The Biochemical Synthesis of

‘Alliin’ by Garlic

Jill Hughes

School of Biological Sciences

University of Liverpool

Hamish Collin, Brian Tomsett, Meriel Jones, Rick Cosstick, Angela Tregova, and Gloria Van der Werff

What is Alliin?

O

CH2=CHCH2SCH2CH(COOH)(NH2)

(+)-S-2-Propenyl-L-cysteine S-oxide

also called alliin, allyl cysteine sulphoxide or allyl CSO

Alliin is a three carbon allyl group linked to the oxidised sulphur atom of the amino acid cysteine

Our aim is to find out where these

carbon skeletons and the sulphur originate!

What is Alliin?

Alliin is one member of a group of related flavour precursors - the S -alk(en)yl-L-cysteine sulphoxides

O

RSCH2CH(COOH)(NH2)

These cysteine sulphoxides are present in varying amounts and proportions in different Allium species

R = generalised alk(en)yl group

The four common CSOs in Allium sp.

O

Alliin CH2=CHCH2SCH2CH(COOH)(NH2) and

S-(E)-1-Propenyl-L-cysteine S-oxide (also called propenyl cysteine sulphoxide, propenyl CSO or isoalliin)

O CH2CH=CH2SCH2CH(COOH)(NH2)

S-Propyl-L-cysteine S-oxide (also called propyl cysteine sulphoxide, propyl CSO or propiin)

O CH2CHCH2SCH2CH(COOH)(NH2)

S-Methyl-L-cysteine S-oxide (also called methyl cysteine sulphoxide, methylCSO or methiin) O

CH3SCH2CH(COOH)(NH2)

Alliinase

When garlic tissue is damaged, the flavour precursors are brought into contact with the enzyme Alliinase

Alliinase is a C-S lyase and breaks the bond within the cysteine moiety

O

2CH2=CHCH2SCH2CH(COOH)(NH2)

O

CH2=CHCH2S SCH2 CH= CH2

+ 2 CH3(CO)(COOH)

alliinase

alliicin2-propenyl-2-propenethiosulphinate

pyruvate

Alliinase

alliicin (and its breakdown products) are responsible for the odour of freshly crushed garlic and the health giving properties

Other cysteine sulphoxides are lysed by alliinase to give their respective volatiles.

O

RSCH2CH(COOH)(NH2)

RS=O + CH3(CO)(COOH)

alliinase

R1S-S-R2 etc.

CSO biosynthetic pathway

SO42- SO3

2- SO22- cysteine

glutathione(γ-glu-cys-gly)

S-methyl-γ-glu-cys

gly

S-methylcysteine

methiin

glu

trans-peptidase

oxidase

S-2-CP-γ-glu-cys

gly

S-trans-1-propenyl-γ-glu-cys

S-trans-1-propenylcysteine

oxidase

trans-peptidaseglu

HCOOH

S-trans-1-propenylcysteine sulphoxide(isoalliin)

S-methylglutathione

S-(2-carboxypropyl)-glutathioneS-allylglutathione

S-allyl-γ-glu-cys

gly

S-allylcysteine

glu

trans-peptidase

oxidase

alliin

Allyl-S(unknown sources)

valine & methacrylateserine

oxidase

S-allylcysteine

S-allyl-cysteine sulphoxide(alliin)

What is known already?

In 1989, Jane Lancaster and her team fed labelled sulphate to cut onion leaves. From her results, she proposed that the cysteine sulphoxides were made by conjugation of the alk(en)yl moiety ( R ) to glutathione.

RSCH2CHCONHCH2COOH

NH2

CO R- cys-gly R-C-

G

l CH2 glu E

CH2

CH(NH )(COOH)

A gamma-glutamyl, cysteine, glycine peptide

How to study a metabolic pathway

Alliin and other CSOs are secondary metabolites. Non-essential for cell function but evolved with a selective advantage

What is the first committed step? Is this linked to sulphur availability, as with

onions, stage of development or tissue type regulated? Is the pathway controlled at the transcription,

post-transcription or translation level?

Where in the cell does this take place? Are gamma-glutamyl peptides

compartmentalised? Can we separate Alliinase containing cells from

Alliin synthesising cells?

What are the metabolites of this biosynthetic pathway? How do we look at a complex network of

interacting pathways without perturbing the system

Initial HPLC approach to identify CSOs and possible intermediates

Summary of preparative workSolvent extraction methods (based on amino acid extraction methods) and HPLC have been developed previously in this laboratory and used to estimate cysteine

sulphoxides in onion (Allium cepa). These methods have been further developed and improved for larger scale analysis of garlic extracts.

