Synthesis of bacillithiol and the catalytic selectivity of FosB-type fosfomycin resistance proteins

Article abstract of DOI:10.1021/ol302327t

Bacillithiol (BSH) has been prepared on the gram scale from the inexpensive starting material, d-glucosamine hydrochloride, in 11 steps and 8-9% overall yield. The BSH was used to survey the substrate and metal-ion selectivity of FosB enzymes from four Gr

Full text of DOI:10.1021/ol302327t

ORGANIC
LETTERS
2012
Vol. 14, No. 20
5207–5209
Synthesis of Bacillithiol and the Catalytic
Selectivity of FosB-Type Fosfomycin
Resistance Proteins
Alexander P. Lamers, Mary E. Keithly, Kwangho Kim, Paul D. Cook, Donald F. Stec,
Kelly M. Hines, Gary A. Sulikowski,* and Richard N. Armstrong*
Departments of Chemistry and Biochemistry, Vanderbilt University, Vanderbilt Institute
of Chemical Biology, Nashville, Tennessee 37235, United States
gary.a.sulikowski@vanderbilt.edu; r.armstrong@vanderbilt.edu
Received August 20, 2012
ABSTRACT
Bacillithiol (BSH) has been prepared on the gram scale from the inexpensive starting material, D-glucosamine hydrochloride, in 11 steps and
8ꢀ9% overall yield. The BSH was used to survey the substrate and metal-ion selectivity of FosB enzymes from four Gram-positive
microorganisms associated with the deactivation of the antibiotic fosfomycin. The in vitro results indicate that the preferred thiol substrate
and metal ion for the FosB from Staphylococcus aureus are BSH and Ni(II), respectively. However, the metal-ion selectivity is less distinct with
FosB from Bacillus subtilis, Bacillus anthracis, or Bacillus cereus.
Low-molecular-weight thiols are critical to living
systems for maintaining a reducing environment in the
cytosol and preventing oxidation of cysteine residues in
proteins. Eukaroyotes typically utilize glutathione for
these purposes while most Gram-positive bacteria and
Archae lack glutathione and instead utilize novel cysteine
derived thiols.1 For example, the N-acetyl cysteine deriva-
tive mycothiol (MSH, Figure 1) is found in most actino-
mycetes including mycobacteria and streptomycetes.2
In 2009 a structurally related thiol, bacillithiol (BSH,
Figure 1), was detected in Gram-positive bacteria, includ-
ing Bacilli and Staphylococcus aureus and then isolated
from Deinococcus radiodurans and characterized as its
S-bimane derivative (BSmB, Figure 1).3,4 Subsequently, a
biosynthetic pathway for BSH was proposed and the first
chemical synthesis was reported.5,6
Although the functions of BSH in biology are not fully
understood, recent reports suggest that it may be the
preferred substrate for the FosB-type fosfomycin resis-
tance proteins.5,6 FosB is a metallo-enzyme, found in
Gram-positive microorganisms, that catalyzes the general
reaction illustrated in Scheme 1 and confers resistance to
the antibiotic, fosfomycin.7 Previous work in our labora-
tory suggested that the preferred thiol substrate and
divalent metal ion might be L-Cys and Mg2þ, respectively.7
The discovery of BSH and the availability in our lab of
FosB enzymes from several Gram-positive microorgan-
isms prompted us to initiate an investigation of their sub-
strate and metal-ion selectivity. In this report we describe
a complete chemical synthesis of BSH from inexpensive
(1) (a) Fahey, R. C. Annu. Rev. Microbiol. 2001, 55, 333. (b) Masip,
L.; Veeravalli, K.; Georgioui, G. Antioxid. Redox Sign. 2006, 8, 753.
(2) Jothivasan, V. K.; Hamilton, C. J. Nat. Prod. Rep. 2008, 25, 1091.
(3) Newton, G. L.; Rawat, M.; La Clair, J. J.; Jothivasan, V. K.;
Budiarto, T.; Hamilton, C. J.; Claiborne, A.; Helmann, J. D.; Fahey,
R. C. Nat. Chem. Biol. 2009, 5, 625.
(4) Review: Helmann, J. D. Antioxid. Redox Sign. 2011, 15, 123.
(5) Biosynthesis studies: (a) Gaballa, A.; Newton, G. L.; Antelmann,
H.; Parsonage, D.; Upton, H.; Rawat, M.; Claiborne, A.; Fahey, R. C.;
Helmann, J. D. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 6482. (b)
Parsonage, D.; Newton, G. L.; Holder, R. C.; Wallace, B. D.; Paige,
C.; Hamilton, C. J.; Dos Santos, P. C.; Redinbo, M. R.; Reid, S. D.;
Claiborne, A. Biochemistry 2010, 49, 8398. (c) Upton, H.; Newton,
G. L.; Gushiken, M.; Lo, K.; Holden, D.; Fahey, R. C.; Rawat, M.
FEBS Lett. 2012, 586, 1004.
(6) Sharma, S. V.; Jothivasan, V. K.; Newton, G. L.; Upton, H.;
Wakabayashi, J. I.; Kane, M. G.; Roberts, A. A.; Rawat, M.; La Clair,
J. J.; Hamilton, C. J. Angew. Chem., Int. Ed. 2011, 50, 7101.
(7) (a) Cao, M.; Bernat, B. A.; Wang, Z. P.; Armstrong, R. N.;
Helmann, J. D. J. Bacteriol. 2001, 183, 2380. (b) Rigsby, R. E.; Fillgrove,
K. L.; Beihoffer, L.; Armstrong, R. N. Methods Enzymol. 2005, 401, 367.
r
10.1021/ol302327t
2012 American Chemical Society
Published on Web 10/03/2012

