regra do isopreno - wallach (1887) - usp · regra do isopreno - wallach (1887) 2-m e tilbuta...

Regra do isopreno - Wallach (1887)

2-metilbuta-1,3-dieno(isopreno)

O nome terpeno deriva da terebentina (turpentine) de

onde foram isolados a cânfora e o a-pineno. As

estruturas foram elucidadas em torno de 1894.

Terpenes

• Terpenes are natural products that are

structurally related to isoprene.

H2C C

CH3

CH CH2 or

Isoprene

(2-methyl-1,3-butadiene)

Terpenes

• Myrcene (isolated from oil of bayberry) is a

typical terpene.

CH2

CH3

CH3C CHCH2CH2CCH

CH2

or

The Isoprene Unit

• An isoprene unit is the carbon skeleton of isoprene

(ignoring the double bonds)

Myrcene contains two isoprene units.

The Isoprene Unit

• The isoprene units of myrcene are joined "head-to-tail."

head tail

tail head

Isoprene

Head

Tail

Head

Tail

Isoprene

Head

Tail

Head

Tail

Isoprene

• Classe Número de átomos de Carbono

• Monoterpeno 10

• Sesquiterpeno 15

• Diterpeno 20

• Sesterpeno 25

• Triterpeno 30

• Tetraterpeno 40

• C10 terpenoids (monoterpenes) components of volatile essences, essential oils

• C15 (sesquiterpenes) components of essential oils, phytoalexins (self defense)

• C20 (diterpenes) gibberellins, resin acids, phytol (chlorophyll side chain)

• C30 (triterpenes) phytosterols, brassinosteroids

• C40 (tetraterpenes)

carotenoids

a-Phellandrene

(eucalyptus)

Menthol

(peppermint)

Citral

(lemon grass)

O

H

OH

a-Phellandrene

(eucalyptus)

Menthol

(peppermint)

Citral

(lemon grass)

O

H

OH

a-Phellandrene

(eucalyptus)

Menthol

(peppermint)

Citral

(lemon grass)

a-Selinene

(celery)

H

a-Selinene

(celery)

H

a-Selinene

(celery)

Vitamin A

OH

Vitamin A

OH

Vitamin A

Squalene

(shark liver oil)

tail-to-tail linkage of isoprene units

O

cânfora

O

carvona

CHO

citronelal

O

eucaliptol(1,8-cineol)

OH

(óleo de pau-rosa) linalol

MONOTERPENOS

SESQUITERPENOS

HO

nerolidol

HH

cariofileno

O

O

O

H

H

OO H

artemisinina

ISOPENTENYL

PYROPHOSPHATE:

THE BIOLOGICAL ISOPRENE

UNIT

The Biological Isoprene Unit

• The isoprene units in terpenes do not come

from isoprene.

• They come from isopentenyl pyrophosphate.

Isopentenyl Pyrophosphate

H2C CCH2CH2OPOPOH

CH3 O O

Isopentenyl pyrophosphate

or OPP

Isopentenyl and Dimethylallyl

Pyrophosphate

• Dimethylallyl pyrophosphate has a leaving group

(pyrophosphate) at an allylic carbon; it is reactive toward

nucleophilic substitution at this position.

Isopentenyl pyrophosphate is interconvertible with

2-methylallyl pyrophosphate.

OPP OPP

Isopentenyl pyrophosphate Dimethylallyl pyrophosphate

CH 3

H SH R

OPP

H

H

CH 3

H SH R

OPP

H

HH w

H 3 C

CH 3

H S

OPP

H+

W

H+

RI P P D M A P P

First specific step in the biosynthesis of terpenoids

- isomerization of IPP into DMAPP-

OPP

HH

H

H

H

HSHS

C

Glu207

OO

S

Cys138

HS

Cys139

H

OPP

HH

H

H

H

HS

C

Glu207

OHO

S

Cys138

HS

Cys139

H

IPPDMAPP

IPP-Isomerase mechanism

THE PATHWAY FROM ACETATE

TO ISOPENTENYL

PYROPHOSPHATE

The Biological Isoprene Unit

CH3COH

O

3 HOCCH2CCH2CH2OH

CH3

OH

O

Mevalonic acid

H2C CCH2CH2OPOPOH

CH3 O O

Isopentenyl pyrophosphate

Biosynthesis of Mevalonic Acid

• In a sequence analogous to the early steps of fatty acid

biosynthesis, acetyl coenzyme A is converted to S-

acetoacetyl coenzyme A.

