Biosynthesis & Biomimetic Total Synthesis -

Primary Metabolism, Enzyme Cofactor Chemistry & Shikimate Metabolites

Alan C. Spiveya.c.spivey@imperial.ac.uk

Format & Scope of Lecture• What is biosynthesis?

– some definitions – phototrophs, chemotrophs; metabolism (catabolism/anabolism), 1° & 2° metabolites• Overview of primary metabolism → secondary metabolites

– photosynthesis & glycolysis → shikimate formation → shikimate metabolites– acetylCoA & the citric acid cycle → α-amino acids → penicillins, cephalosporins, alkaloids– acetylCoA → malonylCoA → fatty acids, prostaglandins, polyketides, macrolide antibiotics– acetylCoA → mevalonate → isoprenoids, terpenoids, steroids, carotenoids

• Biological/biosynthetic reactions – enzyme & cofactor chemistry– free energy source – ATP– C-C & C-O bond formation – CoASH, SAM, DMAP, biotin– oxidation – NAD+, FAD, haem iron oxo monooxygenases– reduction – NADPH– C-N bond formation – pyridoxal

• The shikimate biosynthetic pathway– aromatic amino acids: Phe, Tyr & Trp– ArC3 metabolites – coumarins, lignans & lignins

• mixed shikimate/malonylCoA (polyketide): flavonoids– ArC2, ArC1 & ArC0 metabolites

• mixed shikimate/mevalonate (isoprenoid): ubiquinones, menaquinones & tocopherols

Me MeMe

O

camphor

N

O

O

OH

H CO2H

H

clavulanic acid

N

N

Me

nicotine

O

O

O

OHpatulin

N

MeO

N

H

H

HHO

quinine

NMe

NH

HO2C

H H

lysergic acid

N

N N

N

O

Ocaffeine

O

O

Me

Me

androstenedione

CO2 H2O Pi N2hv

Metabolism & Natural Product Diversity

Phototrophs & Chemotrophs• Living organisms are not at equilibrium. They require a continuous influx of free energy to perform

mechanical work & for cellular growth/repair:

– Phototrophs (e.g. green plants, algae & photosynthetic bacteria): derive free energy from the sun viaphotosynthesis (‘CO2 fixation’):

• 35 × 1015 kg/year by green plants, which constitute 99% of Earths biomass (i.e. 1.7 × 1012 tons of dry matter)• 1g of carbon processed = >6250 litres of air

– Chemotrophs (e.g. animals, fungi): derive free energy by oxidising nutrients (carbohydrates, lipids, proteins) obtained from other organisms, ultimately phototrophs

• some bacteria & fungi require just D-glucose• mammals require sugars, essential amino acids (~half total used) & certain vitamins (enzyme co-factors or precursors)

• Degradation of the nutrients is coupled to the stoichiometric production of ‘high energy’ phosphate compounds, particularly adenosine triphosphate (ATP, see later). All metabolic function is underpinned by ATP energetic coupling.

• By analogy with a money-based economy, the metabolic cost of production of a given metabolite from another can be quantified in terms of ‘ATP equivalents’ defined as the # of moles of ATP consumed/produced per mole of substrate converted in the reaction or sequence

CO2 + H2Ohv

(CHO) + O 2 PHOTOSYNTHESIS

complex metabolites

CatabolismAnabolism

ATP

ADP + P i

NAD(P)H

NAD(P) + H

Nutrients

Energy &Building blocks

Cell components & Growth

simple products

Building Blocks

reductivebiosynthesis

energyrelease

oxidativedegredation

energystorage

CO2 + H2O

hv'CO2 fixation'(photosynthesis)

respiration

N2

'nitrogen fixation'(by diazatrophs, lightening,the Haber process)

CO2 + H2O

Natural product secondary metabolites

O2

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

• generally oxidative processes/sequences (glycolysis, Krebs cycle)– Anabolic or biosynthetic: primarily to create new cellular materials (1° & 2° metabolites)

• generally reductive processes/sequences

• These two types of process are coupled – one provides the driving force for the other:

Types of Metabolite & Biosynthesis• Biosynthesis is the term for the in vivo synthesis of metabolites/natural products:

