OPP OPP

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
CH3C
CH2
CHCH2CH2CCH
or
CH2
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
Monoterpeno
Sesquiterpeno
Diterpeno
Sesterpeno
Triterpeno
Tetraterpeno
Número de átomos de Carbono
10
15
20
25
30
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
(eucalyptus)
(peppermint)
Citral
(lemon grass)
OH
O
H
a-Phellandrene
Menthol
(eucalyptus)
(peppermint)
Citral
(lemon grass)
a-Phellandrene
Menthol
(eucalyptus)
(peppermint)
Citral
(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
cânfora
carvona
O
citronelal
linalol
(óleo de pau-rosa)
eucaliptol
(1,8-cineol)
SESQUITERPENOS
H
H
H
O
O
H
O
HO
HO
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
H2C
O O
CCH2CH2OPOPOH
Isopentenyl pyrophosphate
or
OPP
Isopentenyl and Dimethylallyl
Pyrophosphate
Isopentenyl pyrophosphate is interconvertible with
2-methylallyl pyrophosphate.
OPP
Isopentenyl pyrophosphate
OPP
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
3
H
OP
H
H
R
IP P
H
S
H
H
H
CH
3
w
CH
OP
H
R
H
3
OP
H 3C
S
H
H
S
+
R
D M A P P
IPP-Isomerase mechanism
Cys138
Cys139
S
H
HH
S
H
H
O
Glu 207
IPP
Cys139
S
H
H
OPP
H HS
O C
Cys138
S
HH
H
H
H
OPP
HS
O C
H HS
OH
Glu 207
DMAPP
THE PATHWAY FROM ACETATE
TO ISOPENTENYL
PYROPHOSPHATE
The Biological Isoprene Unit
O
O
3 CH3COH
CH3
HOCCH2CCH2CH2OH
OH
Mevalonic acid
CH3
H2C
O O
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 Sacetoacetyl 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
CH3CCH2CH2OH
CH2COH
O
HO
O
CH3CCH2CSCoA
CH2COH
O
Mevalonic
acid
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
CH2
O
CH3CCH2CH2OPP
CH2
C
O
C
O
• Phosphorylation is followed by a novel elimination
involving loss of CO2 and PO43–.
•• –
O ••
••
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
CH3COH
CH3
HOCCH2CCH2CH2OH
OH
Mevalonic acid
CH3
H2C
O O
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.
CH3
O
CH3COH
H2C
•
•
•
•
•
CCH2CH2OPOPOH
O
•
O O
H
Inibidores da biossíntese de colesterol
(produtos que mimetizam o ácido mevalônico)
HO
R1
HO
H3C
CO2
OH
OH
O
CH3
CO2
mevalonate
O
CH3
R2
R1 = H
R1 = CH3
R1 = H
R1 = H
R2 = H
R2 = CH3
R2 = OH
R2 = CH3
Compactin
Simvastatin (Zocov)
Pravastatin (Precavol)
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
H OH
HO
HO
HO
H
H
HOOC
H
OH
OH
[1- C]-glucose
HO
S
S
TPP
pyruvate
13
N
N
O
H
HO
H
OH
HO
PO
N
S
glyceraldehyde-3-P
O
-CO2
O
HO
HO
PO
1-deoxy-xylulose-5-P
O
N
-TPP
HO
HO
S
OH
OP
HO
OP
O
OH
OPP
IPP
OPP
IPP originated from HMG-CoA
Pyruvate
2 x Acetyl CoA
Glyceraldehyde
3-phosphate
Deoxy xylulose
5-phosphate
Methyl erythritol
4-phosphate
HMG CoA
MVA
MVA pathway
(cytoplasm)
IPP
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
Mitochondrion
Sterols,
Rubber
Haem a
UQ
PQ
Chloroplast
ER
Carotenoids
CHLOROPHYLL
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
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
DMAPP
HR HS
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
lose a proton to give
a double bond.
–H
+
+
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
H2 O
OH
Geraniol
Cyclization
• Rings form by intramolecular carbon-carbon bond
formation.
+
OPP
OPP
E double
bond
Z double
bond
–H
+
+
OH
H2O
H
H
1,2-hydride
shift
Wagner-Meerwein
Rearrangements
H
H
secondary carbocation
H
tertiary carbocation
1,2-methyl
shift
secondary carbocation
H
tertiary carbocation
1,3-hydride
shift
H
H
H
H
tertiary carbocation
ressonance-stabilized
allylic cation
Wagner-Meerwein
Rearrangements
cont.
1,2-alkyl
shift
tertiary carbocation,
but strained
4-membered ring
secondary carbocation.
but reduced ring strain in
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
linalyl-PP
Geranyl cation
(ressonance stabilized allylic cation)
neryl-PP
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
carotyl cation
Z
b
b
E,Z-farnesyl cation
b
H
cis-germacryl cation
cis-humulyl cation
cadinyl 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=CH3 (-amirina)
R=COOH (ácido ursólico)
R=CH3 (a-amirina)
O
O
O
H
O
H
H
H
H
H
HO
HO
H
Diosgenina
Digitoxigenina
H
H
HO
Colesterol
H
OH
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
•• OH
2
HO
H
• Deprotonation and multiple migrations.
H
H
HO
H
Biossíntese de Esteróides
Sequence od W-M 1,2-hydride and
1,2 methyl shifts
H
H
H
H
H
H
HO
O
H
H
H
HO
H
protosteryl cation
squalene oxide
plants
animals
fungi
loss of a proton
gives alkene
H
H
H
HO
H
lanosterol
HO
H
cycloartenol
loss of proton
leads to cyclopropane
Biossíntese de
Triterpenos
H
Chair-chair-chair-boat conformation
H+
H
O
HO
H
H
H
H
H
HO
H
HO
a-amirina
H