Download Lecture 1 - Imperial College London

An Overview of Biosynthesis Pathways –
Inspiration for Pharmaceutical and
Agrochemical Discovery
Alan C. Spivey
[email protected]
Oct 2016
Lessons in Synthesis - Azadirachtin
•
Azadirachtin is a potent insect anti-feedant from the Indian neem tree:
–
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
AcO
H
AcO
tirucallol
(cf. lanosterol)
–
H
OH
H
azadirachtanin A
(a limanoid =
tetra-nor-triterpenoid)
oxidative
cleavage
of C ring
8
AcO
MeO2C
H
OH
O
O
OH
highly hindered C-C bond
for synthesis!
O
azadirachtin
–
–
Intense synhtetic efforts by the groups of Nicolaou, Watanabe, Ley and others since structural elucidation in
1987.
1st total synthesis achieved in 2007 by Ley following 22 yrs of effort
~40 researchers and over 100 person-years of research! – 64-step synthesis
–
–
Veitch Angew. Chem. Int. Ed. 2007, 46, 7629 (DOI) & Veitch Angew. Chem. Int. Ed. 2007, 46, 7633 (DOI)
Review ‘The azadirachtin story’ see: Veitch Angew. Chem. Int. Ed. 2008, 47, 9402 (DOI)
Format & Scope of Presentation
•
Metabolism & Biosynthesis
–
•
Shikimate Metabolites
–
–
•
acetylCoA & the citric acid cycle → -amino acids → alkaloids
Opioids – powerful pain killers
Fatty Acids and Polyketides
–
–
•
photosynthesis & glycolysis → shikimate formation → shikimate metabolites
Glyphosate – a non-selective herbicide
Alkaloids
–
–
•
some definitions, 1° & 2° metabolites
acetylCoA → malonylCoA → fatty acids, prostaglandins, polyketides, macrolide antibiotics
NSAIDs – anti-inflammatory’s
Isoprenoids/terpenes
–
–
acetylCoA → mevalonate → isoprenoids, terpenoids, steroids, carotenoids
Statins – cholesterol-lowering agents
Metabolism and Biosynthesis
Metabolism & Natural Product Diversity
Me
HO2C
NMe
H
H
O
Me
Me
N
N
O
O
H
N
N
H
caffeine
camphor
HO
H
MeO
N
NH
CO2 H2O Pi N2
hv
lysergic acid
N
quinine
Me O
H
OH
O
O
N
O
Me
O
H
CO2H
N
clavulanic acid
Me
N
nicotine
O
O
patulin
OH
androstenedione
Metabolism
•
Metabolism is the term used for in vivo processes by which compounds are degraded,
interconverted and synthesised:
–
Catabolic or degradative: primarily to release energy and provide building blocks
•
–
Anabolic or biosynthetic: primarily to create new cellular materials (1° & 2° metabolites)
•
•
generally oxidative processes/sequences (glycolysis, Krebs cycle)
generally reductive processes/sequences
These two types of process are coupled – one provides the driving force for the other:
Natural product
secondary metabolites
CO 2 + H 2O
complex metabolites
'CO 2 fixation' hv
(photosynthesis)
Nutrients
energy
storage
O2
energy
release
Cell components
& Growth
ADP + P i
Catabolism
Energy &
Building blocks
ATP
oxidative
degredation
NAD(P)H
NAD(P) + H
Anabolism
reductive
biosynthesis
Building Blocks
respiration
'nitrogen fixation'
(by diazatrophs, lightening,
the Haber process)
simple products
CO 2 + H 2O
N2
Primary Metabolism - Overview
Primary metabolites
Primary metabolism
Secondary metabolites
CO2 + H2O
PHOTOSY NTHESIS
HO
HOHO
1) 'light reactions': hv -> ATP and NADPH
2) 'dark reactions': CO2 -> sugars (Calvin cycle)
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
FATT Y ACIDS & POLY KETIDES
prostaglandins
polyacetylenes
aromatic compounds, polyphenols
macrolides
ISOPRENOIDS
terpenoids
steroids
carotenoids
For interesting animations’ of e.g. photosynthesis see: http://www.johnkyrk.com/index.html
Shikimate Metabolites
Shikimate Metabolites
H NH3
O2C
NH3
H NH3
CO2
NH
CO2
H
n
Me
O
(S)-tryptophan
(ArC0)
O
O
(S)-phenylalanine
(ArC3)
OH
(S)-tyrosine
(ArC3)
O
MeO
SHIKIMATE METABOLITES
menaquinone (vitamin K2)
(ArC1)
OH
OH
H
H
HO
OH
O
H
3
O
scopoletin
(ArC3)
O
O
O
Me
Me
H
H
MeO
OH
-tocopherol (vitamin E)
(ArC1)
O
OH
OMe
OMe
HO
OH
OH
epigallocatechin (EGC)
(ArC3)
podophyllotoxin
(ArC3)
The Shikimate Biosynthetic Pathway - Overview
•
Phosphoenol pyruvate & erythrose-4-phosphate → shikimate → chorismate → prephenate:
–
The detailed mechanisms of these steps have been studied intensively. Most are chemically complex and
interesting. For additional details see:
•
•
•
