Chemical and chemoenzymatic syntheses of bacillithiol: A unique low-molecular-weight thiol amongst low G + C gram-positive bacteria

Article abstract of DOI:10.1002/anie.201100196

The full monty: The recently discovered thiol cofactor bacillithiol (BSH), its biosynthetic precursors, and its symmetrical disulfide are prepared in two ways. The fosfomycin resistance protein (FosB) is shown to be a BSH-utilizing enzyme. It displays bac

Full text of DOI:10.1002/anie.201100196

DOI: 10.1002/anie.201100196
Natural Products
Chemical and Chemoenzymatic Syntheses of Bacillithiol: A Unique
Low-Molecular-Weight Thiol amongst Low G + C Gram-Positive
Bacteria**
Sunil V. Sharma, Vishnu K. Jothivasan, Gerald L. Newton, Heather Upton, Judy I. Wakabayashi,
Melissa G. Kane, Alexandra A. Roberts, Mamta Rawat, James J. La Clair, and Chris J. Hamilton*
In eukaryotes and Gram-negative bacteria, the cysteinyl
tripeptide glutathione (GSH, Figure 1) is the predominant
many other important metabolic functions.[1] For instance, the
reversible formation of GS-S-protein disulfides (glutathiony-
lation) is an important post-translational modification for
regulating protein function and protecting exposed cysteine
residues from irreversible oxidative damage.[2] Glutathione-S-
transferases also mediate xenobiotic detoxification by S con-
jugation with GSH. Most Gram-positive bacteria lack GSH,
but instead produce other, distinctly different low-molecular-
weight thiols. Gram-positive high G + C content actinobac-
teria produce mycothiol (MSH, Figure 1), which serves
analogous functions to GSH.[3] Low G + C Gram-positive
bacteria (Firmicutes) produce neither GSH nor MSH and
until recently the identity of their major, cysteine-derived,
low-molecular-weight thiol has been elusive. In 2007, an
unknown 398 Da thiol was observed in Bacillus anthracis cell
extracts[4] and in Bacillus subtilis as a mixed disulfide with the
redox controlled ohr regulator protein (OhrR).[5] The same
thiol was subsequently isolated by treating Deinococcus
radiodurans cell extracts with monobromobimane (mBBr)
from which the structure of bacillithiol (BSH, 1) was then
elucidated as its corresponding fluorescently labeled S-
bimane (mB) derivative BSmB (3, Figure 1).[6]
Figure 1. Structures of bacillithiol (BSH, 1, with its subunits indicated,
and its conversion into its disulfide BSSB, 2), bimane derivative
(BSmB, 3), and related redox thiols GSH and MSH.
BSH (1) occurs in several clinically important pathogens
including B. anthracis, Bacillus cereus, Staphylococcus aureus,
Staphylococcus saprophyticus, and Streptococcus agalactiae,
which do not produce GSH or MSH.[6] Evidence is now
emerging suggesting that BSH (1) plays an important role in
metabolic regulation including sporulation,[7,8] acid and salt
resistance, and detoxification of electrophilic xenobiotics such
as fosfomycin.[7,9] Thiol-S-transferase fosfomycin resistance
genes (fosB) have been detected in many BSH-producing
bacteria.[10] FosB is mechanistically related to the glutathione-
S-transferase, FosA, which catalyzes the S conjugation of
GSH with the epoxide motif of fosfomycin.[11] However,
cysteine and GSH are extremely poor substrates for
FosB.[12,13] BSH deficient mutant strains of B. subtilis[7] and
B. anthracis[9] exhibit markedly increased sensitivity to fosfo-
mycin. This implies that FosB is a bacillithiol-S-transferase.
Additionally, several bacillithiol disulfide (BSSB, Figure 1)
reductases have been proposed,[7] that could help maintain
intracellular BSH:BSSB redox ratios (ca. 200:1),[6] but further
analyses of these requires access to the substrate BSSB (2).