– These are divided into two camps:• Primary metabolites: These are the universal and essential components for the survival of living organisms. e.g. sugars,

amino acids, nucleotides, ‘common’ fats and polymers such as proteins, DNA, RNA, lipids and polysaccharides• Secondary metabolites: Compounds produced by organisms which are not required for survival, many of which have no

apparent utility to the host organism. Frequently a given metabolite will only be produced in a single organism or in a set of closely related organisms. Provide a rich source of pharmacologically active compounds. e.g. shikimate derivatives, alkaloids, fatty acids, polyketides, isoprenoids

– Although the boundary is imprecise the term biosynthesis is most commonly applied, by organic chemists, to the in vivo synthesis of secondary metabolites:

“Now ever since Perkin, failing to make quinine, founded the dyestuffs industry, organic chemists have found the study of ‘natural products’ an inexhaustable source of exercises, which can be performed out of pure curiosity even when paid for in the hope of a more commercial reward. As a result the organic chemist’s view of nature is unbalanced, even lunatic but still in some ways more exciting than that of the biochemist. While the enzymologist’s garden is a dream of uniformity, a green meadow where the cycles of Calvin and Krebs tick round in disciplined order, the organic chemist walks in an untidy jungle of uncouthly named extractives, rainbow displays of pigments, where in every bush there lurks the mangled shapes of some alkaloid, the exotic perfume of some new terpene, or some shocking and explosive polyacetylene. To such a visionary both the diatetrynes* are equal prizes, to be set together as ‘natural products’. We shall do the same, but since to a more sober eye both are in a sense less ‘natural’ than, say, glycine or ATP, we may prefer the term ‘secondary metabolite’.”

Bu’Lock Adv. Appl. Microbiol. 1961, 3, 293

Primary Metabolism - Overview

CO2 + H2O

1) 'light reactions': hv -> ATP and NADH 2) 'dark reactions': CO2 -> sugars (Calvin cycle)

OHOHOHOOH

HO

glucose & other 4,5,6 & 7 carbon sugars

Primary metabolism Secondary metabolites

oligosaccharidespolysaccharidesnucleic acids (RNA, DNA)

phosphoenol pyruvate

glycolysis

CO2

HOOH

OHOOH

HO

POCO2

PO

erythrose-4-phosphate

SHIKIMATE METABOLITEScinnamic acid derivativesaromatic compoundslignans, flavinoids

+

shikimate

aromatic amino acids

aliphatic amino acids

peptidesproteinsCO2

Opyruvate

SCoA

Oacetyl coenzyme A

Citric acidcycle

(Krebs cycle)

ALKALOIDSpenicillinscephalosporinscyclic peptides

tetrapyrroles (porphyrins)

PHOTOSYNTHESIS

saturated fatty acidsunsaturated fatty acidslipids

FATTY ACIDS & POLYKETIDESprostaglandinspolyacetylenesaromatic compounds, polyphenolsmacrolides

ISOPRENOIDSterpenoidssteroidscarotenoids

SCoA

OCO2

malonyl coenzyme A

CoAS

O

O

HO

HOCO2

mevalonateacetoacetyl coenzyme A

Primary metabolites

Biological/Biosynthetic Reactions –Enzyme Catalysis & Cofactors

• Most biosynthetic steps are catalysed by specific, individual enzymes. They generally perform familiar processes such as oxidation, reduction, alkylation, hydrolysis, acylation, hydroxylation, elimination etc.

• Different enzymes carrying out related reactions often employ common co-factors: small organic functional fragments and/or metal ions. e.g.

– FREE ENERGY RELEASING COUPLE: Adenosine triphosphate (ATP)

– C-C & C-O BOND FORMATION: Coenzyme A (CoASH); S-adenosyl methionine (SAM); dimethylallylpyrophosphate (DMAP); biotin

– OXIDATION: NAD(P)+; FAD; Haem iron oxo species (e.g. P450)

– REDUCTION: NAD(P)H; (FADH2)

– C-N BOND FORMATION: Pyridoxal

Free Energy Releasing Couple - ATP• Adenosine triphosphate (ATP)

– phosphorylation of an alcohol by adenosine diphosphate (ADP) is highly exothermic (i.e. liberates energy):

– The phosphorylated alcohol (ROP) is then activated towards nucleophilic displacement:

