biosintesis of terpenoid

8/10/2019 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.

Subsequent steps lead to the important C5 building blocksIPP and DMAPP.IPP is isomerized to DMAPP

by the enzyme isopentenyl pyrophosphate isomerase:

During the past 10 years a very important discovery was made, namely, that in some organisms analternative pathway exists to DMAPP and IPP. This alternative pathway is found in some microorganisms


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as well as the plastids of plants and algae, and is called the MEP (2-methyl-D-erythritol-4-phosphate)-

pathway (or more simply the non-mevalonate pathway), which is initiated from C5-sugars. The

mechanisms of some of the steps in this pathway have not yet been elucidated:

Me

O

COOH

CHO

OH

CH2O-PO3

Me

O

OH

CH2O-PO32-

HO O PO32-

HO

MeHO

OH

IPP

Deoxyxylulose-5-Phosphate

CO2

TPPNADPH

O P2O63-

HO

Me

DMAPP

CTP PPi

O P

HO

MeHO

OH

O

O-

O CMP

ATP

ADP

O P

HO

Me2-O3PO

OH

O

O-

O CMP

CMP

O

PO2

HO

Me O

OH

PO2 O

H+ 2e

-H2O H

+

2e-

H2O Me O P2O63-

Me

O P2O63-

Me

After the formation of IPP and DMAPP, there exists in all organisms a central route to the universal

building blocks needed for mono-, sesqui-, di-, tri and tetra-terpene biosynthesis:

Me

R O-P-P

Me

O-P-P Me

Me Me

O-P-P

Me

Me

R O-P-P

Me

RP-P-O

Me

Me Me Me

Me

MeMeMe

Me

Me O-P-P

Me

O-P-P Me

Me

R

O-P-P

Me Me

O-P-P

Me

O-P-P Me

Me Me Me

O-P-P

Me

+

C

20

Building block

Diterpenes

Geranylgeranyl pyrophosphate (GGPP)

+

Steroids

DMAPP

C

30

Building block

C

15

Building block

C

10

Building block

Triterpenes

Squalene

Sesquiterpenes

Monoterpenes

Farnesyl pyrophosphate (FPP)

+

GPP

Geranyl pyrophosphate (GPP)

+

IPPFPP

IPP

IPP

FPPFPP

TetraterpenesC

40

Building block

Me

R O-P-P

Me

RP-P-O

+

GGPPGGPP

The mechanism and stereochemical course of all these steps was investigated by J. W. Cornforth, whoreceived the Nobel Prize in Chemistry for his work (1975, mit V. Prelog, ETH-ZH). In recent years, direct

access to the biosynthetic genes for many of the enzymes in terpene biosynthesis has provided an enormous

impulse for structural and mechanistic studies. There is also great interest in the design and development of

specific inhibitors, as potential drugs against bacterial and parasitic infections, and in the agrochemical area.


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2.3 The Formation of GPP, FPP und GGPP

The steps from DMAPP and IPP to GPP, FPP and GGPP are catalyzed by so-called prenyl transferases.

These enzymes (35 - 80 kDa) require Metal2+-ions for activity. The Kmvalues are typically


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The resulting !-terpinyl carbocation remains a bound intermediate in many terpene cyclase reactions, and

can react further in many different ways. Each monoterpene cyclase will typically catalyze preferentially

one reaction pathway:

OPP

3-Carene

Sabinene

!-Thujene

"-Terpinene

!-Terpinene

#-Phellandrene

OH

endo-Fenchol-Pinene

!$Pinene

Camphene

OPP

(+)-Bornyl-pyrophosphate

O

1,8-Cineol

OH

!-Terpineol

(-)-Limonene

Terpinolene

!-Terpinyl-Kation

One well studied example is the bornyl pyrophosphate cyclase, which is involved in the biosynthesis of

camphor :

OPP OPP

O

OH

(+)-Camphor(+)-Borneol

Bornyl-PPcyclase

Mechanism of the cyclase reaction:

OPP

OPP OPPOPP

Cyclase

!

enzyme bound intermediates

HS

HR H

H

H

H

H

H

Bornylpyrophosphate

H

H

OPP


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Limonene synthase is another well-studied enzyme. (-)-Limonene is the precursor of menthol and carvone,

which can be isolated from extracts of peppermint, carraway (Carum carvi) and dill.

