Improved synthesis of the super antioxidant, ergothioneine, and its biosynthetic pathway intermediates

Article abstract of DOI:10.1039/c4ob02023e

Ergothioneine and mycothiol are low molecular mass redox protective thiols present in actinomycetes, in particular mycobacteria. We report the improved chemical synthesis of ergothioneine (ESH) and biosynthetic pathway intermediates using either histidine or ESH as the starting material. The detailed mechanism of ESH biosynthesis has not yet been completely elucidated and substrates for enzymes in the pathway will provide valuable tools to aid this study. Particularly interesting is the PLP dependent β-lyase, EgtE, of mycobacteria, having the capability of cleaving the substrate, S-(β-amino-β-carboxyethyl)ergothioneine sulfoxide, to provide ESH. A synthetic route toward ESH pathway intermediates also allowed the preparation of stable isotopically labelled hercynine-d₃ which was enzymatically transformed into ESH-d₃. The deuterated ergothioneine biosynthetic pathway metabolites are valuable tools for future studies.

Full text of DOI:10.1039/c4ob02023e

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Journal Name
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COMMUNICATION
Improved synthesis of the super antioxidant,
ergothioneine and its biosynthetic pathway
intermediates.
Cite this: DOI: 10.1039/x0xx00000x
Received 00th January 2012,
Accepted 00th January 2012
Peguy Lutete Khonde and Anwar Jardine*
DOI: 10.1039/x0xx00000x
www.rsc.org/
function of ESH in humans is currently under the spotlight in
particular its potent antioxidant activity.7 Recent commercial
interest in ESH as a super anti-oxidant molecule has added
even greater value to improved synthetic process development
of this molecule.
Ergothioneine along with mycothiol, are low molecular mass
redox protective thiols present in Actinomycetes, in
particular mycobacteria. We report the improved chemical
synthesis of ergothioneine (ESH) and biosynthetic pathway
intermediates using either histidine or ESH as starting
material. The detailed mechanism of ESH biosynthesis is not
yet completely elucidated and substrates for enzymes in the
pathway will provide valuable tools to aid this study.
Particularly interesting is the PLP dependent βꢀlyase, EgtE,
of mycobacteria, having the capability of cleaving the
substrate, Sꢀ(βꢀaminoꢀβꢀcarboxyethyl)ergothioneine sulfoxide
to proꢀvide ESH. A synthetic route toward ESH pathway
intermediꢀates also allowed the preparation of stable
isotopically labelled hercynineꢀd3 which was enzymatically
In 1956, Heath et al elucidated ESH biosynthesis in Claviceps
purpurea. He demonstrated that histidine or a compound
closely related to histidine might be a precursor of ESH,
subsequent publications disclosed the biosynthetic assembly of
ESH utilizing organisms such as Neurospora crassa and
Mycobacterium smegmatis with the aid of radio isotopic
labelling (14C and 35S).8,9,10
Melville et al further established the participation of the S-(β-
amino-β-carboxyethyl)ergothioneine
sulfoxide
as
an
intermediate in ESH synthesis by incubation of hercynine in
cell-free extracts of Neurospora crassa in the presence of O2
and Fe2+.11
transformed to ESHꢀd3
biosynthetic pathway metabolites are valuable tools for future
studies
. The deuterated ergothioneine
.
It has now been established that ESH is synthesized by the
sequential action of five enzymes, encoded by the genes egtA,
egtB, egtC, egtD and egtE (Scheme 1).12 EgtA is considered to be
a γ-glutamyl cysteine ligase and catalyzes the formation of γ-
glutamylcysteine. Histidine is methylated by an S-
adenosylmethionine (SAM) dependant methyl transferase,
EgtD, to give the trimethyl ammonium betaine, hercynine.
Many gram positive bacteria, such as Mycobacterium
tuberculosis, lack the redox protective molecule, glutathione
and instead produce mycothiol and ergothioneine (ESH) as
their principal low molecular mass thiols.1,2 ESH is
a
thiohistidine betaine derivative with a thiol group at the C2
atom (ε-position) of the imidazole ring (Scheme 1). Present
knowledge indicates that ESH may play a critical role in the in
vivo and in vitro survival of mycobacteria.3 Recently, it was
found that ESH is actively secreted into culture media by
Mycobacterium smegmatis.4
Hercynine
is
then
converted
into
S-(β-amino-β-
carboxyethyl)ergothioneine sulfoxide (II), via an iron (II)-
dependent oxidase (EgtB) which requires oxygen and γ-
glutamylcysteine to produce γ-glutamylcysteinylhercynine (I).