starting materials, a new continuous 31P NMR assay of
FosB enzymes, and its use tofurther elucidate the substrate
and metal-ion selectivity of FosB enzymes from four
Gram-positive microorganisms.
Scheme 2. Synthesis of Bacillithiol
Figure 1. Mycothiol, bacillithiol, S-bimane bacillithiol, and
bacillithiol disulfide.
Scheme 1
TMSOTf activation afforded 3 in yields ranging from 61
to 91% (3:1 R/β), with highest yields observed on a 10 g
reaction scale. Alpha (3a) and beta (3b) glycosides are
separable by chromatography leading to an isolated yield
of 52% for the desired 3a. Reduction of azide 3a was first
examined using polymer-supported triphenylphosphine
followed by treatment of the crude product with 4 N
HCl-dioxane leading to isolation of the corresponding
hydrochloride salt (4•HCl) albeit in low yield (ca. 30%).
It was subsequently determined that hydrogenation (45 psi)
of azide 3a over palladiumꢀcarbon in methanol afforded
crude amine (4) of sufficient purity to be condensed directly
with commercially available Boc protected cysteine disul-
fide (5) to afford disulfide 6 in 80% yield (two steps).
Bacillithiol disulfide was derived from 6 by a two-step de-
protection sequence starting with removal of acetyl groups
(NaOMe, MeOH, ꢀ40 to ꢀ20 °C) to provide disulfide
7 in 92% yield. Next, treatment of 7 with a 50% TFAꢀ
dichloromethane solution served to remove remaining
acid labile protecting groups and following elution through
a strong cation exchange column with 5% acetic acidꢀ
methanol yielded BSSB disulfide as its ammonium acetate
salt (BSSB•2HOAc).
Our synthetic strategy (Scheme 2) targeted the disulfide
BSSB and the use of D-glucosamine as a starting point.
While our synthetic studies were underway, Hamilton and
co-workers6 reported the chemical synthesis of bacillithiol
starting with trichloroacetimidate 1 prepared by the oxida-
tive azidation (CAN, NaN3)8 of D-glucal. We also started
from trichloroacetimidate, 1, but in our case derived 1
from D-glucosamine by way of a known9 diazo transfer-
acetylation reaction (TfN3 then Ac2O) followed by selective
removal of the C1 acetate (NH2NH2, MeOH)10 and tri-
chloroacetimidate formation (Cl3CCN, NaH).11 Coupling
of trichloroacetimidate 1 with di-tert-butylmalate 212 under
(8) Lemieux, R. U.; Racliffe, R. M. Can. J. Chem. 1979, 57, 1244.
(9) (a) Nyffeler, P. T.; Liang, C. H.; Koeller, K. M.; Wong, C. H. J.
Am. Chem. Soc. 2002, 124, 10773. (b) Alper, P. B.; Hung, S. C.; Wong,
C. H. Tetrahedron Lett. 1996, 37, 6029.
(10) Exoffier, G.; Gagnaiere, D.; Utille, J.-P. Carbohydr. Res. 1975,
39, 368.
(11) (a) Lee, S.; Rosazza, J. P. N. Org. Lett. 2004, 6, 365. (b) Rele,
S. M.; Iyer, S. S.; Chaikof, E. L. Tetrahedron Lett. 2007, 48, 5055.
(12) (a) Rotzoll, N.; Dunkel, A.; Hofmann, T. J. Agric. Food Chem.
2005, 53, 4149. (b) Allais, F.; Martinet, S.; Ducrot, P.-H. Synthesis 2009,
3571.
Access to BSH allowed us to screen several FosB
enzymes from Staphylococcus aureus (FosBSA), Bacillus
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Org. Lett., Vol. 14, No. 20, 2012

