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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.
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Terpenes
• Terpenes are natural products that are
structurally related to isoprene.
H2C C
CH3
CH CH2 or
Isoprene
(2-methyl-1,3-butadiene)
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Terpenes
• Myrcene (isolated from oil of bayberry) is a
typical terpene.
CH2
CH3
CH3C CHCH2CH2CCH
CH2
or
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The Isoprene Unit
• An isoprene unit is the carbon skeleton of isoprene
(ignoring the double bonds)
Myrcene contains two isoprene units.
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The Isoprene Unit
• The isoprene units of myrcene are joined "head-to-tail."
head tail
tail head
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Isoprene
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Head
Tail
Head
Tail
Isoprene
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Head
Tail
Head
Tail
Isoprene
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• Classe Número de átomos de Carbono
• Monoterpeno 10
• Sesquiterpeno 15
• Diterpeno 20
• Sesterpeno 25
• Triterpeno 30
• Tetraterpeno 40
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• 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
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a-Phellandrene
(eucalyptus)
Menthol
(peppermint)
Citral
(lemon grass)
O
H
OH
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a-Phellandrene
(eucalyptus)
Menthol
(peppermint)
Citral
(lemon grass)
O
H
OH
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a-Phellandrene
(eucalyptus)
Menthol
(peppermint)
Citral
(lemon grass)
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a-Selinene
(celery)
H
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a-Selinene
(celery)
H
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a-Selinene
(celery)
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Vitamin A
OH
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Vitamin A
OH
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Vitamin A
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Squalene
(shark liver oil)
tail-to-tail linkage of isoprene units
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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
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ISOPENTENYL
PYROPHOSPHATE:
THE BIOLOGICAL ISOPRENE
UNIT
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The Biological Isoprene Unit
• The isoprene units in terpenes do not come
from isoprene.
• They come from isopentenyl pyrophosphate.
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Isopentenyl Pyrophosphate
H2C CCH2CH2OPOPOH
CH3 O O
Isopentenyl pyrophosphate
or OPP
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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
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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-
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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
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THE PATHWAY FROM ACETATE
TO ISOPENTENYL
PYROPHOSPHATE
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The Biological Isoprene Unit
CH3COH
O
3 HOCCH2CCH2CH2OH
CH3
OH
O
Mevalonic acid
H2C CCH2CH2OPOPOH
CH3 O O
Isopentenyl pyrophosphate
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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
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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
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Biosynthesis of Mevalonic Acid
CH3CSCoA
O
CH3CCH2CSCoA
CH2COH
HO O
O
+ CH3CCH2CSCoA
O O
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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
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CH3CCH2CSCoA
CH2COH
HO O
O
CH3CCH2CH2OH
CH2COH
HO
O
Mevalonic
acid
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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–
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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
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Conversion of Mevalonic Acid to
Isopentenyl Pyrophosphate
• The product of this elimination is isopentenyl
pyrophosphate.
CH3CCH2CH2OPP
CH2
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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
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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
•
• • •
•
•
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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)
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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
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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
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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)
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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
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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
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CARBON-CARBON BOND
FORMATION IN TERPENE
BIOSYNTHESIS
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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
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OPP
DMAPP
SN1 Reaction
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H3C
CH3
HS
DMAPP
CH3
HSHR
OPP
IPP
OPP
HSHR
OPP
OPP
Geranyl diphosphate (GPP)
Head-tail condensation
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FORMATION OF THE
MONOTERPENE SKELETONS
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Carbon-Carbon Bond Formation
+ OPP
OPP –
+ OPP
OPP
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After C—C Bond Formation...
• The carbocation can
lose a proton to give
a double bond.
+ OPP
OPP
H – +
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After C—C Bond Formation...
• This compound is called geranyl pyrophosphate. It can undergo
hydrolysis of its pyrophosphate to give geraniol (rose oil).
OPP
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After C—C Bond Formation...
OPP
OH
Geraniol
H2O
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Cyclization
• Rings form by intramolecular carbon-carbon bond
formation.
OPP
OPP
+
E double
bond
Z double
bond
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+
OH
H – +
H2O
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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
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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.
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Bicyclic Terpenes
+ +
+
-Pineno
+
a-Pineno
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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
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FORMATION OF THE
SESQUITERPENE SKELETONS
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• 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
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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
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From 10 Carbons to 15
+ OPP
OPP
Geranyl pyrophosphate
+ OPP
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From 10 Carbons to 15
+ OPP
H – +
OPP
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From 10 Carbons to 15
• This compound is called farnesyl pyrophosphate.
• Hydrolysis of the pyrophosphate ester gives the alcohol
farnesol
OPP
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E
E
HH
E,E-farnesyl cation
germacryl cation
humulyl cation
caryophyllyl cation
eudesmyl cation
guaiyl cation
Pathways for cyclization of farnesyl-PP
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E
Z
a
b
a
a
b
Hb
E,Z-farnesyl cation
bisabolyl cation
carotyl cation
cadinyl cationcis-germacryl cation
cis-humulyl cation
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Formation of the
Diterpene Skeletons
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From 15 Carbons to 20
• Farnesyl pyrophosphate is extended by another isoprene
forming the geranylgeranyl diphostate (GGPP)
OPP
OPP
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OPP
H
H HH
OPP
H H
OPP
H
H
Copalil-pp
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H
OPP
H
H
labdadienil -pp
H
H
OPP
H
H
H
OPP
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TRITERPENOS & ESTERÓIDES
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HO
R
R=COOH (ácido oleanólico)
R=CH3 (-amirina)
HO
R
R=COOH (ácido ursólico)
R=CH3 (a-amirina)
HO
HO
lupeol
eufol
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HO
O
O
H
H
HH
Diosgenina
HO
H
O
O
H
OHH
Digitoxigenina
HO
H
H H
Colesterol
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PPO
OPP
H H
OPP
H
HH
H
HH
H(NADPH)
HH
Squalene
farnesol-PP
farnesol-PP
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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
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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
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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
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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
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Biosynthesis of Cholesterol
• Cyclization is triggered by epoxide ring opening.
O H+
+
HO
H
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Biosynthesis of Cholesterol
• The five-membered ring expands to a six-membered one.
+
HO
H
H
HO
H
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Biosynthesis of Cholesterol
• Cyclization to form a tetracyclic carbocation.
H
HO
H
H
H
HO
protosteryl cation
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Biosynthesis of Cholesterol
• Deprotonation and multiple migrations.
HO
OH2
•• • •
H
H
HO
H
H
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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
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O
Chair-chair-chair-boat conformation
H+
H
HO
H
H
HO
H
H
H
HO
H
H
H
a-amirina
Biossíntese de
Triterpenos