OPP

OH O O

OOOH

GPP (-)-Limonen(-)-trans-Isopiperitenol (-)-Isopiperitenon cis-Isopulegon

(+)-Pulegon(-)-Menthon(-)-Menthol

The main product of the limonene synthase reaction is limonene, but small amounts of myrcene (2%), !-pinene und -pinene (4%) can also be detected:

PPO

GPP

PPO

OPP OPP OPP

OPP

(-)-4S-Limonen

OPP

-HMyrcen

!-Pinen "-Pinen

Sesquiterpene Synthases(Curr. Opin. Struct. Biol. 1998, 8, 695; Chem. Rev.1990, 90, 1089)

All sesquiterpenes are formed from FPP. A large variety of different cyclic sesquiterpenes have been

discovered in Nature.

OPP

!-Cadinene

"-Humulene

E--Farnesene

E-#-Bisabolen

TrichodienePentalenene

OHepi-Cedrol

Vetispiradiene

5-epi-aristolochen

Germacrene C!-Selinene

FPP

Amorpha-4,11-diene

O

H Me

H

O

OMe

O

O

Artemisinin


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The sesquiterpene cyclasesrequire Mg2+as cofactor and use FPP as substrate. The metal is coordinated

both to the protein and to the pyrophosphate group of the substrate. An important point is the stereochemical

course of the cyclization at C1, with some reactions proceeding with retention and others with inversion of

configuration. Mechanism in overview:

OPP OPP

OPP

OPP OPP

FPHPH2 E--Farnesene

1,10 cyclization

Aristolochene- H+

!-Humulene Longifolene

"-Longipinene

"-Ylangene

1,11 cyclization

1,10 cyclization-Bisabolene

1,6- 1,11-

A well studied example is the enzyme trichodiene synthase from the fungus Fusarium sporotrichioides,

which converts FPP into trichodiene:

OPP

OPP OPP

H

!

!

Trichodiene

!H

!

H

!

The aristolochene synthaseisolated from tobacco plants and the vetispiradiene synthasefromHyoscyamusmuticusare two phylogenetically closely related enzymes that catalyze also very closely related reactions. In

both cases, E,E-germacrene-A is formed as a short-lived enzyme-bound intermediate. Studies reported so


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Taxol is an important natural product because of its anti-cancer activity. It was discovered in a National

Cancer Institute program at the Research Triangle Institute in 1967 when it was isolated from the bark of the

Pacific yew tree, Taxus brevifolia and named 'taxol'. When developed commercially by Bristol-Myers

Squibb (BMS) the generic name was changed to 'paclitaxel'. The BMS compound is sold under the

trademark 'Taxol'. Paclitaxel is now used to treat patients with lung, ovarian, breast cancer, head and neck

cancer, and advanced forms of Kaposi's sarcoma. Paclitaxel is also used for the prevention of restenosis.

Paclitaxel works by interfering with normal microtubule growth during cell division.

From 1967 to 1993, almost all the paclitaxel produced was derived from the bark of the Pacific yew, the

harvesting of which kills the tree in the process. In 1992 BMS started to manufacture paclitaxel from 10-

deacetylbaccatin isolated from the needles of the European yew. By the end of 1995, BMS stopped

production from the bark of the Pacific yew, effectively terminating the ecological controversy over its use.

Currently, all paclitaxel production for BMS uses plant cell fermentation technology. This starts from a

specific taxus cell line propagated in aqueous medium in large fermentation tanks. Paclitaxel is thenextracted directly, purified by chromatography and isolated by crystallization.

There is now great interest in trying to reconstitute the entire biosynthetic pathway in vitro. Several of the

enzymes on the pathway have already been cloned and produced by recombinant DNA techniques. A key

step is catalyzed by the taxadiene synthase:

O

N

H OH

O

O

Me

Me

Me

O O

MeOH

O

O

H

HO O

O

Me

O

MeO

PhTaxol

GGPP

The mechanism of the cyclization has been intensively studied:

OPP H

D

DD

H

D

D

HH

2.6. The formation of triterpenes from squalene (Angew. Chem. 2000, 112, 2930)

Squalene is the universal precursor of all triterpenes, including all steroids. In animals, squalene is

converted in only two steps into a steroid called lanosterol. The first step is catalyzed by a monooxygenase,which is a flavo-enzyme not a hemoprotein, but uses molecular oxygen and NADPH to epoxidize squalene:


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O MeMe

Me

Me

Me

Me

Me

Me

Me Me

Me

Me

Me

Me

HO

Me

MeH

H

Squalene Epoxide Lanosterol

Steroide

Me

Me Me Me

Me

MeMeMe

Squalene

NADPH, O2

NADP+, H2O squalene epoxidase

squalene epoxidecyclase

Perhaps of most interest here is how the cyclase enzyme can take squalene epoxide as substrate and release

lanosterol as product. What chemical steps take place at the active site of the enzyme and how is thereaction catalyzed?