Mann Chemical Aspects of Biosynthesis Oxford Chemistry Primer No. 20, 1994 (key details)
Haslam Shikimic Acid – Metabolism and Metabolites Wiley, 1993 (full details and primary Lit. citations)
http://www.chem.qmul.ac.uk/iubmb/enzyme/reaction/misc/shikim.html (interesting web-site with many biosynethtic pathways)
Rational Agrochemical Development – Shikimate
Pathway Intervention
•
The shikimate biosynthetic pathway is not found in animals/humans – only in plants
–
•
selective intervention in these pathways allows development of agrochemicals with minimal human toxicity
Glyphosate (‘Roundup’) – a Monsanto agrochemical is a potent inhibitor of the conversion of
3-phosphoshikimate (3-PS) → 5-enolpyruvylshikimate-3-phosphate (5-EPS-3P)
–
a non-selective herbicide
H NH3
CO2
CO2
CO2
PO
CO2
phosphoenol pyruvate
( PEP)
PO
HO
CO2
PEP
PO
+
OH
PO
OH
O
CO2
O
OH
H
OP
Pi
3-phosphoshikimate
( 3-PS)
O
CO2
glyphosate ( Roundup® )
inhibits this step
PO
N
H2
CO2
OH
( S)-tyrosine
OH
5-enolpyruvylshikimate-3-phosphate
( 5-EPS-3P)
OH
erythrose-4-phosphate
( E-4-P)
PO
H NH3
CO2
( S)-phenylalanine
Chorismate → Tryptophan, Tyrosine & Phenylalanine
•
Chorismate → anthranilate → tryptophan
ribose derived
CO2
O
OH
•
O2C
CO2
'NH3'
NH2
NH3
NH
CO2
chorismate
(S)-tryptophan
[78 ATP equivs] ArC
0
anthranilate
Chorismate → prephenate → tyrosine & phenylalanine
–
NB. The enzyme chorismate mutase [EC 5.4.99.5] which mediates the conversion of chorismate to prephenate
is the only known ‘Claisen rearrangementase’
H NH3
CO2
CO2
Claisen
rearangement
CO2
NAD
H3N H
OH
CO2
chorismate
chorismate
mutase
[EC 5.4.99.5]
'NH3'
OH prephenate
ArC3
OH
O
O
(S)-tyrosine
[62 ATP equivs]
CO2
O2C
O2C
CO2
NADH
H NH3
OH
(S)-arogenate
H2O
CO2
CO2
(S)-phenylalanine
[65 ATP equivs]
ArC3
Tyrosine/Phenylalanine → ArC3 Metabolites
•
Tyrosine & phenylalanine → cinnamate derivatives → ArC3 metabolites
–
coumarins, lignans (stereoselective enzymatic dimerisation) & lignins (stereorandom radical polymerisation)
O
O
scopoletin (a coumarin)
germination stimulant
MeO
ArC3
OH
OH
H
H NH2
H
CO2
phenylalanine
ammonia lyase
(PAL)
CO2
CO2
OH
O
H
O
O
O
H
and
O
H
X
X = OH
X=H
X
cinnamate
derivatives
H
OMe
MeO
MeO
HO
OMe
podophyllotoxin (a lignan)
natural product used to treat worts
OH
ferulate
OMe
H
MeO
NH4
H
O
pinoresinol (a lignan)
2 x ArC3
OMe
HO
HO
O
O
OMe
O
fragment of lignin polymer
'woody' component of cell walls
O
O
MeO
OH
OH
OMe
OH
n x ArC3
Primary Metabolism - Overview
Primary metabolites
Primary metabolism
Secondary metabolites
CO2 + H2O
PHOTOSY NTHESIS
HO
HOHO
1) 'light reactions': hv -> ATP and NADPH
2) 'dark reactions': CO2 -> sugars (Calvin cycle)
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
FATT Y ACIDS & POLY KETIDES
prostaglandins
polyacetylenes
aromatic compounds, polyphenols
macrolides
ISOPRENOIDS
terpenoids
steroids
carotenoids
For interesting animations’ of e.g. photosynthesis see: http://www.johnkyrk.com/index.html
Alkaloids
Alkaloids
•
Definitions:
–
–
originally – ‘a natural product that could be extracted out of alkaline but not acidic water’ (i.e. containing a
basic amine function that protonated in acid)
more generally - ‘any non-peptidic & non-nucleotide nitrogenous secondary metabolite’
H
OH
O
N
O
H
N
H
coniine
N
HO
Me
N
nicotine
OH
H
N
CO2H
H
N
retronecine
clavulanic acid
N
H
H
HO
MeO
H
ALKALOIDS
H
N
sparteine
HO
N
quinine
HO2C
N
H
O
NMe
H
H
N
O
H
H
H
NMe
HO
morphine
H
H O
strychnine
NH
lysergic acid
The Citric Acid Cycle
•
The citric acid (Krebs) cycle is a major catabolic pathway of 1° metabolism that provides two key
building blocks for aliphatic amino acid biosynthesis - oxaloacetate & -ketoglutarate:
CO2 NADH
CO2
SCoA
O
CoA
O
acetyl coenzyme A
pyruvate
OVERAL STOICHIOMETRY
NAD
CO2
1x
CO2
CO2
NADH
CO2
OH
NAD
HO
CO2
O
CO2
CO2
CO2
oxaloacetate
O
'acetate'
CO2
H2O
CO2
cis-aconitate
citrate
HO
+
CO2
H2O
CO2
1x
O2
CO2
CO2
malate
isocitrate
aliphatic amino acids
NAD
H2O
CO2
CO2
CoA
CO2
NADH
GTP
FADH2
CO2
fumarate
O
O
SCoA
CO2
O
O
CO2
NADH
CO2
CO2
2x
FAD
oxalosuccinate
CO2
succinate
Pi + GDP
CO2
CO2
CO2
succinyl-SCoA
CoA
NAD
+
CO2
-ketoglutarate
THE CITRIC ACID CYCLE
12x
ATP
energy!