The exact nature of the latter transformation, in particular the
sulfoxide formation, is still under investigation. Subsequently, a
putative class-II glutamine amidotransamidase, EgtC, mediates
the hydrolysis of the N-terminus glutamic acid, providing S-(β-
amino-β-carboxyethyl)ergothioneine sulfoxide (II). Finally,
EgtE, a pyridoxal 5-phosphate (PLP)-dependent β-lyase gives
the final product, ESH.
A structural variant of ESH, ovothiol A, also serve as an anti-
oxidant albeit in sea urchin eggs as well as in the pathogens,
Leishmania major and Trypanosoma cruzi.5
Humans do not synthesize ESH, but possess an active
transport system, a cation transporter (OCTN1) with high
specificity for its uptake from dietary sources.6 The exact
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DOI: 10.1039/C4OB02023E
Recently, the research focus with regard to these carboxyethyl)ergothioneine sulfoxide (II) was extensively
mercapohistidines has shed light on the mechanism of C-S cleaved to ESH by crude cell free extracts of Neurospora Crassa.
bond formation at the δ- or ε-positions of the imidazole ring.13
OvoA is an iron (II) dependent sulfoxide synthase which
absolute configurations and in some instances, the differences
catalyzes the first step in ovothiol A synthesis and is a homolog
of EgtB. Interestingly, the substrate specificity of EgtB vs. OvoA
in achieving C-S bond formation differs significantly. OvoA is
very selective for its sulfur donor substrate and only accept L-
cysteine while it prefers histidine as co-substrate. However,
EgtB require γ-glutamyl-L-cysteine as sulfur donor.
Furthermore, it is selective toward α-N,N,N-methylation on the
histidine, i.e. hercynine as co-substrate. Surprisingly, OvoA
switches its sulfurization pattern on the histidine ring from the
δ-carbon to the ε-carbon depending on the level of α-N-
methylation.14 Thus, OvoA converts hercynine directly into S-
(β-amino-β-carboxyethyl)ergothioneine sulfoxide (II) and
A large number of naturally occurring sulfoxides have known
in biological activity of both diastereomers have been
determined.16 S-substituted cysteine sulfoxides occur almost
exclusively in the RcSs configuration in nature. It is therefore
possible
that
the
isolated
S-(β-amino-β-
RcSs
carboxyethyl)ergothioneine sulfoxide (II) has
a
configuration. However, the latter sulfoxide absolute chirality
and its importance are yet to be established.
Two different routes to the target compound, S-(β-amino-β-
carboxyethyl)ergothioneine sulfoxide (II) were considered. In
this approach, retrosynthetic cleavage of S-(β-amino-β-
carboxyethyl)ergothioneine sulfoxide (II) gave the β-chloro-
alanine methyl ester and ESH (Scheme 2, S1) Thus, S-
alkylation of a protected chloromethyl alanine ester (2), derived
from serine, provided the core structure (3). The resulting
sulfide (3) was oxidised using either mCPBA or H2O2.
produce
a minor amount of the δ-sulfoxide (ovothiol
substitution pattern) when α-N,N-dimethyl histidine is used as
the co-substrate (Scheme 1).
O
O
O
(S)
N
N
N
ε
Ishikawa et al. suggested that the reaction may possibly occur
via the formation of the cyclic ethylenimine carboxylic acid
intermediate produced by an intramolecular SN2 reaction of the
β-chloroalanine (2), followed by the ring opening induced by
nucleophilic attack of the sulfur atom of ESH, giving the major
product N-Boc methyl ester (3a).
OH
OH
OH
HS
ε
δ
N
NH2
NH2
δ
HN
HN
N
SH
Histidine
Ergothioneine (ESH)
Ovothiol (OSH)
SAM
O
Pyruvate, NH3 [O]
O
EgtD
EgtE, PLP
O
N
N
HO2
C
Cysteine, iron (II), O2
OH
OH
S
Sulfoxidation reaction conditions with H2O2 previously
investigated by Ishikawa et al led to an over oxidation to the
sulfone in our hands and no analytical evidence were
provided.11 In order to prevent over oxidation of the sulfoxide,
N
N
HN
HN
H2
N
OvoA
II
Hercynine
EgtB, Iron (II)
O2
SH
EgtC
O
O
Glutamate
(R)
mCPBA was used.
