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Home , Camphene, Farnesyl pyrophosphate, Geranyl pyrophosphate, Isoprene, Lanosterol, Oxidosqualene cyclase

Biosynthesis of Isoprenoids: (Including & )

Alan C. Spivey [email protected]

Dec 2014 Format & Scope of Lectures

• What are isoprenoids?

– n × C5 diversity: terpenes, steroids, carotenoids & – „the rule‟ – mevalonate & 1-deoxyxylulose pathways to IPP & DMAPP

• Monoterpnes (C10) – regular („head-to-tail‟) via geranyl – irregular: incl. (e.g. seco-loganin)

• (C15) – derived metabolites – cyclases: pentalenene, & 5-epi-aristolochene

• (C20) – gibberellins & taxol

• (C30) – ( → hopene) – steroids (2,3-oxidosqualene → → → estrone)

– ring-opened „steroids‟: vitamin D2 & • Biomimetic cationic cyclisation cascades

• Carotenoids (C40) – b-, & 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:

• (C10); sesquiterpenes (C15); diterpenes (C20); sesterpenes (C25, rare); triterpenes (C30); carotenoids (C40)

H O H OH O

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

O OPP OPP OH O OH dimethylallyl isopentenyl pyrophosphate pyrophosphate HO OH (DMAPP) (IPP) euonyminol (C ) b-carotene (C40) 15 OH OH OH H H OH OH AcO O H OH Ph H O gibberellic acid (C20) (gibberellin A ) H H 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 – of many monoterpenes produced two moles of isoprene:

2x  200°C isoprene (C5) - (C10) • 1940s: – biogenesis of terpenes attributed to oligomers of isoprene – ‘the isoprene rule’ • 1953: – Ruzicka proposes „the biogenetic isoprene rule‟ to accomodate „irregular‟ : • 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) → isopentenyl pyrophosphate (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 - Overview

Primary metabolism Primary metabolites Secondary metabolites

CO2 + H2O

PHOTOSYNTHESIS 1) 'light reactions': hv -> ATP and NADH 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 fatty acids prostaglandins CO2 polyacetylenes O O acetyl coenzyme A aromatic compounds, malonyl coenzyme A macrolides

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

• Mevalonate (MVA) is the first committed step of isoprenoid biosynthesis – this key 6-carbon metabolite is formed from three 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) condensation carboxylation 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:

Claisen condensation CoASH B-Enz SCoA CoAS O SCoA CoAS O SCoA acetoacetyl CoA H O O O O O CoAS acetyl CoA CoAS aldol reaction O

H O 2 CoASH

CoAS HO hydroxymethylglutaryl CoA OPP CO (HMG CoA) H 2 DMAPP D O

IPP isomerase T 2x NADPH 2x NADP HMG CoA reductase (overall anti D2O RDS in cholesterol stereochemistry) CoASH biosynthesis Cys 3x ATP 139 S decarboxylative elimination O OPP D PO Me HO IPP mevalonate (MVA) CO2 H PPO O HO O HR Hs T Pi + CO2 3x ADP Glu 207 O

sequential addition HMG CoA reductase inhibitors -

• HMG CoA → MVA is the rate determining step in the biosynthetic pathway to cholesterol – 33 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 (=®, 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 O O 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 in HMG reduction by NADPH Biosynthesis of IPP & DMAPP – via 1-Deoxyxylulose • the mevalonate route to IPP & DMAPP has been proven in yeast & animals & in some plants & for a long while was believed to be the only pathway to these key intermediates – However, in some bacterial labelling studies: • no incorporation of mevalonolactone was observed • the pattern of label from glucose was inconsistent with derivation via catabolism to acetate • in 1993 an additional pathway to IPP & DMAPP was discovered: – Rohmer et al. Biochem. J. 1993, 295, 517 (DOI)

OH DXP NADPH OH CO2 O OH O OH H synthase + O H HO O OP OH OP pyruvate OH OP OH OP CO2 NADP glyceraldehyde 1-deoxyxylulose DXP 2-methyerythritol 3-phosphate 5-phosphate (DXP) reducto- 4-phosphate isomerase ATP + CTP

ADP + PPi OPP DMAPP NADPH NH2 NADPH OH OH O N OPP HO HO P O HO O O P O OP O O O N O OPP P P IPP NADP O O O 4-hydroxy-DMAPP CMP O O O O NADP