The Biosynthesis of Lysine & Ornithine
•
Lysine & ornithine - the two most significant, non-aromatic -amino acid precursors to alkaloids:
–
–
–
NB. lysine (Lys) is proteinogenic whereas ornithine (Orn) is not
phenylalanine (Phe), tyrosine (Tyr) & tryptophan (Trp) from shikimate are the other important precursors
biosynthesis is via reductive amination of the appropriate -ketoacid mediated by pyridoxal-5’-phosphate
(PLP)
O
O
R
NH2
reductive
amination
NH3
OH
R
O
O
ketoacid
O
R
O
amino acid
NH3
O
H3N
NH3
O O
P
O
O
pyridoxamine
phosphate
O
N
H
tightly
bound to
enzyme
O O
P
O
O
Me
O
CHO
O
N
H
pyridoxal
phosphate
lysine (Lys)
[50 ATP equivs]
Me
NH3
O
H3N
NH3
O
O
citric
acid
cycle
O
O
O
O
-ketoglutarate
oxidative
deamination
OVERALL: TRANSAMINATION
O
O
O
glutamic acid (Glu)
O
ornithine (Orn)
[<44 ATP equivs (=Arg)]
PLP Chemistry – Transamination & Racemisation
•
Transamination – LHS → RHS (reductive amination); RHS → LHS (oxidative deamination):
Enz-NH2
OP
NH3
O
N
H
pyridoxamine
phosphate
R
CO2
O
R
CO2
N
H
OP
H
Enz-NH3
R
CO2
N
OP
O
H2O
imine
formation
N
H
H
O
N
H
transaminase
Enz-NH2
H
R
CO2
N
OP
N
H
H
O
Enz
imine OP
exchange
R
H
CO2
NH3
N
H
O
N
H
pyridoxal phosphate
bound as imine
PLP Chemistry – Decarboxylation
•
Decarboxylation:
OP
Enz
N
H
O
R
H
Enz-NH2
CO2
H
O
R
NH3
OP
imine
exchange
N
H
pyridoxal phosphate
bound as imine
R
O
N
Enz-NH2
Enz-NH3
H
O
N
OP
H
R
H
O
H
N
OP
H
R
N
H
N
H
NH3
decarboxylase
•
Enz
OP
O
CO2
N
H
imine
exchange
N
H
O
N
H
pyridoxal phosphate
bound as imine
Decarboxylation of lysine & ornithine:
PLP dependant
decarboxylase
HO2C
NH2 NH2
CO2
PLP
H2N
lysine
NH2
RHN
NH2
NH2 NH2
ornithine
O
cadaverine
PLP dependant
decarboxylase
HO2C
RHN
PLP
H2N
CO2
N
R
iminium salt
NH2
putrescine
RHN
NH2
RHN
O
N
R
iminium salt
piperidine
alkaloids
pyrrolidine
alkaloids
Lysine-derived Piperidine Alkaloids – Hemlock!
Socrates drinking poison hemlock, 399 B.C.
"The Death of Socrates" by Jacques-Louis David (1787)
Piperidine Alkaloids – Pelletierine & Coniine
•
Pelletierine:
lysine
O
hydrolysis
O
N
R
from
lysine
•
O
N
R
SCoA
O
acetoacetylCoA
SCoA
O
N
R
CO2
R = H pelletrine
R = Me, N-methylpelletrine
Coniine:
– in 399 BC Socrates was sentenced to death for impiety and executed by being forced to drink a
potion made from poison hemlock. The toxic component in hemlock is coniine. Although by
analogy with the above pathway, biosynthesis from lysine might be suspected, it is in fact of fatty
acid origin
3x
O
O
SCoA
CO2
malonylCoA
O
SCoA
O
[O]
O
SCoA
SCoA
acetylCoA
NADPH
fatty acid
NADPH
O
N
H H
coniine
NH2
PLP
reductive amination
N
NADP
coniceine
H2O
O
NH3
CO2
CO2
NADP
O
O
Tyrosine-derived Alkaloids - Opium Alkaloids
Benzylisoquinoline Alkaloids
papaverine
morphine
Benzylisoquinoline Alkaloids – Ring Formation
•
Benzylisoquinoline alkaloids constitute an extremely large and varied group of alkaloids
–
many, particularly the opium alkaloids (e.g. papaverine, morphine) are biosynthesised from two molecules of
tyrosine via nor-laudanosoline:
–
Mechanism of Pictet Spengler reaction:
HO
DHPP
+
dopamine
HO
HO
HO
H
NH
HO2C
HO
OH
OH
HO2C
NH
NH
HO
OH
OH
HO2C
OH
OH
nor-laudanosoline-1-carboxylic acid
Benzylisoquinoline Alkaloids - Papaverine
•
Papaverine: analgesic contsituent of the opium poppy (Papaver somniferum):
–
biosynthesis:
–
NB. The prefix nor means without a methyl group. Laudanosoline, reticuline and laudanosine are the N-methyl
compounds
Oxidative Phenolic Coupling –
Morphine & Synthetic Opioids
•
Morphine: analgesic & sedative contsituent of the opium poppy (Papaver somniferum):
–
biosynthesis: o-/p- oxidative phenolic coupling of reticuline:
–
–
Morphine acts by activating the opiate receptors in the brain (IC50 3 nM)
The natural ligands for these receptors are peptides: e.g. Leu-enkephalin (Tyr–Gly–Gly–Phe–Leu) (IC50 12 nM)
Dimeric Indole Alkaloids – Vinca extracts
Dimeric Indole Alkaloids
vinblastine (R = Me)
vincristine (R = CHO)
Potent anti tumour alkaloids used in cancer chemotherapy
Tryptamine + Secolaganin → Strictosidine
•
Most alkaloids of mixed Tryptophan/mevalonate biogenesis (>1200) are derived from strictosidine:
–
Strictosidine is derived from the condensation of tryptamine with the iridoid C10 monoterpene secologanin:
OPP
O
H
HO
CO2
HO
H
O
MeO2C
mevalonate (x2)
geranyl pyrophosphate
see isoprenoids
OGlu
tryptophan
enzymatic
Pictet-Spengler
reaction
secologanin
CO2
NH3
N
H
H
H
H2O
PLP
NH2
MeO2C
OGlu
isoprene
O
strictosidine
N
H
CO2
tryptophan
–
NH
N
H H
tryptamine
Mechanism of Pictet-Spengler reaction:
•
via spirocyclic intermediate then Wagner-Meerwein 1,2-alkyl shift:
NH2
N
H
H
+
H
MeO2C
tryptamine
N
H
O
NH
H
OGlu
O
secologanin
H
MeO2C
O
OGlu
N
H
H
MeO2C
NH
NH
N
Wagner-Meerwein H H
H
OGlu 1,2-alkyl shift
H
H
O
spirocyclic intermediate
MeO2C
O
N
H H
NH
H
OGlu
H
MeO2C
O
strictosidine
OGlu
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
H
H
H
O
MeO2C
ajmalicine
(vinca)
OAc
N
H
OH
Me CO2Me
MeO
N
N
H H
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
H
MeO2C
camptothecin
H
N
H
aspidospermine
(vinca)
OGlu
O
strictosidine
(isovincoside)
N
H
HO
H
MeO
N
Me
N
H
N
O
N
quinine
H
O
N
O
H
gelsemine
(oxindole)
H
H
H O
strychnine
(strychnos)
Primary Metabolism - Overview
Primary metabolites
Primary metabolism
Secondary metabolites
CO2 + H2O
PHOTOSY NTHESIS
HO
HOHO
1) 'light reactions': hv -> ATP and NADPH
2) 'dark reactions': CO2 -> sugars (Calvin cycle)
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
FATT Y ACIDS & POLY KETIDES
prostaglandins
polyacetylenes
aromatic compounds, polyphenols
macrolides
ISOPRENOIDS
terpenoids
steroids
carotenoids
For interesting animations’ of e.g. photosynthesis see: http://www.johnkyrk.com/index.html
Fatty Acids
Fatty Acid Primary Metabolites
•
OH
OH
glycerol
Primary metabolites:
–
fully saturated, linear carboxylic acids & derived (poly)unsaturated derivatives:
•
•
•
•
OH
constituents of essential natural waxes, seed oils, glycerides (fats) & phospholipids
structural role – glycerides & phospholipids are essential constituents of cell membranes
energy storage – glycerides (fats) can also be catabolised into acetate → citric acid cycle
biosynthetic precursors – for elaboration to secondary metabolites
3x fatty acids
OCOR1
OCOR2
OCOR3
glycerides
SATURATED ACIDS [MeCH2(CH2CH2)nCH2CO2H (n = 2-8)]
e.g.