Recent studies have shown that the biosynthesis of BSH
(1), is initiated by a retaining glycosyltransferase (BshA) that
catalyses the glycosylation of l-malic acid with UDP-GlcNAc
to afford d-GlcNAc-l-Mal (4, Figure 2).[7,9] An N-acetyl
hydrolase (BshB) then liberates the free amine d-GlcN-l-
low-molecular-weight thiol. It plays a critical role in main-
taining an intracellular reducing environment and serves
[*] Dr. S. V. Sharma, Dr. V. K. Jothivasan, Dr. A. A. Roberts,
Dr. C. J. Hamilton
School of Pharmacy, University of East Anglia
Norwich NR4 7TJ (UK)
E-mail: c.hamilton@uea.ac.uk
G. L. Newton
Department of Chemistry and Biochemistry
University of California, San Diego (USA)
H. Upton, J. I. Wakabayashi, M. G. Kane, Dr. M. Rawat
Department of Biology, California State University-Fresno (USA)
Dr. J. J. La Clair
Xenobe Research Institute, San Diego (USA)
[**] We acknowledge financial support from the BBSRC Grant BB/
H013504/1, the Overseas Research Studentship Awards Scheme,
the University of East Anglia, the National Institutes of Health Grant
AI-72133, and the Engineering and Physical Sciences Research
Council (EPSRC). We also thank the Mass Spectrometry Service
Centre, Swansea and Dr. Su at UC San Diego for their invaluable
assistance.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201100196.
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Towards this goal, we report complementary chemical and
chemoenzymatic methods for the synthesis of BSH (1), its
biosynthetic intermediates d-GlcNAc-l-Mal (4) and d-GlcN-
l-Mal (5, Figure 2), and its symmetrical disulfide BSSB (2,
Figure 1).
Initial efforts focused on the per-benzylated glycoside 9d
(Scheme 1). Me3SiOTf promoted coupling of per-benzylated
trichloroacetimidate donor 7a[14] with dibenzylmalate 8c
provided glycoside 9d as an anomeric mixture. However,
the desired a-glycoside could not be separated by chroma-
tography. Considering the difficulties encountered with
debenzylation of fully protected BSH precursors (see
below) this route was pursued no further.[14]
Figure 2. Biosynthetic pathway to bacillithiol (BSH, 1).
Under the same coupling conditions, per-acetylated donor
7b[15] delivered a-glycosides 9a–9c from the corresponding
dimethyl-, diallyl-, or dibenzyl-l-malate esters, 8a–8c, in good
yields and with excellent anomeric selectivity (a:b ratios in
most cases greater than 95:5). Anomerically pure samples of
9a–9c were readily obtained by recrystallization or flash
chromatography. Reduction of the azide moiety in 9a–9c
afforded the free amines 10a–10c and subsequent coupling
with cysteinyl pentafluorophenyl ester 11 provided the fully
protected BSH derivatives 12a–12c. Deprotection strategies
for each of 12a–12c were explored as follows. Attempts to
Mal (5).[7,9] Gene knockout studies in B. subtilis have identi-
fied a bacillithiol synthase (BshC),[7] which mediates the
condensation of d-GlcN-l-Mal (5) with l-cysteine to deliver
BSH (1), but the limited availability of 5, has limited studies
of this enzyme.
To date, BSH (1) has only been isolated as BSmB (3) from
D. radiodurans in a yield of approximately 50 mglꢀ1 of cell
culture.[6] A synthetic route to BSH (1), as well as its
biosynthetic precursors 4 and 5, is clearly needed to further
study its biosynthesis and to reveal its metabolic functions.
Scheme 1. Synthesis of BSH (1) along with its conversion into BSSB (2) and fluorescently labeled BSmB (3) is achieved by a route that also
provides access to biosynthetic precursors 4 and 5. Reagents and conditions: a) CAN (2.6 equiv), NaN3 (2.0 equiv), CH3CN, then H2O; b) NaH,
Cl3CCN, THF, 08C, 7a 24% and 7b 67% over two steps; c) Me3SiOTf (0.5 equiv), CH2Cl2, ꢀ308!ꢀ108C, 3–5 h, 9a 72% (only a), 9b 84% (a:b
95:5), 9c 90% (a:b 96:4), 9d 37% (a:b 31:69); d) Ph3P (3 equiv), THF, H2O (8:1), room temperature, 15 h; e) 11 (1.2 equiv), HOBT (2 equiv),
Et3N (2 equiv), DMF, room temperature, 6–18 h, 12a 66%, 12b 35%, 12c 37% over two steps; f) [Pd(PPh3)4] (10 mol%), imidazole (10 equiv),
THF, room temperature, 14 h; g) NaOMe, MeOH, 08C, 75 min, 68% over two steps; h) TFA, Et3SiH, CH2Cl2 (80:2:20), 08C, 20 min, 99%;
i) aq NH4HCO3, 1 h, 99%; j) mBBr, NaHCO3, CH3CN, H2O, 2 h, 90%; k) Ac2O, Pd/C, Et3SiH, EtOAc, room temperature, 12 h; l) NaOMe, MeOH,
08C, 50% over two steps; m) Pd/C, H2 (50 psi), EtOAc, aqHCl; n) NaOMe, MeOH, 08C, 48% over two steps; o) BshA, l-malate, UDP-GlcNAc,
0.1m NaCl, 10 mm MgCl2, 1 mm 2-mercaptoethanol, 25 mm HEPES pH 7.5, 20 h 378C, 93%; p) BshB, pH 7, 23 h 378C, 90%; q) 5, Boc-
Cys(Trt)CO2H, HATU, EtNiPr2, DMF, 15%. Abbreviations: DMF=N,N-dimethylformamide, HATU=2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetrame-
thyluronium hexafluorophosphate, HOBT=1-hydroxybenzotriazole, UDP=uridine 5’-diphosphate, TFA=trifluoroacetic acid, Boc=tert-butyloxy-
carbonyl, HEPES (buffer: 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid), CAN=cerium(IV) ammonium nitrate, OTf=triflate (CF3SO3).
