biosintesis of terpenoid

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    2. Biosynthesis of Natural Products - Terpene Biosynthesis

    2.1 Introduction

    Terpenes are a large and varied class of natural products, produced primarily by a wide variety of plants,

    insects, microoroganisms and animals. They are the major components of resin, and of turpentine produced

    from resin. The name "terpene" is derived from the word "turpentine". Terpenes are major biosynthetic

    building blocks within nearly every living creature. Steroids, for example, are derivatives of the triterpene

    squalene. When terpenes are modified, such as by oxidation or rearrangement of the carbon skeleton, the

    resulting compounds are generally referred to as terpenoids. Some authors will use the term terpene toinclude all terpenoids. Terpenoids are also known as Isoprenoids.

    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, and

    in traditional and alternative medicines such as aromatherapy. Synthetic variations and derivatives of natural

    terpenes and terpenoids also greatly expand the variety of aromas used in perfumery and flavors used infood additives. Recent estimates suggest that over 30'000 different terpenes have been characterized from

    natural sources.

    Early on it was recognized that the majority of terpenoid natural products contain a multiple of 5C-atoms.

    Hemiterpenesconsist of a single isoprene unit, whereas the monoterpenesinclude e.g.:

    CH2OH

    CH2OH

    OH

    CHO

    CHO O

    O

    Camphor!-Pinene

    Citronellal

    MentholCitralGeraniolNerolLimonensMyrcens

    Monoterpenes

    Terpenes with 15 C-atoms are known as sesquiterpenes:

    CH2OH

    O

    Farnesol Bisabolene Cadinene Selinene Vetivone

    HO

    Patchoulol(Perfume)

    O

    COOH

    OH Abscisic acid(Phytohormone)

    O

    O

    O

    COOMe

    OH

    Pentalenolactone(Antibiotic)

    Sesquiterpenes

    The terpenes containing, or originating from precursors, containing 20 C-atoms are known as diterpenes,

    those with 30 C-atoms as triterpenesand those with 40 C-atoms as tetraterpenes:

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    Diterpenes

    CH2OH

    CH2

    OH

    Vitamin A(Retinol)

    Phytol

    AcO O OH

    O

    OAcOBzOH

    OPh

    O

    OH

    NH

    O

    Ph

    H

    Taxol (anti-cancer)

    Casbene(Phytoalexin)

    HO

    O

    COOH

    Giberellic acid(Phytohormone)

    OH

    O

    Triterpene Squalene

    HO

    H

    HH

    HO

    H

    OH

    H

    HO

    H

    H

    COOH

    O

    O

    H

    OH

    HH

    OCH2OH

    Cholesterol(Membrane component) Cholic acid

    Cortisone(Hormone)

    H

    O

    OH

    HH

    H

    HO

    OH

    HH

    H

    O

    HH

    OTestosterone(Hormone) stradiol

    (Hormone)Progesterone(Hormone)

    In contrast to other classes of terpenes that vary greatly in structure and molecular size, the steroids

    constitute a family of terpenes with a common structural feature, namely, the steroid ring system:

    Tetraterpene

    -Carotene(Pigment, Provitamin A)

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    Mixed origin

    N N

    NN

    Me

    Me

    MeMe

    Mg

    OCOOMeO

    O

    Chlorophyll-a(Photosynthesis)

    O

    O 18

    Plastoquinone(Electron transport)

    O

    OH

    C5H11

    Tetrahydrocannabinol(Cannabis sativa)

    Polymer

    OH

    Rubber(Heva brasilensis)

    500-5000

    Ruzicka (ETH-ZH) recognized already in the 1920's that most terpenes appear to be constructed from a

    multiple of linked isoprene units. This is called the isoprene rule.

    The isoprene rule(cf. Birch, Polyketide Hypothesis) was of great value also in the structure determination

    of new terpenoids isolated from Nature. However, isoprene itself is not the building block used by Nature toconstruct terpenes.

    CH2OH

    OH

    O OH

    Vitamin ACadinene

    Grandisol

    Camphor

    Menthol

    2.2 The Mevalonate Pathway

    It was only much later (ca. 1955) shown that the biosynthesis of terpenes does indeed occur starting fromisoprene-like C5 building blocks. Labelling experiments, using 14C-labelled acetic acid, showed early on

    that the steroid skeleton is constructed from this building block, but not simply through regular head-to-tail

    coupling reactions:

    Me COOH

    HO

    MeH

    Me

    Me Me

    Me

    HH

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    A breakthrough came around 1955 with the discovery of mevalonic acid (MVA), which was isolated from

    concentrated yeast extracts at the end of the beer brewing process. It was also shown that 14C-labelled forms

    of MVA are efficiently and specifically incorporated into cholesterol. Another important discovery was the

    isolation and structure determination of squalene from sharks (Squalus), which was also shown to be an

    efficient biosynthetic intermediate in steroid biosynthesis :

