A mild and selective protecting and reversed modification of thiols

Article abstract of DOI:10.1016/j.tetlet.2016.05.027

One selective thiol-protecting study has been investigated for a wide range of thiols including general thiols and thiols containing multiple functional groups. The reactions of bromomaleimides and thiols under the mild condition afforded the protected products in excellent yields. The thiols can be recovered very quickly using dithiothreitol (DTT) under the mild condition.

Full text of DOI:10.1016/j.tetlet.2016.05.027

Tetrahedron Letters 57 (2016) 2660–2663
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Tetrahedron Letters
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A mild and selective protecting and reversed modification of thiols
a,c,
Xiangmin Li a, Hongxian Li a, Wei Yang b, Jinchen Zhuang a, Hao Li a, , Wei Wang
⇑
⇑
a State Key Laboratory of Bioengineering Reactor, Shanghai Key Laboratory of New Drug Design, and School of Pharmacy, East China University of Science and Technology, 130
Mei-long Road, Shanghai 200237, China
b Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
c Department of Chemistry and Chemical Biology, University of New Mexico, Albuquerque, NM 87131-0001, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
One selective thiol-protecting study has been investigated for a wide range of thiols including general thi-
ols and thiols containing multiple functional groups. The reactions of bromomaleimides and thiols under
the mild condition afforded the protected products in excellent yields. The thiols can be recovered very
quickly using dithiothreitol (DTT) under the mild condition.
Received 9 April 2016
Revised 5 May 2016
Accepted 9 May 2016
Available online 9 May 2016
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
Thiols
Bromomaleimide
Protecting
Deprotecting
Selective
Thiols are the most important nucleophilic residues for study-
ing peptides and proteins in chemical biology.1 Synthesis of
thiol-containing biomolecules is an important, yet challenging
work which usually was puzzled by the formation of disulfide bond
and unexpected acetylation or alkylation.2,3 In the past few dec-
ades, selective chemical modification, fluorescent labeling, and
detection of thiols in proteins is widely used in a range of funda-
mental biological and biophysical studies.4 Significant research
efforts have been realized that the optical probes for various bio-
logical thiols to achieve high sensitivity, low cost, and ease of
detection have been developed.4 Meanwhile, identification of
reagents that enable blocking or labeling of protein thiols with
high selectivity and conversion yields has attracted great atten-
tion.5 Common thiol-protecting groups such as thioethers (trityl,
benzyls, and t-butyl),6 thioesters,7 and disulfides8 have limited
scope of applications due to either unsatisfactory stability profiles
or the harsh deprotecting conditions. The acetamidomethyl (Acm)
protecting group developed by Hirschmann and co-workers has
been shown to be useful in the synthesis of peptides.9 Unfortu-
nately, the reagents could be dimmed by the use of toxic heavy
metals in deprotection process. For the purpose of protecting Cys
side chain in peptides and proteins, Liu and co-workers developed
a thiol protecting group called Hqm group.10 It’s a good-quality
protecting group, however, several synthetic steps were required
for synthesis and protecting processes. In order to protect the thi-
ols in peptide condensation reactions, phenacyl and N-methyl-
phenacyloxycarbamidomethyl were developed to improve
thioether-based thiol-protecting groups by Hojo and co-workers.11
Nevertheless, the carbonyl of these protecting groups may react
with amino residues of peptides, which need to be protected before
the thiol-protecting process. Other protecting groups such as p-
toluenesulfonylacetylene12 and quinolone13 have limited utiliza-
tion due to the instability in the presence of amino or the high pro-
tecting temperature.
Maleimides have been proved to be one of the most widely used
reactive motifs for cysteine modification.1 Bromomaleimides,
developed by Baker and co-workers, react rapidly with protected
cysteine to afford thiomaleimides.14 Recently, bromomaleimides
have been successfully applied in the synthesis of polymers,15 pro-
tein labeling,16 and peptide platforms17 with protected cysteines.
All cysteines used in these methods were protected cysteines. To
the best of our knowledge, the efficient protecting and reversed
modification of general thiols or thiols with active functional
groups has not been studied. Herein, we wish to focus on bromo-
maleimides as selective thiol-protecting reagents to a wide range
of thiols. Furthermore, DTT has been applied to an efficient depro-
tecting reagent of protected thiols under the mild condition.
The protecting group bromo-N-R-maleimide 1 could be easily
synthesized from the corresponding N-R-maleimides in two
steps.18 In the initial experiment, bromo-N-methylmaleimide 1a
⇑
Corresponding authors. Tel./fax: +86 (21) 6425 3299 (H.L.); tel.: +1 (505) 277
0756; fax: +1 (505) 277 2609 (W.W.).
E-mail addresses: hli77@ecust.edu.cn (H. Li), wwang@unm.edu (W. Wang).
http://dx.doi.org/10.1016/j.tetlet.2016.05.027
0040-4039/Ó 2016 Elsevier Ltd. All rights reserved.