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remove the O-acetate and methyl ester protecting groups
from 12a under a variety of conditions (i.e., Ba(OH)2, LiOH,
KOH, Et3N) proved problematic owing to cleavage of the
malate aglycone (see Table 6 in the Supporting Information).
Attempts to cleanly remove the benzyl esters from 12c under
a various reducing conditions were also unsuccessful (Table 7
in the Supporting Information).
A successful route to BSH (1) was finally established via
12b (Scheme 1) whose allyl esters were readily removed using
catalytic [Pd(PPh3)4] in the presence of excess imidazole.[16]
Subsequent removal of the acetate protecting groups under
mild Zemplꢀn conditions provided 13. Unlike the hydrolysis
of 12a, this two-step process avoided loss of the malate
aglycone. The acid-labile protecting groups on the cysteine
moiety of 13 enabled the thiol to be unveiled in the final step
under non-basic conditions preventing purification complica-
tions that would otherwise be anticipated due to partial
oxidation of BSH (1) to BSSB (2).[17] Using this sequence,
BSH (1) was obtained from 12b in 67% yield over three steps.
When dissolved in aqueous NH4HCO3,[18] BSH (1) was
readily oxidized to provide BSSB (2) in quantitative yield.
A sample of BSH (1) was converted to its S-bimane derivative
BSmB (3) for comparison with an authentic sample isolated
from D. radiodurans cell lysates.[6] A combination of com-
parative NMR spectroscopy, circular dichroism (CD), and
HPLC analyses of synthetic and natural BSmB (3) were in
agreement, thereby confirming the structure of BSH (1).
This synthesis also provided access to the biosynthetic
intermediates d-GlcNAc-l-Mal (4) and d-GlcN-l-Mal (5),
which were effectively prepared from 9c (Scheme 1). An
alternative chemoenzymatic route to BSH (1) was also
developed (Scheme 1). Incubation of recombinant BshA
(B. subtilis) with stoichiometric ratios of commercially avail-
able l-Mal and UDP-GlcNAc afforded d-GlcNAc-l-Mal (4)
in excellent yield after purification by HPLC. Recombinant
BshB (B. anthracis)[9] was then used to N-deacetylate 4
delivering d-GlcN-l-Mal (5). Enzymatically synthesized d-
GlcN-l-Mal (5) was further elaborated to intercept our total
synthetic route to BSH (1). Chemical coupling of 5 with Boc-
Cys(Trt)-OH provided pure 13 after HPLC purification,
which was spectroscopically identical to that prepared
through the total synthetic route.
Figure 3. a) Mechanism of FosB-catalyzed inactivation of fosfomycin;
b) time course for SaFosB-catalyzed fosfomycin-S-conjugate formation.
Reagents and conditions: 20 mm HEPES (pH 7), 1 mm MgCl2, 2 mm
~
*
fosfomycin, 500 nm FosB, and 1 mm BSH ( ) or 1 mm Cys ( ), 228C.
cysteine and synthetic BSH (1) in FosB-catalyzed S conjuga-
tion with fosfomycin. The results clearly demonstrate that
BSH (1) is the preferred thiol substrate (Figure 3b) for FosB,
which has long been known to confer fosfomycin resistance in
many clinically important Staphylococci.[10]
Herein, we have reported the first preparations and
spectroscopic characterization of BSH (1) as its native free
thiol and further confirmed the structure of BSH (1) and
BSmB (3) as originally assigned.[6] Access to BSH (1), BSSB
(2), as well as the biosynthetic intermediates d-GlcNAc-l-
Mal (4) and d-GlcN-l-Mal (5) will now enable the detailed
elucidation of the biosynthesis and metabolic functions of this
unique biothiol amongst numerous bacteria of medical and
industrial significance.
Received: January 10, 2011
Revised: March 22, 2011
Keywords: Firmicutes · fosfomycin · natural products ·
.
thiol-s-transferase · total synthesis
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