MethodTissue is extracted overnight in 12:5:3 Methanol:Chloroform:WaterAfter addition of an equal volume of 9:11 Chloroform:Water, the aqueous extract is freeze dried, 50l is applied to a Phenomenex MAX-RP HPLC column with 0.03M HCl mobile phase run at 0.9ml/min@RT

Standards for HPLC

As many as possible:

Synthesised and confirmed by NMR and mass spectrometry

Purchased - amino acids, glutathione, gamma glutamyl cysteine

Gifts - gamma glutamyl allyl cysteine (Thomas Haffner)

Modified - oxidation of CPC and gamma glutamyl cysteine

Synthesis of standards

Alliin synthesised from Allyl cysteine made in the laboratory from cysteine and Allyl bromide (method based of Stoll and Seebeck, 1949)

Propyl cysteine sulphoxide and n-Butyl cysteine sulphoxide similarly synthesised from Propyl cysteine (made in the laboratory from cysteine and 1-bromo-propane) and n-Butyl cysteine (made in the laboratory from cysteine and 1-bromo-butane)

Methyl cysteine sulphoxide and Ethyl cysteine sulphoxide synthesised by oxidation of methyl cysteine and ethyl cysteine (Sigma chemicals) respectively

Carboxy-propyl cysteine (CPC) synthesised from cysteine and methacrylic acid by a method based on that described by Schoberl, 1947 and Schoberl and Wagner, 1960

Propenyl cysteine sulphoxide

Synthesised by oxidation of propenyl cysteine.

O

CH2CH=CH2SCH2CH(COOH)(NH2)

Propenyl cysteine was synthesised by ‘base isomerisation’ with tertiary butoxide of allyl cysteine by a method based on that described by Carson and Wong(1963). This method is described for the production of cis- propenyl cysteine sulphoxide, however it should theoretically to produce both ‘cis’ and ‘trans’ isomers.

It was decided to search the reaction products for the biological ‘trans’ isomer. This was successful and this synthetic method has been used, together with repeated preparative HPLC, to purify (+)-S-1-E- propenyl-L- cysteine sulphoxide from the reaction products.

Confirmation of structure has been made by NMR and Mass Spectroscopy and comparison with synthetic alliin and both onion and garlic extracts (HPLC).

Synthesis of standards

The structure and purity of synthesised compounds have been confirmed by NMR and Mass Spectroscopy.

The chemical synthetic methods described will enable future synthesis of these and similar compounds using isotopically labelled starting material

Retention times of standard compounds

0.03M HCl, 0.9ml/min

Serine 3.03

Methyl cysteine sulphoxide 3.28

Glutamic acid 3.53

Cysteine 3.58

Ethyl cysteine sulphoxide 3.94

Methyl cysteine 4.64

Allyl cysteine sulphoxide 4.92

CPC'oxidised' 5.29 & 5.54

Propenyl cysteine sulphoxide 5.75

Propyl cysteine sulphoxide 6.4

Valine 6.43

Glutathione 7.1

Gamma glutamyl cysteine 8.45

Gamma glutamyl allyl cysteine'oxidised' 8.61 &10.08

Ethyl cysteine 8.79

n-Butyl cysteine sulphoxide14.95 & 15.69

Allyl cysteine 15.16

CPC'oxidised' 15.89

Propenyl cysteine 19.51 & 20.8

Propyl cysteine 25.16

Gamma glutamyl allyl cysteine 44

Garlic tissue analysis

The HPLC flavour precursor profiles of various Allium species and garlic tissue types have been produced.

Alliin is present in garlic leaf, bulb and roots but is not observed in significant levels in undifferentiated garlic callus

Upon differentiation of the callus, the new roots start to produce alliin.

Precursor feeding experiments 1

Based on ‘precursor feeding to onion callus’ experiments by Selby et.al. We are starting with garlic callus

Garlic callus (variety ‘Printanor’) is now routinely cultured in this laboratory and available in sufficient quantities for precursor feeding experiments.

There is little background interference in undifferentiated callus

It is relatively easy to introduce the substrate

HPLC traces of garlic tissue

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Printanor clove

Printanor callus

Printanor roots fromdifferentiating callus

Printanor plant roots

Printanor leaves

Time

alliin

isoalliin

isoalliin

alliin

isoalliin

isoalliin

Note: The same amount of tissue was extracted for each of these traces

Absorbance

215nm

alliin

alliin

alliin

Concentration of flavour precursors in garlic tissue

Tissue Precursor concentration (mM)

Alliin Isoalliin

Clove 50 3

Callus 1 not detectable

Callus root 3 1

Root 5 not detectable

Leaf 50 10

Precursor feeding experiments 2

In initial experiments both undifferentiated and differentiating callus have been maintained for up to 15 days on a phytogel/MS medium, with and without sulphate, containing a range of potential precursors to the synthesis of Alliin (at different concentrations).

This method of substrate feeding will only give a positive result if: the substrate gets into the cell enzymes are present that utilise the

substrate the product is not further metabolised

So far we have shown that both Allyl cysteine and Allyl thiol can be taken

up by callus and converted to Alliin

Precursor feeding experiments 3

This work is in progress

It is possible to extend these preliminary experiments to other tissues using labelled precursors and linking HPLC to Mass Spectroscopy.