Oxidosqualene-Lanosterol-Cyclase

In higher organisms the steroid skeleton is produced through the action of a membrane-bound enzyme. In

the course of the transformation, a series of ring-forming steps and rearrangement reactions take place:

Me

Me

O

Me

Me

Me

Me

MeMe

MeMe

Me

HO

Me H

MeH H

MeMe

Me

HO

Me

Me

H

Me

H

Me

Me

H

Me

H

MeO

Me

Me

HO

MeH

MeH H

Lanosterol

X = OEnzyme

BH X

H

Br

O F

ON

Ro 48-8071an inhibitor

MeMe

Me

Me

Me

HO

Me H

MeH

H

Me

Me

MeMe

MeMe

Me

HO

Me H

MeH H

H

MeMe

Me

MeMe

Me

HO

Me H

MeH H

H

MeMe

Me

HO

Me

Me

H

Me

H

Me

Me

H

H

?


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The cyclase must bind the substrate in the correct folded conformation to allow a stereoelectronically

assisted series of rapid ring closure steps, and form the product with the correct relative and absolute

configuration. All the intermediate carbocation intermediates must be shielded from reaction either with

water or with the protein. Finally, the correct proton must be removed to terminate the reaction. The H-atom

shifts and Wagner-Meerwein rearrangements occur along a kinetically and thermodynamically preferred

pathway, until the end product is reached. At the end of 2004 a group at Hoffmann-La Roche in Basel

succeeded for the first time in crystallizing the enzyme (Nature, 2004, 432, 118).

Left:Ribbon diagram of human OSC. a, The C and N termini and several sequence positions are labelled. Theinner barrel helices are coloured yellow. The bound inhibitor (black) indicates the location of the active site. b,The orientation of OSC relative to one leaflet of the membrane, whose polar and nonpolar parts are depicted inlight blue and light yellow respectively. Internal surfaces and channels of OSC are shown with the followingcolour code: blue, positive; red, negative; cyan, hydrogen-bond donor; magenta, other polar. Ro 48-8071 binds inthe central active-site cavity. Two channels lead to the enzyme surface: one is hydrophobic to the membraneinsertion site and one is polar. The fragment of lipid (blue) binds to the hydrophobic substrate entrancechannel. A -OG molecule belonging to a crystal neighbour (black) interacts with the membrane-insertinghydrophobic surface.

Right: Stereoview of the electron density representing the bound substrate. Residues in the enzyme within 5are shown. A/B-Rings: The cationic intermediates may be stabilized by cation-$interactions with the aromatic

rings of Trp387, Phe444 and Trp581. The catalytic Asp455 is activated by Cys 456 and Cys 533. The Tyr 98side chain sterically hinders the B-ring from assuming the favourable chair conformation. C/D-Rings: Phe 696and His232 can stabilize the positive charge at the C20 cation by cation-$interactions. His 232 is the nearestbasic residue that could deprotonate the C8/9 lanosterol cation.


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From lanosterol, the pathway for steroid biosynthesis continues on to cholesterol. Three methyl groups must

be removed, one double bond is reduced and another is shifted. Cholesterol then becomes a branch point in

steroid biosynthesis, serving as a precursor from which other steroids are produced:

!"

!" !"

!"

!"

!"

#$

!"

!"#

#

Lanosterol

!" !"

!"

#$

!"

!"#

#

Cholesterol

##

#

In plants the oxidosqualene cyclase does not form lanosterol, but rather cycloartenol, which is then the

precursor for the formation of other plant steroids:

Me

Me

O

Me

Me

MeBH Me Me

Me

Me

MeMe

MeMe

Me

HO

Me H

MeH H

H

MeMe

MeHO

Me

Me

H

Me

H

Me

Me

H

H

Me

Me

HO

Me

Me

H

Me

H

Me

Me

H

Cycloartenol

Bacterial squalene cyclase catalyzes a different cyclization cascade, which is mechanistically related, but

not so complicated. Now squalene (not the epoxide) is bound in a specific conformation, which allows a

rapid series of cyclization steps to occur. The process is now started by protonation of the terminal double

bond (Chem.Biol. 2000, 7, 643):

H

H

Squalene-HopeneCyclase

This cyclase is a homodimeric, soluble enzyme. The active site is a buried cavity, which binds squalene in

the preferred conformation. Most probably the side chain of Asp376 acts as a general acid catalyst to startthe cyclization cascade.


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biosintesis of terpenoid

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