Angewandte
Chemie
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 Parkinsons 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]
[*] 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
[**] 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.
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).
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
1
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Angew. Chem. Int. Ed. 2012, 51, 1 – 5
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; 1 a–e). The
ambident sulfinate anion behaves primarily as a soft nucle-
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Communications
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 formation.
buffer (50 %; PBS = phosphate buffered saline) and CH3CN
(50 %; Table 2). Interestingly, the methyl ester 7 a reacted
with 1 b, but the intermediate species was not converted into
the N-sulfonylbenzisoxazolone 9 a (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
MeOH
CH3CN
CF3CO2H
HCO2H
CH3CO2H
CH3CO2H
CH3CO2H
CH3CO2H
CH3CO2H
6a
6a
6a
6b
6b
6b
6b
> 98
98
95
97
> 98
85
89
[a] Determined by 1H NMR analysis after a 10 min reaction time.
DMF = N,N’-dimehthylformamide, DMSO = dimethylsulfoxide. R for 1 a
and 1 b as depicted in Figure 1.
2
Ü
Ü
Encouragingly, in the presence of weak acids, we found
that both aryl (1 a) and alkyl sulfinates (1 b) 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 deprotonated 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 (7 a–b) and tested their reactivity with methane
sulfinic acid (1 b) in a solvent system containing pH 7.0 PBS
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Entry
C-Nitroso compound
R1
R2
R
1
2
3
4
5
6
7
7a
7b
7c
7c
7d
7d
7d
Me
Et
Me
Me
Me
Me
Me
H
H
NO2
NO2
H
H
H
1b
1b
1b
1a
1b
1a
1c
Yield [%][a]
(9 a)
(9 b)
84 (9 c)]
88 (9 d)
84 (9 e)
89 (9 f)
81 (9 g)
[a] Yields of isolated products after purification by silica gel chromatography. R for 1 a–c as depicted in Figure 1.
ering that the analogous result was obtained with ethyl ester
7 b (Table 2, entry 2), we speculated that low conversion
reflects the relatively weak acidity of the sulfonyl hydroxylamine 10 (see Scheme 2). Thus, to increase the acidity of this
group, a nitro substitutent was introduced on the phenyl ring
(7 c). Compound 7 c 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 (7 d), in which the scaffold is elaborated with
a carboxylic acid group. Compound 7 d 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 7 d 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
1
H NMR spectroscopy and LC-MS. The rate of ligation was
2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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H
H
H
H
CO2H
CO2H
CO2H
RSO2
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Angewandte
Chemie
measured using a large excess of 1 b over the nitroso
compound, so that pseudo-first-order rate constants could
be obtained (Table 3 and see Figures S3–S8 in the Supporting
Table 3: Rate constants for the reaction of 2-nitroso benzoic ester
derivatives with methane sulfinic acid.
with sulfinic acids assures short reaction times. Furthermore,
the resulting sulfonyl adducts (9 e–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 5: Reactivity of 7 f toward biological functional groups in buffer.
Entry
C-Nitroso
compound
pH
k ( 10
1
2
3
4
5
6
7
7d
7d
7d
7e
7f
7f
7f
6
7
8
7
5
6
7
0.2
1.1
2.3
0.6
16
54
131
3
s 1)
Information). In accord with the proposed mechanism
(Scheme 2), the rate of reaction between 7 d and 1 b
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 (7 e–f).
Although the ethyl ester 7 e gave similar reactivity to that of
7 d (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
Table 4: Reactivity of 2-nitroso terephthalic acid phenyl ester in buffer.[a]
Entry
Biofunctional group
Result[a]
1
11
no reaction
2
12
no reaction
3
13
no reaction
4
14
cystine with reduced
products of 7 f
5
15
no reaction
6
16
no reaction
7
17
no reaction
8
18
no reaction
9
19
no reaction
[a] Reactions were analyzed by LC-MS. Cbz = benzyloxycarbonyl.
RSO2
Product
Yield [%]
1
2
3
4
5
1a
1b
1c
1d
1e
9f
9e
9g
9h
9i
quantitative
quantitative
quantitative
quantitative
quantitative
[a] R for 1 a–e as depicted in Figure 1.
simple (1 a–b) as well as more elaborate sulfinic acids (1 c–e).
In all cases, LC-MS analysis verified formation of a single
product with an m/z corresponding to the N-sulfonylbenzisoxazolone adduct (see Figure S9 in the Supporting Information). As compared to the methyl ester 7 d, 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
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
(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-hydroxylsulfenamide which condenses with a second thiol to yield Nhydroxylamine and disulfide products (Scheme S1). Consistent 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).
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 1 b (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, 1 b was
converted into the ligation product 9 e almost quantitatively
Experimental Section
See the Supporting Information for experimental details.
Received: March 6, 2012
Revised: April 6, 2012
Published online: && &&, &&&&
.
Keywords: chemoselectivity · nitrogen heterocycles · oxidation ·
protein modifications · sulfur
Scheme 3. Control experiments for the ligation of sulfinic acid by 7 f in
the presence of excess thiols.
4
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Ü
(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 1 b 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 9 e 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 15 c (obtained by selfcondensation 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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[1] B. C. Dickinson, C. J. K. Chang, Nat. Chem. Biol. 2011, 7, 504 –
511.
[2] B. Halliwell, J. M. C. Gutteridge, Free Radicals in Biology and
Medicine, 4th ed., Oxford University Press, New York, 2007.
[3] K. G. Reddie, K. S. Carroll, Curr. Opin. Chem. Biol. 2008, 12,
746 – 754.
[4] C. Jacob, G. I. Giles, N. M. Giles, H. Sies, Angew. Chem. 2003,
115, 4890 – 4907; Angew. Chem. Int. Ed. 2003, 42, 4742 – 4758.
[5] C. E. Paulsen, T. H. Truong, F. J. Garcia, A. Homann, V. Gupta,
S. E. Leonard, K. S. Carroll, Nat. Chem. Biol. 2011, 8, 57 – 64.
[6] M. Hamann, T. Zhang, S. Hendrich, J. A. Thomas, Methods
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[7] T. Murakami, M. Nojiri, H. Nakayama, M. Odaka, M. Yohda, N.
Dohmae, K. Takio, T. Nagamune, I. Endo, Protein Sci. 2000, 9,
1024 – 1030.