X. Li et al. / Tetrahedron Letters 57 (2016) 2660–2663
2661
Table 1
(Table 1, entry 5). Other organic or inorganic bases, such as DMAP,
DIPEA and NaHCO3 were also suitable for this reaction. On the
basis of these exploratory studies, we probed various bromoma-
leimides derived thiol-protecting groups with N-Boc-Cys-OMe
under the treatment of Et3N in THF. It was found that the reactions
with small protecting groups, such as H, Me and Et, took place very
quickly to give the products in high yields (Table 1, entries 1, 6 and
7). In contrast, the bromomaleimide with bigger substitutes, such
as bromo-N-phenylmaleimide 1d and bromo-N-benzylmaleimide
1e, afforded the corresponding products in lower yields (Table 1,
entries 8 and 9). Longer reaction time could not raise the reaction
yields. We envisioned that both steric and electronic effects of sub-
stituents account for the yield differences. Based on the above
results, the optimized reaction condition was found: THF as sol-
vent, Et3N as base, bromo-N-methylmaleimide 1a or bromoma-
leimide 1b as the protecting reactants.
Optimization of reaction conditionsa
O
O
3 equiv Et3N
BocHN
+
R
N
R
SH
N
BocHN
Solvent
20 min, rt
S
COOMe
Br
O
COOMe
O
2a
1
3
Entry
R
Solvent
Yieldb (%)
1
2
3
4
5c
6
7
8
9
Me
Me
Me
Me
Me
H
Et
Ph
Bn
THF
MeOH
Toluene
CH2Cl2
H2O
THF
THF
THF
THF
99
96
93
95
91
97
92
83
77
With the optimal reaction condition in hand, we turned our
attention to probing the scope of different thiols (Table 2). Satisfac-
torily, almost all reactions finished within half an hour to give the
protected products in excellent yields (Table 2). Linear thiols
reacted smoothly and finished in 20 min to afford the products in
excellent yields (3b–3d). Branched isobutylthiol had similar reac-
tivity as linear thiols (3e). Steric effect had great effect on this reac-
tion. Bulky cyclohexanethiol gave high yields while longer reaction
time was needed (3f). More bulky t-butylthiol needed 6 h to finish
the reaction (3g). Interestingly, triphenylmethylthiol reacted much
faster than t-butylthiol under the same reaction condition even
though its steric effect was greater than t-butylthiol (3h, 30 min).
The heterocyclicthiols, such as thiophene-2-thiol and 1-methyl-
1H-imidazole-2-thiol, afforded the corresponding products with
high yields (3i–3j).
a
Reaction condition: to a solution of 2a (61.8 mg, 0.263 mmol) in solvents (5 mL)