Its milder nature and potential for
OH
HO
N
H
controlled sulfoxidation compare to hydrogen peroxide is
advantageous. The sulfide methyl ester (3a) was subjected to S-
oxidation using one equivalent of mCPBA to afford the
sulfoxide methyl ester (4a). The synthetic product is most
likely a mixture of RcSs and RcRs diastereomers. The lowest
steric energy conformations (total energy 34.37 kcal/mol) of the
sulfide methyl ester (3a) indicated potential face selectivity
toward sulfoxidation, which could lead predominantly to the SR
diastereoisomer sulfoxide derivative (S1.3 & S1.4). 1H NMR
spectra of the sulfoxide methyl ester (4b) displayed evidence of
diastereoselectivity (ca. 3:1 ratio) (Fig. S1.4.2-S1.4.3). However,
only a crystal structure, supported by CD (circular dichroism)
spectra, will help establish the absolute configuration of the
major chiral sulfoxide and also that of the natural sulfoxide (II).
Deliberate oxidation of sulfide (3b), (4b) or sulfoxide (5a) to the
sulfone (III) was achieved with excess oxidant.
NH2
NH2
O
H
N
HO
CO2
H
(S)
γ−glutamylcysteine
(R)
O
O
O
S
HN
N
I
N
O
HO
Scheme 1 ESH biosynthesis
While the enzymes EgtB and EgtC have been expressed in a
functional form, EgtE is still elusive and none of the enzymes
have been thoroughly studied due to the lack of readily
available substrate intermediates.
Here we report the improved synthesis of the ESH and its
biosynthetic
precursor,
S-(β-amino-β-
carboxyethyl)ergothioneine sulfoxide (II). The latter sulfoxide
is the substrate for the mycobacterial enzyme, EgtE. However,
the absolute chirality of the sulfoxide is not known for the
natural substrate or the synthetic one. Prior synthesis of
intermediate (II) was reported in 1974 and were elaborate and
irreproducible and resulted in a low overall yield of 8.5%.15 The
authors reported only the position of the aromatic proton
resonance and no further structural confirmation. An optical
rotation, [α]D +74.4 (c = 0.5, H2O), was reported and could not
be reconciled with the authentic natural product [α]D +9.1 (c =
0.5, H2O). However, the m.p. of both natural and synthetic
product was recorded as 188-190 °C. None-the-less, it was
Finally, attempted global deprotection of the Boc group and
hydrolysis of the methyl ester under aqueous acidic conditions
gave only the methyl ester sulfide (3b) or methyl ester sulfoxide
(4b) from (3a) and (4a) respectively. Unsuccessful acid, base or
esterase mediated ester hydrolysis obligated reconsideration of
the synthetic route. Stable amino acid methyl esters have been
reported before.17,18 An allyl ester derivative of β-chloroserine
provided the N-Boc allyl ester sulfide (3c) after S-alkylation.
Sulfoxidation followed by mild RhCl(PPh3)3 catalysed allyl ester
cleavage19 and acid mediated Boc protecting group removal
gave the target S-(β-amino-β-carboxyethyl)ergothioneine
sulfoxide (II) in a moderate overall yield of 63 %.
claimed
that
synthetic
S-(β-amino-β-
2 | J. Name., 2012, 00, 1-3
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DOI: 1C0.O10M39M/CU4ONBI0C2A0T23IOE N
Cysteine HCl, H2O 1 hr at 0 °C, (5a); (xii) 3-mercaptopropionic acid, HCl
/ H2O, 19 hrs reflux.
With the second retrosynthetic approach, cleavage of the
histidine moiety of S-(β-amino-β-carboxyethyl)ergothioneine
sulfoxide (3) gave cysteine and 2-bromohercynine (Scheme 2).
This route has received the most attention as it provides the
required sulfurization of histidine to provide the commercially
important ESH. 2-Bromohercynine was synthesized via the
Erdelmeier method.20 Thus, S-alkylation of 2-bromohercynine,
provided the S-(β-amino-β-carboxyethyl)ergothioneine sulfide
(5a) (Scheme 2). However, a large quantity of salt by-product
had to be removed which hampered purification. Furthermore,
the treatment of the S-(β-amino-β-carboxyethyl)ergothioneine
sulfide (5a) with 3-mercaptopropionic acid at 90 °C for 18 hr
gave ESH. The conversion of the sulfide (5a) to the bis-
benzyloxy N-Boc protected ester (5b), allowed organic
extraction and removal of salts to give a clean benzyl ester (5b).