CO2H
8
CO2H
capric acid (C8, n = 3)
1
CO2H
1
16
CO2H
lauric acid (C12, n = 4)
1
12
1
14
CO2H
10
CO2H
caprylic acid (C8, n = 2)
1
1
18
MONO-UNSATURATED ACID DERIVATIVES (MUFAs) e.g.
myristic acid (C14, n = 5)
palmitic acid (C16, n = 6)
stearic acid (C18, n = 7)
9
9
CO2H
CO2H
1
1
palmitoleic acid (C16, Z -9)
16
oleic acid (C18, Z-9)
18
(>80% of fat in olive oil )
POLY-UNSATURATED ACID DERIVATIVES (PUFAs)
e.g.
8
5
CO2H
1
20
11
14
8
5
CO2H
arachidonic acid (AA)
1
(C20, Z -5, Z -8, Z -11, Z -14)
11
14
17
eicosapentaenoic acid (EPA)
(C20, Z-5, Z -8, Z-11, Z -14, Z -17)
20
(in cod liver oil)
Fatty Acids Derivatives – Secondary Metabolites
•
Secondary metabolites
–
further elaborated derivatives of polyunsaturated fatty acids (PUFAs)
•
e.g. polyacetylenes & ‘eicosanoids’ (prostaglandins, thromboxanes & leukotrienes)
Biosynthesis of Fatty Acids – Iterative Oligomerisation
•
fatty acids are biosynthesised from acetyl CoA as a starter unit by iterative ‘head-to-tail’
oligomerisation involving:
–
–
•
condensation with malonyl CoA as an extender unit (with loss of CO2) – a decarboxylative Claisen
condensation
3-step reduction of the resulting ketone → methylene
after n = 2-8 iterations the C8-20 saturated fatty acid is released from the enzyme(s):
Biosynthesis of Fatty Acids – Overview of FAS
•
The in vivo process by which all this takes place involves a ‘molecular machine’ - Fatty Acid
Synthase (FAS)
–
–
–
Type I FAS: single multifunctional protein complex (e.g. in mammals incl. humans)
Type II FAS: set of discrete, dissociable single-function proteins (e.g. in bacteria)
All FASs comprise 8 components (ACP & 7× catalytic activities): ACP, KS, AT, MT, KR, DH, ER & [TE] :
O
O
O
O
S
AT
CoAS
O
O
MT
SH
KS
S
S
O
decarboxylative
Claisen
condensation
SH
S
reduction
SH
1) KR
2) DH
3) ER
n = 2-8
cycles
ACP
n
O
S
SH
O
S
SH
SH
S
translocation
O
TE
OH
O
KS ACP
KS ACP
KS ACP
CoAS
n
SH
O
O
O
KS
ACP
KS ACP
KS ACP
KS = keto synthase (also known as CE = condensing enzyme); AT = acetyl transferase; MT = malonyl transferase;
KR = keto reductase; DH = dehydratase; ER = enoyl reductase; TE = thioesterase; ACP = acyl carrier protein
O
Human Fatty Acid Synthase (FAS)
•
the first three-dimensional structure of human fatty acid synthase (272 kDa) at 4.5 Å resolution by Xray crystallography:
–
Maier, Jenni & Ban Science 2006, 311, 1258 (DOI) ; also Fungal FAS @ 3.1 Å resolution see: Jenni et al.
