Methylsulfonyl benzothiazole (MSBT): A selective protein thiol blocking reagent

Article abstract of DOI:10.1021/ol301370s

A new thiol blocking reagent, methylsulfonyl benzothiazole, was discovered. This reagent showed good selectivity and high reactivity for protein thiols.

Full text of DOI:10.1021/ol301370s

ORGANIC
LETTERS
2012
Vol. 14, No. 13
3396–3399
Methylsulfonyl Benzothiazole (MSBT): A
Selective Protein Thiol Blocking Reagent
Dehui Zhang,†,§ Nelmi O. Devarie-Baez,†,§ Qian Li,‡ Jack R. Lancaster Jr.,‡ and
Ming Xian*,†
Department of Chemistry, Washington State University, Pullman, Washington 99164,
United States, and Departments of Anesthesiology, Physiology & Biophysics, and
Environmental Health Sciences, and Center for Free Radical Biology,
University of Alabama at Birmingham, Birmingham, Alabama 35294, United States
mxian@wsu.edu
Received May 17, 2012
ABSTRACT
A new thiol blocking reagent, methylsulfonyl benzothiazole, was discovered. This reagent showed good selectivity and high reactivity for protein
thiols.
Protein cysteine residues are targets of numerous post-
translational modifications (PTM) that are essential to
maintain cell redox homeostasis as well as signaling.1
Modifications at cysteine residues are caused by their
interaction with reactive oxygen/nitrogen species (ROS/
RNS) in response to cellular oxidative damage.1a,2 Most of
these modifications, which comprise the thiol proteome,
are reversible and encompass a range of functional groups
with very distinctive chemistry including mixed disulfides
(RS-SR0, SR0 low molecular weight thiols), nitrosothiols
(RS-NO), sulfenic acids (RSOH), sulfinic acids (SO2H),
sulfonic acids (SO3H), S-lipidation (palmytoylation,
RS-COR), and perthiols (RS-SH). This diversity of func-
tionality has possessed some difficulty in selectively deter-
mining each modification.3 Nevertheless many advances
have been made in this field, in particular employing
chemical methods to detect specific thiol modifications.3,4
In these methods, a common step involves selective block-
ing of unmodified thiols (reduced thiols) (Scheme 1).
Scheme 1. Chemical Approach to Study Protein Posttransla-
tional Modifications (PTM) at Cys
To a great extent, the efficiency of these assays relies on
the efficiency of the thiol blocking step. Many research
efforts have been made to identify reagents that enable
blocking or labeling of protein thiols with high selectivity
and conversion yields.5 Among those, thiol-alkylation
reagents such as iodoacetamides (IAM) and N-substituted
maleimides (NSM) are by far the mostcommonly usedand
† Washington State University.
‡ University of Alabama at Birmingham.
§ These authors contributed equally.
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r
10.1021/ol301370s
Published on Web 06/08/2012
2012 American Chemical Society

