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.

CH3

H2C C CH CH2 or

Isoprene (2-methyl-1,3-butadiene) Terpenes

• Myrcene (isolated from oil of bayberry) is a typical terpene.

CH3 CH2

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 Isoprene Head

Tail Head Tail Isoprene Head

Tail • 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 OH O

H

a-Phellandrene Menthol Citral (eucalyptus) (peppermint) (lemon grass)

OH O

H

a-Phellandrene Menthol Citral (eucalyptus) (peppermint) (lemon grass)

a-Phellandrene Menthol Citral (eucalyptus) (peppermint) (lemon grass) H a-Selinene (celery)

H

a-Selinene (celery)

a-Selinene (celery) OH

Vitamin A

OH

Vitamin A

Vitamin A tail-to-tail linkage of isoprene units

Squalene (shark liver oil) MONOTERPENOS OH O O CHO O cânfora carvona citronelal linalol eucaliptol (óleo de pau-rosa) (1,8-cineol)

SESQUITERPENOS

H

H H O O H HO O H O nerolidol O cariofileno 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

CH3 O O

H2C CCH2CH2OPOPOH or OPP

Isopentenyl pyrophosphate Isopentenyl and Dimethylallyl Pyrophosphate Isopentenyl pyrophosphate is interconvertible with 2-methylallyl pyrophosphate.

OPP OPP

Isopentenyl pyrophosphate Dimethylallyl pyrophosphate

• Dimethylallyl pyrophosphate has a leaving group (pyrophosphate) at an allylic carbon; it is reactive toward nucleophilic substitution at this position. First specific step in the biosynthesis of terpenoids - isomerization of IPP into DMAPP-

+ H W CH CH 3 3 H w CH H OPP 3 H OPP OPP H 3 C H H R H S H R H S H H S + H R I P P D M A P P IPP-Isomerase mechanism

Cys138 Cys138 Cys139 Cys139 S S H S H H S H H H H H H H H OPP H H OPP H HS S H HS O OH O C O C

Glu207 Glu207

DMAPP IPP THE PATHWAY FROM ACETATE TO ISOPENTENYL PYROPHOSPHATE The Biological Isoprene Unit

O O CH3

3 CH3COH HOCCH2CCH2CH2OH

OH

Mevalonic acid CH3 O O

H2C CCH2CH2OPOPOH

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.

O O

CH3CCH2CSCoA S-Acetoacetyl coenzyme A Biosynthesis of Mevalonic Acid O O O

CH3CCH2CSCoA + CH3CSCoA

• 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. Biosynthesis of Mevalonic Acid O O O

CH3CCH2CSCoA + CH3CSCoA

HO O

CH3CCH2CSCoA

CH2COH O Biosynthesis of Mevalonic Acid

• Next, the acyl coenzyme A function is reduced. • The product of this reduction is mevalonic acid.

HO O

CH3CCH2CSCoA

CH2COH O HO Mevalonic CH3CCH2CH2OH acid CH2COH O

HO O

CH3CCH2CSCoA

CH2COH O Conversion of Mevalonic Acid to Isopentenyl Pyrophosphate

2– HO OPO3

CH3CCH2CH2OH CH3CCH2CH2OPP

CH2COH CH2COH O O

• The two hydroxyl groups of mevalonic acid undergo phosphorylation. Conversion of Mevalonic Acid to Isopentenyl Pyrophosphate

3– 2– OPO3 OPO3

CH3CCH2CH2OPP CH3CCH2CH2OPP •• • – CH2 CH2 C O • •• O C O O

• Phosphorylation is followed by a novel elimination 3– involving loss of CO2 and PO4 . Conversion of Mevalonic Acid to Isopentenyl Pyrophosphate

CH3CCH2CH2OPP

CH2

• The product of this elimination is isopentenyl pyrophosphate. Biosynthetic pathway is based on experiments with 14C-labeled acetate

O O CH3

CH3COH HOCCH2CCH2CH2OH

OH

Mevalonic acid CH3 O O

H2C CCH2CH2OPOPOH

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.

O CH3 O O

CH3COH H2C CCH2CH2OPOPOH

• • O • • • • H Inibidores da biossíntese de colesterol (produtos que mimetizam o ácido mevalônico)

HO - HO - CO CO2 2 H3C OH OH R1 O mevalonate O CH3 CH3

R2

R1 = H R2 = H Compactin R1 = CH3 R2 = CH3 Simvastatin (Zocov) R1 = H R2 = OH Pravastatin (Precavol) R1 = H R2 = CH3 Lovastatin (Mevacor) 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 H O O OH H N H O N HO HO HOOC HO H O S H OH S H OH TPP H 13 pyruvate [1- C]-glucose -CO2 HO O

HO HO N PO glyceraldehyde-3-P S 1-deoxy-xylulose-5-P O H

OH N HO -TPP HO PO HO S OH OP

HO OP OPP OPP IPP originated from HMG-CoA O OH IPP Pyruvate 2 x Acetyl CoA Glyceraldehyde 3-phosphate