Although the continuous 31P NMR assay is quick and
convenient, it is not suitable with paramagnetic metals or
for performing detailed steady-state kinetics. Nevertheless,
approximate minimum turnover numbers (kcat) can be
estimated from the initial rates of the reactions. The apparent
kcat for FosBSA with BSH and Ni(II) is ∼30 sꢀ1 but with
Mg2þ drops to ∼1sꢀ1. TheFosBSA activity in the presence of
Ni(II) also drops to ∼0.9 sꢀ1 when L-Cys is the substrate. The
results with Mg2þ are consistent with previous observations.6
The apparent kcat values for FosBBS with BSH and Ni(II)
or Mg2þ are ∼6 and 3 sꢀ1, respectively. With L-Cys, the kcat
values are much lower, on the order of 0.05 to 0.2 sꢀ1
.
In summary, BSH is clearly the preferred in vitro thiol
substrate for FosB enzymes from several Gram-positive
microorganisms. However, the metal-ion preferences of
the enzymes are not so clear and bear further investigation
particularly with respect to what the preferred metal is
in vivo. It is interesting to note that the preferred metal in
the reaction catalyzed by the anthrax enzyme (FosBBA) is
Mg2þ (Figure S2A, Supporting Information). The avail-
ability of synthetic BSH opens the door to more detailed
kinetic and metal-ion preference studies of the enzymes
as well as structural investigation of the FosB•BSH and
FosB•product complexes.
By way of the described 10-step reaction sequence, we
have prepared over 1 g of BSSB•2HOAc. Reduction of
BSSB disulfide to afford BSH is easily accomplished by
stirring with [tris(2-carboxyethyl)phosphine] (TCEP) re-
ducing gel in water. The bromobimane derivative (BSmB)
was prepared, and its properties were compared favorably
by LC-MS and NMR analysis to those of an authentic
sample of BSmB.3
Figure 2. Time course of the FosB catalyzed-addition of BSH or
L-Cys to fosfomycin in the presence of Mg2þ or Ni(II). Results
for FosBSA and FosBBS are shown in panels A and B, respec-
tively. Reactions were run at 25 °C in 20 mM HEPES (pH 7.0)
with 4 mM fosfomycin and 0.5 μM enzyme in the presence of (9)
2 mM BSH and 10 μM Ni(II), (2) 2 mM BSH and 1 mM Mg2þ
,
(1) 2 mM L-Cys and 10 μM Ni(II), or ([) 2 mM L-Cys and 1 mM
Mg2þ
.
cereus (FosBBC), Bacillus anthracis (FosBBA), and Bacillus
subtilis (FosBBS). Time courses for the reactions were
followed by integrated peak intensities in a continuous
31P NMR assay, which is quite convenient with diamag-
netic metals. The assay results for the FosBSA and FosBBC
enzymes with combinations of Ni(II), Mg2þ, L-Cys, and
BSH are illustrated in Figure 2. The results indicate that
there is a very clear preference of the FosBSA enzyme for
BSH and Ni(II). We detected no activity with L-Cys in
the presence of Mg2þ. The enzymes from bacilli also show
a substantial preference for BSH but exhibit far less
discrimination between the two metals (Figure 2B) and
Figure S2 (Supporting Information). In other experiments
we have found Zn(II) is an excellent inhibitor of the FosB
enzymes (data not shown).
Acknowledgment. This work was supported by grants
from the National Institutes of Health R01 GM030910,
F32 GM093507, T32 ES007028, and P30 ES000267 and
the Vanderbilt Institute of Chemical Biology. We grate-
fully acknowledge Professor Robert Fahey (UC-San
Diego) for a sample of mB-bacillithiol.
Supporting Information Available. Experimental pro-
cedures and full spectroscopic data for all new com-
pounds; enzyme expression, purification, and assays.
This material is available free of charge via the Internet
at http://pubs.acs.org.
The authors declare no competing financial interest.
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