– So, overall the endothermic process ROH + Y- → RY + OH- has been achieved by ‘coupling’ the process to the ‘hydrolysis of ATP’

– The situation is analogous to the use of tosylate activation to achieve nucleophilic displacement of an alcohol

– In general, the exothermicity associated with phosphorylation shifts the equilibria of ‘coupled’ process by a factor of ~108

N

NN

N

O

NH2

OHOH

OPOPOPOOOOOO

HO

PPPO-

N

NN

N

O

NH2

OHOH

OPOPHO

OOOO

PPO-

+ ROH PRO

OO

OH+ ∆G° = -31 kJmol-1

enzyme

Mg2+ or Mn2+

adenosine triphosphate (ATP) ADP

-OP

Nu + ROP R-Nu + OP OP = Pi = orthophosphate = P OHO

OO

Acylation & C-C Bond Formation α to C=O – CoASH• Coenzyme A (CoASH)

– Coenzyme A acts as an acyl transfer/α-carbon activation reagent by forming reactive acyl thioesters:

– Acyl CoA derivatives can act as nucleophiles or electrophiles depending on the circumstances– These modes of reactivity inherent properties of alkyl thioesters:

• The good leaving group ability of RS- (cf. RO-) reflects: pKa (RSH) ~10 cf. pKa (ROH) ~16• The enhanced acidity of protons α to the carbonyl of thioesters cf. normal esters reflects the poor orbital overlap

between the lone pairs on sulfur (nS) [cf. nO] and the carbonyl anti bonding molecular orbital π*C=O

N

NN

N

O

NH2

OHO

OPOPO

OOOO

coenzyme A (CoASH)

H

O

NH

O

NH

HS

PHO

OO

HO

pantothenate

X

OR

SCoA

OR

Y

Y

OR

SCoA

OR

ACYL TRANSFER

R'-LG

R'

α-CARBON ALKYLATION

SCoA

OR

ALDOL REACTIONS

SCoA

OR CLAISEN-type C-ACYLATION

O

OH

SCoAO

O

C O

XR'

nOp

π*C=O

OX

R'

nX-π*C=O resonance makes carbonyl less susceptible to enolisationSulfur is in the 2nd period

so its lone pair has poor size/energy match with the π*C=O orbitalHence: pKa(RCH2COSR’) ~20 cf. RCH2COOR’ ~25

i.e. α to a thioester is similar to α to a ketone

RR

H

H

H

H

B:

OHX

R'

RH

+ BH

Methylation/Dimethylallylation – SAM & DMAP• S-Adenosyl methionine (SAM)

– SAM acts as a versatile O-, C-, N- & S- methylating reagent in vivo

– Equivalent to performing an SN2 methylation using MeI in the laboratory

• Dimethylallyl pyrophosphate (DMAP)– DMAP acts a dimethylallylating reagent – the pyrophosphate (+ Mg2+/Mn2+) is an excellent leaving group

– Equivalent to performing an SN2 allylation using allyl bromide in the laboratory

N

NN

N

O

NH2

OHOH

SO2C

S-adenosyl methionine (SAM)

Me

NH3

Nu

Nu-Me

methionine

e.g.

R2N

O

O

Me SR2

OMe

Me SR2

Me SR2

O

Me

OH

Me

R2NMe

Nu

OPOPO

OOOO

dimethylallyl pyrophosphate (DMAP)

Nu

Carboxylation – Biotin

• Biotin– Biotin in the presence of bicarbonate, ATP and Mg2+ enables nucleophile carboxylation in vivo:

– a very similar reaction can be carried out in the laboratory• H. Sakurai et al. ‘α-Carboxylation reaction of carbonyl compounds with bromomagnesium ureide-carbon dioxide

adducts’ Tetrahedron Lett. 1980, 21,1967

biotin (R = carrier protein)

Nu

NHHN

S

O

HH

O

NHR

NHN

S

O

HH

O

NHR

HCO3ATPMg2+

O

OMg

Nu O

O

HN NH

O 1) MeMgBr (2 eq)2) CO2

N N

OMgBr

BrMg N N

O

O

O

O

OBrMg MgBr

white powder

O

DMF110C

O O

O

CH2N2

[74%]