Deoxy xylulose 5-phosphate

Methyl erythritol 4-phosphate HMG CoA

MVA

MVA pathway Non-MVA pathway (cytoplasm) IPP (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

Mitochondrion ER Also Haem a Sterols, UQ Rubber Gibberellins, ABA, CKs

tRNAs PQ Carotenoids phytoalexins CHLOROPHYLL Chloroplast 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

OPP + OPP

• The key process involves the double bond of isopentenyl pyrophosphate acting as a nucleophile toward the allylic carbon of dimethylallyl pyrophosphate. SN1 Reaction OPP

DMAPP Head-tail condensation

CH3 CH3 OPP H3C

HS HR HS DMAPP IPP

OPP OPP HR HS

OPP

Geranyl diphosphate (GPP) FORMATION OF THE MONOTERPENE SKELETONS Carbon-Carbon Bond Formation

OPP + OPP

– OPP

+ OPP After C—C Bond Formation...

OPP

• The carbocation can + – H lose a proton to give a double bond.

+ OPP After C—C Bond Formation...

OPP

• This compound is called geranyl pyrophosphate. It can undergo hydrolysis of its pyrophosphate to give geraniol (rose oil). After C—C Bond Formation...

OPP

H2O

OH Geraniol Cyclization

• Rings form by intramolecular carbon-carbon bond formation.

+

OPP

OPP

E double Z double bond bond + – H

+ OH

H2O H 1,2-hydride H shift H Wagner-Meerwein H Rearrangements secondary carbocation tertiary carbocation

1,2-methyl H shift H

secondary carbocation tertiary carbocation

1,3-hydride shift

H

H H H tertiary carbocation ressonance-stabilized allylic cation Wagner-Meerwein

1,2-alkyl Rearrangements shift cont.

tertiary carbocation, secondary carbocation. but strained but reduced ring strain in 4-membered ring 5-membered ring

H

H H H

a series of concerted 1,2 hydride and methyl shifts Bicyclic Terpenes

+ + +

+ a-Pineno -Pineno OPP E

OPP OPP OPP Z

OPP

OPP Geranyl-PP neryl-PP linalyl-PP

Geranyl cation (ressonance stabilized allylic cation) neryl cation (ressonance stabilized allylic cation)

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

OPP

• This compound is called farnesyl pyrophosphate. • Hydrolysis of the pyrophosphate ester gives the alcohol farnesol Pathways for cyclization of farnesyl-PP

H E H guaiyl cation germacryl cation

E

E,E-farnesyl cation eudesmyl cation humulyl cation

caryophyllyl cation bisabolyl cation

a E a a Z carotyl cation b b

E,Z-farnesyl cation b H

cadinyl cation cis-germacryl cation

cis-humulyl cation Formation of the Diterpene Skeletons From 15 Carbons to 20

OPP

OPP

• Farnesyl pyrophosphate is extended by another isoprene forming the geranylgeranyl diphostate (GGPP) H H H

H H

OPP

OPP H

OPP

H

H

Copalil-pp OPP OPP H H H H H H

OPP

H

H

labdadienil-pp TRITERPENOS & ESTERÓIDES HO HO eufol lupeol

R R

HO HO

R=COOH (ácido oleanólico) R=COOH (ácido ursólico) R=CH (-amirina) 3 R=CH3 (a-amirina) O O

O H

O

H H H H H OH HO HO H Diosgenina Digitoxigenina

H

H H

HO

Colesterol PPO

farnesol-PP

farnesol-PP

OPP H H

H H OPP

H H

H H

H (NADPH)

H H

Squalene Enz -OPP OPP

OPP pirofosfato de farnesila pirofosfato de farnesila

OPP OPP H Enz

*H

NADP*H esqualeno

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)

OPP OPP

phytoene PSY and SqS share domain in common 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.

O2, NADH, enzyme

O Biosynthesis of Cholesterol

O

• 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

Chair-boat-chair-boat conformation Biosynthesis of Cholesterol

HO +

H

• Cyclization is triggered by epoxide ring opening.

H+ O Biosynthesis of Cholesterol

HO +

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

HO H Biosynthesis of Cholesterol H

HO

protosteryl cation H • Cyclization to form a tetracyclic carbocation. H

HO H Biosynthesis of•• Cholesterol • • OH2

HO H H • Deprotonation and multiple migrations. H

HO H Biossíntese de Esteróides Sequence od W-M 1,2-hydride and 1,2 methyl shifts

H H H H H

H H O HO HO H H H protosteryl cation

squalene oxide animals plants fungi

loss of a proton gives alkene H H

H loss of proton leads to cyclopropane

HO H HO H

lanosterol cycloartenol Biossíntese de Triterpenos

H Chair-chair-chair-boat conformation

H+

O H

HO H

H

H

H H

HO HO H a-amirina H