Global deprotection of the N-Boc benzyl ester (5b) was
achieved by hydrogenation (Pd/C) in the presence of TFA
under 50 psi hydrogen pressure to give pure S-(β-amino-β-
carboxyethyl)ergothioneine sulfide (5a). Biphasic sulfoxidation
of the sulfide (5a) with mCPBA in a DCM/water mixture gave
the S-(β-amino-β-carboxyethyl)ergothioneine sulfoxide (II) in a
low overall yield of 36 %.
The diastereomers of all S-substituted cysteine sulfoxides
1
display H NMR spectra with a characteristic ABX pattern for
1
the S(O)CH2CH(NH2) methylene protons. H NMR spectra of
the S-(β-amino-β-carboxyethyl)ergothioneine sulfide (5a),
sulfoxide (II) and sulfone (III) was consistent with the reported
structures. Diagnostic ABX coupling patterns were observed for
the α- and β-protons, thus giving rise to sets of doublets at
about 4.0-4.5ppm and 3.3-3.6ppm respectively.
Coupling
constants JAX, JBX and JAB were approx. 10, 5 and 14 Hz
1
respectively. H NMR cosy spectra as well as 13C NMR aided
complete assignment of structure.
Improved synthesis of ESH
Here we report the improved synthesis of the ESH via the
biosynthetic
precursor,
S-(β-amino-β-
carboxyethyl)ergothioneine sulfide. The latter sulfide and its
sulfoxide (II) are substrates for the mycobacterial enzyme, EgtE
that produces ESH. Our process starts with a commercially
available N-benzyl protected histidine rather than the
unprotected form (key difference). We discovered that the
bromination of the N-benzyl protected hercynine intermediate
was achieved with N-bromosuccinimide (NBS) to give a more
stable N-benzyl protected 2-bromohercynine derivative using
DMF as solvent in 90% yield. The advantage of the latter
intermediate is that subsequent process steps is near
quantitative, relatively simple, all at room temperature,
shortened and allow an overall synthesis yield of 80%. Thus,
our process is at least 2 times better in overall yield than any
prior patented or published process.20 The high yields of the
intermediate products also allow viable isotopic labelling steps
to be performed. Isotopes are usually very expensive and are
advantaged by high yield conversions. The final step involves a
biomimetic pyridoxal phosphate (PLP) mediated cleavage of
the sulfide or sulfoxide substrates as well as with crude
enzymatic extracts of M. smegmatis to give ESH. (Scheme 3)
O
Cl
OR1
O
O
NHR2
N
OH
N
HS
2a - 2c
R1O2C
R2HN
OR3
S
N
HN
N
HN
(i)
Ergothioneine (1)
R1
R2
=
=
-CH3 or allyl
Boc
3a : R1=Me, R2=Boc, R3=H
3b : R1=Me, R2=R3=H
(ii)
3c : R1=Allyl, R2=Boc, R3=H
(iii)
(iv)
5a : R1=R2=R3=H
5b : R1=Bn, R2=Boc, R3=Bn
O
(v)
(vi)
O
N
S
R1O2C
R2HN
OR3
N
HN
(xii)
(iv)
(xi)
4a : R1=Me, R2=Boc, R3=H
4b : R1=Me, R2=R3=H
O
(iv)
O
N
OH
Br
N
N
OH
CR3
aq acid, baseX
HN
HS
or esterase
N
HN
2-bromohercynine
ESH (1) : R=H
ESH-d3 (10) ; R=D
O
O
N
S
HO2
H2
C
(vii)
OH
crude enzyme
N
(x)
O
O
HN
N
O
O
O
N
OH
II
N
N
N
(ii)
(i)
N
OH
OH
NHBoc
N
OH
CR3
OH
N
RS
NH2
N
N
N
N
HN
13
HN
(vi)
12
11
Quantitative
Quantitative
hercynine : R=H
7 : R=D
9 : R=t-But
(iii)
N
O
O
O
N
(viii)
O
O
HO2C
OH
(ix)
S
O
NH2
N
HN
OH
N
N
OH
H2N
O
HO
S
(iv)
(vi)
N
N
N
HN
15
N
OH
OH
O
HS
III
(v)
14
NR2
NH2
HN
HN
Isolated yield= 90 %
Crude yield= 93%
histidine : R=H