[8] X. Fu, S. Y. Kassim, W. C. Parks, J. W. Heinecke, J. Biol. Chem.
2001, 276, 41279 – 41287.
[9] J. Blackinton, M. Lakshminarasimhan, K. J. T. Ahmad, E.
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Chem. 2009, 284, 6476 – 6485.
[10] W. T. Lowther, A. C. Haynes, Antioxid. Redox Signaling 2011,
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[11] B. Biteau, J. Labarre, M. B. Toledano, Nature 2003, 425, 980 –
984.
[12] C. Jacob, A. L. Holme, F. H. Fry, Org. Biomol. Chem. 2004, 2,
1953 – 1956.
[13] E. S. Witze, W. M. Old, K. A. Resing, N. G. Ahn, Nat. Methods
2007, 4, 798 – 806.
[14] H. A. Woo, S. W. Kang, H. K. Kim, K. S. Yang, H. Z. Chae, S. G.
Rhee, J. Biol. Chem. 2003, 278, 47361 – 47364.
[15] C. F. Babbs, M. J. Gale, Anal. Biochem. 1987, 163, 67 – 73.
[16] D. F. Detar, J. Am. Chem. Soc. 1956, 78, 3911.
[17] J. M. Hooker, E. W. Kovacs, M. B. Francis, J. Am. Chem. Soc.
2004, 126, 3718 – 3719.
[18] J. Patt, M. Patt, J. Labelled Compd. Radiopharm. 2002, 45, 1229 –
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[19] M. Baidya, S. Kobayashi, H. Mayr, J. Am. Chem. Soc. 2010, 132,
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[20] E. Bamberger, H. Buesdorf, B. Szolayski, Chem. Ber. 1899, 32,
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[21] A. Darchen, C. Moinet, J. Chem. Soc. Chem. Commun. 1976,
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[22] A. Guilbaud-Criqui, C. Moinet, Bull. Soc. Chim. Fr. 1992, 129,
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Protein Modifications
&&&&—&&&&
Chemoselective Ligation of Sulfinic Acids
with Aryl-Nitroso Compounds
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
Making a comeback: The inefficient condensation of sulfinic acid and aryl nitroso
compounds has been transformed into
a chemoselective process which 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.
2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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M. Lo Conte,
K. S. Carroll*
These are not the final page numbers!
Supporting Information
Wiley-VCH 2012
69451 Weinheim, Germany
Chemoselective Ligation of Sulfinic Acids with Aryl-Nitroso
Compounds**
Mauro Lo Conte and Kate S. Carroll*
anie_201201812_sm_miscellaneous_information.pdf
General Experimental
All reactions were performed under a nitrogen atmosphere in oven-dried glassware. Reagents
and solvents were purchased from Sigma or other commercial sources and were used without
further purification. Analytical thin layer chromatography (TLC) was carried out using Analtech
Uniplate silica gel plates and visualized using a combination of UV and potassium permanganate
staining. Flash chromatography was performed using silica gel (32-63 µM, 60 Å pore size) from
Sorbent Technologies Incorporated. NMR spectra were obtained on a Bruker Avance 400 (400
MHz for 1H; 100 MHz for
13
C). 1H and
13
C NMR chemical shifts are reported in parts per
million (ppm) referenced to the residual solvent peak. Low-resolution electrospray ionization
(ESI) mass spectra were obtained with an Agilent 6120 Single Quadrupole LC/MS.
1 Experimental Procedures and Spectroscopic Data
Benzenesulfinic acid sodium salt (1a), Methanesulfinic acid sodium salt (1b), hypotaurine (1d),
lysine (11), serine (12), tyrosine (13), cysteine (14) and cysteine sulfonic acid (18) are
commercially available. 1-methyl 4-nitrosobenzoate (5),[1] 1-methyl 2-nitrosobenzoate (7a),[2] 1ethyl 2-nitrosobenzoate (7b),[2] the cyclic sulfenamide 16,[4] and trityl S-nitrosothiol (19)[3] were
synthesized according to procedures established in the literature; physical properties were
consistent with the previously reported values cited above.
Sulfenic acid 15a was generated in situ by the decomposition of β-sulfinyl propionic acid ester
15b[5] in 0.2 M PBS pH 7.5 at RT.
General Procedure 1: Synthesis of methyl 4-(N-hydroxymethylsulfonamido)benzoate (6a)
Benzenesulfinic acid (5.0 mg, 0.030 mmol) was added to a solution of 1-methyl 4nitrosobenzoate (4.3 mg, 0.027 mmol) dissolved in the appropriate deuterated solvent (700 µL)
containing 2 equiv of an organic acid (see Table 1). After 10 min at RT, the reaction was
monitored by 1H-NMR. 1H NMR (DMSO-d6, 400 MHz): δ 7.89 (d, J = 8.8 Hz, 2H), 7.72 (t, J =
2 7.2 Hz, 1H), 7.54 (t, J = 8.4 Hz, 2H), 7.46 (dd, J = 8.0, 1.2 Hz, 2H), 3.83 (s, 3H). ESI-LRMS
calcd. for C14H15NO5S (M+H) 308.1, found 308.1.
General Procedure 2: Synthesis of methyl 4-(N-hydroxyphenylsulfonamido)benzoate (6b)
Sodium methane sulfinic acid 46% (6.7 mg, 0.030 mmol) was added to a solution of 1-methyl 4nitrosobenzoate (4.3 mg, 0.027 mmol) in the appropriate deuterated solvent (700 µL) containing
2 equiv of an organic acid (see Table 1). After 10 min at RT, the reaction was monitored by 1HNMR. 1H NMR (DMSO-d6, 400 MHz): δ 8.07 (dd, J = 8.0, 1.8 Hz, 2H), 7.58 (dd, J = 8.0, 2.0
Hz, 2H), 3.95 (s, 3H), 2.85 (s, 3H). ESI-LRMS calcd. for C9H12NO5S (M+H) 246.0, found
246.1.