CH3CCH2CSCoA

O O

S-Acetoacetyl

coenzyme A

Biosynthesis of Mevalonic Acid

• In the next step, S-acetoacetyl coenzyme A reacts with

acetyl coenzyme A.

• Nucleophilic addition of acetyl coenzyme A (probably via

its enol) to the ketone carbonyl of S-acetoacetyl coenzyme

A occurs.

CH3CSCoA

O

+ CH3CCH2CSCoA

O O

Biosynthesis of Mevalonic Acid

CH3CSCoA

O

CH3CCH2CSCoA

CH2COH

HO O

O

+ CH3CCH2CSCoA

O O

Biosynthesis of Mevalonic Acid

• Next, the acyl coenzyme A function is reduced.

• The product of this reduction is mevalonic acid.

CH3CCH2CSCoA

CH2COH

HO O

O

CH3CCH2CSCoA

CH2COH

HO O

O

CH3CCH2CH2OH

CH2COH

HO

O

Mevalonic

acid

Conversion of Mevalonic Acid to

Isopentenyl Pyrophosphate

• The two hydroxyl groups of mevalonic acid undergo

phosphorylation.

CH3CCH2CH2OH

CH2COH

HO

O

CH3CCH2CH2OPP

CH2COH

O

OPO3

2–

Conversion of Mevalonic Acid to

Isopentenyl Pyrophosphate

• Phosphorylation is followed by a novel elimination

involving loss of CO2 and PO43–.

CH3CCH2CH2OPP

CH2

O

OPO3

2–

C O ••

••

• • –

CH3CCH2CH2OPP

CH2

OPO3

3–

O C O

Conversion of Mevalonic Acid to

Isopentenyl Pyrophosphate

• The product of this elimination is isopentenyl

pyrophosphate.

CH3CCH2CH2OPP

CH2

Biosynthetic pathway is based on

experiments with 14C-labeled acetate

CH3COH

O

HOCCH2CCH2CH2OH

CH3

OH

O

Mevalonic acid

H2C CCH2CH2OPOPOH

CH3 O O

Isopentenyl pyrophosphate

Biosynthetic pathway is based on

experiments with 14C-labeled acetate

• Citronellal biosynthesized using 14C-labeled acetate as the

carbon source had the labeled carbons in the positions

indicated.

CH3COH

O

H2C CCH2CH2OPOPOH

CH3 O O

O

H

•

• • •

•

•

OH

CO2-HO

R2

O

OR1

CH3CH3

OH

CO2-HO

H3C

R1 = H R2 = H Compactin

R1 = CH3 R2 = CH3 Simvastatin (Zocov)

R1 = H R2 = OH Pravastatin (Precavol)

R1 = H R2 = CH3 Lovastatin (Mevacor)

mevalonate

Inibidores da biossíntese de colesterol

(produtos que mimetizam o ácido mevalônico)

Rohmer & coworkers, Braunschweig (1980s)

eubacterial sterol (hopanoids) synthesis

metabolic profiling with 13C-NMR

fed [13C]acetate and looked at label in endproducts and

intermediates

pattern did not match that of mevalonate pathway

also mevilonin insensitive

substrates

glyceraldehyde 3P and pyruvate efficiently incorporated

phosphorylated 5C sugar is intermediate

1-deoxyxylulose-5P (DXP)