6 : R=Me
mercapto-histidine (8)
O
O
S
(vii)
NH2
O
N
OH
HO
O
NH2
O
N
N
OH
N
HN
II
Isolated yield= 70 %
HO
S
O
Scheme 2. Reagents and conditions: (i) Et3N / H2O 5 h at 30°C, (3a);
(ii) N-Boc deprotection: TFA, DCM / H2O, 0-5°C (3b), (4b) or (II) (iii) a)
RhCl(PPh3)3, EtOH/ H2O (1:1) reflux b) TFA, DCM / H2O, 0-5°C (5a); (iv)
mCPBA, H2O / DCM 1:1, 5 hr at 25°C (4a) or (II) (v) a) Boc2O / NaOH,
H2O / CH3CN, rt overnight b) BnBr / DMF, rt overnight (vi) Pd/C, 3 eq
TFA, H2 50 psi, rt 12h (vii) H2O2, 3 hr at rt (III) (viii) a) CH2O, sodium
triacetoxyborohydride / CH3CN, 18-24 hrs at rt (6), b) NH4OH, CH3 I or
CD3I / MeOH, 24 h at rt, (7); (ix) a) t-BuOH / HCl, refluxed for 3-4 hrs,
S-t-butyl mercaptohistidine; b) CH2O, sodium triacetoxyborohydride /
THF, 6-8 hrs at 10 °C, (9); (x) a) NH4OH, CD3I / MeOH 24 h, rt; b)
HCl, 2-mercaptoprionic acid refluxed for 21 h (quantitative); (xi)
HN
III
Isolated yield= 61 %
O
O
N
OH
HS
N
HN
ESH
(v)
Scheme 3. Improved synthesis of sulfoxide (II), sulfone (III) and
ESH. : (i) TFA, DCM, rt overnight; (ii)CH2O, NaBH(OAc)3, CH3CN, 24
h at rt; (iii) MeI, THF, 24 h at rt; (iv) (a) NBS (2.5 eq), DMF, dark at rt,
b) L-Cysteine (2.5), DMF, 24 h at rt; (v) p-toluene sulfonic acid (cat),
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H2O2 (2.4 eq); (vi) boric acid (cat), H2O2 (4.8 eq); (vii) C-S enzyme lyase
or PLP non enzymatic cleavage.
the crude M. smegmatis enzymes (dialyzed extract)
transformed the hercynine-d3 (7) into ESH-d3 (10). This proved
that the isolated crude enzyme extract were fully functional in
the reconstitution of ESH synthesis.
Previously, the biosynthesis pathway of ESH has been
elucidated utilising radiolabeled intermediates. Advances in
synthetic procedures now made it possible to synthesize the
same intermediates incorporating stable isotopes. These
intermediates are valuable internal standards in the
quantitation of pathway metabolites during external stimuli or
drug treatment. Here we also report the synthesis of ESH-d3
(10) and demonstrate its biosynthesis from deuterated
hercynine.
(β-amino-β-carboxyethyl)ergothioneine
biosynthetically produced the highest concentration of ESH
(22.6 ng/ml) (Fig. 2a). The (β-amino-β-
carboxyethyl)ergothioneine sulfide (15) appeared to be almost
as good substrate as the (β-amino-β-
sulfoxide
(II)
a
carboxyethyl)ergothioneine sulfoxide (II) (19.2 ng/ml ESH). It
is well known that PLP-dependent transformations can also
undergo enzyme free conversion albeit with a much slower rate
and specificity.22 Thus, the non-enzymatic treatment of the
sulfide (15) with 50 mM PLP at 37 °C resulted in an efficient
formation of ESH (96.3 ng/ml) (Fig. S4.5.1). However, under
the same conditions, the sulfoxide (II) produced no ESH at all
(Fig. S4.5.2).
Hercynine-d3 (7) was synthesized in a two-step reaction
starting with the commercially available L-histidine (Scheme
2). The first step involved reductive amination using aqueous
formaldehyde and sodium triacetoxyborohydride to give N,N-
dimethyl histidine (6). The second step involved the
quaternarization of the crude N,N-dimethyl histidine (6) using
methyl-d3 iodide under basic conditions to give the hercynine-
d3 (7).
