1. 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

2. What is Alliin? O  CH 2 =CH  CH 2  S  CH 2  CH(COOH)(NH 2 ) (+)-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!

3. What is Alliin? zAlliin is one member of a group of related flavour precursors - the S -alk(en)yl-L-cysteine sulphoxides O  R  S  CH 2  CH(COOH)(NH 2 ) zThese cysteine sulphoxides are present in varying amounts and proportions in different Allium species R = generalised alk(en)yl group

4. The four common CSOs in Allium sp.  O Alliin  CH 2 =CH  CH 2  S  CH 2  CH(COOH)(NH 2 ) and  S-(E)-1-Propenyl-L-cysteine S-oxide (also called propenyl cysteine sulphoxide, propenyl CSO or isoalliin) O  CH 2  CH=CH 2  S  CH 2  CH(COOH)(NH 2 ) zS-Propyl-L-cysteine S-oxide (also called propyl cysteine sulphoxide, propyl CSO or propiin) O  CH 2  CH  CH 2  S  CH 2  CH(COOH)(NH 2 ) zS-Methyl-L-cysteine S-oxide (also called methyl cysteine sulphoxide, methylCSO or methiin) O  CH 3  S  CH 2  CH(COOH)(NH 2 )

5. Alliinase zWhen garlic tissue is damaged, the flavour precursors are brought into contact with the enzyme Alliinase zAlliinase is a C-S lyase and breaks the bond within the cysteine moiety O  2CH 2 =CH  CH 2  S  CH 2  CH(COOH)(NH 2 ) O  CH 2 =CH  CH 2  S  S  CH 2  CH= CH 2 + 2 CH 3 (CO)(COOH) alliinase alliicin 2-propenyl-2-propenethiosulphinate pyruvate

6. Alliinase zalliicin (and its breakdown products) are responsible for the odour of freshly crushed garlic and the health giving properties zOther cysteine sulphoxides are lysed by alliinase to give their respective volatiles. O  R  S  CH 2  CH(COOH)(NH 2 ) R  S=O + CH 3 (CO)(COOH) alliinase R 1  S-S- R 2 etc.

7. CSO biosynthetic pathway SO 4 2- SO 3 2- SO 2 2- 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- peptidase glu HCOOH S-trans-1-propenylcysteine sulphoxide (isoalliin) S-methylglutathioneS-(2-carboxypropyl)-glutathioneS-allylglutathione S-allyl-γ-glu-cys gly S-allylcysteine glu trans- peptidase oxidase alliin Allyl-S (unknown sources) valine & methacrylate serine oxidase S-allylcysteine S-allyl-cysteine sulphoxide (alliin)

8. 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. R  S  CH 2  CH  CO  NH  CH 2  COOH  NH 2  CO R- cys-gly R-C-G   l CH 2 glu E  CH 2  CH(NH )(COOH) A gamma-glutamyl, cysteine, glycine peptide

9. How to study a metabolic pathway Alliin and other CSOs are secondary metabolites. Non-essential for cell function but evolved with a selective advantage zWhat is the first committed step? yIs this linked to sulphur availability, as with onions, stage of development or tissue type yregulated? yIs the pathway controlled at the transcription, post-transcription or translation level? zWhere in the cell does this take place? yAre gamma-glutamyl peptides compartmentalised? yCan we separate Alliinase containing cells from Alliin synthesising cells? zWhat are the metabolites of this biosynthetic pathway? yHow do we look at a complex network of interacting pathways without perturbing the system

10. Initial HPLC approach to identify CSOs and possible intermediates Summary of preparative work Solvent 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. Method Tissue is extracted overnight in 12:5:3 Methanol:Chloroform:Water After addition of an equal volume of 9:11 Chloroform:Water, the aqueous extract is freeze dried, 50  l is applied to a Phenomenex MAX-RP HPLC column with 0.03M HCl mobile phase run at 0.9ml/min@RT