was added Et3N (0.789 mmol) and 1 (0.263 mmol) and the resulting mixture was
stirred for 20 min at rt.
b
Isolated yields.
0.5 mL DMF as base and 4.5 mL H2O was used.
c
and commercially available N-Boc-Cys-OMe 2a were tested in the
model reaction (Table 1). Treatment of 2a (0.263 mmol) and 1a
(0.263 mmol) with 3 equiv of Et3N in THF (5 mL) resulted in a rapid
and complete reaction in 20 min with excellent yields (99%, Table 1,
entry 1). Different solvents and bases were tested for the reactions.
The reactions in other solvents, such as MeOH, toluene, CH2Cl2, and
water, also gave the products in excellent yields (>93%) within
20 min (Table 1, entries 2–4). To our delight, it was convinced that
the protecting process was easy to implement at room tempera-
ture, which even took place in water as solvent and DMF as base
Table 3
Thiol-protecting reactions for thiols with functional groupsa
Table 2
Thiol-protecting reactions for general thiolsa
O
O
3 equiv Et3N
THF, rt
O
N
R1
O
N
R1
+
R2 SH
R2
S
Br
3 equiv Et3N
THF, rt
N
R1
O
N
R1
+
O
R2 SH
R2
S
1a
1b
: R1 = H
2a
3
Br
O
O
: R1 = Me
1a
: R1 = H
2a
3
1b
: R1 = Me
O
O
O
HO
O
N Me
O
O
N
Me
N
H
S
S
HO
S
O
N
Me
N
Me
O
O
NH2
N
Me
3m
BocHN
3l
3k
S
S
10
S
COOMe
96% yield[b]
10 min
96% yield[b]
93% yield[b]
O
O
O
3b
3c
30 min
30 min
3a
97% yield[b]
15 min
96% yield[b]
20 min
O
O
O
O
99% yield[b]
Me
O
O
N
HOOC
N
H
N
20 min
N
Me
N Me
N
Me
H2N
O
O
HO
S
N
S
S
S
O
O
O
O
Me
3p
N
Me
N
Me
N
Me
3n
3o
91% yield[b,d]
15 min
92% yield[b,d]
30 min
Ph
S
96% yield[b,c]
6 h
S
S
O
O
O
3d
3e
3f
COOH
O
O
94% yield[b]
20 min
94% yield[b]
20 min
96% yield[b]
30 min
O
H2N
N
H
N
Me
O
O
O
O
HN
HOOC
NH2
O
S
S
N
Ph
O
O
N
Me
Ph
Ph
N
Me
N Me
HOOC
N
H
O
N
Me
3q
3r
N
S
S
S
S
98% yield[b,c]
S
96% yield[b,c]
10 h
O
O
O
3j
3g
3h
3i
6 h
90% yield[b]
30 min
94% yield[b]
6 h
97% yield[b]
30 min
97% yield[b]
10 min
a
Reaction condition: To a solution of 2 (0.263 mmol) in THF (5 mL) was added
Et3N (0.789 mmol) and 1 (0.263 mmol) and the mixture was stirred for the indi-
a
Reaction condition: To a solution of 2 (0.263 mmol) in THF (5 mL) was added
cated time at rt.
b
Et3N (0.789 mmol) and 1 (0.263 mmol) and the mixture was stirred for the indi-
Isolated yields.
H2O was used as solvent without the addition of Et3N.
NaHSO4 (2.63 mmol) and anhydrous MgSO4 (1 g) was added into the mixture.
c
cated time at rt.
b
d
Isolated yields.