Characteristic HRMS peaks indicating M+3 for
hercynine-d3 (7) were obtained (S3.2.1).
ESH-d3 (10) was synthesized in two sequential reaction steps
starting with the S-tert-butyl protected 2-mercapto-histidine
(9), derived from mercapto histidine (8).21 Selective N-
methylation with methyl-d3 iodide, followed by S-tert-butyl
deprotection using 2-mercaptopropionic acid (tert-butyl
scavenger) in HCl gave ESH-d3 (10). Characteristic HRMS peaks
indicating M+3 for the ESH-d3 (10) were obtained (S3.2.2).
We compared the enzymatic and non-enzymatic PLP
mediated synthesis of ESH from the synthetic sulfoxide (II).
To this end, crude M. smegmatis cell free extracts were isolated
from cultures grown and harvested at the late exponential
phase, characterised by high enzymatic activity.10
Figure 2a In vitro reconstitution of ESH; 100 µl reactions containing
20 mM Tris HCl pH= 7.4, 20 mM NaCl, 0.2 Mm FeSO4.7 H2O, 0.5 mM
mercaptoethanol, 83 µl of crude M. smeg enzymes and 50 mM of either
(a) S-(β-amino-β-carboxyethyl)ergothioneine sulfide (15); (b) S-(β-
amino-β-carboxyethyl)ergothioneine sulfoxide (II) and (c) control. The
crude enzyme reactions were incubated for 1 day at 37° C followed by
analysis by LC/MS.
The crude enzymatic transformation of the ESH biosynthetic
pathway precursors, including sulfide and sulfoxide variants
were evaluated by the concomitant production of ESH in excess
of basal levels as determined by LCMS. ESH precursor
metabolites hercynine-d3 (7), hercynyl cysteine methyl ester
sulfoxide (4b), (β-amino-β-carboxyethyl)ergothioneine sulfide
(15) and (β-amino-β-carboxyethyl)ergothioneine sulfoxide (II)
were incubated with the crude cell free extract at 37° C, pH =
7.4 for 1 day, and analyzed by LCMS.
The pKa value of the amino acid α-hydrogen usually is in the
range 20-30 and many enzymes effectively increase the α-
hydrogen acidity with the aid of coenzyme, PLP. PLP-
dependent enzymes exist in their native state as an internal
aldimine (Schiff base) (S-5.1-5.3) with the ε-amino group of a
lysine residue present in the catalytic site. The amino acid
substrate displaces the lysine from the internal aldimine to
form a new aldimine, termed external aldimine. The formation
of such an external aldimine can reduce the pKa value of the α-
proton of the substrate from 30 to as low as 6.22
The control reaction containing only the crude M. smegmatis
cell free extract was also treated under the same conditions as
the four metabolites. The concentration of ESH thus obtained
was 5.70 (±0.30) ng /ml, which was equated to that of
endogenous ESH. This concentration was above the limit of
detection (0.78 ng/ml), thus any increase in the concentration
of ESH in the experiment above 1 ng/ml is considered
significant enough to be ascribed to basal levels or
biotransformation of the respective substrates by the crude
endogenous enzymes of the ESH pathway.
PLP-dependent enzymes have been differentiated on the
basis of their tertiary structures, more specifically according to
fold type I–V. EgtE is classified as a fold type V which is found
mainly in enzymes that catalyze β-replacement and β-
elimination reactions. In the absence of a crystal structure of
EgtE, structure homology analysis amongst mycobacteria is not
possible. However, a putative β‐lyase from E. tasmaniensis
appeared to catalyze this step but has a sequence similarity
with EgtE of M.Smeg and M.tb of only 14%.12 Thus, the proposed
mechanism of this C-S β‐lyase utilizing (β-amino-β-
carboxyethyl)ergothioneine sulfide (15) as substrate was based
on that of the well characterized, cystalysin from Treponema
denticola which shares 31% sequence identity with the β‐lyase
When hercynine-d3 (7) was used as substrate, as expected, no
change in baseline ESH was observed. However, as expected,
the LCMS analysis of the mixture revealed the production of
ESH-d3 (10). The HRMS (ESI+) displayed a peak at m/z 233.1161
corresponding to [M+d3]+ (S4.2.1). Peak values at m/z 230.0958
[M]+ or at m/z 231.0980 [M+H]+ belonging to the natural ESH
were not present in the chromatogram. This clearly shows that
4 | J. Name., 2012, 00, 1-3
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DOI: 1C0.O10M39M/CU4ONBI0C2A0T23IOE N
from E. tasmaniensis.23 It may well be that actinomycetes has
similar β‐lyases at work.24
5.