OH OH NB. CMP = cytidine monophosphate CTP = cytidine triphosphate cytosine

• The pathway is prevalent in many pathogenic and so its inhibition represents an exciting opportunity for antiinfective therapeutic development: Rohdich J. Org. Chem. 2006, 71, 8824 (DOI) Chorismate → Coenzymes Q & Vitamins E & K

• Chorismate → p- & o-hydroxybenzoic acids → coenzymes Q & vitamins E & K – NB. ‘Mixed’ biosynthetic origin: shikimate/mevalonate (isoprenoid)

H O C CO2 CO2 2 CO2 H H O O CO2 OH OH O O 3 O 3

H OH 1 2 CO2 CO2 R , R = H or Me CO2 isoprenoid 1 2 prephenate R R OH OH OH OH OH homogentisate (vitamins E) electron transport, antioxidant ArC1

CO2 OH2 CO2 CO2 O isoprenoid MeO Me

O CO2 ubiquinones (coenzymes Q) H OH R MeO R MeO H -soluble electron transport CO n chorismate 2 OH OH OH O O R ArC0 pyruvate

-ketoglutarate (KG) KG H R isoprenoid O SCoA n CO2 R CO O C CO 2 2 2 OH O Me O OH O menaquinones (vitamins K) CO2 cofactors for plasma proteins O O essential for blood clotting O HO O CO2 CO ArC1 isochorismate 2 CO2 O Hemi-Terpenes – ‘Prenylated ’

• 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 ’ – ‘SN2’-like alkylation; ‘reverse prenylation’ – ‘SN2’-like alkylation

• e.g. (recall the ergot alkaloids) – a „normal prenylated‟ (with significant subsequent processing) • e.g. roquefortine (recall diketopiperazine alkaloids) – a „reverse prenylated‟ alkaloid

DMAPP 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 (blue cheese mould) lysergic acid (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‟

• head-to-tail C5 oligomers are the key precursors to isoprenoids – (C10) is formed by SN1 alkylation of DMAPP by IPP → monoterpenes – farnesyl (C15) & geranylgeranyl (C20) pyrophosphates are formed by further SN1 alkylations with IPP:

via OPP inversion OPP SN1 H OPP HR H* OPP

DMAPP HS

OPP *H H OPP evidence for S 1: N OPP MONOTERPENES (C10)

HS HR OPP intimate pair gerenyl pyrophosphate (C ) OPP 10 IPP F C F C 3 3 IPP ionisation is 1,000,000 times slower OPP

SESQUITERPENES (C15) TRITERPENES (C30) farnesyl pyrophosphate (C15)

IPP OPP

DITERPENES (C20) CAROTENOIDS (C40) gerenylgeranyl pyrophosphate (C20) Monoterpenes from Parsley & Sage

apiol -pinene Parsley (Petroselinum sativum)

thujone Sage (Salvia officinalis) Monoterpenes from Rosemary & Thyme

borneol cineol Rosemary (Rosmarinus officinalis)

Thyme (Thymus vulgaris) & 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) Monoterpenes – -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 3 × Mg2+ – all three pyrophosphates are substrates for cyclases via an -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 -terpinyl cation allylic cation intimate ion pair 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

limonene - 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 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 -terpinyl cation E1 elimination H d = H H = 1,2-hydride shift e = b-pinene H O H E1 elimination = =

-pinene H thujone Irregular Monoterpenes

• Non-‟head-to-tail‟ linkage of IPP &/or DMAPP leads to ‘irregular’ monoterpenes – e.g. daisy (Compositae) & chrysanthemum metabolites:

[O] H H OH CO2H chrysanthemic acid H O 2 Enz-B: O OPP a [O] HH b OPP OPP OPP a 2x DMAPP artemisia ketone cyclopropylmethyl cations are relatively stable because of the high p-character of the -bonds

b

H O 2 HO

santolina alcohol

– Natural crysanthemic acid derivatives are referred to as pyrethrins and are natural insecticides – Synthetic analogues of crysanthemic acid are referred to as pyrethroids. e.g. bifenthrin:

Cl O Bifenthrin F3C potent insecticide (via ATPase inhibition) O 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 - 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 -> (NB. Non-cationic) OH

O HO2C NH2 [O] P450 N H formation H O ATP N NH HO HO [O] P OP 450 tryptophan H H H H H OGlu OGlu OGlu OGlu OGlu 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 loganin enzymatic Pictet-Spengler unusual fragmentation reaction Strictosidine → Vinca, Strychnos, 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 (strychnos) (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

