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Biosynthesis – Inspiration for Drug Discovery
Biosynthesis of Isoprenoids
Alan C. Spivey
[email protected]
Dec 2008
Format & Scope of Lecture
•
What are isoprenoids?
–
–
–
•
Monoterpnes (C10)
–
–
•
farnesyl pyrophosphate derived metabolites
Diterpenes (C20)
–
•
regular (‘head-to-tail’) via geranyl pyrophosphate
apparently irregular ‘iridoids’ (e.g. seco-loganin)
Sesquiterpenes (C15)
–
•
n × C5 diversity: terpenes, steroids, carotenoids & natural rubber
‘the isoprene rule’
mevalonate pathway to IPP & DMAPP
taxol
Triterpenes (C30)
–
–
steroids (2,3-oxidosqualene → lanosterol → cholesterol → estrone)
ring-opened ‘steroids’: vitamin D2 & azadirachtin
Isoprenoids
•
isoprenoids are widely distributed in the natural world
–
–
particularly prevalent in plants and least common in insects; >30,000 known
composed of integral numbers of C5 ‘isoprene’ units:
•
monoterpenes (C10); sesquiterpenes (C15); diterpenes (C20); sesterpenes (C25, rare); triterpenes (C30); carotenoids (C40)
H
O
OH
H
O
HO
OH
O
OH
thujone
(C10)
HO
humulone (2x C5)
lavandulol
(C10)
borneol
(C10)
(Z)-α-bisabolene
(C15)
H
O O
OH
n
O
H
ISOPRENOIDS
5
natural rubber (~10 x C5)
H
artemisinin (C15)
O
O
OPP
OPP
β -carotene (C40)
H
AcO
H
H
H
HO
OH
cholesterol
(C27 but C30-derived)
O
taxol (C20)
OH
HO
BzO AcO
H
O
HO
H
O
O
OH
euonyminol (C15)
H
O
HO
HO
O
Ph
BzHN
O
isopentenyl
pyrophosphate
(IPP)
dimethylallyl
pyrophosphate
(DMAPP)
OH
OH
CO2H
OH
OH
OH
OH
gibberellic acid (C20)
(gibberellin A3)
Historical Perspective – ‘The Isoprenoid Rule’
•
Early 1900s:
–
–
common structural feature of terpenes – integral # of C5 units
pyrolysis of many monoterpenes produced two moles of isoprene:
Δ
2x
200°C
α-pinene (C10)
•
1940s:
–
•
biogenesis of terpenes attributed to oligomers of isoprene – ‘the isoprene rule’
1953:
–
Ruzicka proposes ‘the biogenetic isoprene rule’ to accomodate ‘irregular’ terpenoids:
•
•
i.e. that terpenes were derived from a number of biological equivalents of isoprene initially joined in a ‘head-to-tail’
manner & sometimes subsequently modified enzymatically to provide greater diversity of structure
1964:
–
Nobel prize awarded to Bloch, Cornforth & Popjak for elucidation of biosynthetic pathway to cholesterol
including the first steps:
•
•
isoprene (C5)
acetate → mevalonate (MVA) → isopentenylpyrophosphate (IPP) & dimethylallyl pyrophosphate (DMAPP)
1993:
–
Rohmer, Sahm & Arigoni elucidate an additional pathway to IPP & DMAPP:
•
pyruvate + glyceraldehyde-3-phosphate → 1-deoxyxylulose-5-phosphate → IPP & DMAPP
Primary Metabolism - Overview
Primary metabolites
Primary metabolism
Secondary metabolites
CO2 + H2O
PHOT OSY NTHESIS 1) 'light reactions': hv -> AT P and NADPH
2) 'dark reactions': CO2 -> sugars (Calvin cycle)
HO
HOHO
oligosaccharides
polysaccharides
nucleic acids ( RNA, DNA)
O
HO
OH
glucose
& other 4,5,6 & 7 carbon sugars
glycolysis
CO2
SHIKIMAT E METABOLIT ES
cinnamic acid derivatives
aromatic compounds
lignans, f lavinoids
PO
CO2
PO
phosphoenol pyruvate
+
HO
HO
O
OH
erythrose-4-phosphate
OH
OH
shikimate
aromatic amino acids
CO2
aliphatic amino acids
O
pyruvate
SCoA
O
acetyl coenzyme A
CoAS
tetrapyrroles (porphyrins)
Citric acid
cycle
