Transcript allyl cysteine sulphoxide

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=CHCH2SCH2CH(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

RSCH2CH(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=CHCH2SCH2CH(COOH)(NH2)
and
 S-(E)-1-Propenyl-L-cysteine S-oxide (also
called propenyl cysteine sulphoxide, propenyl CSO
or isoalliin)
O

CH2CH=CH2SCH2CH(COOH)(NH2)
 S-Propyl-L-cysteine S-oxide (also called propyl
cysteine sulphoxide, propyl CSO or propiin)
O

CH2CHCH2SCH2CH(COOH)(NH2)
 S-Methyl-L-cysteine S-oxide (also called methyl
cysteine sulphoxide, methylCSO or methiin)
O

CH3SCH2CH(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=CHCH2SCH2CH(COOH)(NH2)
alliinase
O

CH2=CHCH2S SCH2 CH= CH2
alliicin
2-propenyl-2-propenethiosulphinate
pyruvate
+ 2 CH3(CO)(COOH)
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

RSCH2CH(COOH)(NH2)
alliinase
+
RS=O
R1S-S-R2
etc.
CH3(CO)(COOH)
CSO biosynthetic
pathway
SO42serine
SO32-
SO22-
cysteine
valine &
methacrylate
Allyl-S
(unknown sources)
S-allylglutathione
glutathione
(γ-glu-cys-gly)
S-(2-carboxypropyl)-glutathione S-methylglutathione
gly
S-allyl-γ-glu-cys
gly
S-2-CP-γ-glu-cys
gly
S-methyl-γ-glu-cys
HCOOH
glu
glu
transpeptidase
S-allylcysteine
S-trans-1-propenyl-γ-glu-cys
glu
transpeptidase
S-allylcysteine
transpeptidase
S-methylcysteine
S-trans-1-propenylcysteine
oxidase
oxidase
alliin
S-allyl-cysteine sulphoxide
(alliin)
oxidase
oxidase
S-trans-1-propenylcysteine sulphoxide methiin
(isoalliin)
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.
A gamma-glutamyl,
cysteine,
glycine peptide
RSCH2CHCONHCH2COOH

NH2

CO
R- cys-gly
R-C-G


l
CH2
glu
E

CH2

CH(NH )(COOH)
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 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
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

CH2CH=CH2SCH2CH(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-Lcysteine 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 sulphoxide
14.95 & 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
alliin
Printanor clove
1
0
0
8
0
6
0
isoalliin
4
0
2
0
0
2.
00
4.
00
6.
00
8.
00
1
0
0
Printanor callus
8
0
6
0
4
0
2
0
0
0.
00
2.
00
4.
00
6.
00
8.
00
alliin
Printanor roots from
differentiating callus
8
0
6
0
isoalliin
4
0
2
0
0
0.
00
2.
00
4.
00
6.
00
8.
00
alliin
alliin
1
0
0
Printanor plant roots
8
0
isoalliin
6
0
4
0
2
0
0
0.
00
2.
00
4.
00
alliin
6.
00
8.
00
1
0
0
isoalliin
8
0
6
0
Absorbance
215nm
4
0
2
0
0
2
.
0
0
4
.
0
0
Time
6
.
0
0
8
.
0
0
Note: The same amount of tissue was extracted for each of these traces
Printanor leaves
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.