The Non-mevalonate or DXP pathway

O

H

HO

H

HO

H

OHOHH

H

OH

HOOCO

S

N

S

NHO

O OH

-CO2

S

NHOHO

PO

OHO

H

S

N

HO

HO

OH

OP

-TPP

O

OH

HO

PO

H

OP

HO

O OH

OPP

IPP

1-deoxy-xylulose-5-P

[1-13

C]-glucosepyruvate

glyceraldehyde-3-P

TPP

OPP

IPP originated from HMG-CoA

2 x Acetyl CoA

HMG CoA

MVA

IPP

MVA pathway

(cytoplasm)

Pyruvate

Glyceraldehyde

3-phosphate

Deoxy xylulose

5-phosphate

Methyl erythritol

4-phosphate

Non-MVA pathway

(plastids)

Sources of Isoprenoids in Higher Plants

• Higher plants like red (and brown) algae

• Cytosolic mevalonate pathway

• sterols (C30), ubiquinone

• certain sesquiterpenoid (C15) phytoalexins

• plastidic DXP pathway

• carotenoids, phytol, PQ

• mono- and di-terpenes (C10 & C20), and other

sesquiterpenes

Subcellular Location of Isoprenoid synthesis

Chloroplast

ER Mitochondrion

Sterols,

Rubber

CHLOROPHYLL

PQ Carotenoids

UQ Haem a

Also

Gibberellins, ABA,

CKs

tRNAs

phytoalexins

volatile oils

Feed IPP to cps, mitos or ER

incorporated into organelle-specific isoprenoids

CARBON-CARBON BOND

FORMATION IN TERPENE

BIOSYNTHESIS

Carbon-Carbon Bond Formation

• The key process involves the double bond of isopentenyl

pyrophosphate acting as a nucleophile toward the allylic carbon

of dimethylallyl pyrophosphate.

+ OPP

OPP

OPP

DMAPP

SN1 Reaction

H3C

CH3

HS

DMAPP

CH3

HSHR

OPP

IPP

OPP

HSHR

OPP

OPP

Geranyl diphosphate (GPP)

Head-tail condensation

FORMATION OF THE

MONOTERPENE SKELETONS

Carbon-Carbon Bond Formation

+ OPP

OPP –

+ OPP

OPP

After C—C Bond Formation...

• The carbocation can

lose a proton to give

a double bond.

+ OPP

OPP

H – +

After C—C Bond Formation...

• This compound is called geranyl pyrophosphate. It can undergo

hydrolysis of its pyrophosphate to give geraniol (rose oil).

OPP

After C—C Bond Formation...

OPP

OH

Geraniol

H2O

Cyclization

• Rings form by intramolecular carbon-carbon bond

formation.

OPP

OPP

+

E double

bond

Z double

bond

+

OH

H – +

H2O

HH

H

H

1,2-hydrideshift

H

1,3-hydrideshift

H H Htertiary carbocation

ressonance-stabilized allylic cation

HH

1,2-methylshift

tertiary carbocation

tertiary carbocation

secondary carbocation

secondary carbocation

Wagner-Meerwein

Rearrangements

1,2-alkylshift

tertiary carbocation, but strained4-membered ring

secondary carbocation.but reduced ring strain in5-membered ring

H

H

H

H

a series of concerted 1,2 hydrideand methyl shifts

Wagner-Meerwein

Rearrangements

cont.

Bicyclic Terpenes

+ +

+

-Pineno

+

a-Pineno

OPP OPP OPP

OPP

OPP

OPP

E

Z

Geranyl cation(ressonance stabilized allylic cation) neryl cation

(ressonance stabilized allylic cation)

neryl-PPlinalyl-PP

Geranyl-PP

FORMATION OF THE

SESQUITERPENE SKELETONS

• Probably substrate specific

• ie different ones to make GPP, FPP, GGPP

• GPP synthase in peppermint (Burke et al, 1999)

• heterodimer of 28 & 37 kDa

• plastid located

• GGPP synthases in Arabidopsis (Okada et al, 2000)