1-methyl 2-amino 4-nitrobenzoate (22) A solution of 10% sulfuric acid in methanol (10 mL) was slowly added to a solution of 2-amino
4-nitrobenzoic acid (1.8 g, 20 mmol) in methanol (20 mL). The reaction was warmed to 80 °C
and maintained at this temperature for 3 d. The reaction was cooled to room temperature and
diluted with a saturated solution of sodium hydrogen carbonate (50 mL). The precipitate was
filtered, washed several times with water, and dried by high vacuum to yield 22 (2.01 g,
3 quantitative) as a bright yellow solid. 1H NMR (DMSO-d6, 400 MHz): δ 8.58 (d, J = 2.4 Hz,
1H), 8.09 (dd, J = 9.2, 2.8 Hz, 2H), 7.83 (s, 1H), 6.90 (d, J = 8.8 Hz, 1H), 3.86 (s, 3H).
13
C
NMR (DMSO-d6, 100 MHz): δ 166.39, 155.77, 135.17, 128.88, 128.35, 116,81, 107.56, 52.29.
ESI-LRMS calcd. for C8H9N2O3 (M+H) 197.1, found 197.1.
1-methyl 2-nitroso 4-nitrobenzoate (7c)
A solution of Oxone® (2.55 g, 4.2 mmol) in water (15 mL) was added to a solution of 22 (275
mg, 1.4 mmol) in CH2Cl2 (5 mL). The reaction was vigorously stirred at room temperature until
LC-MS analysis indicated complete consumption of the starting material (2 d). The product was
extracted with additional CH2Cl2, and the organic phase was dried with MgSO4, filtered, and
concentrated. The residue was crystallized from chloroform to yield 7c as a yellow solid (281
mg, 96% of yield). 1H-NMR (DMSO-d6, 400 MHz, mixture of monomer and dimer 1:1): δ 9.00
(s, 1H), 8.85 (s, 1H) 8.66 (dd, J = 8.8, 2.0 Hz, 1H), 8.41 (d, J = 2.0 Hz, 1H), 8.13 (d, J = 9.4 Hz,
1H), 6.65 (d, J = 8.4 Hz, 1H), 4.10 (s, 3H), 4.06 (s, 3H).
13
C NMR (DMSO-d6, 100 MHz,
mixture of monomer and dimer): δ 162.09, 148.68, 143.79, 134.66, 129.88, 127.25, 125.96,
125.87, 125.55, 114.15, 53.81, 53.63. ESI-LRMS calcd. for C8H7N2O5 (M+H) 211.0, found
211.0.
4 1-methyl 2-nitrosoterephthalate (7d)
A solution of Oxone® (15.8 g, 25.6 mmol) in water (150 mL) was added to a solution of 1methyl 2-aminoterephthalate (2.5 g, 12.8 mmol) in CH2Cl2 (40 mL). The reaction was vigorously
stirred at room temperature until LC-MS analysis indicated complete consumption of the starting
material (2 h). The precipitate was filtered, washed with water and desiccated to yield the
nitroso-compound 7d (2.39 g, yield of 89%) as a pale yellow solid. 1H NMR (DMSO-d6, 400
MHz, mixture of monomer and dimer 1:1): δ 8.34 (d, J = 7.6 Hz, 1H), 8.33-7.97 (m, 4H), 7.43
(s, 1H), 3.99 (s, 3H), 3.96 (s, 3H).
13
C NMR (DMSO-d6, 100 MHz, mixture of monomer and
dimer): δ 166.68, 165.45, 165.01, 163.15, 161.88, 140.64, 136.38, 135.89, 134.04, 133.11,
132.18, 131.48, 130.18, 127.80, 125.51, 113.82, 53.44, 53.37. ESI-LRMS calcd. for C9H6NO5
(M-H) 208.0, found 208.1.
N-acetyl-L-cystine methyl ester (17)
Sodium iodine (9 mg) and hydrogen peroxide 30% in water (0.69 mL, 6 mmol) were added to a
solution of N-acetyl-L-cysteine (1 g, 6 mmol) in water (5 mL). The mixture was stirred until an
orange-brown color formed (1 h), at which point it was concentrated. The residue was dissolved
in methanol (45 mL), and thionyl chloride (1.32 mL, 18 mmol) was dropwise added to the
former solution. The reaction was stirred for 5 h and the mixture was then concentrated. The
5 crude material was purified by flash chromatography (on silica gel, AcOEt/hexanes 1:1 to 3:1) to
yield 17 (1678 mg, 79% of yield) as a white solid. 1H NMR (CDCl3, 400 MHz): δ 6.51 (d, J =
6.8 Hz, 1H), 4.89-4.85 (m, 1H), 3.77 (s, 3H), 3.22-3.15 (m, 2H) 2.06 (s, 3H). 13C NMR (CDCl3,
100 MHz): δ 171.02, 170.21, 52.88, 51.84, 40.83, 23.18. ESI-LRMS calcd. for C12H21N2O6S2
(M+H) 353.1, found 353.1.
Sodium N-acetyl-L-cysteine sulfinic acid methyl ester (1c) Hydrogen peroxide 30% in water (60 µL, 2 mmol) was added dropwise at 0 °C to a solution of
disulfide 17 (184 mg, 0.52 mmol) in acetic acid (3 mL) The reaction was warmed to room
temperature and stirred for 3 hours. The mixture was diluted with ethyl acetate (25 mL) and
washed with saturated NaHCO3 solution (2 X 15 mL). The organic layer was dried over MgSO4,
filtered, and concentrated. The crude material was dissolved in methanol (5 mL), and sodium
thiophenolate (68 mg, 0.52 mmol) was then added. The reaction was stirred for 1 h and then
concentrated. The crude material was re-suspended in acetone and filtered to yield 1c (58 mg,
48% of yield) as a white solid. 1H NMR (D2O, 400 MHz): δ 4.58 (dd, J = 10.0, 4.8 Hz, 1H), 3.80
(s, 3H), 2.88 (dd, J = 14.4, 10.4 Hz, 1H), 2.68 (dd, J = 13.6, 4.8 Hz, 1H), 2.06 (s, 3H). 13C NMR
(D2O, 100 MHz): δ 174.08, 173.12, 60.95, 53.20, 49.18. ESI-LRMS calcd. for C6H10NO5S (MH) 208.0, found 208.0.