Science 2007, 316, 254 & 288
Structural overview. (A) Front view: FAS consists of a lower part comprising the KS (lower body) and MAT domains (legs) connected at
the waist with an upper part formed by the DH, ER (upper body), and KR domains (arms). (B) Top view of FAS with the ER and KR
domains resting on the DH domains. (C) Bottom view showing the arrangement of the KS and MAT domains and the continuous
electron density between the KS and MAT domains
FATTY ACID BIOSYNTHESIS (type II FAS)
ACP1
AT1
KS1
Cys
KR1
DH1
ER1
ACP2
MT2
SH
Pantetheine
SH
NB. the following sequence of slides have been adapted from: http://www.courses.fas.harvard.edu/%7echem27/
SH
FATTY ACID BIOSYNTHESIS
ACP1
AT1
KS1
Cys
KR1
DH1
ER1
ACP2
MT2
SH
Pantetheine
O
SH
Me
S
Co
Acetyl-CoA
• AT1 loads acetyl group onto KS1
SH
FATTY ACID BIOSYNTHESIS
ACP1
AT1
KS1
Cys
O
Pantetheine
SH
KR1
DH1
ER1
ACP2
MT2
S
Me
SH
FATTY ACID BIOSYNTHESIS
ACP1
AT1
KS1
Cys
O
Pantetheine
KR1
DH1
ER1
ACP2
S
Me
SH
O
-O
O
S
MT2
Co
Malonyl-CoA
• AT1 loads malonyl group onto ACP1
SH
FATTY ACID BIOSYNTHESIS
ACP1
AT1
KS1
Cys
O
Pantetheine
S
O
DH1
ER1
ACP2
MT2
S
Me
SH
O
O
KR1
-
FATTY ACID BIOSYNTHESIS
ACP1
AT1
KS1
Cys
O
Pantetheine
KR1
DH1
ER1
ACP2
S
Me
CO2
S
O
O
O
MT2
-
• KS1 catalyzes Claisen condensation
SH
FATTY ACID BIOSYNTHESIS
ACP1
AT1
KS1
Cys
KR1
DH1
ER1
ACP2
MT2
SH
Pantetheine
S
O
O
Me
SH
FATTY ACID BIOSYNTHESIS
ACP1
AT1
KS1
Cys
Pantetheine
SH
KR1
S
DH1
ER1
ACP2
MT2
Me
O
O
SH
• KR1 catalyzes reduction of ketone
FATTY ACID BIOSYNTHESIS
ACP1
AT1
KS1
Cys
Pantetheine
SH
KR1
S
DH1
ER1
ACP2
MT2
Me
O
OH
SH
FATTY ACID BIOSYNTHESIS
ACP1
AT1
KS1
Cys
Pantetheine
SH
KR1
DH1
S
ER1
ACP2
MT2
Me
O
OH
SH
• DH1 catalyzes dehydration of alcohol
FATTY ACID BIOSYNTHESIS
ACP1
AT1
KS1
Cys
Pantetheine
SH
KR1
DH1
S
ER1
ACP2
MT2
Me
O
SH
FATTY ACID BIOSYNTHESIS
ACP1
AT1
KS1
Cys
Pantetheine
SH
KR1
DH1
ER1
S
ACP2
MT2
Me
O
SH
• ER1 catalyzes reduction of alkene
FATTY ACID BIOSYNTHESIS
ACP1
AT1
KS1
Cys
Pantetheine
SH
KR1
DH1
ER1
S
ACP2
MT2
Me
O
SH
FATTY ACID BIOSYNTHESIS
ACP1
AT1
KS1
Cys
Pantetheine
KR1
DH1
ER1
ACP2
MT2
KS2
Cys
SH
S
O
Me
SH
• KS2 catalyzes translocation to module 2
SH
KR
H1
FATTY ACID BIOSYNTHESIS
ER1
ACP2
MT2
KS2
Cys
Pantetheine
S
O
Me
SH
KR2
DH2
ER2
TE
Ser
OH
H1
FATTY ACID BIOSYNTHESIS
ER1
ACP2
MT2
KS2
Cys
KR2
DH2
ER2
TE
S
O
Pantetheine
Me
SH
O
-O
O
S
Co
Malonyl-CoA
• MT2 loads malonyl group onto ACP2
Ser
OH
H1
FATTY ACID BIOSYNTHESIS
ER1
ACP2
MT2
KS2
Cys
S
O
Pantetheine
Me
S
O
O
O
-
KR2
DH2
ER2
TE
Ser
OH
H1
FATTY ACID BIOSYNTHESIS
ER1
ACP2
MT2
KS2
Cys
KR2
DH2
ER2
TE
S
O
Pantetheine
Me
S
CO2
O
O
O
-
• KS2 catalyzes Claisen condensation
Ser
OH
H1
FATTY ACID BIOSYNTHESIS
ER1
ACP2
MT2
KS2
Cys
Pantetheine
S
O
O
Me
SH
KR2
DH2
ER2
TE
Ser
OH
H1
FATTY ACID BIOSYNTHESIS
ER1
ACP2
MT2
KS2
Cys
Pantetheine
SH
KR2
DH2
ER2
TE
Ser
S
Me
O
O
• KR2 catalyzes reduction of ketone
OH
H1
FATTY ACID BIOSYNTHESIS
ER1
ACP2
MT2
KS2
Cys
Pantetheine
SH
KR2
DH2
ER2
TE
Ser
S
Me
O
OH
OH
H1
FATTY ACID BIOSYNTHESIS
ER1
ACP2
MT2
KS2
Cys
Pantetheine
SH
KR2
DH2
S
ER2
TE
Me
O
OH
• DH2 catalyzes dehydration of alcohol
Ser
OH
H1
FATTY ACID BIOSYNTHESIS
ER1
ACP2
MT2
KS2
Cys
Pantetheine
SH
KR2
DH2
S
ER2
Me
O
TE
Ser
OH
H1
FATTY ACID BIOSYNTHESIS
ER1
ACP2
MT2
KS2
Cys
Pantetheine
SH
KR2
DH2
TE
ER2
S
Me
O
• ER2 catalyzes reduction of alkene
Ser
OH
H1
FATTY ACID BIOSYNTHESIS
ER1
ACP2
MT2
KS2
Cys
Pantetheine
SH
KR2
DH2
ER2
S
TE
Me
O
Ser
OH
H1
FATTY ACID BIOSYNTHESIS
ER1
ACP2
MT2
KS2
Cys
Pantetheine
KR2
DH2
TE
ER2
Ser
SH
S
O
Me
• TE catalyzes transesterification
OH
H1
FATTY ACID BIOSYNTHESIS
ER1
ACP2
MT2
KS2
Cys
KR2
DH2
ER2
TE
SH
O
O
Pantetheine
SH
Me
Ser
H1
FATTY ACID BIOSYNTHESIS
ER1
ACP2
MT2
KS2
Cys
KR2
DH2
ER2
TE
SH
O
O
Pantetheine
SH
Me
• TE catalyzes hydrolysis
Ser
H
OH
H1
FATTY ACID BIOSYNTHESIS
ER1
ACP2
MT2
KS2
Cys
KR2
DH2
ER2
TE
Ser
SH
OH
Pantetheine
SH
OH
O
Me
Biosynthesis of Unsaturated Fatty Acids
•
two mechanisms are known for the introduction of double bonds into fatty acids:
