or cycloartenol, the structural precursors to all the steroids. squalene Farnesyl pyrophosphate lanosterol cycloartenol

64 Representative Triterpene tail-to-tail linkage of isoprene units Squalene (shark liver oil)

65 O O O-P-O-P-O O O O O O O O-P-O-P-O O O GPP Squalene O-P-O-P-O O O O O O-P-O-P-O O O O O O-P-O-P-O FPP O O O O O-P-O-P-O FPP O O O O O-P-O-P-O FPP O O Squalene

66 Structure ----Triterpenids Acyclic triterpenids squalene Bicyclic triterpenids α-carotene β-carotene

67 Structure ----Triterpenids Tetracyclic triterpenids Dammarane Tirucallane Lanostane H H H H H H dammarane H H lanostane H H tirucallane Cycloartane Cucurbitane H H H H cycloartane H cucurbitane

68 Structure ----Triterpenids Pentacyclic triterpenoids Oleanane Ursane Lupane H H H H H H H oleanane H H ursane H H lupane

69 Citral C 10 H 16 O It is the most important member of the most of the acyclic monoterpenoides. Because the structure of most of the other compounds in this group are based on that of citral. Its is widely distributed and occurs to an extent of 60-80% in lemon grass oil. It is a liquid which has the smell of lemons.

70 Structure of Citral. The molecular formula of citral is C 10 H 16 O analogue of saturated hydrocarbons CnH2n+2 C 10 H 10x2+2 = C 10 H 22 IHD: 22-16/2 = 3 From IHD it is found that the citral contains 3 double bonds

71 Cat. Hydrogentation On cat. Hydrogenation 2 moles of hydrgone were consumed. This showed that the molecule contain 2 C=C bonds CHO CHO Cat. Hydrogenation 2 moles

72 On bromination also two molecule of bromine were consumed. This also proved that molecule contain 2 C=C bonds. OHC Br CHO Br Br 2 2 moles Br Br

73 Citral was converted into an oxime on treatment with NH 2 - OH. This reaction showed that molecule contains an oxo group. CHO H 2 N=OH CH=N-OH 2 moles

74 Citral is reduced to an alcohol geraniol ( C 10 H 18 O). Geraniol is a primary alcohol and the formation a primary alcohol form carbonyl compound confirmed that the citral contains an aldehyde functional group. CHO CH 2 -OH Na/Hg geraniol

75 Oxidation of citral with Ag 2 O gives geranic acid C 10 H 16 O 2 and no loss of carbon atom takes place. This further proves that oxo group in citral is an aldehyde group. CHO CO 2 H Ag 2 O geranic acid

76 Reaction with potassium hydrogen sulphate On heating with KHSO4 citral form P-Cymene. This reaction was used by semmler to determine the position of methyl and isopropyl group in the skeleton structure 1 CHO This reaction showed that the citral molecule is acyclic in which two isoprene Units are joined in head to tail manner 1 KHSO4 P.cymene

77 The examination of the formula of citral shows that 2 geometrical isomers are possible The functional group may be Cis or Trans. CHO CHO H H (a) Trans (b) Cis These structure are supported by NMR spectroscopy

78 Synthesis of citral can be carried out by the following synthetic route. Br Br Na HC COMe COMe CH COMe COMe Br NaOH O Zn/I-CH 2-CO 2 Et H Reformatsky Rtn OH CO 2 Et AC 2 O CHO Ca salt (HCO 2 )Ca -H 2 O Co 2 Et

79 CHO CHO Cat. Hydrogenation 2 moles C 10 H 16 O 2Br 2 C 10 H 16 Br 4 O

80 CHO H2N=OH CH=N-OH 2 moles

81 Citral is reduced to an alcohol geraniol ( C10H18O). Geraniol is a primary alcohol and the formation a primary alcohol form carbonyl compound confirmed that the citral contains an aldehyde functional group. CHO Na/Hg CH 2 -OH geraniol

82 Essential Oils

83 Essential Oils Terpenes and terpenoids are the primary constituents of the essential oils of many types of plants and flowers. Essential oils are used widely as natural flavor additives for food, as fragrances in perfumery, in aroma therapy, and in traditional and alternative medicines. Synthetic variations and derivatives of natural terpenes and terpenoids also greatly expand the variety of aromas used in perfumery and flavors used in food additives.

84 ISOLATION & SEPARATION TECHNIQUES Essential oils containing mono- and sesquiterpenoids are obtained by water and or steam distillation of the part such as flowers, leaves or stems, where the essential oils occur in more concentrated form. Due to the heat lability of certain constituents of essential oils different distillation methods have to be used for different raw materials which are briefly described below:

85 Distillation Today, most common essential oils, such as lavender, peppermint, and eucalyptus, are distilled. Raw plant material, consisting of the flowers, leaves, wood, bark, roots, seeds, or peel, is put into a n alem bic (distillation apparatus) over water. As the water is heated the steam passes through the pla nt material, vaporizing the volatile compounds. The vap ors flow through a coil where they condense back to liquid, which is then collected in the receiving ve ssel.