(Krebs cycle)
SCoA
CO2
O
malonyl coenzyme A
HO
O
O
acetoacetyl coenzyme A
peptides
proteins
ALKALOIDS
penicillins
cephalosporins
cyclic peptides
HO
CO2
mevalonate
saturated f atty acids
unsaturated f atty acids
lipids
FAT T Y ACIDS & POLY KET IDES
prostaglandins
polyacetylenes
aromatic compounds, polyphenols
macrolides
ISOPRENOIDS
terpenoids
steroids
carotenoids
Biosynthesis of Mevalonate
•
Mevalonate (MVA) is the first committed step of isoprenoid biosynthesis
–
this key 6-carbon metabolite is formed from three molecules of acetyl CoA via acetoacetyl CoA:
Primary metabolism
CO2
Secondary metabolism
GLYCOLYSIS
pyruvate
O
CoASH
CO2
NAD
NADH
oxidative decarboxylation
SCoA
CITRIC ACID CYCLE
acetyl CoA
O
SCoA
Claisen
condensation
O
aldol
reaction
then [R]
CO2
CoASH
CoAS
O
O
acetoacetyl CoA
2x CoASH
SCoA
2x NADPH O
acetyl CoA carboxylase
(biotin-dependent)
carboxylation
2x NADP
HO
CO2
HO
mevalonate (MVA)
SCoA
CO2
O
malonyl CoA
saturated fatty acids
unsaturated fatty acids
lipids
FATTY ACIDS & POLYKETIDES
prostaglandins
polyacetylenes
aromatic compounds, polyphenols
macrolides
ISOPRENOIDS
terpenoids
steroids
carotenoids
Biosynthesis of IPP & DMAPP - via Mevalonate
•
IPP & DMAPP are the key C5 precursors to all isoprenoids
–
the main pathway is via: acetyl CoA → acetoacetyl CoA → HMG CoA → mevalonate → IPP → DMAPP:
HMG CoA reductase inhibitors - Statins
•
HMG CoA → MVA is the rate determining step in the biosynthetic pathway to cholesterol
–
•
33 enzyme mediated steps are required to biosynthesise cholesterol from acetyl CoA & in principle the
inhibition of any one of these will serve to break the chain. In practice, control rests with HMG-CoA reductase
as the result of a variety of biochemical feedback mechanisms
‘Statins’ inhibit HMG CoA reductase and are used clinically to treat hypercholesteraemia - a
causative factor in heart disease
–
e.g. mevinolin (=lovastatin®, Merck) from Aspergillus terreus is a competitive inhibitior of HMG-CoA reductase
HO
CoAS HO
H
R N
CO2
CO2
O
CoAS
H
NADPH
H2N
O
HMG CoA
H
Zn2
NADPH
HO
O
CO2
HO
NADP
NADP
mevalonate (MVA)
OH
CoASH
OH
HO
O
O
O
O
O
CO2
O
OH2
CO2
H
O
OH
PhHN
i
Pr
OH
N
LIPITOR
in vivo
Ph
H
F
mevinolin (=lovastatin®)
PRO-DRUG
NB. type I (iterative) PKS natural product
H
ACTIVE DRUG
mimic of tetrahedral intermediate
in HMG reduction by NADPH
H
HO
cholesterol
Hemi-Terpenes – ‘Prenylated Alkaloids’
•
•
DMAPP is an excellent alkylating agent
C5 units are frequently encountered as part of alkaloids (& shikimate metabolites) due to ‘latestage’ alkylation by DMAPP
–
–
the transferred dimethyl allyl unit is often referred to as a ‘prenyl group’
‘normal prenylation’ – ‘SN2’-like alkylation; ‘reverse prenylation’ – ‘SN2’-like alkylation
•
•
e.g. lysergic acid (recall the ergot alkaloids) – a ‘normal prenylated’ alkaloid (with significant subsequent processing)
e.g. roquefortine – a ‘reverse prenylated’ alkaloid
DMAPP
O
OPP
HO
N
H
cf. SN2
N
Me
'reverse'
prenylation
3
tyrosine
N
H
lysergic acid
(halucinogen)
–
OPP
H
'normal'
prenylation
4
H
histidine
N
H
NH
tyrosine
cf. SN2'
O
DMAPP
N
H
N
N
N H
O
H
roquefortine
(blue cheese mould)
review: R.M. Williams et al. ‘Biosynthesis of prenylated alkaloids derived from tryptophan’ Top. Curr. Chem.