6 N-methanesulfonyl 5-nitrobenzo[c]isoxazol-3(1H)-one (9c) Methane sulfinic acid sodium salt 46% (98 mg, 0.55 mmol) was added to a solution of methyl 2nitrosobenzoate (85 mg, 0.4 mmol) in acetonitrile / PBS 0.2 M pH=7 1:1 (8 mL). The reaction
was stirred for 1 h, and then the organic solvent was evaporated under reduced pressure. The
product was extracted with ethyl acetate (2 x 5 mL), and the organic layers were dried with
MgSO4, filtered, and concentrated. The residue was crystallized from methanol to yield 9c (86
mg, yield of 84%) as a white solid. 1H NMR (DMSO-d6, 400 MHz): δ 8.49-8.41 (m, 2H), 7.98
(d, J = 8.0 Hz, 1H), 3.01 (s, 3H).
13
C NMR (DMSO-d6, 100 MHz): δ 165.19, 146.14, 145.10,
127.85, 127.15, 125.01, 123.39, 33.06. ESI-LRMS calcd. for C8H7N2O6S (M+H) 259.0, found
259.0.
N-benzenesulfonyl 5-nitrobenzo[c]isoxazol-3(1H)-one (9d) Benzenesulfinic acid sodium salt (73 mg, 0.44 mmol) was added to a solution of methyl 2nitrosobenzoate (85 mg, 0.4 mmol) in acetonitrile / PBS 0.2 M pH=7 1:1 (8 mL). The reaction
was stirred for 1 h, and then the organic solvent was evaporated under reduced pressure. The
product was extracted with ethyl acetate (2 x 5 mL), and the organic layers were dried with
7 MgSO4, filtered, and concentrated. The residue was crystallized from ethanol to yield 9d (113
mg, yield of 88%) as a white solid. 1H NMR (DMSO-d6, 400 MHz): δ 8.48 (d, J = 2.8 Hz, 1H),
8.27 (dd, J = 9.2, 2.8 Hz, 1H), 7.76 (t, J = 7.2 Hz, 1H), 7.57 (t, J = 7.8 Hz, 2H), 7.44 (d, J = 7.8
Hz, 2H), 7.22 (d, J = 9.2 Hz, 1H), 3.01 (s, 3H).
13
C NMR (DMSO-d6, 100 MHz): δ 165.05,
146.11, 145.18, 134.58, 131.89, 129.23, 129.02, 128.03, 126.68, 124.99, 123.43, 33.0. ESILRMS calcd. for C13H9N2O6S (M+H) 321.0, found 321.0.
N-methanesulfonyl 5-carboxylic benzo[c]isoxazol-3(1H)-one (9e) Methane sulfinic acid sodium salt 46% (122 mg, 0.55 mmol) was added to a solution of 1-methyl
2-nitrosoterephthalate (105 mg, 0.5 mmol) in acetonitrile / PBS 0.2 M pH=7 1:1 (10 mL). The
reaction was stirred for 1 h, then the organic solvent was evaporated under reduced pressure. The
pH of the resulting solution was adjusted to 3 using 1 M HCl solution. The precipitate was
filtered and washed with water and methanol to yield 9e (108 mg, yield of 84%) as a white solid.
1
H NMR (DMSO-d6, 400 MHz): δ 8.20-8.13 (m, 3H), 3.56 (s, 3H).
13
C NMR (DMSO-d6, 100
MHz): δ 165.54, 164.71, 148.32, 138.52, 128.77, 126.89, 117.11, 116.70, 35.18. ESI-LRMS
calcd. for C9H6NO6S (M-H) 256.0, found 255.9.
8 N-benzenesulfonyl 5-carboxylic benzo[c]isoxazol-3(1H)-one (9f) Benzenesulfinic acid sodium salt (91 mg, 0.55 mmol) was added to a solution of 1-methyl 2nitrosoterephthalate (105 mg, 0.5 mmol) in acetonitrile / PBS 0.2 M pH=7 1:1 (10 mL). The
reaction was stirred for 1 h, and the organic solvent was then evaporated under reduced pressure.
The pH of the resulting solution was adjusted to 3 using 1 M HCl solution. The precipitate was
filtered and washed with water and methanol to yield 9f (145 mg, yield of 89%) as a white solid.
1
H NMR (DMSO-d6, 400 MHz): δ 8.31(s, 1H), 8.05-8.03 (m, 1H), 7.91-7.89 (m, 1H), 7.77-7.75
(m, 1H), 7.64-7.54 (m, 4H). 13C NMR (DMSO-d6, 100 MHz): δ 165.44, 164.48, 148.83, 138.67,
136.48, 129.27, 129.01, 117.25, 116.92. ESI-LRMS calcd. for C14H8NO6S (M-H) 318.0, found
318.0. (S)-N-((2-acetamido-3-methoxy-3-oxopropyl)sulfonyl) 5-carboxylic benzo[c]isoxazol-3-one
(9g) The protected cysteine sulfinic acid 1c (64 mg, 0.28 mmol) was added to a solution of 1-methyl
2-nitrosoterephthalate (105 mg, 0.25 mmol) in acetonitrile / PBS 0.2 M pH=7 1:1 (5 mL). The
reaction was stirred for 1 h, then the organic solvent was evaporated under reduced pressure. The
residue was purified by flash chromatography (on silica gel, AcOEt/hexanes/methanol 3:7:1) to
9 yield 9g (78 mg, 81% of yield) as a white solid. 1H NMR (DMSO-d6, 400 MHz): δ 8.15-7.98 (m,
3H), 4.23 (dd, J = 8.0, 5.2 Hz, 1H), 3.56 (s, 3H), 2.65 (dd, J = 13.0, 8.6 Hz, 1H), 2.32 (dd, J =
12.8, 5.2 Hz, 1H), 2.12 (s, 3H).
13
C NMR (DMSO-d6, 100 MHz): δ 174.08, 173.12, 164.23,
146.77, 144.65, 127.17, 126.92, 124.81, 123.03, 58.73, 51.36, 46.04. ESI-LRMS calcd. for
C14H13N2O9S (M-H) 385.0, found 385.0.
4-carboxylic isatoic anhydride (23) Trichloromethyl chloroformate (0.6 mL, 5 mmol) was gently added to a solution of 2-amino
terephthalic acid (930 mg, 5 mmol) in dry 1,4-dioxane (15 mL). The reaction was warmed until
reflux temperature, stirred for 4 h, and then concentrated to yield 23 (428 mg, yield of 72%) as a
white solid. 1H NMR (DMSO-d6, 400 MHz): δ 13.61 (br, 1H), 11.90 (s, 1H), 8.01 (d, J = 10.8
Hz, 1H), 7.72-7.70 (m, 2H). 13C NMR (DMSO-d6, 100 MHz): δ 165.87, 159.46, 146.92, 137.87,
129.40, 123,42, 116.14, 113.55. ESI-LRMS calcd. for C9H4NO5 (M-H) 206.0, found 206.0.