86 Distillation Most oils are distilled in a single process. One except ion is Ylang- ylang (Cananga odorata), which takes 2 2 hours to complete through a fractional distillation.

87 Distillation The recondensed water is referred to as a hydrosol, herbal di stillate or plant water essence, which may be sold as another fra grant product. Popular hydrosols are rose water, lavender water, lemon balm, a nd orange blossom water. The use of herbal distillates in cosmetics is increasing. Some plant hydrosols have unpleasant smells and are therefore not sold.

88 Expression Most citrus peel oils are expressed mechanically, or cold-pressed. Due to the large quantities of oil in citrus peel and the relatively low cost to grow and harvest the raw materials, citrus-fruit oils are cheaper than most other essential oils. Lemon or sweet orange oils that are obtained as by-products of the citrus industry are even cheaper. Prior to the discovery of distillation, all essential oils were extracted by pressing.

89 Solvent extraction Most flowers contain too little volatile oil to undergo expression and their chemical components are too delicate and easily denatured by the high heat used in steam distillation. Instead, a solvent such as hexane or supercritical carbon dioxide is used to extract the oils Extracts from hexane and other hydrophobic solvent are called concretes, which is a mixture of essential oil, waxes, resins, and other lipophilic (oil soluble) plant material.

90 Solvent extraction Although highly fragrant, concretes contain large quantities of non-fragrant waxes and resins. As such another solvent, often ethyl alcohol, which only dissolves the fragrant low-molecular weight compounds, is used to extract the fragrant oil from the concrete The alcohol is removed by a second distillation, leaving behind the absolute

91 ISOLATION & SEPARATION TECHNIQUES Terpenoids Following methods are employed for the extraction of mono-, sesqui-, di-, tri-, and tetraterpenoids. Air dried powdered part of the plant is extracted by percolation or soxhlet extraction successively with organic solvents with increasing polarity such as petroleum ether, benzene, diethyl ether, chloroform, ethyl acetate, acetone, ethanol, methanol and water. The extraction efficiency can be increased with the decrease in the time of the process by stirring the pulverized plant material using mechanical stirrer with the chosen solvent and filtering it to obtain the extract.

92 STRUCTURE ELUCIDATION Physical Mehtods 1. Molecular formula 2. Specific rotation 3. Refractive index Spectral Methods for Structure Determination 1. UV 2. IR 3. MS 4. NMR

93 Physical Mehtods 1. Molecular formula Determination of the molecular formula of an isolated pure terpenoid is done by finding out the empirical formula and molecular weight. Empirical formula can be found out by elemental analysis.while molecular weight can be determined by vapour density, elevation of boiling point and depression of freezing point. 2. Specific rotation Specific rotation of a compound is measured to ascertain the optical activity exhibited by it. It helps to distinguish between optical isomers. 3. Refractive index It is measured to calculate the value of molecular refraction, which is useful to find out the nature of the carbon skeleton especially in the case of sesquiterpenoids.

94 Spectral Methods 1. UV Functional groups, present in terpenoids, which absorb in the UV range between nm are termed as chromophores.however UV data becomes valuable only when the terpenoid molecule contains conjugated double bonds and/or α,β-unsaturated carbonyl group. 2. IR This method is routinely used for the identification as well as the structure elucidation of new terpenoids. 3. MS FAB-MS affords the e xact molecular ion peak along with diagnostic fragmentation patterns of the terpenoid molecule. It is an important tool for the structure determination.

95 4. NMR Spectral Methods NMR spectroscopy comprising of both PMR and CMR is in fact one of the Most important tools furnishing a good teal of information required for the structure elucidation. The combination of 1D selective and 2D NMR techniques such as COSY, TOCSY, ROESY,2D IN-ADEQUATE, HMQC, HMBC COLOC, HOHAHA, HETCOR and selective INEPT are of great value for the structure elucidation of various terpenoids including the saponins and glyosides of a number of sugar moieties.

96 EXAMPLE C 10 H 16 O b.p.77 UV:236nm IR:1665,1625,1603,1398, 1190,1117cm -1. MS:m/z 69(100),41,84,94,109,67,83,81 1 H-NMR:1.65(6H,d, C-7 methyls),2.15(3h,s,c-3 Me), 5.0(1H,t, H-6), 5.8(1H,d,H- 2), 9.84(1H,d,H-1). 13 C-NMR:190(C-1),127.5(C-2), 162.1(C-3),40.5(C-4),26.5(C-5), 123.5(C-6),132.3(C-7),25.3(C-8), 17.4(C-9),17.0(C-10). geranial CHO

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