2000, 209, 97-173 (DOI)
Linear C5n ‘head-to-tail’ Pyrophosphates
•
head-to-tail C5 oligomers are the key precursors to isoprenoids
–
–
geranyl pyrophosphate (C10) is formed by SN1 alkylation of DMAPP by IPP → monoterpenes
farnesyl (C15) & geranylgeranyl (C20) pyrophosphates are formed by further SN1 alkylations with IPP:
Monoterpenes – α-Terpinyl Cation Formation
•
geranyl pyrophosphate isomerises readily via an allylic cation to linalyl & neryl pyrophosphates
–
–
(E)
the leaving group abilty of pyrophosphate is enhanced by coordination to Mg2+ ions
all three pyrophosphates are substrates for cyclases via an α-terpinyl cation:
OPP
O
O P
O
gerenyl
pyrophosphate
O
O
Mg
P O
O
Mg
OPP
linalyl
pyrophosphate
OPP
(Z)
OPP
cyclase
=
OPP
neryl
pyrophosphate
initial
chiral centre
allylic cation
intimate ion pair
α-terpinyl cation
MONOTERPENES (C10)
Monoterpenes – Fate of the α-Terpinyl Cation
•
•
The α-terpinyl cation undergoes a rich variety of further chemistry to give a diverse array of
monoterpenes
Some important enzyme catalysed pathways are shown below
–
NB. intervention of Wagner-Meerwein 1,2-hydride- & 1,2-alkyl shifts
α-terpineol
limonene
hydrolysis
trapping by PPO
OH
E1 elimination
trapping with
water
a
OPP
c
d
c
=
He
OPP
b
OPP
1,2-alkyl shift
H
=
H
=
=
=
β -pinene
H
E1 elimination
thujone
E1 elimination
E1 elimination
d
H
O
O
camphor
camphene
trapping by alkene
at 'blue' carbon
(Markovnikov)
e
OH
borneol
OPP
bornyl
pyrophosphate
H
trapping by alkene
at 'red' carbon
(anti-Markovnikov)
H2O
1,2-hydride shift
H
c
d
a α-terpinyl cation
[O]
H
=
b
H
-
H
=
=
α-pinene
Limonene & Carvone
Chiroscience plc. (now Dow Inc.)
1. S-(-)-limonene (lemon)
4. R-(-)-carvone (spearmint)
2. R-(+)-limonene (orange)
5. S-(+)-carvone (caraway)
3. RS-(±)-limonene (pleasant)
6. RS-(±)-carvone (disgusting)
Apparently Irregular Monoterpenes
•
Apparently irregular monoterpenes can also occur by non-cationic cyclisation of geranyl PP
derivatives followed by extensive rearrangement
–
e.g. iridoids – named after Iridomyrmex ants but generally of plant origin and invariably glucosidated
•
e.g. seco-loganin (recall indole alkaloids) is a key component of strictosidine - precorsor to numerous complex
medicinally important alkaloids:
H2O
OPP
H
OPP
O
O
2x NADP
NADPH
[O]
P450
OH
2x NADPH
10
O
OH
OH
NADP
[O]
P450
10
PPi
geranyl PP
O
OH
O
=
10-oxo geranal
unusual reductive ring-closure -> cyclopentane
(NB. Non-cationic)
OH
HO2C
tryptophan
isoprene
NH2
N
H
tryptamine
NH
N
H H
H
H
MeO2C
O
strictosidine
[O] P450
O
H
OGlu
H
H2O
enzymatic
Pictet-Spengler
reaction
MeO2C
ATP
HO
O
[O] P450
OP
OGlu
glycoside
formation
H
HO
H
OGlu
H2O
MeO2C
secologanin
O
ADP
H
MeO2C
O
loganin
unusual fragmentation
O
OGlu
methylation
SAM
HO2C
OGlu
O
Strictosidine → Vinca, Strychnos, Quinine etc.