1-ethyl 2-aminoterephthalate (24)
A solution of 4-carboxylic isatoic anhydride (23) (530 mg, 2.5 mmol) in ethanol was stirred at
reflux for 2 h, and then cooled to room temperature and concentrated. The residue was
crystallized from chloroform to yield 24 (462 mg, yield of 86%) as a bright yellow solid. 1H
10 NMR (DMSO-d6, 400 MHz): δ 7.75 (d, J = 9.8 Hz, H), 7.42-7.39 (m, 1H) 7.03 (dd, J = 8.4, 1.6
Hz, 1H), 6.76 (br, 1H), 4.24 (q, J = 14.4, 6.8 Hz, 2H), 1.30 (t, J = 7.2 Hz, 3H).
13
C NMR
(DMSO-d6, 100 MHz): δ 167.40, 167.06, 151.05, 137.05, 130.68, 117.77, 114.93, 111.33, 60.18,
14.19. ESI-LRMS calcd. for C10H10NO4 (M-H) 208.1, found 208.0.
1-ethyl 2-nitrosoterephthalate (7e)
A solution of Oxone® (3.26 g, 5.30 mmol) in water (30 mL) was added to a solution of 1-ethyl
2-aminoterephthalate (554 mg, 2.65 mmol) in CH2Cl2 (5 mL). The reaction was vigorously
stirred at room temperature until LC-MS analysis indicated complete consumption of the starting
material (1.5 h). The precipitate was filtered, washed with water, and desiccated to yield the
nitroso-compound 7e (428 mg, yield of 72%) as a pale yellow solid. 1H NMR (DMSO-d6, 400
MHz, mixture of monomer and dimer 1:1): δ 8.48-7.95 (m, 5H), 7.49 (s, 1H), 4.51-4.36 (m, 4H),
1.38-131 (m, 6H). 13C NMR (DMSO-d6, 100 MHz, mixture of monomer and dimer): δ 166.19,
165.46, 165.06, 162.72, 161.98, 140.54, 136.40, 135.87, 134.22, 132.94, 132.13, 131.20, 128.96,
128.18, 125.49, 114.34, 62.36, 13.96, 13.91. ESI-LRMS calcd. for C10H8NO5 (M-H) 222.0,
found 222.0.
11 1-phenyl 2-aminoterephthalate (25)
Phenol (681 mg, 7.25 mmol) and triethylamine (1.34 mL, 9.66 mmol) were added to a solution
of 4-carboxylic isatoic anhydride (23) (1000 mg, 4.83 mmol) in dry 1,4-dioxane. The mixture
was stirred at reflux for 5 h, and was then cooled to room temperature and concentrated. The
residue was diluted with water (15 mL), and the pH was adjusted to 3. The precipitate was
filtered and washed with water (10 mL) and acetone (5 mL) to yield 25 (872 mg, yield of 70%)
as a bright yellow solid. 1H NMR (DMSO-d6, 400 MHz): δ 13.13 (s, 1H), 8.01 (d, J = 8.4 Hz,
1H), 7.51-7.43 (m, 3H), 7.51-7.43 (m, 3H), 7.11 (d, J = 8.0 Hz, 1H), 6.91 (s, 2H).
(DMSO-d6, 100 MHz):
13
13
C NMR
C NMR (DMSO-d6, 100 MHz): δ 166.94, 165.57, 151.85, 150.45,
136.11, 131.41, 129.52, 125.87, 122.13, 117.97, 114.82, 110.38 δ. ESI-LRMS calcd. for
C14H10NO4 (M-H) 256.1, found 256.0.
1-phenyl 2-nitrosoterephthalate (7f)
A solution of Oxone® (3.74 g, 6.08 mmol) in water (35 mL) was added to a solution of 1-phenyl
2-aminoterephthalate (825 mg, 3.04 mmol) in CH2Cl2 (5 mL). The reaction was vigorously
stirred for 16 h and then a second quantity of Oxone® (1.87 g, 3.04 mmol) was added. After an
additional 7 h, the LC-MS analysis indicated complete consumption of the starting material. The
precipitate was filtered, washed with water, and desiccated to yield the nitroso-compound 7f
12 (710 mg, yield of 86%) as a brown orange solid. 1H NMR (DMSO-d6, 400 MHz, mixture of
monomer and dimer 1:1): δ 8.20-7.98 (m, 4H), 7.74 (s, 1H), 7.60-7.08 (m, 11H).
13
C NMR
(DMSO-d6, 100 MHz, mixture of monomer and dimer): δ 165.32, 164.80, 164.69, 161.95,
161.80, 135.97, 134.68, 130.53, 130.42, 129.83, 129.49, 126.58, 121.58, 121.50, 121.42, 116.12.
ESI-LRMS calcd. for C14H8NO5 (M-H) 270.1, found 270.0.
N-Boc-glutathione methyl ester oxidized (26) Sodium iodine (2 mg) and hydrogen peroxide 30% in water (195 µL, 1.7 mmol) were added to a
solution of N-(tert-butoxycarbonyl)-glutathione dimethyl ester[6] (740 mg, 1.7 mmol) in THF (10
mL). The mixture was stirred until formation of an orange-brown coloration (1 h), then
concentrated. The crude material was purified by flash chromatography (on silica gel, AcOEt) to
yield 26 (683 mg, 86% of yield) as a white foam. 1H NMR (CDCl3, 400 MHz): δ 8.49 (br, 1H),
6.94 (d, J = 9.2 Hz, 1H), 5.46 (t, J = 9.8 Hz, 1H), 5.38 (d, J = 9.2 Hz, 1H), 4.33-4.27 (m, 1H),
4.09 (dd, J = 17.6, 5.6 Hz, 1H), 3.92 (dd, J = 18.0, 5.6 Hz, 1H), 3.80 (s, 2H), 3.58 (s, 3H), 3.34
(s, 3H), 2.33 (t, J = 8.4 Hz, 2H), 2.17-2.07 (m, 1H), 1.97-1.89 (m, 1H), 1.36 (s, 9H). 13C NMR
(CDCl3, 100 MHz): δ 172.93, 172.52, 170.83, 169.91, 155.96, 80.05, 53.44, 53.03, 52.85, 52.39,
52,30, 45.96, 41.31, 32.25, 28.47, 28.29. ESI-LRMS calcd. for C34H56N6NaO16S2 (M+Na)
891.3, found 891.3.