• 1 & 3 go to cps, 2 & 4 go to ER and 6 is in mitos

• 1 & 6 ubiquitously expressed

• 2, 3 & 4 in flower, root and flower respectively

Cyclases or Terpene Synthases

• Responsible for cyclization of isoprenoid

• C10 to monoterpenes

• C15 to sesquiterpenes

• C20 to diterpenes

• Similar reaction to prenyltransferases

• loss of PP group produces carbocation

• this attacks a double bond

• multiple double bonds mean alternative products

• Plant cyclases have sequence similarity

• differ from fungal or animal enzymes

From 10 Carbons to 15

+ OPP

OPP

Geranyl pyrophosphate

+ OPP

From 10 Carbons to 15

+ OPP

H – +

OPP

From 10 Carbons to 15

• This compound is called farnesyl pyrophosphate.

• Hydrolysis of the pyrophosphate ester gives the alcohol

farnesol

OPP

E

E

HH

E,E-farnesyl cation

germacryl cation

humulyl cation

caryophyllyl cation

eudesmyl cation

guaiyl cation

Pathways for cyclization of farnesyl-PP

E

Z

a

b

a

a

b

Hb

E,Z-farnesyl cation

bisabolyl cation

carotyl cation

cadinyl cationcis-germacryl cation

cis-humulyl cation

Formation of the

Diterpene Skeletons

From 15 Carbons to 20

• Farnesyl pyrophosphate is extended by another isoprene

forming the geranylgeranyl diphostate (GGPP)

OPP

OPP

OPP

H

H HH

OPP

H H

OPP

H

H

Copalil-pp

H

OPP

H

H

labdadienil -pp

H

H

OPP

H

H

H

OPP

TRITERPENOS & ESTERÓIDES

HO

R

R=COOH (ácido oleanólico)

R=CH3 (-amirina)

HO

R

R=COOH (ácido ursólico)

R=CH3 (a-amirina)

HO

HO

lupeol

eufol

HO

O

O

H

H

HH

Diosgenina

HO

H

O

O

H

OHH

Digitoxigenina

HO

H

H H

Colesterol

PPO

OPP

H H

OPP

H

HH

H

HH

H(NADPH)

HH

Squalene

farnesol-PP

farnesol-PP

OPP

OPP

Enz

Enz

OPPH

OPP

NADP*H

esqualeno

*H

pirofosfato de farnesila pirofosfato de farnesila

-OPP

Um processo idêntico ocorre na formação de tetraterpenos

a partir de diterpenos

Coupling Enzymes • Head-to-head condensation

• Squalene synthase for sterols (C30), phytoene

synthase for carotenoids (C40)

PSY and SqS share domain in common

OPP OPP

phytoene

Arab PSY

Syn. PSY

Arab SqS

prenyltransferase

domain

Biosynthesis of Cholesterol

• Cholesterol is biosynthesized from the triterpene squalene.

In the first step, squalene is converted to its 2,3-epoxide.

O

O2, NADH, enzyme

Biosynthesis of Cholesterol

• To understand the second step, we need to look at squalene

oxide in a different conformation, one that is in a geometry

suitable for cyclization.

O

O

Chair-boat-chair-boat conformation

Biosynthesis of Cholesterol

• Cyclization is triggered by epoxide ring opening.

O H+

+

HO

H

Biosynthesis of Cholesterol

• The five-membered ring expands to a six-membered one.

+

HO

H

H

HO

H

Biosynthesis of Cholesterol

• Cyclization to form a tetracyclic carbocation.

H

HO

H

H

H

HO

protosteryl cation

Biosynthesis of Cholesterol

• Deprotonation and multiple migrations.

HO

OH2

•• • •

H

H

HO

H

H

O HO

H

HH

HHO

H

HH

H

H

HOH

H

HOH

H

H

H

cycloartenollanosterol

squalene oxide

protosteryl cation

Sequence od W-M 1,2-hydride and1,2 methyl shifts

animalsfungi

plants

loss of a proton gives alkene

loss of protonleads to cyclopropane

Biossíntese de Esteróides

O

Chair-chair-chair-boat conformation

H+

H

HO

H

H

HO

H

H

H

HO

H

H

H

a-amirina

Biossíntese de

Triterpenos