•
The diversity of alkaloids derived from strictosidine is stunning and many pathways remain to be fully
elucidated:
N
OH
N
H
N
H
MeO2C
N
MeO
Me
H
N
N
H H
H
H
H
O
MeO2C
ajmalicine
(vinca)
OAc
OH
CO2Me
H
H
MeO2C
OH
vinblastine
(vinca)
MeO
N
N
H H
yohimbine
O
N
N
N
N
H H
O
OH O
3
NH
H
H
camptothecin
H
H
N
H
aspidospermine
(vinca)
OGlu
O
MeO2C
strictosidine
(isovincoside)
N
H
HO
H
MeO
N
Me
N
H
N
O
N
quinine
O
N
O
H
gelsemine
(oxindole)
H
H
H
H O
strychnine
(strychnos)
Sesquiterpenes – Farnesyl Pyrophosphate (FPP)
•
‘SN2’-like alkylation of geranyl PP by IPP gives farnesyl PP:
pro-R hydrogen is lost
OPP
OPP
HS HR
(E)
OPP
geranyl PP
•
(E)
OPP
E,E-farnesyl PP (FPP)
IPP
just as geranyl PP readily isomerises to neryl & linaly PPs so farnesyl PP readily isomerises to
equivalent compounds – allowing many modes of cyclisation & bicyclisation
(E)
(E)
OPP
O
O P
E,E-FPP
(E)
E,Z-FPP
O
(Z)
O
O
Mg
P O
O
cyclases
OPP
Mg
6-memb
10-memb
11-memb
ring cyclised
'CATIONS'
- further cyclisation
- 1,2-hydride & alkyl shifts
vast array of
mono- & bicyclic
SESQUITERPENES
- trapping with H2O
- elimination to alkenes
OPP
NB. control by:
1) enzyme to enforce conformation & sequestration of reactive intermediates
2) intrinsic stereoelectronics of participating orbitals
nerolidyl PP
allylic cation
intimate ion pair
Diterpenes - Taxol
Diterpenes – Geranylgeranyl PP → Taxol
•
Taxol is a potent anti-cancer agent used in the treatment of breast & ovarian cancers
–
–
•
comes from the bark of the pacific yew (Taxus brevifolia)
binds to tubulin and intereferes with the assembly of microtubules
biosynthesis is from geranylgeranyl PP:
cembrene
a
cyclisation
b
AcO
14-exo
OPP
geranylgeranyl PP
H
a
H
O
BzNH
O
taxol
H
OPP
HO
sesquiterpene
(isoprenoid)
OH
Ph
b
O
HO
BzO AcO
O
β-amino acid
(shikimate)
–
–
for details see: http://www.chem.qmul.ac.uk/iubmb/enzyme/reaction/terp/taxadiene.html
home page is: http://www.chem.qmul.ac.uk/iubmb/enzyme/
•
•
recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the
Nomenclature and Classification of Enzyme-Catalysed Reactions
based at Department of Chemistry, Queen Mary University of London
Triterpenes – FPP → Squalene
•
triterpenes (C30) arise from the ‘head to head’
coupling of two fanesyl PP units to give
squalene catalysed by squalene synthase:
OPP
FPP (acceptor)
OPP
FPP (donor)
–
–
squalene was first identified as a steroid precursor
from shark liver oil
the dimerisation proceeds via an unusual mechanism
involving electrophilic cyclopropane formation rearrangement to a tertiary cyclopropylmethyl cation
and reductive cyclopropane ring-opening by NADPH
(NB. exact mechanism disputed)
EnzB:
H H
OPP
OPP
blocked by squalestatins
squalene synthase
H
presqualene PP
OPP
–
Zaragozic acids (squalestatins) mimic a
rearrangement intermediate and inhibit squalene
synthase. They constitute interesting leads for
development of new treatments for
hypercholesteraemia & heart disease (cf. statins)
H
H
OPP
NADPH
NADP
O
O
HO2C
zaragozic acid A HO2C
(squalestatin S1)
OH
O
O
CO
2H
OH
+ PPi
H H
OAc
squalene
Triterpenes – Squalene → 2,3-Oxidosqualene
•
squalene is oxidised to 2,3-oxidosqualene by squalene oxidase – which is an O2/FADH2dependent enzyme:
squalene
oxidase
O2 + FADH2
O
squalene
•
H2O + FAD
2,3-oxidosqualene
the key oxidant is a peroxyflavin:
reductive recycling
O
O
H
N
O
O
HN
NH
NR
O
O
O
HN
H
N
O
O
HO
H N
NH
NR
H
N
O
O
N
N
NR
O
peroxyflavin
hydroxyflavin
O
N
NR
H2O
FADH2
H
N
FAD
Oxidosqualene-Lanosterol Cyclase – Mechanism
•
oxidosqualene-lanosterol cyclase catalyses the formation of lanosterol from 2,3-oxidosqualene:
–
–
–
this cascade establishes the characteristic ring system of ALL steroids
ring-expansion sequence to establish the C ring