13 Sodium N-Boc-gluthathione sulfinic acid methyl ester (1e) Hydrogen peroxide 30% in water (140 µL, 4.5 mmol) was added dropwise at 0 °C to a solution
of glutathione disulfide 26 (500 mg, 0.56 mmol) in acetic acid (4 mL) The reaction was stirred
for 24 hours at room temperature and then diethyl ether (30 mL) was added and the formed
precipitated was filtered and washed with diethyl ether (10 mL). The crude material was
dissolved in methanol (5 mL) and then sodium thiophenolate (64 mg, 0.52 mmol) was added.
The reaction was stirred for 1 h and then concentrated. The crude material was re-suspended in
acetone and then filtered to yield glutathione sulfinic 1e (113 mg, 45% of yield) as a white solid.
1
H NMR (DMSO-d6, 400 MHz): δ 8.41 (t, J = 11.6 Hz, 1H), 8.20 (d, J = 8.0 Hz, 1H), 7.25 (t, J
= 8.0 Hz, 1H), 4.63-4.55 (m, 1H), 4.19-3.94 (m, 1H), 3.85 (d, J = 8.2 Hz, 2H) 3.63 (s, 3H), 3.33
(s, 3H), 3.09 (dd, J = 13.6, 5.2 Hz, 1H), 2.85 (dd, J = 13.6, 9.6 Hz, 1H), 2.23 (t, J = 8.0 Hz, 2H)
1.98-1.87 (m, 1H), 2.17-2.07 (m, 1H), 1.82-1.72 (m, 1H), 1.38 (s, 9H).
13
C NMR (D2O, 100
MHz): δ 173.92, 172.19, 171.77, 170.21, 157.32, 60.05, 53.65, 52.79, 52.11, 50.60, 52,30, 41.32,
30.77, 30.24, 25.20. ESI-LRMS calcd. for C17H28N3O10S (M-H) 466.1, found 466.0.
14 5-carboxylic benzo[c]isoxazol-3(1H)-one (21b)
Sodium ascorbate (118 mg, 0.6 mmol) was added to a solution of nitroso compound 7f (136 mg,
0.5 mmol) in PBS pH 9 (5 mL). The resulting solution was stirred for 5 h, and a 1 M solution of
NaHSO4 (10 mL) was then added. The precipitate was filtered, washed with water, and
desiccated to yield 21b (76 mg, yield of 84%) as a bright yellow solid. 1H NMR (DMSO-d6, 400
MHz): δ 7. 90 (d, J = 9.8 Hz, 1H), 7.82-7.74 (m, 2H), 3.34 (br, 1H). 13C NMR (DMSO-d6, 100
MHz): δ 165.13, 159.72, 152.64, 137.53, 131.05, 117.27, 115.77, 113.22. ESI-LRMS calcd. for
C8H4NO4 (M-H) 178.0, found 178.0.
Aza-adduct 21d.
The benzoisoxazolone 21b (32 mg, 16.8 µmol) was added to a solution of nitroso compound 7f
(42 mg, 15.3 µmol) in PBS pH 7 (5 mL). The resulting solution was stirred for 2 h, and a 1 M
solution of NaHSO4 (2 mL) was then added. The orange precipitate was filtered and washed with
water and CH2Cl2. The crude material was purified by flash chromatography on silica gel
(eluting with EtOAc/Hexanes 1:1) to yield 21d as an orange-brown solid (48 mg, 67 %). 1H
NMR (DMSO-d6, 400 MHz,): δ 13.58 (br, 1H), 8.61 (s, 1H) 8.37 (d, J = 8.0 Hz, 1H), 8.29 (d, J
15 = 7.2 Hz, 1H), 8.09 (d, J = 8.0 Hz, 1H), 8.01 (d, J = 6.4 Hz, 1H), 7.75 (s, 1H), 7.54-7.24 (m,
5H). 13C NMR (DMSO-d6, 100 MHz): δ 167.09, 165.63, 164.68, 162.81, 151.79, 150.15, 134.55,
134.23, 130.88, 130.80, 129.96, 128.94, 126.13, 122.97, 121.85, 121.45, 118.12, 117.97, 116.11,
114.41 ESI-LRMS calcd. for C22H13N2O9 (M-H) 449.1, found 449.2.
Measurement of N-sulfonyl hydroxylamine formation from RNO and RSO2Na (Table 1).
Benzene or methanesulfinic acid sodium salt (0.03 mmol, 43 mM final concentration) was added
to a solution of 1-methyl 4-nitrosobenzoate (0.027 mmol, 40 mM final concentration) dissolved
in the appropriate deuterated solvent (700 µL) containing 2 equiv (0.06 mmol, 86 mM final
concentration) of an organic acid. After 10 min at RT, the extent of reaction was determined by
1
H-NMR.
Measurement of reactivity of 2-nitroso benzoic acid derivatives towards sulfinic acids
(Table 2).
The appropriate sulfinic acid (55 mM final concentration) was added to a solution of 2-nitroso
benzoic acid derivative (50 mM final concentration) in acetonitrile/0.2 M PBS (1:1) pH=7. The
reaction was stirred for 1 h at RT, and then the organic solvent was evaporated under reduced
pressure. The crude material was purified in according to the preparative procedure above and
the yield refers to the amount of final product recovered.
Measurement of the rate constant of the reaction between nitroso compounds 7d-f and
methanesulfinic acid (Table 3).
16 Because the limiting step is the cyclization of the intermediate 10 (Figure S3), the constant rate
of reaction was measured in a pseudo-first-order condition using an excess of methanesulfinic
acid sodium salt (5 equiv).
General procedure for measuring the rate constant for compounds 7d and 7e.