–
–
in BACTERIA: anaerobic [O] → monounsaturated FAs (MUFAs)
in MAMMALS, INSECTS & PLANTS: aerobic [O] → MUFAs & polyunsaturated FAs (PUFAs)
Rational Anti-inflammatory Development –
Prostaglandin & Thromboxane Pathway Intervention
•
•
prostaglandins & thromboxanes are derived from further oxidative processing of arachiodonic acid
both are important hormones which control e.g. smooth muscle contractility (blood pressure),
gastric secretion, platelet aggregation & inflammation (<nM activity)
–
various pharmaceuticals including corticosteroids & asprin inhibit biosynthethetic steps in these pathways
Polyketides
Polyketides
•
the structural variety of polyketide secondary metabolites is very wide:
–
NB. starter units marked in red; extender units in bold black; post oligomerisation appended groups in blue
Me
HO
O
CO2H
OH
6-methylsalicylic acid
(antibiotic)
OMe O
OH
O
CO2H
OH
Me
orsellinic acid
O
CO2H
2
H
O
MeO
OH
citrinin
(kidney toxin
'yellow rice disease')
O
Cl
Griseofulvin
(treatment for ring
worm infections)
O
O
O
O
OH
MeO
O
O
O
H
O
POLYKETIDES
OH
HO
O
OH
OH
OH
O
rapamycin
(immunosuppressant)
NB. a mixed polypropionate/acetate
aflatoxin B1
(mycotoxic carcingen)
O
O
O MeO
OH H
O
H
O
MeO
O
N
CO2H
OH O
actinorhodin
(antibiotic)
OMe
HO
O
OH
6-deoxyerythronolide B
NB. a polypropionate
HO
O
O
OH
O
erythromycin A
(antibiotic)
NMe2
O
O
Me
O
O
Me
O
O
OMe
OH
mevinolin
(=lovastatin®)
(anti-cholesterol)
O
Biosynthesis of Polyketides – Oligomerisation Steps
•
polyketides are biosynthesised by a process very similar to that for fatty acids
–
the key differences are:
•
•
–
greater variety of starter units, extender units & termination processes
absent or incomplete reduction of the iteratively introduced -carbonyl groups: ie. each cycle may differ in terms
of KR, DH & ER modules & stereochemistry
this leads to enormous diversity...
Biosynthesis of Polyketides – Overview of PKS
•
the in vivo process of polyketide synthesis involves PolyKetide Synthases (PKSs):
–
PKSs (except Type II, see later) comprise the same 8 components as FASs. i.e. (ACP & 7× catalytic
activities): ACP, KS, AT, MT, [KR, DH, ER & TE]
Type I PKSs: single (or small set of) multifunctional protein complex(es)
–
•
•
–
modular (microbial) - each ‘step’ has a dedicated catalytic site (→ macrolides)
iterative (fungal) – single set of catalytic sites, each of which may operate in each iteration (cf. FASs) (→
aromatics/polyphenols - generally)
Type II PKSs: single set of discrete, dissociable single-function proteins
•
iterative (microbial) - each catalytic module may operate in each iteration (cf. FASs) (→ aromatics/polyphenols)
O
Type I (modular & iterative):
[Type II see later]
O
O
O
S
SH
AT
SH
MT
KS
S
O
decarboxylative
Claisen
condensation
SH
S
1) ± KR
2) ± DH
3) ± ER
n
cycles
ACP
O
O
n
O
S
SH
O
S
SH
SH
S
translocation
O
TE
OH
O
KS ACP
KS ACP
SH
O
n
S
reduction ?
O
O
O
O
KS ACP
CoAS
O
CoAS
O
O
O
CE
ACP
KS ACP
KS = keto synthase; AT = acetyl transferase; MT = malonyl transferase;
KR = keto reductase; DH = dehydratase; ER = enoyl reductase; TE = thioesterase; ACP = acyl carrier protein
KS ACP
O
POLYKETIDE BIOSYNTHESIS [Type I – (modular)]
ACP0
AT0
ACP1
AT1
KS1
Cys
Pantetheine
DH1
ER1
ACP2
SH
Pantetheine
Pantetheine
SH
KR1
SH
NB. the following sequence of slides has also been adapted from: http://www.courses.fas.harvard.edu/%7echem27/
A
POLYKETIDE BIOSYNTHESIS
ACP0
AT0
ACP1
AT1
KS1
Cys
Pantetheine
KR1
DH1
ER1
SH
Pantetheine
Pantetheine
SH
Me
SH
O
S
ACP2
Co
Propionyl-CoA
• AT0 loads starting group (propionyl) onto ACP0
A
POLYKETIDE BIOSYNTHESIS
ACP0
AT0
ACP1
AT1
KS1
Cys
Pantetheine
O
Me
DH1
ER1
ACP2
SH
Pantetheine
Pantetheine
S
KR1
SH
A
POLYKETIDE BIOSYNTHESIS
ACP0
AT0
ACP1
AT1
KS1
Cys
Pantetheine
Pantetheine
S
O
KR1
DH1
ER1
ACP2
SH
Pantetheine
Me
SH
• KS1 catalyzes translocation to module 1
A
POLYKETIDE BIOSYNTHESIS
ACP0
AT0
ACP1
AT1
KS1
Cys
Pantetheine
SH
ER1
ACP2
Pantetheine
Me
SH
DH1
S
O
Pantetheine