MgBrO O

OMe

Oxidation – NAD+

• Nicotinamide-adenine dinucleotide (NAD+) [and its phosphorylated analogue (NADP+)] are mediators of biological oxidation (e.g. alcohol to ketone oxidation)

– In general, the couple NAD+/NADH is used by enzymes in catabolic oxidation (degradation)– The reagent is a stereospecific hydride acceptor:

– Different enzymes show different absolute specificities but are generally specific for the pro-R or pro-S hydrogens both for removal and delivery

– The Oppenauer oxidation is a similar (non-stereoselective) laboratory reaction:• for asymmetric variants see: K. Nishide et al. Chirality 2002, 14, 759

N

NN

NO

NH2

OROH

OPOPO

OOOO

nicotinamide-adenine dinucleotide (NAD+) R = HNB. NADP+: R = PO3H-

O OHOH

N

O

NH2+ H

- H N

O

NH2

HRHS

NB. H = H + 2e

R OH

H H

R O

H

stereospecific:

N

ONH2

N

ONH2

HH

HR O

H+ H

NADH

NAD+ NADH

R OH

H H

R O

HAl(OtBu)3

H

OAl

O

RH OtBu

OtBuO

HO H

++ Al(OtBu)3

Oxidation – FAD• Flavin adenine dinucleotide (FAD) is also a mediator of biological oxidation (e.g. alcohol to

ketone oxidation, alkane dehydrogenation to alkene)– Unlike NAD+, which readily diffuse from enzyme to enzyme, FAD is usually tightly bound to a given enzyme

– Re-oxidation of the FADH2 back to FAD is generally by molecular oxygen via single electron transfers(SETs). The intermediate superoxide radical anion and peroxyflavin can also mediate hydroxylationand epoxidation reactions:

N

NN

NO

NH2

OHOH

OPOPO

OOOO

flavine-adenine dinucleotide (FAD)

+ H2

- H2

R OH

H H

R O

H

FADH2

OH

OH

OHN

NN

HN OO

NHN

NH

HN OO

NN N

HN OO

FAD FADH2

NHN NH

HN OO

isoalloxazine

FADH2

NN

N

HN OO

NHN

NH

HN OO

- H

NHN

N

HN OO

O O

O ON

HNN

HN OO

NHN

N

HN OO

OO - H

NN

N

HN OO

OO

O O+

FADsuperoxideradical anion

peroxyflavin

peroxidedianion

Biomimetic Oxidation using FAD Models• FAD model system

– S. Shinkai Chem. Lett. 1982, 812 & Bull. Soc. Chim. Fr. 1983, 56, 1694

• Peroxyflavin model systems– J. E. Bäckvall Chem. Eur. J. 2001, 7, 297

OH

ZrCl4

O

N NN N

N OO

~100%

M4+

(1 eq)

N NHN NH

N OO

H H(1 eq)

intermolecular oxidation

NPh

NB. simple N-alkylated nicotinamide salts (cf. NAD+) perform poorlye.g.

CONMe2

X

NHEtN

N

N OO

FFN N

O(2.8 mol%)

H2O2 (2.6 eq), MeOH, 50 min

NHEtN

N

N OO

FF

via

OOH

>90%

Oxidation – Haem Iron oxo Species (P450)• Haem iron oxo species e.g. in cytochrome P450 (a ubiquitous heam monooxygenase) are also

mediators of biological oxidation (e.g. phenolic coupling, epoxidation, hydroxylation):

– The porphyrin ring acts as a tetradentate ligand for the octahedral iron. The two axial positions are occupied by an enzyme amino acid ligand (typically a histidine nitrogen) and hydroxy/hydroperoxy residue respectively

– Ferricyanide effects similar oxidative processes in the laboratory (e.g. phenolic coupling)• D.H.R. Barton & G.W. Kirby Proc. Chem. Soc. 1960, 392

N N

N N

CO2HHO2C

FeV

L

haem Fe(V) oxo species

N N

N N

CO2HHO2C

FeIII

L

haem Fe(III) species

N N

N N

CO2HHO2C

FeIV

L

OHH

O2

OHH

HHO+

O

O

MeO

NMeK3Fe(CN)6

OH

MeO

NMe0.92% (!)HO

O

MeO

NMeHO

narwedinedesmethyl belladine

Reduction - NADPH• Dihydro-nicotinamide-adenine dinucleotide phosphate (NADPH) [and its de-phosphorylated

analogue (NADH)] are mediators of biological reduction (e.g. ketone to alcohol reduction) – In general, the couple NAPH/NADP+ is used by enzymes in anabolic reduction (biosynthesis)– The reagent is a stereospecific hydride donor:

– As for the reverse process, different enzymes show different absolute specificities but are generally specific for the pro-R or pro-S hydrogens both for removal and delivery

– NADPH acts like a biochemical equivalent of ‘laboratory’ metal hydride reductants (e.g. LiAlH4, NaBH4) or their chiral equivalents (e.g. CBS-borane):

N

NN

NO

NH2

OROH

OPOPO

OOOO

Hydro-nicotinamide-adenine dinucleotide (NADPH) R = PO3H-

NB. NADH: R = H

O OHOH

N

O

NH2

+ H

- H

N

O

NH2

NB. H = H + 2e

R O

H H

stereospecific:

N

ONH2

N

ONH2

HH

R O

H

HRHS

R OH

H HH

NADP+

NADPH NADP+

O B-Me CBS·BH3(10 mol%)0.6 eq BH3·DMS

CH2Cl2, rt, 45 minN

O

H PhPh

BMeBH3

H

B-Me CBS·BH3

HO

100% yield, 95% ee

Biomimetic Reduction using NADH Models• J.G. deVries, R.M. Kellog J. Am. Chem. Soc. 1979, 101, 2759

• M. Seki et al. J. Am. Chem. Soc. 1981, 103, 4613

N

O

NH

H2NOC

N

O

H

CONH2O

O

OEt

O

OEtOHH

67% yield, 98% ee

Mg(ClO4)2, MeCN:CH3Cl (3:1), rt, 1h

H HH H

O

O

OEtO

OEtHHO

61% yield, 86% ee

Mg(ClO4)2, MeCN, rt, 72h

NH

OO

O

HN

OO

OO

N

RHR HHH

Transamination - PLP• Pyridoxine (vitamin B6) → pyridoxal-5’-phosphate (PLP)

– PLP forms imines (Schiffs bases) with primary amines. This forms the basis of in vivo transamination of α-ketoacids to give α-amino acids (& also racemisation/decarboxylation processes, see ‘alkaloids’)

– The α-carbon protonation is stereospecific and gives the (S) configured chiral centre – Jørgensen has developed a catalytic enantioselective laboratory equivalent of this process:

• K.A. Jørgensen et al. Chem. Comm. 2003, 2602

HO

Pyridoxine (vitamin B6)N

OH

OH

pyridoxaminephosphate

Enz-NH2

NH

O

NH3OP

R CO2

R CO2

O

NH

O

NOP

R CO2

NH

O

NOP

R CO2

H

NH

O

NOP

R CO2H

NH3

HNH

O

NOPH H

Enz-NH3 Enz-NH2

H

Enz

H2Opyridoxal phosphate

bound as imine

H

transaminase

imineexchange

imineformation

N

NH2

NH

O

O

OMe

+N

ZnN

OO

TfO OTf

1) Zn-cat (20 mol%) MeNO2, 40h, rt

2) Boc2O, Et3N, MeOH 50°C

Zn-catNH

O

OMe

N

O

+

15% yield, 37%ee

NHBocH

Primary Metabolism - Overview

CO2 + H2O

1) 'light reactions': hv -> ATP and NADH 2) 'dark reactions': CO2 -> sugars (Calvin cycle)

OHOHOHOOH

HO

glucose & other 4,5,6 & 7 carbon sugars

Primary metabolism Secondary metabolites

oligosaccharidespolysaccharidesnucleic acids (RNA, DNA)

phosphoenol pyruvate

glycolysis

CO2

HOOH

OHOOH

HO

POCO2

PO

erythrose-4-phosphate

SHIKIMATE METABOLITEScinnamic acid derivativesaromatic compoundslignans

+

shikimate

aromatic amino acids

aliphatic amino acids

peptidesproteinsCO2

Opyruvate

SCoA

Oacetyl coenzyme A

Citric acidcycle

(Krebs cycle)

ALKALOIDSpenicillinscephalosporinscyclic peptides

tetrapyrroles (porphyrins)