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 Sesquiterpene Cyclases

Christianson et al. Curr. Opin. Struct. Biol. 1998, 695 (DOI) Cyclases – Control of Cyclisation

• Functional aspects of cyclases:

– Templating: Active site provides a template for a specific conformation of the flexible linear isoprenoid starting material. – Triggering: Cyclase initiates carbocation formation. • Metal-assisted leaving group departure (e.g. pyrophosphate aided by Mg2+) • C=C bond protonation (e.g. squalene-hopene cyclase, see later). • Epoxide protonation (e.g. , see later). – Chaperoning: Chaperones conformations of carbocationic intermediates through the reaction sequence, ordinarily leading to one specific product. – Sequestering: Sequesters the carbocation intermediates by burying the substrate in a hydrophobic cavity that is generally -inaccessible. Carbocations are concomitantly stabilized by the presence of aromatic residues in the active site that exert their effects via cation-p interactions

R cation - p   on edges stabilisation RECALL: =   on faces -1  R R (~40-80 kJmol ) Enz – Adapted from: Christianson et al. Curr. Opin. Struct. Biol. 1998, 695 (DOI)

• BUT: – individual terpene cyclases can give multiple products, see: Matsuda J. Am. Chem. Soc. 2007, 129, 11213 (DOI)

Sesquiterpene Cyclase Crystal structures

pentalenene synthase 5-epi-aristolochene synthase aristolochene synthase

→ pentalenolactone (antibiotic) → fungal (myco)toxins (e.g. bipolaroxin, PR-toxin) Aristolochene & 5-Epi-Aristolochene Synthases

• molecular modelling studies indicate that the shape of the active sites determines the conformation of FPP and thus the stereochemistry of the final product

– Penicillium roqueforti aristolochene synthase – Felicetti & Cane J. Am. Chem. Soc. 2004, 126, 7212 (DOI)

– tobacco 5-epi-aristolochene synthase – Starks, Back, Chappell & Noel Science 1997, 277, 1815 (DOI)

Diterpenes - Gibberellins

Dwarf rice seedlings (on the left) have a defect in the gibberellin- dependent signalling mechanism

gibberellin A20 effects of gibberellin A1 and brassinolide on rice seedlings Diterpenes – Geranylgeranyl Pyrophosphate

• SN2 alkylation of farnesyl PP by IPP gives geranylgeranyl PP:

OPP OPP H H OPP OPP

FPP IPP gerenylgeranyl PP (C ) 20 • geranylgeranyl PP readily cyclises to give numerous multicyclic diterpenes – e.g. gibberellins – plant growth hormones • NB. cyclisation initiated by alkene protonation NOT loss of PPO-

1,2-alkyl H OPP bicyclisation OPP cyclisation cyclisation shift

H H H H H 6-endo 6-exo 6-endo H H H H H gera nylgeranyl PP labdadienyl PP

elimination

H OH H pinacol 4x [O] H O rearrangement (?) H HO

CO H H O 2 H H OH gibberellic acid HO C CHO H 2 HO2C OP kaurene (gibberellin A3)

• review: L.N. Mander „Twenty years of gibberellin research‟ Nat. Prod. Rep. 2003, 20, 49-69 (DOI) 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

b-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 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

OPP • triterpenes (C30) arise from the ‘head to head’ coupling of two fanesyl PP units to give FPP (acceptor) OPP squalene catalysed by squalene synthase: FPP (donor) EnzB: H H – squalene was first identified as a precursor OPP from shark liver oil

OPP – the dimerisation proceeds via an unusual mechanism squalene synthase blocked by squalestatins

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

H – Zaragozic acids (squalestatins) mimic a H rearrangement intermediate and inhibit squalene synthase. They constitute interesting leads for OPP development of new treatments for NADPH hypercholesteraemia & heart disease (cf. statins) NADP + PPi H H O squalene O OH OAc HO C 2 O zaragozic acid A HO2C O (squalestatin S1) For an interesting account of the elucidation of this pathway see: OH CO2H Poulter J. Org. Chem. 2009, 74, 2631 (DOI) 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 therefore a peroxyflavin:

reductive recycling

H H H O N O O N O O N O H O O N O O O HO NH O NH N HN HN H N N N NR NR NR NR H2O O

FADH 2 peroxyflavin hydroxyflavin FAD Modes of Cyclisation of Squalene

• all triterpenes (steroids, hopanoids etc.) are formed by the action of cyclase on either squalene or 2,3-oxidosqualene – i.e. different methods of ‘triggering’ cyclisation

