Biosynthesis – Inspiration for Drug Discovery

Biosynthesis of Isoprenoids

Alan C. Spivey [email protected]

Dec 2008 Format & Scope of Lecture

• What are isoprenoids?

– n × C5 diversity: terpenes, steroids, carotenoids & natural rubber – ‘the isoprene rule’ – mevalonate pathway to IPP & DMAPP

• Monoterpnes (C10) – regular (‘head-to-tail’) via geranyl pyrophosphate – apparently irregular ‘iridoids’ (e.g. seco-loganin)

• Sesquiterpenes (C15) – farnesyl pyrophosphate derived metabolites

• Diterpenes (C20) – 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 H OH O

OH HO O OH thujone borneol lavandulol HO (C10) (C10) (C10) (Z)-α-bisabolene humulone (2x C ) 5 (C ) 15 H O O OH H n O 5 ISOPRENOIDS H artemisinin (C15) natural rubber (~10 x C5) O

O OPP OPP OH O OH dimethylallyl isopentenyl pyrophosphate pyrophosphate HO OH (DMAPP) (IPP) euonyminol (C ) β-carotene (C40) 15 OH OH OH H H OH OH AcO O H OH Ph H O gibberellic acid (C20) (gibberellin A ) HH BzHN O HO 3 H CO H HO O 2 HO O HO cholesterol BzO AcO O (C27 but C30-derived) taxol (C20) 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:

2x Δ

200°C

isoprene (C5) 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: • 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 metabolism Primary metabolites Secondary metabolites

CO2 +H2O PHOTOSYNTHESIS 1) 'light reactions': hv -> ATP and NADPH 2) 'dark reactions': CO2 -> sugars (Calvin cycle)

HO O oligosaccharides HOHO polysaccharides HO nucleic acids (RNA, DNA) OH glycolysis glucose & other 4,5,6 & 7 carbon sugars

CO2 SHIKIMATE METABOLITES PO cinnamic acid derivatives CO2 aromatic compounds + HO HO OH lignans, flavinoids PO O OH OH phosphoenol pyruvate erythrose-4-phosphate shikimate

ALKALOIDS aromatic amino acids peptides penicillins CO 2 proteins cephalosporins aliphatic amino acids cyclic peptides O pyruvate tetrapyrroles (porphyrins) Citric acid cycle (Krebs cycle) saturated fatty acids FATTY ACIDS & POLYKETIDES SCoA SCoA unsaturated f atty acids prostaglandins lipids CO2 polyacetylenes O O aromatic compounds, polyphenols acetyl coenzyme A malonyl coenzyme A macrolides

CoAS O HO ISOPRENOIDS terpenoids CO2 O HO steroids carotenoids acetoacetyl coenzyme A mevalonate 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 Secondary metabolism

CO2 pyruvate GLYCOLYSIS O

CoASH CO2 oxidative decarboxylation NAD NADH

SCoA acetyl CoA CITRIC ACID CYCLE O

SCoA acetyl CoA carboxylase O CO2 Claisen CoASH (biotin-dependent) carboxylation condensation CoAS O

aldol O SCoA saturated fatty acids FATTY ACIDS & POLYKETIDES acetoacetyl CoA unsaturated fatty acids reaction CO2 prostaglandins then [R] O lipids polyacetylenes SCoA 2x CoASH malonyl CoA aromatic compounds, polyphenols 2x NADPH macrolides O 2x NADP

HO ISOPRENOIDS CO2 terpenoids HO steroids mevalonate (MVA) 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 CO2 NADPH Zn2 H CO2 HO R N O CoAS O CO2 H H HO O NADPH HMG CoA H2N NADP NADP mevalonate (MVA) OH CoASH OH CO2 HO H O OH O CO2 i 2 O Pr OH O OO O OH PhHN N LIPITOR in vivo Ph H F mevinolin (=lovastatin®) H H HO ACTIVE DRUG cholesterol PRO-DRUG mimic of tetrahedral intermediate NB. type I (iterative) PKS natural product in HMG reduction by NADPH Hemi-Terpenes – ‘Prenylated Alkaloids’