PHOTOSYNTHESIS

saturated fatty acidsunsaturated fatty acidslipids

FATTY ACIDS & POLYKETIDESprostaglandinspolyacetylenesaromatic compounds, polyphenolsmacrolides

ISOPRENOIDSterpenoidssteroidscarotenoids

SCoA

OCO2

malonyl coenzyme A

CoAS

O

O

HO

HOCO2

mevalonateacetoacetyl coenzyme A

Primary metabolites

Shikimate Metabolites

SHIKIMATE METABOLITES

NHNH3

O2C

(S)-tryptophan

CO2

OH

NH3H

CO2

NH3H

(S)-tyrosine (S)-phenylalanine

MeOOMe

OMe

OO

O

OH

H

H

H

O

O

O

MeOOH

scopoletin

OH

OH

3

Me

α-tocopherol (vitamin E)

Me

podophyllotoxin

menaquinone (vitamin K2)

O

OMe

Hn

OH

O

HO OH

epigallocatechin (EGC)

OHHO

HOH

Biosynthesis of Shikimate

• Phosphoenol pyruvate & erythrose-4-phosphate are converted to shikimate and its phosphatederivative:

– The detailed mechanisms of these steps have been studied intensively. Most are chemically complex and interesting. For more details see:

• J. Mann Chemical Aspects of Biosynthesis Oxford Chemistry Primer No. 20, 1994 (key details)• E. Haslam Shikimic Acid – Metabolism and Metabolites Wiley, 1993 (full details and primary Lit. citations)

phosphoenol pyruvate(PEP)

OOH

HO

PO

CO2

PO

erythrose-4-phosphate+

PO

CO2

HOOH

OH

O PO

CO2

HOO

OH

O

CO2

HO OH

O

OH

CO2

OOH

OH

HOCO2

OOH

OH

dehydroshikimate 5-dehydroquinate

CO2

HOOH

OH

shikimate

NADPH

NAD

NADH

NADP

Pi

DAHP

non-concertedsyn-elimination

CO2

POOH

OH

shikimate phosphate

ATP

ADP H2O

Pi

H

Shikimate → Tryptophan, Tyrosine & Phenylalanine• Shikimate phosphate → chorismate → anthranilate → tryptophan

• Chorismate → prephenate → tyrosine & phenylalanine– NB. The enzyme chorismate mutase which mediates the conversion of chorismate to prephenate is the only

known ‘Claisen rearrangementase (!)’

PiCO2

POOH

OH

shikimate phosphate

CO2

PO

PEP

+

CO2

POOH

O CO2

HPi CO2

OHO CO2

chorismate

CO2

anthranilate

NH2 NHNH3

O2C

(S)-tryptophan[78 ATP equivs]5-EPS-3-P

non-concerted1,4-anti-elimination

'NH3'

ArC0

ribose derived

glyphosate(Roundup)

PO NH2

CO2

NAD

NADH

CO2

OHO CO2

chorismate

Claisenrearangement

O2C

OHprephenate

O

CO2

CO2

H2O CO2

CO2

OH

NH3H

CO2

NH3H

(S)-tyrosine[62 ATP equivs]

(S)-phenylalanine[65 ATP equivs]

O2C

OH(S)-arogenate

CO2

'NH3'

H3N H

ArC3

ArC3

Tyrosine/Phenylalanine → ArC3 Metabolites• Tyrosine & phenylalanine → cinnamate derivatives → ArC3 metabolites

– coumarins, lignans (stereoselective enzymatic dimerisation) & lignins (stereorandom radical polymerisation)

CO2

X

NH3H

X

H

X = OHX = H

CO2

O

O

MeOOH

scopoletin (a coumarin)germination stimulant

MeOOMe

OMe

OO

O

OH

H

H

H

podophyllotoxin (a lignan)natural product used to treat worts

Oand

OH

CO2

MeO

ferulate

O

O

OH

OMe

HO

MeO

pinoresinol (a lignan)

PAL

phenylalanineammonia lyase

fragment of lignin polymer'woody' component of cell walls

OOMe

OHMeO

O

O

OMeOH

O

OMeO

HOHO

OH

n x ArC3

2 x ArC3

ArC3

HH

H

H

Biomimetic Lignan Synthesis• Oxidative dimerisation of cinnamyl alcohols gives symmetric furanofuran lignans

