Chemoselective ligation of sulfinic acids with aryl-nitroso compounds

Article abstract of DOI:10.1002/anie.201201812

Making a comeback: The inefficient condensation of sulfinic acid and aryl nitroso compounds has been transformed into a chemoselective process that converts sulfinic acid into stable cyclic sulfonamide analogues (see scheme). This ligation proceeds rapidly under aqueous conditions in high yield, and lays the groundwork for the development of sulfinic acid detection methods in biological systems. Copyright

Full text of DOI:10.1002/anie.201201812

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DOI: 10.1002/anie.201201812
Protein Modifications
Chemoselective Ligation of Sulfinic Acids with Aryl-Nitroso
Compounds**
Mauro Lo Conte and Kate S. Carroll*
In memory of William S. Allison
Hydrogen peroxide (H2O2) acts as a second messenger during
cell signaling and, at low levels, regulates an array of
physiological functions.[1] Conversely, excessive H2O2 can
lead to oxidative stress, which is a chronic state implicated in
the etiology or progression of human diseases, including
cancer.[2] Owing to the high nucleophilicity of the thiol group,
reactive cysteine residues in proteins can be modified by H2O2
to form sulfenic acid (RSOH).[3] This cysteine oxoform can be
reduced back to the thiol group or be further oxidized to
sulfinic (RSO2H) and sulfonic acid (RSO3H) (see Figure S1 in
the Supporting Information). Each of these species exhibits
unique chemical properties and affords a versatile mechanism
to alter protein function.[4]
Although the regulatory function of protein sulfenic acids
is now established,[5] little is known about the role of sulfinic
acids. Indeed, this modification was long dismissed solely as
an artifact of protein isolation. However, mounting evidence
indicates that cysteine is oxidized to sulfinic acid in cells to
a greater extent, and is more controlled, than first thought.
For example, quantitative amino acid analysis of soluble
proteins from normal rat liver indicates that approximately
5% of cysteine residues exist in this oxidation state.[6] Sulfinic
acid modification (with concomitant regulation) is also
associated with a growing list of proteins, including nitrile
hydratase,[7] matrilysin,[8] and the Parkinsonꢀs disease protein,
DJ-1.[9] Peroxiredoxins are also highly susceptible to sulfinic
acid formation at their catalytic cysteine and leads to a loss in
peroxidase activity.[10] Cysteine sulfinic acid is not reduced by
typical cellular reductants such as glutathione and thus, this
derivative was considered to be biologically irreversible.
Recently, this viewpoint was revised when an enzyme called
sulfiredoxin was found to reduce the sulfinic form of certain
peroxiredoxins.[11] The discovery of a sulfinic acid reductase
suggests a more fundamental role for this modification,
thereby leading Jacob and colleagues to propose a new
paradigm for protein regulation by H2O2 known as the
“sulfinic acid switch”.[12]
Robust methods for detecting sulfinic acid are required to
understand the physiological and pathological function of this
modification. Sulfinic acid derivatives can be detected by an
increase in cysteine residue mass of 32 Da,[13] however, there
is increasing concern with this potential indicator given that
modification of proteins by hydrogen sulfide (H2S) leads to
a persulfide species (RSSH) with the same nominal mass shift.
Antibodies directed against the sulfinic acid form of specific
proteins are known,[14] but are not suited for global profiling
studies. Aryl diazonium salts have been used for the
quantitation of methanesulfinic acid.[15] Nevertheless, this
system suffers from several significant limitations inherent to
the instability of diazonium salts in aqueous solution[16] and
formation of stable adducts with tyrosine[17] and cysteine.[18]
Herein, we describe a novel selective ligation reaction of
sulfinic acids with potential utility for detection of protein
sulfinylation in biological systems.
With a pKa of approximately 2, sulfinic acids are fully
deprotonated at physiological pH (Figure 1; 1a–e). The
ambident sulfinate anion behaves primarily as a soft nucle-
Figure 1. Resonance and structures of sulfinate anions in this study.