11. Standards for HPLC zAs many as possible: zSynthesised and confirmed by NMR and mass spectrometry zPurchased - amino acids, glutathione, gamma glutamyl cysteine zGifts - gamma glutamyl allyl cysteine (Thomas Haffner) zModified - oxidation of CPC and gamma glutamyl cysteine

12. Synthesis of standards zAlliin synthesised from Allyl cysteine made in the laboratory from cysteine and Allyl bromide (method based of Stoll and Seebeck, 1949) zPropyl 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) zMethyl cysteine sulphoxide and Ethyl cysteine sulphoxide synthesised by oxidation of methyl cysteine and ethyl cysteine (Sigma chemicals) respectively zCarboxy-propyl cysteine (CPC) synthesised from cysteine and methacrylic acid by a method based on that described by Schoberl, 1947 and Schoberl and Wagner, 1960

13. Propenyl cysteine sulphoxide zSynthesised by oxidation of propenyl cysteine. O  CH 2  CH=CH 2  S  CH 2  CH(COOH)(NH 2 ) zPropenyl 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. zIt 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. zConfirmation of structure has been made by NMR and Mass Spectroscopy and comparison with synthetic alliin and both onion and garlic extracts (HPLC).

14. Synthesis of standards zThe structure and purity of synthesised compounds have been confirmed by NMR and Mass Spectroscopy. zThe chemical synthetic methods described will enable future synthesis of these and similar compounds using isotopically labelled starting material

15. Retention times of standard compounds 0.03M HCl, 0.9ml/min Serine3.03 Methyl cysteine sulphoxide3.28 Glutamic acid3.53 Cysteine3.58 Ethyl cysteine sulphoxide3.94 Methyl cysteine4.64 Allyl cysteine sulphoxide4.92 CPC'oxidised'5.29 & 5.54 Propenyl cysteine sulphoxide5.75 Propyl cysteine sulphoxide6.4 Valine6.43 Glutathione7.1 Gamma glutamyl cysteine8.45 Gamma glutamyl allyl cysteine'oxidised'8.61 &10.08 Ethyl cysteine8.79 n-Butyl cysteine sulphoxide14.95 & 15.69 Allyl cysteine15.16 CPC'oxidised'15.89 Propenyl cysteine19.51 & 20.8 Propyl cysteine25.16 Gamma glutamyl allyl cysteine44

16. Garlic tissue analysis zThe HPLC flavour precursor profiles of various Allium species and garlic tissue types have been produced. zAlliin is present in garlic leaf, bulb and roots but is not observed in significant levels in undifferentiated garlic callus zUpon differentiation of the callus, the new roots start to produce alliin.

17. Precursor feeding experiments 1 Based on ‘precursor feeding to onion callus’ experiments by Selby et.al. We are starting with garlic callus zGarlic callus (variety ‘Printanor’) is now routinely cultured in this laboratory and available in sufficient quantities for precursor feeding experiments. zThere is little background interference in undifferentiated callus zIt is relatively easy to introduce the substrate

18. HPLC traces of garlic tissue 4. 0 0 6. 0 0 8. 0 0 0 2 0 4 0 6 0 8 0 1 0 0 Printanor clove Printanor callus Printanor roots from differentiating callus Printanor plant roots Printanor leaves Time alliin isoalliin alliin isoalliin Note: The same amount of tissue was extracted for each of these traces Absorbance 215nm alliin

19. Concentration of flavour precursors in garlic tissue TissuePrecursor concentration (mM) AlliinIsoalliin Clove503 Callus1not detectable Callus root31 Root5not detectable Leaf5010

20. Precursor feeding experiments 2 zIn 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). zThis method of substrate feeding will only give a positive result if: ythe substrate gets into the cell yenzymes are present that utilise the substrate ythe product is not further metabolised zSo far we have shown that both Allyl cysteine and Allyl thiol can be taken up by callus and converted to Alliin

21. 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.