2662
X. Li et al. / Tetrahedron Letters 57 (2016) 2660–2663
O
leimides and water-soluble thiol substrates in water under
vigorous stirring. These substrates contain free amine group which
can be used as base instead of Et3N in the reaction. When the reac-
tion completed, water was removed under vacuum and the excess
protecting reagent was washed by CH2Cl2. To our delight, the
desired thiol protected products were obtained in excellent yields
although much longer reaction time was needed (3q–3r).
O
1) 6 equiv Et3N
CH2Cl2
MeOOC
N R
SH
HCl
MeOOC
N
R
+
S
2) 3 equiv Boc2O
rt, 10 h
NH2
Br
O
NHBoc
O
3a, R = Me, 91%
3a'
1a,
R = Me
1b, R = H
2s
, R = H, 83%
Scheme 1. Thiol-protecting with amino-protecting reactions.
Next, we wanted to investigate two protecting reactions to pro-
tect thiol and amine using a ‘one-pot’ process. In our exploratory
study, the commercially available methyl 2-amino-3-mercapto-
propanoate hydrochloride 2s was chosen as a model substrate for
the proposed one-pot protecting reactions (Scheme 1). After the
addition of Et3N into the mixture of substrates in CH2Cl2 for
20 min, Boc2O was added and stirred for another 10 h at rt to give
the desired product. Both bromomaleimide and bromo-N-methyl-
maleimide can be used as thiol protecting groups to afford prod-
ucts 3a and 3a’ in high yields.
In our effort on the construction and expanding scope of the
synthetically useful thiol protecting method, we also wish to
develop an efficient deprotecting method. In Baker and co-workers’
work, expensive TCEP in buffer was applied to reverse the modifi-
cation of cysteine and protein.14 Later on, cheaper 2-mercap-
toethanol (BME) and 1,2-ethanedithiol (EDT) have been applied
to the cleavage strategy. However, large scale of BME (100 equiv)
and longer reaction time of EDT (24 h) was needed.16 We tested
a series of potential deprotection reagents such as triphenylphos-
phine, tributylphosphine and DTT. The results showed that DTT
in THF and water, under the treatment of Et3N, was very efficient
for cleaving C–S bond. Several selective substrates were chosen
for the deprotection study and the results are listed in Table 4. In
Having tested the reactivity of general thiols with bromoma-
leimides, next we questioned whether thiols containing functional
groups also had good selectivities. Various thiols with different
functional groups were investigated in the thiol protecting reac-
tions (Table 3). Thiol substrates containing hydroxyl, phenolic
hydroxyl, and phenylamino groups gave the only thiol protected
products in excellent yields without any other byproduct (3k–
3m). Heterocyclic 5-amino-1,3,4-thiadiazole-2-thiol afforded the
mono thiol addition product 3n in high yields. Both thiol and car-
boxyl are common groups in drug molecules. In order to investi-
gate the effects of carboxyl on the thiol-protecting reactions, we
selected tiopronin 2o and captopril 2p as models for the research.
The reactions proceeded very quickly to give single thiol protected
products in excellent yields (3o–3p). Encouraged by the promising
results, we moved on to investigate the protecting method for
water-soluble substrates, such as cysteine and glutathione.
Water-soluble substrates containing mercapto, carboxy, and amino
could not easy to be protected by conventional methods as a result
of low solubility in organic solvents. Inspired by the result of good
reaction in water in the optimization process, we mixed bromoma-
Table 4
Deprotection of maleimide protected thiolsa
O
DTT, Et3N
THF, H2O
N
R
R
SH
R
S
O
2
3
Entry
1
3
DTT (equiv)
1.1
TEA (equiv)
0.1
t (min)
Yieldb (%)
O
N
Me
H
BocHN
15
92
S
O
O
COOMe
3a
N
BocHN
HO
2
3.0
1.5
15
83
S
O
3a'
O
COOMe
N
Me
3
4
3.0
1.1
3.0
3.0
30
15
95
96
S
O
3l
O
Ph
Ph
Ph
N
Me
S
O
3h
COOH
O
O
H2N
N
Me
91c
HN
5
3.0
3.0
30
S
O
HOOC
N
H
O
3r
a
Reaction condition: To a solution of 3 (0.2 mmol) in THF (8 mL) and H2O (2 mL) was added dropwise DTT and Et3N in THF (2 mL) over 5 min. Then the solution was stirred
at rt for the indicated time.
b
Isolated yields.
c
The yield was determined by 1H NMR using CH2Br2 as internal standard.

X. Li et al. / Tetrahedron Letters 57 (2016) 2660–2663
2663
the deprotecting reaction of 3a, the loading of DTT can be as low as
References and notes
1.1 equiv and only 0.1 equiv of Et3N was needed. For other sub-
strates, no more than 3 equiv of DTT and Et3N was used to give
the corresponding deprotected thiols in high to excellent yields.
It should be noted that all reactions completed within 30 min.
With the demonstrated results, we have illustrated DTT as a
synthetically useful deprotecting reagent for thiol protecting
method.
In summary, bromomaleimides have been used as an efficient
and selective thiol-protecting group, which is applicable for a wide
range of thiols including general thiols and thiols containing mul-
tiple functional groups. The reaction proceeded very fast under the
mild condition to give protected thiols in excellent yields. Further-
more, the protected group can be easily removed under the mild
condition using low equivalent of DTT to release the sulfhydryl
moiety.
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Supplementary data
Supplementary data (all experimental procedures, spectro-
scopic data) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.tetlet.2016.05.027.
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