Boc=tert-butoxycarbonyl.
ophile. Sulfur attack is favored and proceeds toward the more
thermodynamically stable sulfone.[19] The key challenge is to
develop a ligation method for sulfinic acid that is orthogonal
to cysteine, related oxyacids, and other common biological
functionalities. With these considerations, we focused on the
reaction of C-nitroso compounds (2) with aryl sulfinic acids to
provide an N-sulfonyl hydroxylamine (3; Scheme 1).
[*] Dr. M. Lo Conte, Dr. K. S. Carroll
Department of Chemistry, The Scripps Research Institute
130 Scripps Way, Jupiter, FL 33458 (USA)
E-mail: kcarroll@scripps.edu
Reports of this condensation date back to the end of the
19th century,[20] however this topic remains surprisingly
understudied. For instance, reactions with aromatic sulfinic
acids and C-nitroso compounds have been reported,[21] but the
reactivity of alkyl sulfinic acids have not been explored. With
[**] The authors acknowledge funding from the Camile Henry Dreyfus
Teacher Scholar Award (to K.S.C.) and the American Heart
Association Scientist Development Award (0835419N to K.S.C.).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201201812.
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Scheme 1. Condensation reaction between a C-nitroso compound and
aryl sulfinic acid.
respect to our goal for sulfinic acid ligation, it is also
important to note that the resulting adduct is unstable in
basic solutions. In fact, the N-sulfonyl hydroxylamine 3 may
be deprotonated (4) and readily dissociate back into the
starting materials.[21] In this context, we investigated the
reactivity of p-nitroso methyl benzoate (5) with sulfinic acids
(Table 1).
Scheme 2. Proposed mechanism of N-sulfonylbenzisoxazolone forma-
tion.
buffer (50%; PBS = phosphate buffered saline) and CH3CN
(50%; Table 2). Interestingly, the methyl ester 7a reacted
with 1b, but the intermediate species was not converted into
the N-sulfonylbenzisoxazolone 9a (Table 2, entry 1). Consid-
Table 1: N-sulfonyl hydroxylamine formation from RNO and RSO2Na.
Table 2: Reactivity of 2-nitroso benzoic acid derivatives towards sulfinic
acids.
Entry
RSO2Na
Solvent
Acid
Product
Yield [%][a]
1
2
3
4
5
6
7
1a
1a
1a
1b
1b
1b
1b
DMSO
DMSO
DMSO
DMSO
DMF
CF3CO2H
HCO2H
6a
6a
6a
6b
6b
6b
6b
>98
98
95
97
>98
85
C-Nitroso compound
Yield [%][a]
À
Entry
RSO2
CH3CO2H
CH3CO2H
CH3CO2H
CH3CO2H
CH3CO2H
R1
R2
R3
1
2
3
4
5
6
7
7a
7b
7c
7c
7d
7d
7d
H
H
NO2
NO2
H
H
H
H
H
CO2H
CO2H
CO2H
Me
Et
1b
1b
1b
1a
1b
1a
1c
À (9a)
À (9b)
84 (9c)]
88 (9d)
84 (9e)
89 (9 f)
81 (9g)
MeOH
CH3CN
Me
Me
Me
Me
Me
89
[a] Determined by 1H NMR analysis after a 10 min reaction time.
DMF=N,N’-dimehthylformamide, DMSO=dimethylsulfoxide. R for 1a
and 1b as depicted in Figure 1.
H
H
[a] Yields of isolated products after purification by silica gel chroma-
tography. R for 1a–c as depicted in Figure 1.
Encouragingly, in the presence of weak acids, we found
that both aryl (1a) and alkyl sulfinates (1b) reacted rapidly
with high yields in a wide variety of organic solvents. Given
that these condensation reactions only proceed in aqueous
media of very low pH (0–3),[21] we next needed to develop
a strategy to convert the sulfonyl hydroxylamine adduct into
a more stable product. We hypothesized that the deproton-
ated form of 4 is a potential nucleophile, which in the
presence of an electrophilic center on the aromatic group
could trap the oxyanion by intramolecular rearrangement. In
support of this proposal, Moinet et al. have described the
intramolecular cyclization reaction of N-sulfonyl aromatic
hydroxylamines with an ester group in the ortho position
(e.g., 8; Scheme 2).[22] Since cyclization proceeds through
acid-catalyzed transesterification, long reaction times (24 h)
are required to obtain moderate yields. However, we
reasoned that the reaction kinetics could be improved by
performing the ligation in neutral or slightly basic conditions.