Ariyanayagam, M.R.; Fairlamb A.H. Molecular and
Biochemical Parasitology, 2001, 115, 189–198.
6. Grundemann D.; Harlfinger S.; Golz S.; Geerts A.; Lazar A.;
Berkels R.; Jung N.; Rubbert A.; Schomig E. PNAS, 2005,
102, 5256-5261.
We also observed a rather facile non-enzymatic (PLP only) β-
elimination
with
(β-amino-β-carboxyethyl)ergothioneine
sulfide (15). However, the conversion of the natural substrate,
(β-amino-β-carboxyethyl)ergothioneine sulfoxide (II) to ESH is
not that straight forward. Elimination of an unstable (β-amino-
β-carboxyethyl)ergothioneine sulfinate (IV) is envisaged,
whereby self-condensation leads to the thiosulfinate, which in
turn, decompose to ESH and an equivalent amount of a
7.
Cheah I.K.; Halliwell B. Biochim Biophys Acta. 2012, 1822,
784-793.
8. Heath H.; Wildy, J., Biochem. J., 1957, 65, 220-222.
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249, 4420-4427.
12. Seebeck F.P. J. Am. Chem. Soc. 2010, 132, 6632–6633. Also
Blast sequence alignment analysis of M. Smeg EgtE against
the β-lyases of T. denticola and E. tasmaniensis (not
shown).
relatively
stable
(β-amino-β-carboxyethyl)ergothioneine
sulfinic acid (V). The latter sulfinic acid can subsequently be
reduced to the thiol, ESH, with the aid of excess
mercaptoethanol, but may be difficult under the current
experimental conditions.25 Note that the (β-amino-β-
carboxyethyl)ergothioneine sulfoxide (II) did not produce ESH
(in the absence of mercaptoethanol).
13. Mashabela G.T.M.; Seebeck F.P. Chem. Commun., 2013, 49,
7714-7716.
Efforts are underway to purify and crystallize EgtE, which
would allow a better understanding of enzyme-substrate
binding, specificity and the potential for inhibitor design. An
EgtD deletion mutant of M. smegmatis and a mycothiol-
deficient mutant did not affect their susceptibility to
antibiotics.4 However, the ESH/mycothiol-deficient double
mutant was significantly more sensitive to peroxide than either
of the single mutants lacking either ESH or mycothiol,
suggesting that both thiols play a role in protecting M.
smegmatis against oxidative stress. Thus, an inhibitor of ESH
synthesis will be valuable in drug susceptibility studies of
mycobacteria.
14. Song H.; Leninger M; Lee N; Liu P. Organic Letters, 2013,
15, 4854–4857.
15. Ishikawa, Y.; Israel S.E.; Melville D.B. J. Biol. Chem., 1974,
249, 4420-4427.
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2004, 6, 4359-4361.
20. a) Erdelmeier, I.; Daunay, S.; Lebel, R; Farescour, L.; Yadan
J-C. Green Chem., 2012, 14, 2256-2265. b) Irene Erdelmeier,
The method of synthesizing ergothioneine and analogs.
US 20120136159 A1.
21. Trampota, M.; United States Patent, 2010, US 7,767,826,
B2.
22. Toth, K.; Richard, J.P. J. Am. Chem. Soc., 2007, 129, 3013–
3021.
Supporting Information
Experimental materials, methods, and figures. This material is
available free of charge via the Internet at http://pubs.acs.org.”
23. Krupka, H.I.; Huber R.; Holt S.C.; Clausen T. EMBO J.
2000, 19, 3168-3178.
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Bioorganic & Medicinal Chemistry Letters, 2010, 20, 444–
447.
AUTHOR INFORMATION
Corresponding Author
anwar.jardine@uct.ac.za
Notes
The authors declare no competing financial interest.
TOC Graphic
ACKNOWLEDGMENT
I would like to acknowledge Dr Z. MacDonald for mass
spectroscopy support and the University of Cape Town
Research and postgraduate committees for funding as well as
Carine Sao and Dr B Baker for mycobacterium cultures.
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