• DMAPP is an excellent alkylating agent

• C5 units are frequently encountered as part of alkaloids (& shikimate metabolites) due to ‘late- stage’ 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 histidine O H OPP OPP Me O HO N DMAPP H 'reverse' 'normal' H N prenylation NH prenylation 4 3 tyrosine N N

tyrosine cf. SN2' N H cf. S 2 H O N N N N H H H roquefortine lysergic acid (blue cheese mould) (halucinogen)

– 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 – 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 (E) O O Mg O P O P O O O gerenyl Mg pyrophosphate

OPP

linalyl pyrophosphate

OPP (Z) OPP cyclase

= MONOTERPENES (C10) OPP neryl pyrophosphate initial chiral centre α-terpinyl cation allylic cation intimate ion pair 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

limonene α-terpineol hydrolysis [O] trapping by PPO- H H OH OH OPP OPP O bornyl borneol camphor E1 elimination trapping with pyrophosphate a b water =

c OPP OPP trapping by alkene H E elimination c 1 = d at 'red' carbon 1,2-alkyl shift c d (anti-Markovnikov) H e = camphene b trapping by alkene H2O at 'blue' carbon (Markovnikov) a α-terpinyl cation E1 elimination H d = H H = 1,2-hydride shift e = β-pinene H O H E1 elimination = =

α-pinene H thujone 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 H OPP OPP O O 2x NADP NADPH [O] OH = O

P450 OH O OH 2x NADPH O NADP OH 10 10 [O] PP geranyl PP i 10-oxo geranal P450

unusual reductive ring-closure -> cyclopentane (NB. Non-cationic) OH

O HO2C NH2 [O] P450 glycoside N H formation H tryptamine O ATP N NH HO HO [O] P OP 450 tryptophan H H H H H OGlu OGlu OGlu OGlu OGlu methylation H H O H SAM isoprene O O ADP O O MeO C H2O MeO2C H O MeO C HO C 2 2 MeO2C 2 2 strictosidine secologanin loganin enzymatic Pictet-Spengler unusual fragmentation reaction 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 N H H H N H N H N H H N O H H H MeO2C MeO2C N OAc ajmalicine H MeO H OH Me (vinca) CO2Me MeO2C vinblastine OH (vinca) yohimbine MeO O N N H N 3 NH O N H H H H OGlu N H OH O H O aspidospermine camptothecin MeO2C (vinca) H strictosidine (isovincoside) N H N Me HO H MeO N H O N H H N O H O quinine N O H strychnine (strychnos) gelsemine (oxindole) Sesquiterpenes – Farnesyl Pyrophosphate (FPP)

• ‘SN2’-like alkylation of geranyl PP by IPP gives farnesyl PP:

pro-R hydrogen is lost

OPP OPP HS HR OPP OPP (E) (E)

geranyl PP IPP E,E-farnesyl PP (FPP)

• just as geranyl PP readily isomerises to neryl & linaly PPs so farnesyl PP readily isomerises to equivalent compounds – allowing many modes of cyclisation & bicyclisation

OPP (E) (E) O O Mg O O P P O E,E-FPP O O Mg

- further cyclisation (E) 6-memb - 1,2-hydride & alkyl shifts (Z) cyclases vast array of 10-memb mono- & bicyclic 11-memb SESQUITERPENES ring cyclised E,Z-FPP OPP - trapping with H2O 'CATIONS' - elimination to alkenes

OPP NB. control by: 1) enzyme to enforce conformation & sequestration of reactive intermediates nerolidyl PP 2) intrinsic stereoelectronics of participating orbitals 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 O sesquiterpene H OH OPP a b Ph (isoprenoid) H BzNH O geranylgeranyl PP H taxol OPP HO 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 (C ) arise from the ‘head to head’ 30 OPP coupling of two fanesyl PP units to give squalene catalysed by squalene synthase: FPP (acceptor) OPP FPP (donor)

– squalene was first identified as a steroid precursor EnzB: from shark liver oil HH OPP

– the dimerisation proceeds via an unusual mechanism OPP

involving electrophilic cyclopropane formation - squalene synthase blocked by squalestatins rearrangement to a tertiary cyclopropylmethyl cation and reductive cyclopropane ring-opening by NADPH H (NB. exact mechanism disputed) presqualene PP

OPP – Zaragozic acids (squalestatins) mimic a rearrangement intermediate and inhibit squalene H synthase. They constitute interesting leads for H development of new treatments for OPP hypercholesteraemia & heart disease (cf. statins) NADPH