– review: R,C.D. Brown, N.A. Swain Synthesis 2004, 811

O

O

OH

OMe

HO

MeOsyringaresinol

IN VIVO: oxidasesee: N.G. Lewis et al. Science 1997, 275, 362

IN VITRO: CuSO4 (cat.), O2(g), acetone-H2O [90%]see: B. Vermes et al. Phytochem. 1991, 30, 3087

HO

MeO

OH

2 x coniferyl alcohol

O

OMe

O

MeO

OH

HO

O

OMe

O

MeO

OH

HO

OMe

OMe

OMe

HH

H

H

OMe

OMe OMe

OMe

Tyrosine/Phenylalanine → Flavonoids• 4-Hydroxycinnamic acid → flavonoids: flavanones, flavanonols, flavones & anthocyanins

– Glycosides of these ArC3 metabolites (esp. anthocyanins) constitute coloured pigments in flowers and insects. They also confer bitter and astringent flavours (e.g. tannins in tea are polymerised flavonoids)

– NB. ‘Mixed’ biosynthetic origin: shikimate/malonylCoA (polyketide)

OH

CO2

OH

O CoA

OH

O

OO

O

CoA

SCoAO CO2

malonyl coenzyme A

3 x

OH

O

OH

HO OH

chalconesynthase

H

OH

O

O

HO OH

flavanones

ORRO

OH

O

O

HO OH

flavones

ORRO

OH

O

O

HO OH

flavanonols

ORRO

ArC3OH

ArC3

ArC3

OH

O

HO OH

anthocyanins

ORRO

OH

ArC3

Chorismate → Coenzymes Q & Vitamins E & K• Chorismate → p- & o-hydroxybenzoic acids → coenzymes Q & vitamins E & K

– NB. ‘Mixed’ biosynthetic origin: shikimate/mevalonate (isoprenoid)

CO2

OHO CO2

chorismateCO2

O

pyruvate

H

CO2

OH

CO2

OHR

OHRMeO

O

O

Me

MeO

MeO

Hn

ubiquinones (coenzymes Q)lipid-soluble electron transport

O2C

OH

prephenate

O

CO2CO2

OH

O

H

CO2

OH

OO

CO2

OH

CO2 OH

CO2

homogentisateOH

OHH

3

R

OH

OH

3

R1

tocopherols (vitamins E)electron transport, antioxidant

CO2

O CO2

isochorismate

OH

OH2

menaquinones (vitamins K)cofactors for plasma proteins

essential for blood clottingCO2

O

O SCoA

HO

OHCO2

O

OR

O

OMe

Hn

R2 R1, R2 = H or Me

R

isoprenoid

isoprenoid

isoprenoid

ArC1

ArC0

ArC1

H

O2CO

CO2 CO2

α-ketoglutarate (αKG)

CO2

O

CO2

αKG

Primary Metabolism - Overview

CO2 + H2O

1) 'light reactions': hv -> ATP and NADH 2) 'dark reactions': CO2 -> sugars (Calvin cycle)

OHOHOHOOH

HO

glucose & other 4,5,6 & 7 carbon sugars

Primary metabolism Secondary metabolites

oligosaccharidespolysaccharidesnucleic acids (RNA, DNA)

phosphoenol pyruvate

glycolysis

CO2

HOOH

OHOOH

HO

POCO2

PO

erythrose-4-phosphate

SHIKIMATE METABOLITEScinnamic acid derivativesaromatic compoundslignans

+

shikimate

aromatic amino acids

aliphatic amino acids

peptidesproteinsCO2

Opyruvate

SCoA

Oacetyl coenzyme A

Citric acidcycle

(Krebs cycle)

ALKALOIDSpenicillinscephalosporinscyclic peptides

tetrapyrroles (porphyrins)

PHOTOSYNTHESIS

saturated fatty acidsunsaturated fatty acidslipids

FATTY ACIDS & POLYKETIDESprostaglandinspolyacetylenesaromatic compounds, polyphenolsmacrolides

ISOPRENOIDSterpenoidssteroidscarotenoids

SCoA

OCO2

malonyl coenzyme A

CoAS

O

O

HO

HOCO2

mevalonateacetoacetyl coenzyme A

Primary metabolites