200 mM of phosphate buffered saline at pH 6, 7, and 8 was prepared in D2O. 0.056 mmol of
probe was dissolved in 1mL of deuterated PBS at pH 6, 7, or 8 (final concentration solution A =
56 mM). 0.7 mmol of methanesulfinic sodium salt in 200 mM deuterated PBS at the appropriate
pH was prepared (final concentration solution B = 700 mM). The reaction was carried out by
mixing 500 µL of solution A and 200 µL of solution B in a NMR tube (final concentration of
probe 40 mM) at 25 °C. Directly following the addition, the tube was placed in a Bruker Avance
400 NMR apparatus at 25 °C, and 1H NMR spectra were obtained at various times. Figure S3
presents an example of the calculation of the kinetics for entry 1-Table 3. The 1H NMR spectra
of the reaction between the nitroso probe 7d and methanesulfinic acid at t = 0, 50 and 180 min
are partially shown in Figure S4. The spectrum at 50 min clearly indicates complete conversion
of the nitroso probe (disappearance of signal δ = 4.01 ppm) in the opening sulfonic adduct 10
(see signals δ = 3.93, 3.11 ppm). At the same time, new signals arose belonging to the cyclic
product (δ = 3.18 ppm) and to methanol (δ = 3.34 ppm). After 180 min, most of compound 10
was converted into the sulfonamide 9e. By comparing the integrals of the CH3O signal at 3.93
ppm (CH3O- of compound 10) with the integrals of the signal at 3.18 ppm (CH3SO2- of product
9e), the total molar fraction of the products can be determined. Subsequently, the molar fraction
of the product can be plotted as a function of the reaction time resulting in the conversion plot of
17 the reaction (Figure S5). From the conversion plot (Figure S5), the first-order rate plot can be
deduced by fitting the data into the following equation:
ln [A] = -kt + ln [A]0
Herein, k = first-order rate constant (s-1), t = reaction time (s), [A]0 = the initial concentration of
substrate A (M). The initial concentration of the nitroso compound 7d was 4·10-5 M. Using this
initial concentration and the data obtained from Figure S5 (up to 80% of conversion), the firstorder rate plot was constructed as shown in Figure S6. The k was obtained from the slope of the
straight line to afford k = 1.05 × 10-3 s-1. Additional data for compounds 7d and 7e are shown in
Figure S7.
General procedure for measuring the rate constant for compound 7f.
The reaction between compound 7f and methanesulfinic acid was too rapid to be followed by
NMR. Therefore, we measured the rate constant k by LC-MS. The reaction was quenched at
different points in time using an excess of sodium ascorbate (5 equiv). Indeed, the nitroso group
is very sensitive to reducing agents and is immediately reduced to hydroxylamine, which cannot
react with sulfinic acids. The N-sulfenyl adduct was not degraded in the presence of ascorbate.
The reaction rate was measured in a pseudo-first-order condition, using a large excess of sulfinic
acid (5 equiv). Three stock solutions of the nitroso compound were prepared by dissolving 13.5
mg of 7f in 1 mL of 200 mM PBS at pH 5, 6, and 7 (final concentration 50 mM). Three stock
solutions of methanesulfinic acid were prepared by dissolving 57.1 mg of methanesulfinic acid
46% in 1 mL of 200 mM PBS at pH 5, 6, and 7 (final concentration 250 mM). 10 µL (1 equiv) of
50 µM probe solution and 10 µL (5 equiv) of a 250 mM methanesulfinic acid sodium salt
solution were added to 80 µL of PBS 200 mM at the appropriate pH. After 5, 10, 15, 20, 30 sec,
18 5 µL (5 equiv) of a solution of 500 mM of sodium ascorbate was added to the reaction. 20 µL of
each of the final solutions were diluted with 480 µL of acetonitrile/water 3:1 (0.1% of formic
acid) and analyzed by LC-MS. The concentration of the product 9e was determined from the
peak area of the product (retention time 2.62 min). Subsequently, the molar fraction of the
product was plotted as seen previously in order to obtain k (Figure S8).
General procedure to examine the reactivity of 1-phenyl 2-nitrosoterephthalate (7f) toward
sulfinic acids in aqueous media at pH 7 (Table 4).
A 50 mM stock solution of the nitroso compound was prepared by dissolving 13.5 mg of 7f in 1
mL of 200 mM PBS at pH 7. A 100 mM stock solution of the sulfinic acids was prepared in 200
mM PBS at pH 7. 10 µL (1 equiv, 5 mM final concentration) of 7f solution and 5 µL (1 equiv, 5
mM final concentration) of the sulfinic acid solution were added to 85 µL of PBS 200 mM at pH
7. After 10 min, 10 µL of each of the final solutions were diluted with 490 µL of
acetonitrile/water 3:1 (0.1% of formic acid) and analyzed by LC-MS (Figure S9).
General procedure to examine the reactivity of 1-phenyl 2-nitrosoterephthalate (7f) toward
potentially reactive biological species in aqueous media at pH 7 (Table 5).
A 50 mM stock solution of the nitroso compound was prepared dissolving 13.5 mg of 7f in 1 mL
of 200 mM PBS at pH 7. A 50 mM stock solution of the analyte was prepared in 200 mM PBS at
pH 7 (except for entry 19, where the compound was dissolved in DMSO). 10 µL (1 equiv, 5 mM
final concentration) of probe solution and 10 µL (1 equiv, 5 mM final concentration) of the
analyte solution were added to 85 µL of PBS 200 mM at pH 7 (except for entry 19, where the
experiment was performed at pH 7.5). After 1 h, 5 µL (5 equiv) of a solution of 500 mM of
19 sodium ascorbate were added to the reaction. 10 µL of each of the final solutions were diluted
with 490 µL of acetonitrile/water 3:1 (0.1% of formic acid) and analyzed by LC-MS (Figures
S13 and S16).
1
H NMR time course experiment of 7d Stability.
200 mM PBS (pH 7) was prepared in D2O. 7d (6 mg, 29 µmol) was dissolved in PBS at pH 7
D2O (700 µl). 1H-NMR was obtained at 3 min, 1, 2, 4, 8, and 24 h. The nitroso-compounds 7d is
stable under experimental conditions (Figure S2).
1
H NMR time course experiment of 7f stability.
200 mM PBS (pH 7) was prepared in D2O. 7f (8 mg, 30 µmol) was dissolved in PBS at pH 7
D2O (750 µl). 1H-NMR was obtained at 10 min, 1, 2, 4, 8, and 24 h. The nitroso-compounds 7f is
quite stable until 4 h in PBS after which time it slowly started to decompose (Figure S10).
1H NMR Time course experiment for 9e Stability.