O NADP + PPi

O OH H H OAc squalene HO C 2 O zaragozic acid A HO2C O (squalestatin S1) OH CO2H Triterpenes – Squalene → 2,3-Oxidosqualene

• squalene is oxidised to 2,3-oxidosqualene by squalene oxidase – which is an O2/FADH2- dependent enzyme:

squalene oxidase

O2 + FADH2

O

squalene H2O + FAD 2,3-oxidosqualene

• the key oxidant is a peroxyflavin:

reductiverecycling

H H H O N O O NO O NO H O O NO O O HO NH O NH N HN HN H N N N NR NR NR NR H2O O

FADH2 peroxyflavin hydroxyflavin 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-boat- chair 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) Δ24 hydrogenation H 2) 14α DEMETHYLATION H 14 8 H = 1 HO 5 3) 4α & 4β DEMETHYLATION 4 HH HO 4) Δ8 to Δ5 rearrangement flat, rigid structure H HO lanosterol cholesterol

24 1) Δ hydrogenation 2) 14α DEMETHYLATION 2x O NADPH 2 P450 H O2 P450 HCO2H NADPH H H H H 24 14 H :BEnz H 2x H2O NADP O HO O NADP O FeIII

3) 4α & 4β DEMETHYLATION

4x O2 P450 4x O2 P450 NADPH

4 HO O O O O O HO H H H CO H H HH 4x H2O 2 4x H2O CO2 OO H OO NADP

8 5 4) Δ to Δ rearrangement NAD NADH 8 isomerase 5 H H H H H NADH NAD [-> vitamin D] 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 O O 2x O P O P NAD H 2 450 H 2 450 H H H H H H

HH HH O HH HH 2x H2O NADH HO HO HO O cholesterol progesterone

[O]

X O III Fe O O EnzB: O H H OOH 2 P450 O 2x O2 P450 H H HH HH H HH O HO HCO H 2 O O androstendione (X = O) estrone 2x H2O (œstrone) testosterone (X = H, β−OH) DEMETHYLATIVE aromatisation by 'aromatase' enzyme Steroid Ring Cleavage - Vitamin D & Azadirachtin

7 • vitamin D2 is biosynthesised by the photolytic cleavage of Δ -dehydrocholesterol by UV light: – a classic example of photo-allowed, conrotatory electrocyclic ring-opening: OH OH NADP H H UV light (hv) H H [O] H H

electrocyclic 1,5-hydride HH NADPH HH H H H 7 ring-opening shift HO HO HO cholesterol Me [6π] Me H ψ 4 HO HO OH H conrotatory SOMO vitamin D2 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: – exact biogenesis unknown but certainly via steroid modification:

O MeO C OAc O 2 H O OH O H O OH 12 O O C 11 O 14 OH oxidative 8 O H 7 cleavage highly hindered C-C bond HO OH AcO OH AcO OH for synthesis! H H of C ring H MeO2C O AcO H tirucallol azadirachtanin A azadirachtin (cf. lanosterol) (a limanoid = tetra-nor-triterpenoid) Primary Metabolism - Overview

Primary metabolism Primary metabolites Secondary metabolites

CO2 +H2O PHOTOSYNTHESIS 1) 'light reactions': hv -> ATP and NADPH 2) 'dark reactions': CO2 -> sugars (Calvin cycle)

HO O oligosaccharides HOHO polysaccharides HO nucleic acids (RNA, DNA) OH glycolysis glucose & other 4,5,6 & 7 carbon sugars

CO2 SHIKIMATE METABOLITES PO cinnamic acid derivatives CO2 aromatic compounds + HO HO OH lignans, f lavinoids PO O OH OH phosphoenol pyruvate erythrose-4-phosphate shikimate

ALKALOIDS aromatic amino acids peptides penicillins CO 2 proteins cephalosporins aliphatic amino acids cyclic peptides O pyruvate tetrapyrroles (porphyrins) Citric acid cycle (Krebs cycle) saturated f atty acids FATTY ACIDS & POLYKETIDES SCoA SCoA unsaturated f atty acids prostaglandins lipids CO2 polyacetylenes O O aromatic compounds, polyphenols acetyl coenzyme A malonyl coenzyme A macrolides

CoAS O HO ISOPRENOIDS terpenoids CO2 O HO steroids carotenoids acetoacetyl coenzyme A mevalonate