OH H H H squalene OH cyclases H H H HOPANOIDS triggering by H H H ALKENE protonation H H hopene diplopterol H squalene squalene oxidase

H H oxidosqualene H cyclases H STEROIDS triggering by H O EPOXIDE protonation HO HO HO H b-amyrin H H lanosterol 2,3-oxidosqualene

STEROID/ NOMENCLATURE: b-face = top face (as drawn here) = hydrogen up = hydrogen down -face = bottom face (as drawn here) (on b-face) (on -face) Squalene-Hopene Cyclase (SHC)

• Squalene-Hopene Cyclase (SHC) catalyses the formation of hopene from squalene: – what does the enzyme have to do to achieve such exquisite regio- & stereoselectivity over the formation of 9 new stereogenic centres? • enforce an appropriate conformation of squalene • activate the C2 alkene by protonation

• shield reactive cations from nucleophiles (e.g. H2O) using aromatic residues (cation-p) • position a general base precisely to facilitate the terminal elimination

– until recently, the „appropriate‟ conformation was believed to be the formally appealing ‘all-chair’ conformation allowing for a concerted cationic ring-closure cascade (shown below) – remaining stereocontrol would then be taken care of by intrinsic stereoelectronics • i.e. correct orbital overlap

– however, this requires anti-Markovnikov regioselectivity for the C & D ring-closures...

C & D ring-closure by 2x anti-Markovnikov additions? H

H SHC C D EnzB-H H H 5x ring-closures :BEnz H 6-endo, 6-endo, 6-endo, 6-endo, 5-endo ? squalene hopene via chair-chair-chair-chair conformation ? Squalene-Hopene Synthase (SHC)

H

squalene H+ 6-endo 6-endo 6-endo ? 6-endo 5-endo

hopene

X-ray crystal structure: Wendt, Poralla & Schulz Science, 1997, 277, 1811 (DOI) Squalene-Hopene Synthase - Mechanism

• The process is apparently more complex & has more recently been shown to involve: – 2x Markovnikov ring-closure/1,2-alkyl-shift ring-expansion sequences to establish the C & D rings – lessons? • the conformation enforced by the enzyme is NOT strictly an all-chair one! (although probably very close) • the process is NOT concerted, discrete cationic intermediates are involved • stereoelectronics dictate the regio- & stereoselectivity

Markovnikov ring expansion 1) alkene protonation ring-closure (5 -> 6) (5-memb ring) EnzB-H H 1,2-alkyl shift H 5-endo 2) 2x Markovnikov ring-closures squalene (6-memb rings) 6-endo, 6-endo Markovnikov 5-endo ring-closure (5-memb ring) :BEnz H H ring H Markovnikov H H expansion ring-closure H H (5 -> 6) E1 elimination (5-memb ring) H 1,2-alkyl shift H 5-endo H H

H H hopene

– review: Wendt et al. Angew. Chem. Int. Ed. 2000, 39, 2812 (DOI) & Wendt ibid 2005, 44, 3966 (DOI) Oxidosqualene-Lanosterol Cyclase (OSC)

• oxidosqualene-lanosterol cyclase catalyses the formation of lanosterol from 2,3-oxidosqualene: – this cascade establishes the characteristic ring system of ALL steroids – until recently, as for SHC, the enzyme was believed to enforce a chair-boat-chair conformation to allow a concerted cationic ring-closure cascade followed by a series of suprafacial 1,2-shifts (shown below)

– however, this also requires anti-Markovnikov regioselectivity for the C ring-closure...

C ring-closure by anti-Markovnikov addition?