To evaluate this hypothesis, we synthesized ortho-nitroso
benzoic esters (7a–b) and tested their reactivity with methane
sulfinic acid (1b) in a solvent system containing pH 7.0 PBS
ering that the analogous result was obtained with ethyl ester
7b (Table 2, entry 2), we speculated that low conversion
reflects the relatively weak acidity of the sulfonyl hydroxyl-
amine 10 (see Scheme 2). Thus, to increase the acidity of this
group, a nitro substitutent was introduced on the phenyl ring
(7c). Compound 7c was converted in good yield into the
sulfonyl adducts (Table 2, entries 3 and 4), consistent with our
hypothesis. To improve solubility in water, while preserving
reactivity, we then synthesized 2-nitroso terephthalic acid
methyl ester (7d), in which the scaffold is elaborated with
a carboxylic acid group. Compound 7d showed excellent
solubility under neutral pH conditions and robust reactivity
toward a variety of sulfinic acids (Table 2, entries 5–7), as
envisioned. Importantly, the nitroso 7d proved stable in
neutral aqueous conditions (t1/2 ꢀ 24 h; Figure S2). Together,
these studies highlight the potential compatibility of this
reaction with biological systems.
Next, we investigated the kinetics for this conversion by
1H NMR spectroscopy and LC-MS. The rate of ligation was
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measured using a large excess of 1b over the nitroso
compound, so that pseudo-first-order rate constants could
be obtained (Table 3 and see Figures S3–S8 in the Supporting
with sulfinic acids assures short reaction times. Furthermore,
the resulting sulfonyl adducts (9e–i) were stable in buffer
(Figure S11) and unreactive, even with nucleophiles, such as
lysine and cysteine (Figure S12).
To further test the suitability of our sulfinic acid ligation
approach, we examined the potential cross-reactivity between
7 f and a variety of biological functional groups (Table 5). The
phenyl ester 7 f did not react with the lysine e-amino group
Table 3: Rate constants for the reaction of 2-nitroso benzoic ester
derivatives with methane sulfinic acid.
Table 5: Reactivity of 7 f toward biological functional groups in buffer.
Entry
C-Nitroso
pH
k (ꢀ10À3 sÀ1
)
compound
Entry
Biofunctional group
Result[a]
1
2
3
4
5
6
7
7d
7d
7d
7e
7 f
7 f
7 f
6
7
8
7
5
6
7
0.2
1.1
2.3
0.6
16
1
2
11
12
no reaction
no reaction
54
131
3
4
13
14
no reaction
cystine with reduced
products of 7 f
Information). In accord with the proposed mechanism
(Scheme 2), the rate of reaction between 7d and 1b
accelerated with increasing pH (Table 3, entries 1–3). To
further optimize this reaction, we evaluated the rate constants
for 2-nitroso terephthalic acid ester derivatives (7e–f).
Although the ethyl ester 7e gave similar reactivity to that of
7d (Table 3, entry 4), the phenyl ester 7 f exhibited excellent
conversion rates, even at lower pH (Table 3, entries 5–7).
With the improved compound 7 f in hand, we then
explored its reactivity with sulfinic acids of increasing
complexity in PBS buffer (Table 4). We were delighted to
observe complete ligation of 7 f within 10 minutes, with
5
15
no reaction
6
7
16
17
no reaction
no reaction
8
9
18
19
no reaction
no reaction
Table 4: Reactivity of 2-nitroso terephthalic acid phenyl ester in buffer.[a]
[a] Reactions were analyzed by LC-MS. Cbz=benzyloxycarbonyl.