KR1
A
POLYKETIDE BIOSYNTHESIS
P0
AT0
ACP1
AT1
KS1
Cys
KR1
DH1
ER1
ACP2
AT2
S
O
Pantetheine
Me
SH
O
SH
-O
O
Me
S
Co
Methylmalonyl-CoA
• AT1 loads methylmalonyl group onto ACP1
SH
P0
POLYKETIDE BIOSYNTHESIS
AT0
ACP1
AT1
KS1
Cys
KR1
DH1
ER1
ACP2
AT2
S
O
Pantetheine
Me
S
O
SH
O
SH
Me
O
-
P0
POLYKETIDE BIOSYNTHESIS
AT0
ACP1
AT1
KS1
Cys
KR1
DH1
ER1
ACP2
AT2
S
O
Pantetheine
Me
CO2
S
O
SH
O
Me
O
-
• KS1 catalyzes Claisen condensation
SH
P0
POLYKETIDE BIOSYNTHESIS
AT0
ACP1
AT1
KS1
Cys
KR1
DH1
ER1
ACP2
AT2
SH
Pantetheine
S
O
SH
SH
* Me
O
Me
Stereocenter
P0
POLYKETIDE BIOSYNTHESIS
AT0
ACP1
AT1
KS1
Cys
Pantetheine
SH
KR1
DH1
ER1
ACP2
AT2
Me
*
S
O
O
Me
SH
SH
• KR1 catalyzes reduction of ketone
P0
POLYKETIDE BIOSYNTHESIS
AT0
ACP1
AT1
KS1
Cys
Pantetheine
SH
KR1
DH1
ACP2
AT2
Me
*
S
O
* Me
OH
Stereocenter
SH
ER1
SH
P0
POLYKETIDE BIOSYNTHESIS
AT0
ACP1
AT1
KS1
Cys
KR1
SH
Pantetheine
DH1
ER1
ACP2
AT2
Me
*
S
O
* Me
OH
SH
SH
• no DH1 activity
P0
POLYKETIDE BIOSYNTHESIS
AT0
ACP1
AT1
KS1
Cys
KR1
SH
Pantetheine
DH1
ER1
ACP2
AT2
Me
S
*
O
* Me
OH
SH
SH
• no ER1 activity
POLYKETIDE BIOSYNTHESIS
ACP1
AT1
KS1
Cys
KR1
DH1
ER1
ACP2
AT2
KS2
Cys
SH
S
O
Pantetheine
HO
*
*
Me
Me
SH
• KS2 catalyzes translocation to module 2
SH
KR
H1
POLYKETIDE BIOSYNTHESIS
ER1
ACP2
AT2
KS2
Cys
Pantetheine
O
HO
*
Me
SH
DH2
ER2
TE
Ser
S
*
KR2
OH
Me
Biosynthesis of Erythromycin – Type I(modular) PKS
•
6-deoxyerthronolide is a precursor to erythromycin A – bacterial antibiotic (Streptomyces erythreus):
–
–
propionate based heptaketide; 3 multifunctional polypeptides (DEBS1, DEBS2 & DEBS3, all ~350 kDa)
Katz et al. Science 1991, 252, 675 (DOI); Staunton, Leadley et al. Science 1995, 268, 1487 (DOI); Khosla et al. J.
Am. Chem. Soc. 1995, 9105 (DOI); review: Staunton & Weissman Nat. Prod. Rep. 2001, 18, 380 (DOI)
DEBS1
module 1
DEBS2
module 2
module 3
DEBS3
module 4
module 5
module 6
O
KR
AT
KS AT
DH ER KR
KS AT
S
S
O
KR
KS AT
S
KS AT
KR
KR
KS AT
S
S
O
KS AT
S
S
O
O
O
HO
HO
O
HO
HO
O
HO
HO
O
HO
HO
TE
O
O
HO
HO
OH
release
O
OH
O
OH
HO
loading
O
O
OH
HO
HO
HO
OH
O
O
OH
OH
= Acyl Carrier Protein
erythronolide B
TE
= Thioesterase
Type II PKSs – Enzyme Clusters (Microbial)
•
Type II PKSs: single set of discrete, dissociable single-function proteins (ACP & 6× catalytic
functions): ACP, KS, KS, [KR, DH, ER, & TE] [NB. NO acetyl or malonyl transferases (AT, MT)]
–
•
•
iterative - each catalytic module may operate in each iteration (cf. FASs) (→ aromatics/polyphenols)
these clusters (generally) use malonate as BOTH starter & extender unit
their ACP proteins are able to load malonate direct from malonyl CoA (no MT required)
the starter malonate is decarboxylated by ‘ketosynthase’  (KS) to give S-acetyl-ACP
the extender malonates undergo decarboxylative Claisen condensations by ketosynthase  (KS)
–
–
•
these clusters rarely utilise KR, DH or ER activities and produce ‘true’ polyketides:
O
CONH2
Type II (iterative):
O
S
O
SH
O
S
O
O
S
SH
CoAS
O
O
O
O
S
S
O
CoAS
KS ACP
KS ACP
O
KS ACP
S
O
KS ACP
O
n
O
1) ± KR
2) ± DH
3) ± ER
S
SH
O
O
S
SH
SH
S
translocation
O
TE
OH
O
n
cycles
O
n
SH
reduction
(rarely)
SH
O
decarboxylative
Claisen
condensation
KS ACP
KS ACP
CONH2
O
O
O
KS ACP
KS ACP
KS ACP
KS = 'keto synthase ' (=decarboxylase!); KS = 'keto synthase ' (=ketosynthase!); KR = keto reductase;
DH = dehydratase; ER = enoyl reductase; TE = thioesterase; ACP = acyl carrier protein
O
Biosynthesis of Actinorhodin – Type II PKS
•
actinorhodin – octaketide bacterial antibiotic (Streptomyces coelicolor)
–
Hopwood Chem. Rev. 1997, 97, 2465 (DOI)
7x
O
O
CoAS
O
CoAS
O
O
PKS
[ACP, KS , KS]
O
O
O
O
O
O
O
O
O
1
8x CO2
octaketide
O
O
16
cyclase
O
O
O
O
O
16
SEnz
O
O
16
O
1
OH
KR
SEnz
O
HO
O
O
SEnz
1
OH
aromatase
OH
OH
O
O
O
OH
16
2
H
OH O
actinorhodin
–
O
O
octaketide synthesis then cyclisation? (as shown above)
hexaketide synthesis then cyclisation then two further rounds of extension?
indications can sometimes be gleaned from biomimetic syntheses...