O OSC 6-endo, 6-endo, 6-endo, 5-endo ? 4x ring-closures EnzB-H chair-boat-chair conformation ? O

2,3-oxidosqualene

H H H prosterol cation C HO 2x 1,2-hydride shifts 2x 1,2-Me shifts NB all bolded bonds are anti-peri planar H HO elimination H lanosterol Oxidosqualene-Lanosterol Cyclase – Mechanism

• This process has also been shown to involve a Markovnikov ring-closure/1,2-alkyl-shift ring- expansion sequence to establish the C ring – again, the conformation enforced by the enzyme is NOT strictly a chair-boat-chair one (although probably close), the process is NOT concerted, discrete cationic intermediates are involved & stereoelectronics dictate the regio- & stereoselectivity

Markovnikov ring 1) epoxide opening ring-closure expansion (5-memb ring) (5 -> 6) H 1,2-alkyl shift 2) 2x Markovnikov 5-endo H H ring-closures HO EnzB-H O HO HO (6-memb rings) Markovnikov 6-endo, 6-endo 2,3-oxidosqualene 5-endo ring-closure (5-memb ring)

1) 1,2-hydride shift H H H H 2) 1,2-hydride shift E1 elimination

3) 1,2-Me shift H H 4) 1,2-Me shift HO HO HO :BEnz (ALL suprafacial) H H H lanosterol protosterol cation

– “The enzyme’s role is most likely to shield intermediate carbocations… thereby allowing the hydride and 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: Cholesterol → Human Sex Hormones

• cholesterol is the precursor to the human sex hormones – progesterone, & 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

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

[O]

X O III Fe O O EnzB: O H H O OH 2 P450 O 2x O2 P450 H H H H H H H H H O HO HCO H 2 O O androstendione (X = O) estrone 2x H2O (œstrone) testosterone (X = H, bOH) DEMETHYLATIVE aromatisation by '' enzyme

NB. The involvement of a peroxyacetal during aromatase demethylation has recently been disputed, see: Guengerich J. Am. Chem. Soc. 2014, 136, 15036 (DOI). Steroid Ring Cleavage - Vitamin D & Azadirachtin

7 • vitamin D2 is biosynthesised by the photolytic cleavage of  -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 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) Biomimetic Cationic Cyclisations - Progesterone

• in 1971, W.S. Johnson utilized a biomimetic polyolefin cyclization in a pioneering & elegant total synthesis of the hormone progesterone – the substrate‟s preference for the ‘chair-chair-chair’ conformation provided the progesterone core with impressive stereoselectivity – the cascade was initiated by protonation of a tert-alcohol

O O O O O O O O O H

Cl(CH ) Cl 2 2 H H K CO o 2 3 TFA, 0 C H2O, MeOH OH [72%] H

O O O 1) O3, MeOH o CH2Cl2, -70 C H H H H2O/5% KOH, rt 2) Zn, AcOH H H H H [51%] H H [88%] O O O progesterone

– Johnson, Gravestock & McCarry J. Am. Chem. Soc. 1971, 93, 4332 (DOI) Biomimetic Cationic Cyclisations – Enantioselective

• Yamamoto has achieved several enantioselective cationic cascade cyclisations using a chiral

„Lewis acid assisted Brønsted acid‟ (LBA) prepared by mixing binol & SnCl4:

1) (R)-LBA (2eq) toluene, -78°C, 3d = . H H 2) BF OEt H 3 2 MeNO2 H H [65%, 77% ee] O SnCl O 4 iPr

(R)-LBA OH (R)-LBA (2eq) CH Cl , -78°C, 3d O = O H 2 2 O = H H [56%, 42% ee] H + minor diastereomers

– Yamamoto et al. J. Am. Chem. Soc., 1999, 121, 4906 (DOI) – Yamamoto et al. J. Am. Chem. Soc. 2001, 123, 1505 (DOI) Carotenoids – b-Carotene & vitamin A1

• Carotenoids (C40) are coloured pigments made by photosynthetic plants & certain algae, bacteria & fungi. Dietary ingestion by birds and further processing gives rise to bright feather pigments etc. – biosynthesised by head-to-head coupling of two geranylgeranyl PP units to give lycopersene:

OPP

GGPP (acceptor) OPP GGPP (donor) prephytoene synthase

H prephytoene PP (cf. presqualene PP) PPO NADPH NADP + PPi

lycopersene

[O]

b-carotene

– subsequent oxidative degradation (cf. ozonolysis!) gives retinal (mediator of vision) & vitamin A1:

11 (E) O OH

retinal vitamin A1 Primary Metabolism - Overview

Primary metabolism Primary metabolites Secondary metabolites

CO2 + H2O

PHOTOSYNTHESIS 1) 'light reactions': hv -> ATP and NADH 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 fatty acids prostaglandins CO2 lipids polyacetylenes O O acetyl coenzyme A aromatic compounds, polyphenols malonyl coenzyme A macrolides

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