the process is NOT concerted, discrete cationic intermediates are involved & stereoelectronics dictate
the regio- & stereoselectivity although the enzyme undoubtedly lays a role in pre-organising the ~chair-boatchair conformation
–
“The enzyme’s role is most likely to shield intermediate carbocations… thereby allowing the hydride and
methyl group migrations to proceed down a thermodynamically favorable and kinetically facile cascade”
•
Wendt et al. Angew. Chem. Int. Ed. 2000, 39, 2812 (DOI) & Wendt ibid 2005, 44, 3966 (DOI)
Lanosterol → Cholesterol – Oxidative Demethylation
•
Several steps are required for conversion of lanosterol to cholesterol:
24
1) Δ
H
4
HO
1) Δ
24
hydrogenation
2) 14α DEMETHYLATION
H
14
8
1
24
HO
3) 4α & 4β DEMETHYLATION
5
H
4) Δ to Δ rearrangement
8
H
=
H
5
H
flat, rigid structure
HO
lanosterol
cholesterol
2) 14α DEMETHYLATION
hydrogenation
NADPH
H
2x O2
P450
H O2
P450
HCO2H
H
H
NADPH
H
24
14
2x H2O
NADP
O
H
O
HO
O
H
:BEnz
NADP
FeIII
3) 4α & 4β DEMETHYLATION
4x O2
HO
4x O2
P450
4
O
H
4x H2O
O
H
O
O
CO2
O
H
4x H2O
H
4) Δ to Δ rearrangement
8
O
H
5
isomerase
H
5
H
H
O
H
O
O
CO2
NADH
NAD
8
NADPH
P450
H
H
NADH
[-> vitamin D]
NAD
HO
H
NADP
H
Cholesterol → Human Sex Hormones
•
cholesterol is the precursor to the human sex hormones – progesterone, testosterone & estrone
–
–
the pathway is characterised by extensive oxidative processing by P450 enzymes
estrone is produced from androstendione by oxidative demethylation with concomitant aromatisation:
OH
OH
H
2x O2
P450
H
H
H
O
O2
P450
H
H
H
HO
2x H2O
cholesterol
H
O
NAD
H
H
H
O
H
HO
H
H
H
NADH
HO
H
O
progesterone
[O]
X
O
FeIII
EnzB:
H
H
H
O
O
O
OH
O2
P450 O
H
H
H
HO
estrone
(œstrone)
HCO2H
O
2x O2
H
H
P450
H
H
O
DEMETHYLATIVE aromatisation by 'aromatase' enzyme
H
O
2x H2O
androstendione (X = O)
testosterone (X = H, β−OH)
Steroid Ring Cleavage - Vitamin D & Azadirachtin
•
vitamin D2 is biosynthesised by the photolytic cleavage of Δ7-dehydrocholesterol by UV light:
–
a classic example of photo-allowed, conrotatory electrocyclic ring-opening:
OH
OH
NADP
H
UV light (hv)
H
H
H
H
[O]
H
H
H
H
NADPH
HO
electrocyclic
ring-opening
H
7
HO
Me
[6π]
H
•
H
Me H
ψ4
SOMO
conrotatory
HO
HO
vitamin D2
exact biogenesis unknown but certainly via steroid modification:
O
O MeO2C
O
O
OAc
H
O
OH
C
H
11
12
OH
O
14
O
7
HO
H
OH
H
AcO
tirucallol
(cf. lanosterol)
H
AcO
OH
H
azadirachtanin A
(a limanoid =
tetra-nor-triterpenoid)
oxidative
cleavage
of C ring
8
AcO
MeO2C
H
OH
O
azadirachtin
O
O
OH
highly hindered C-C bond
for synthesis!
OH
calcium ion chelator
D vitamins are involved in calcium absorption; defficiency leads to rickets (brittle/deformed bones)
Azadirachtin is a potent insect anti-feedant from the Indian neem tree:
–
H
HO
cholesterol
–
1,5-hydride
shift
H
Primary Metabolism - Overview
Primary metabolites
Primary metabolism
Secondary metabolites
CO2 + H2O
PHOTOSY NTHESIS 1) 'light reactions': hv -> AT P and NADPH
2) 'dark reactions': CO2 -> sugars (Calvin cycle)
HO
HOHO
oligosaccharides
polysaccharides
nucleic acids ( RNA, DNA)
O
HO
OH
glucose
& other 4,5,6 & 7 carbon sugars
glycolysis
CO2
SHIKIMATE METABOLITES
cinnamic acid derivatives
aromatic compounds
lignans, f lavinoids
PO
CO2
PO
phosphoenol pyruvate
+
HO
HO
O
OH
erythrose-4-phosphate
OH
OH
shikimate
aromatic amino acids
CO2
aliphatic amino acids
O
pyruvate
SCoA
O
acetyl coenzyme A
CoAS
tetrapyrroles (porphyrins)
Citric acid
cycle
(Krebs cycle)
SCoA
CO2
O
malonyl coenzyme A
HO
O
O
acetoacetyl coenzyme A
peptides
proteins
ALKALOIDS
penicillins
cephalosporins
cyclic peptides
HO
CO2
mevalonate
saturated f atty acids
unsaturated f atty acids
lipids
FAT TY ACIDS & POLY KETIDES
prostaglandins
polyacetylenes
aromatic compounds, polyphenols
macrolides
ISOPRENOIDS
terpenoids
steroids
carotenoids