16
O
CO2H
OH
O
cyclase
1
SEnz
timing of 1st cyclisation and mechanism of control of chain length uncertain
•
•
–
O
O
O
O
1
SEnz
Scope of Structures - Type II PKS
•
microbial polyphenolic metabolites:
pentaketides (5x C2)
OMe O
OH
O
O
OH
octaketides (8x C2)
emodin
eugenone
MeO
HO
OMe
O
hexaketides (6x C2)
nonaketides (9x C2)
OH
O
OH
O
O
O
NH2
tetracycline
plumbagin
Me
OH
heptaketides (7x C2)
H
OH
H
O
MeO
O
O
OH
NMe2
O
OH
decaketides (10x C2)
rabelomycine
rubrofusarin
OH
•
OH
O
many display interesting biological activities...
OH
O
OH
Primary Metabolism - Overview
Primary metabolites
Primary metabolism
Secondary metabolites
CO2 + H2O
PHOTOSY NTHESIS
HO
HOHO
1) 'light reactions': hv -> ATP and NADPH
2) 'dark reactions': CO2 -> sugars (Calvin cycle)
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
FATT Y ACIDS & POLY KETIDES
prostaglandins
polyacetylenes
aromatic compounds, polyphenols
macrolides
ISOPRENOIDS
terpenoids
steroids
carotenoids
For interesting animations’ of e.g. photosynthesis see: http://www.johnkyrk.com/index.html
Isoprenoids
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)--bisabolene
(C15)
H
O O
OH
n
O
H
ISOPRENOIDS
natural rubber (~105x C5)
H
artemisinin (C15)
O
O
OPP
OPP
 -carotene (C40)
H
AcO
H
H
HO
cholesterol
(C27 but C30-derived)
BzHN
OH
O
taxol (C20)
OH
HO
BzO AcO
H
O
HO
H
O
O
OH
euonyminol (C15)
H
O
HO
HO
O
Ph
H
O
isopentenyl
pyrophosphate
(IPP)
dimethylallyl
pyrophosphate
(DMAPP)
OH
OH
CO2H
OH
OH
OH
OH
gibberellic acid (C20)
(gibberellin A3)
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:
Rational Anti-cholesterol Development - Statins
•
•
HMG CoA → MVA is the rate determining step in the biosynthetic pathway to cholesterol
‘Statins’ inhibit HMG CoA reductase and are used clinically to treat hypercholesteraemia - a
causative factor in heart disease, see: Wu et al. Tetrahedron 2015, 71, 8487 (DOI)
–
–
e.g. mevinolin (=lovastatin®, Merck) from Aspergillus terreus is a competitive inhibitior of HMG-CoA reductase
e.g. lipitor (Atorvastatin calcium, Pfizer) is also a competitive inhibitior of HMG-CoA reductase and the worlds
biggest selling drug [first drug to reach $10 billion sales (2004: $10.8 bn]
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 – -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 -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
-terpinyl cation
MONOTERPENES (C10)
Monoterpenes – Fate of the -Terpinyl Cation
•
•
The -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
-terpineol
limonene
hydrolysis
trapping by PPO
OH
E1 elimination
trapping with
water
a
OPP
c
d
c
=
He
OPP
b
OPP
H
trapping by alkene
at 'red' carbon
(anti-Markovnikov)
1,2-alkyl shift
H
=
H
H
O
=
=
=
-pinene
H
E1 elimination
thujone
E1 elimination
E1 elimination
d
e
O
camphor
camphene
trapping by alkene
at 'blue' carbon
(Markovnikov)
H2O
1,2-hydride shift
OH
borneol
OPP
bornyl
pyrophosphate
c
d
a -terpinyl cation
[O]
H
H
=
b
H
-
H
=
=
-pinene
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 – 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
 -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
OH
O
HO2C
zaragozic acid A HO2C
(squalestatin S1)
O
O
OH CO2H
+ PPi
H H
OAc
squalene
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
24 hydrogenation
H
8
1
4
HO

24
2) 14 DEMETHYLATION
H
14
HO
3) 4 & 4DEMETHYLATION
5
H
 to  rearrangement
8
H
=
H
5
H
flat, rigid structure
HO
lanosterol
cholesterol
2) 14 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) 4 & 4DEMETHYLATION
4x O2
HO
4x O2
P450
4
O
H
4x H2O
O
H
O
O
CO2
5
4x H2O
isomerase
H
5
H
H
O
H
O
O
CO2
NADH
NAD
8
NADPH
O
H
H
 to  rearrangement
8
O
H
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]
O
FeIII
EnzB:
H
H
H
X
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, OH)
Steroid Ring Cleavage - Vitamin D & Azadirachtin
•
•
vitamin D2 is biosynthesised by the photolytic cleavage of 7-dehydrocholesterol by UV light:
–
a classic example of photo-allowed, conrotatory electrocyclic ring-opening:
–
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:
–
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
AcO
H
AcO
tirucallol
(cf. lanosterol)
H
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!
Summary of Presentation
•
Metabolism & Biosynthesis
–
•
Shikimate Metabolites
–
–
•
acetylCoA & the citric acid cycle → -amino acids → alkaloids
Opioids – powerful pain killers
Fatty Acids and Polyketides
–
–
•
photosynthesis & glycolysis → shikimate formation → shikimate metabolites
Glyphosate – a non-selective herbicide
Alkaloids
–
–
•
some definitions, 1° & 2° metabolites
acetylCoA → malonylCoA → fatty acids, prostaglandins, polyketides, macrolide antibiotics
NSAIDs – anti-inflammatory’s
Isoprenoids/terpenes
–
–
acetylCoA → mevalonate → isoprenoids, terpenoids, steroids, carotenoids
Statins – cholesterol-lowering agents