(11); alcohol-containing amino acids, such as serine (12) and
tyrosine (13), were also inert (Table 5, entries 1–3 and see
Figure S13 in the Supporting Information). Thiol compounds
are known to reduce aromatic C-nitroso compounds.[23] In this
reaction, the thiol attacks the nitroso to yield an N-hydroxyl-
sulfenamide which condenses with a second thiol to yield N-
hydroxylamine and disulfide products (Scheme S1). Consis-
tent with this mechanism, treatment of cysteine (14) with 7 f
generated cystine and reduced nitroso products (Table 5,
entry 4; Scheme S2 and Figure S13). Importantly, however,
no stable adduct was formed between 7 f and cysteine, and
reduced nitroso species did not react with biological moieties
(Figures S14 and S15).
À
Entry
RSO2
Product
Yield [%]
1
2
3
4
5
1a
1b
1c
1d
1e
9 f
9e
9g
9h
9i
quantitative
quantitative
quantitative
quantitative
quantitative
[a] R for 1a–e as depicted in Figure 1.
simple (1a–b) as well as more elaborate sulfinic acids (1c–e).
In all cases, LC-MS analysis verified formation of a single
product with an m/z corresponding to the N-sulfonylbenz-
isoxazolone adduct (see Figure S9 in the Supporting Infor-
mation). As compared to the methyl ester 7d, phenyl ester 7 f
was slightly less stable under aqueous conditions (t1/2 ꢀ 8 h;
Figure S10). Nonetheless, the higher chemical reactivity of 7 f
If we apply this method to biological systems, glutathione
(GSH) is likely to be present in many-fold excess, relative to
protein sulfinic acids, even under oxidative stress conditions.
Thus, a major concern is that excess thiol could reduce 7 f and
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preclude ligation to sulfinic acids. To address this question,
the sulfinic acid 1b (1 equiv) was treated with 7 f (100 equiv)
in the presence of a 50-fold excess of cysteine or GSH
[Eq. (1); Scheme 3 and see Table S1 in the Supporting
Information]. Under these reaction conditions, 1b was
converted into the ligation product 9e almost quantitatively
Experimental Section
See the Supporting Information for experimental details.
Received: March 6, 2012
Revised: April 6, 2012
Published online: May 29, 2012
Keywords: chemoselectivity · nitrogen heterocycles · oxidation ·
.
protein modifications · sulfur
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Scheme 3. Control experiments for the ligation of sulfinic acid by 7 f in
the presence of excess thiols.
(98%) after 30 minutes. An alternative strategy, commonly
employed in analysis of related redox modifications, is to
alkylate free thiols with N-ethylmaleimide (NEM).[24] In this
manner, a mixture of sulfinic acid 1b and GSH (1:50) was
treated with NEM for 15 minutes prior to the addition of 7 f
(1 equiv). Notably, this reaction sequence also led to the
desired ligation product 9e in excellent yield (96%) [Eq. (2);
Scheme 3 and see Table S2].
Finally, we investigated the reactivity of 7 f with oxidized
thiol species, including sulfenic acid, sulfenamide, disulfide,
sulfonic acid, and nitrosothiol (Table 5, entries 5–9). In all
cases, LC-MS analysis showed no evidence for the ligation
product (see Figure S16 in the Supporting Information). Even
sulfenic acid, which is known for its ambiphilic reactivity, did
not react with 7 f. Rather, thiosulfinate 15c (obtained by self-
condensation of sulfenic acid; Scheme S3) was detected as the
exclusive product in this reaction.
To summarize, we have developed a robust ligation
reaction that selectively converts sulfinic acid moieties into
stable conjugates. To the best of our knowledge, this is the first
report of a one-step method to selectively convert alkyl
sulfinic acids into stable conjugates in aqueous media under
physiological pH. In view of data obtained in several control
experiments, we expect that the selective and facile reaction
presented herein can serve as the foundation for the future
development of methods to detect sulfinic acid formation in
biological systems. Current studies focus on this area and the
details of these ongoing efforts will be reported in due course.
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