their reactivity profiles have been extensively studied.6 It is
known that, under certain conditions, IAM and NSM can
modify other reactiveaminoacids (e.g., Lysand His).7 Asa
consequence, it has been suggested that the selection of the
thiol blocking reagent should not be arbitrary. Due to the
disparate reactivity of various thiols influenced by their
localization within the protein and physiological environ-
ment, one must consider the unique property of target
protein and necessary experimental conditions to select
proper thiol blocking agents.8
tested several commercially available 2-halogenated benzo-
thiazoles (3aꢀ3c). These compounds are known to react
with thiol at high temperature and under strong basic
conditions.10 However, under mild and biologically mimic
conditions, these substrates displayed very poor reactivity
and only a trace amount of the desired product 2a was
formed. The reaction using 2-diazo substrate 3d resulted
in a complicated mixture of products, and only a small
amount of 2a was produced (judging by TLC and crude
NMR). Interestingly, 2-methylsulfonyl benzothiazole
(MSBT) showed very high reactivity toward 1a, with
almost quantitative formation of 2a within 20 min. To
the best of our knowledge, this was the first example
illustrating the excellent reactivity of MSBT toward al-
kylthiols in aqueous solutions.
On the basis of all the above-mentioned, the develop-
ment of new thiol blocking reagents that possess distinctive
reactivity profiles from currently known compounds is
needed. Our consideration in this subject was to explore
molecules that could react with free thiols via nucleophilic
aromatic substitution (NAS). We developed this idea from
previous work in our laboratory that studied the reactions
of 2-mercaptobenzothiazole (2-SHBT) toward sulfen-
amides and alkyl disulfides.9 The results revealed that
2-SHBT was inert to disulfide exchange reactions but
showed significant reactivity against more reactive electro-
philes, i.e. sulfenamides. In contrast, other aromatic thiols
such as thiophenol, 2-mercaptopyridine, and 2-mercapto-
pyrimidine were found reactive to both alkyl disulfides and
sulfenamides. It should be noted that the disulfide ex-
change is a dynamic equilibrium and thus the progress of
the reaction is controlled by both the electrophilicity/
nucleophilicity of the starting disulfide/thiol pair as well
as those generated. These results suggest that the electron
withdrawing effect of the benzothiazole ring decreases the
reactivity of the corresponding thiol and inhibits disulfide
formation. We envisioned that by placing a leaving group
at the C-2 position, benzothiazole might be vulnerable for
nucleophilic attack by thiols via the NAS mechanism.
With this idea in mind, we designed a series of experiments
to examine whether the benzothiazole moiety could be
employed as an electrophilic trap for thiols and Cys
residues. Here we report our results.
Table 1. Reactions of Benzothiazole Substrates with Model
Substrate 1a
We first examined the reactivity of various benzothia-
zole substrates containing different leaving groups at the
C-2 position (Table 1). A cysteine derivative 1a was used as
a thiol model. In a typical experimental setting, to a
solution of 1a in 1:2 THF/phosphate buffer (200 mM,
pH = 7.4) were added 2 equiv of benzothiazole substrate
respectively. The reaction was monitored by TLC. We
We next investigated whether the pronounced reactivity
of MSBT 3e toward thiols could also occur with other
potential nucleophilic species found in proteins. A series of
amino acid derivatives (4aꢀ4f) were then tested under the
same conditions. As shown in Scheme 2, side chain func-
tionalities of serine, tyrosine, tryptophan, and methionine
are inert to MSBT (5 equiv). In addition, lysine and
histidine substrates (4e and 4f) did not react with MSBT
to form any product (monitored by TLC), even after 4 h.
These outcomes were expected, as known reactions of
MSBT with amines and alcohols require high temperature
and/or strong basic media.11 Nevertheless, these results
suggested that MSBT is a thiol-selective blocking reagent.
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Org. Lett., Vol. 14, No. 13, 2012
3397