    HO

    MeH

    Me

    Me Me

    Me

    HH

    Me COOH

    Me OH

    HOOCOH

    MeMe

    Me

    Me

    Me

    Me

    Me

    Me Me

    Me

    Me

    Me

    HO

    Me

    MeH

    H

    Me

    Me

    In the mean time, all the steps from acetyl-CoA to cholesterol have been established and most of the

    enzymes involved in the pathway have been isolated and studied. The pathway from acetyl-CoA to MVA,

    and on to the various classes of terpenes has now been discovered in almost all living organisms, and is

    known as the mevalonate pathway:

    Me

    O

    SCoA Me

    O

    SCoA Me SCoA

    O O

    Me

    O

    SCoA

    Me OH

    O OH O SCoA

    Me OH

    O OH

    Me

    OH

    Me O-P-P

    Me

    O-P-P

    P

    O

    O-O

    CO2

    +

    + CoASH

    3 ADP

    3 ATP C5

    building blocks

    Isopentenyl pyrophosphate (IPP)

    Dimethylallyl pyrophosphate (DMAPP)

    (R)-Mevalonic acid

    Reduction 2x with NADPH

    ++

    -P- =

    The enzyme 3-hydroxy-3-methylglutaryl-CoA synthase catalyzes an Aldol-type reaction that is unusal from

    a regiochemical viewpoint:Me OH

    O OH O SCoA

    + CoASHMe SCoA

    O O

    Me

    O

    SCoA

    +

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    Mechanism:

    SH SCoA

    O

    S

    O

    CoASH

    S

    O

    H

    B

    S

    O

    SCoA

    O O

    A H

    S SCoA

    O OHO Me

    H2O

    HO SCoA

    O OHO Me+ HMGS

    Through crystallographic studies, also with substrates bound at the active site, a good model for the reaction

    mechanism has been established. The structures have also shown which residues at the active site are most

    likely involved in catalysis (Vgl PNAS2004, 101, 16442):

    A. Acetoacetyl-CoA and Acetyl-Cys, andB. HMG-CoA in the active site

    In the next step of the mevalonate pathway, the CoAS-thioester group is reduced in a reaction requiring twoequivalents of NADPH. The reaction proceeds in two steps (thioester aldehyde alcohol). Many

    inhibitors of this enzymic reaction have been discovered, and several of these (called statins) are now

    important pharmaceutical products. The statins (or HMG-CoA reductase inhibitors) form a class of

    hypolipidemic drugs used to lower cholesterol levels in people with, or at risk of, cardiovascular disease.

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    They lower cholesterol by inhibiting the enzyme HMG-CoA reductase (HMGR), which is the rate-limiting

    enzyme of the mevalonate pathway of cholesterol synthesis.

    In the 1970's the Japanese microbiologist Akira Endo first discovered natural products with a powerful

    inhibitory effect on HMGR in a fermentation broth of Penicillium citrinum, during the search for

    antimicrobial agents. The first product was named compactin (ML236B or mevastatin). Animal trials

    showed very good inhibitory effects, however, in a long term toxicity study in dogs toxic effects were

    observed at higher doses. In 1978, Alfred Alberts and colleagues at Merck Research Laboratoriesdiscovered a new natural product in a fermentation broth of Aspergillus terreus, their product showed good

    HMGR inhibition and they named the product mevinolin, which later became known as lovastatin.

    !

    !

    "# $

    !

    $! !

    "#

    Compactin (IC50= 23 nM)

    $!

    "#

    !

    %&'(

    !

    !

    HMG-CoA (Km= 4 M)

    $! !

    !

    !$

    )

    *

    Fluvastatin (IC50= 28 nM)

    )!

    $! !

    !!$

    *

    Cerivastatin (IC50= 10 nM)

    !

    !

    "# $

    !

    $! !

    "#

    "#

    Mevinolin(Lovastatin)

    %&'(

    &!!$!

    !$$

    The essential structural components of all statins are a dihydroxyheptanoic acid unit and a ring system with

    different substituents. The statin pharmacophore is a modified hydroxyglutaric acid component, which is

    structurally similar to the endogenous substrate HMG-CoA and the mevaldyl-CoA intermediate in the

    enzymic reaction. The statin pharmacophore binds to the same active site as the substrate HMG-CoA and

    inhibits the HMGR enzyme. It has also been shown that the HMGR is stereoselective and as a result all

    statins need to have the 3R,5R absolute configuration.

    Subseq