protein thiols are variable within distinctive protein do-
mains and pH fluctuates in different cell compartments.12
Because the reactivity of many electrophiles toward SH
depends on the concentration of thiolate, disruption in the
pH will affect the equilibrium between thiol (RSH) and
thiolate (RSꢀ) and therefore change the thiol blocking
efficiency.13 To study this problem, we prepared a water-
soluble MSBT reagent (MSBT-A). This compound al-
lowed us to study pH effects in high aqueous buffer
containing systems. The results are summarized in Table 3.
In acidic media (pH = 6.2), MSBT-A reacted with 1a
slowly and only a small amount of product was obtained
after 20 min. At pH 7.0 or 7.4, the reaction went well and
afforded the blocking product in good yield in 20 min.
When the pH was 9.0, the reactivity of 1a was greatly
enhanced and the reaction was completed in a few minutes.
Interestingly, when this reaction was performed in pure
organic solvents such as pure THF, we did not observe
the formation of 2a even after 1 h. With these results we
concluded that the reactions between MSBT substrates
and RSH largely rely on the thiolateconcentration. Similar
reactivity profiles were observed with both IAM and NSM
derivatives.13,14
Scheme 2. Control Experiments
Table 2. Reactions of MSBT with RSH Substrates
Table 3. pH Dependence of MSBT Reaction
pH
time
% yield
6.2
7.0
7.4
9.0
20 min
20 min
20 min
5 min
34
87
95
95
We also tested the stability of the thiol-blocking adducts
(RS-Bt) under common conditions used in protein labeling
experiments. For example, tris(2-carboxyethyl)phosphine
(TCEP) or dithiothreitol (DTT) are often used for protein
thiol reduction or quenching an excess of thiol-blocking
reagent. We found that RS-Bt adducts did not show any
reaction or decomposition in the presence of an excess of
TCEP or DTT (see Supporting Information for details).
Finally we tested the capability of MSBT and MSBT-A
in blocking protein thiol residues. Glyceraldehyde 3-phos-
phate dehydrogenase (GAPDH), whose biological func-
tion has been shown to be mediated by its cysteine thiol
modifications,15 was used as the model. Briefly (Figure 1A),
reduced GADPH was treated with vehicle, MSBT,
MSBT-A, or a common thiol blocking reagent methyl
In order to explore the generality of an MSBT mediated
thiol-blocking reaction, a series of cysteine derivatives
(1aꢀ1g) were tested. As shown in Table 2, in all the cases
the reaction went smoothly and the desired products were
obtainedinhigh yields. Wedidnotobserve any byproducts
in these reactions.
To further expand our understanding of the reactivity
profile of MSBT, we studied the effect of pH on the
reaction. This was driven by the fact that pKa values of
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Org. Lett., Vol. 14, No. 13, 2012

antibody. As shown in Figure 1B, MSBT and MSBT-A
compared to MMTS exhibited excellent thiol blocking
activity. This result confirmed the efficiency of MSBT
substrates in thiol specific blocking.
In summary, we have discovered a new reagent, MSBT,
capable of blocking protein thiols selectively and effec-
tively. As observed in other thiol blocking reagents, the
reactivity profile of MSBT as a function of pH suggests
that the rate of reaction depends on thiolate concentration.
We expect MSBT substrates will find applications in
protein chemistry.
Figure 1. Thiol blocking capability of MSBT and MSBT-A on
GAPDH compared to MMTS. (A) Schematic representation of
the assay using blocking reagents (MSBT, MSBT-A, MMTS)
and vehicle. (B) Western blot results.
Acknowledgment. This work is supported in part by
the NIH (R01GM088226 to M.X. and R01CA131653 to
J.R.L.) and Burroughs Wellcome Fund (Collaborative
Research Travel Grant to M.X.).
methanethiosulfonate (MMTS)16 and then excess reagents
were removed by desalting. The protein sample was then
exposed to a thiol labeling reagent N-[6-(biotinamido)-
hexyl]-30-(20-pyridyldithio)propionamide (biotin-HPDP).
Biotin labeled GAPDH was detected by nonreducing
SDS-PAGE followed by Western blot using the antibiotin
Supporting Information Available. Spectroscopic and
analytical data and selected experimental procedures.
This material is available free of charge via the Internet
at http://pubs.acs.org.
(15) (a) Chakravarti, R.; Kulwan, S. A.; Fox, P. L.; Stuehr, D. J.
Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 18004. (b) Kornberg, M. D.;
Sen, N.; Hara, M. R.; Juluri, K. R.; Nguyen, J. V. K.; Snowman, A. M.;
Law, L.; Hester, L. D.; Snyder, S. H. Nat. Cell Biol. 2010, 12, 1094.
(16) Jaffrey, S. R. Methods Enzymol. 2005, 396, 105.
The authors declare no competing financial interest.
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