200 mM PBS (pH 7) was prepared in D2O. 9e (7.5 mg, 30 µmol) was dissolved in PBS at pH 7
D2O (750 µl). 1H-NMR was obtained at 3 min, 1, 2, 4, 8, and 24 h. The nitroso-compounds 9e is
stable under experimental conditions (Figure S11).
General procedure to examine the stability of the reaction products 9e, 21b and 21c toward
potentially reactive biological species in aqueous media at pH 7 (Figures S12, S14 and S15).
A 100 mM stock solution of the product (9e, 21b or 21c) was prepared in 200 mM PBS at pH 7.
A 100 mM solution of the nucleophile (cysteine or lysine) was prepared in 200 mM PBS at pH 7.
20 10 µL (1 equiv) of probe solution and 20 µL (2 equiv) of the nucleophile solution were added to
70 mL of PBS 200 mM at pH 7. After 1 h, 10 µL of each of the final solutions were analyzed by
LC-MS.
Competitive reaction of methane sulfinic acid with cysteine towards 7f (Scheme 3 and
Table S1).
A 200 mM stock solution of the nitroso compound was prepared by dissolving 27 mg of 7f in 0.5
mL of 200 mM PBS at pH 7. A 50 mM stock solution of the methanesulfinic acid was prepared
in 200 mM PBS at pH 7. A 200 mM stock solution of the cysteine solution was prepared in 200
mM PBS at pH 7. A 200 mM stock solution of the glutathione solution was prepared in 200 mM
PBS at pH 7. 50 µL (100 equiv, 100 mM final concentration) of 7f, 1 µL (1 equiv, 1 mM final
concentration) of methane sulfinic acid and increasing amount of cysteine (14) or glutathione
(27) solution (0, 1, 2, 5, 10, 20, 50 equiv; 0 – 50 mM final concentration) were combined, and
the final solution volume was brought to 100 µL with PBS 200 mM at pH 7. After 30 min at RT,
25 µL of the final solutions was diluted with 175 µL of acetonitrile/water 3:1 (0.1% of formic
acid) and analyzed by LC-MS. The aforementioned method was adapted from reference [7]. Methane sulfinic acid ligation with 7f, after treatment with N-ethylmaleimide (NEM) of a
mixture of glutathione and methane sulfinic acid. (Scheme 3 and Table S2).
A 50 mM stock solution of the nitroso compound was prepared by dissolving 13.5 mg of 7f in 1
mL of 200 mM PBS at pH 7. A 50 mM stock solution of the methanesulfinic acid was prepared
in 200 mM PBS at pH 7. A 200 mM stock solution of the glutathione solution was prepared in
200 mM PBS at pH 7. A 200 stock mM solution of N-ethylmaleimide was prepared in 200 mM
21 PBS at pH 7. 25 µL (50 equiv, 50 mM final concentration) of NEM solution, 1 µL (1 equiv, 1
mM final concentration) of methanesulfinic acid and increasing amount of glutathione (27)
solution (0, 1, 2, 5, 10, 20, 50 equiv; 0 – 50 mM final concentration) were combined, and the
final solution volume was brought to 98 µL with PBS 200 mM at pH 7. After 15 min at RT, 2 µL
(1 equiv) of probe solution was added and the final solution was incubated for additional 30 min.
25 µL of the final solutions was diluted with 175 µL of acetonitrile/water 3:1 (0.1% of formic
acid) and analyzed by LC-MS. 22 Table S1. Competitive reaction between methanesulfinic acid and thiols with 7f.
NO
Me
O
S
CO2Ph
OH
7f
1b
mixture
O
S O
N
O
H3C
HO2C
HO2C
PBS pH 7
RSH
O
[a]
9e
RSH 27
(equiv)
-
% of 9e formation[b]
1
RSH 14
(equiv)
-
2
1
-
98
3
2
-
98
4
5
-
97
5
10
-
100
6
20
-
100
7
50
-
99
8
-
1
97
9
-
2
100
10
-
5
95
11
-
10
98
12
-
20
97
13
-
50
98
Entry
100
[a]
Cysteine (14) and glutathione (27) were employed as representative thiols (RSH).
[b]
Sulfinic acid 1b (1 mM, 1 equiv) was combined with thiol 14 or 22 (0 – 50 equiv) and treated
with 7f (100 equiv) for 30 min. The percentage of 9e formation was determined by LC-MS (SD
+/- 5%).
23 Table S2. Sulfinic acid ligation with 7f, after treatment with N-ethylmaleimide (NEM) of a
mixture of glutathione and methanesulfinic acid.
O
Me
mixture
O
S
NO
CO2Ph
N
OH
1b
NEM
7f
GSH
PBS pH 7
15 min
PBS pH 7
30 min
HO2C
O
27
9e
% of 9e formation[a]
1
27
(equiv)
0
2
1
90
3
2
91
4
5
89
5
10
89
6
20
92
7
50
96
Entry
O
S O
N
O
H3C
HO2C
O
94
[a]
Sulfinic acid 1b (1 mM, 1 equiv) was combined with glutathione 27 (0 – 50 equiv) and treated
with NEM (50 equiv) for 15 min. Next, 7f (1 equiv) was added and the reaction was incubated
for additional 30 min. The percentage of 9e formation was determined by LC-MS (SD +/- 5%).
Note: The use of 7f in excess over thiols is applicable to sulfinic acid ligation in recombinant
proteins and lysates (Table S1). The application of NEM to intact cells to alkylate free thiols is
also well established.[8] Thus, NEM pretreatment and subsequent incubation with stoichiometric
7f (Table S2) is relevant to sulfinic acid ligation in recombination proteins, lysates, and cellular
experiments.
24 Scheme S1. Proposed mechanism of thiol-reduction of C-nitroso-compounds.
Scheme S2. Reaction products obtained from 7f on reduction with cysteine.
25 Scheme S3. Products obtained from reaction of 7f with sulfenic acid 15b generated in situ.
26 Figure S1. Modifications of cysteine that have been confirmed in vivo.
27 Figure S2. 1H NMR of 7d (40 mM) in PBS-D2O solution (pH 7) at 3 min, 1, 2, 4, 8, and 24 h.
28 Figure S3. Proposed mechanism of N-sulfonylbenzyisoxazolone formation.
29 Figure S4. 1H NMR spectra of the reaction between 7d and methanesulfinic acid at a) t = 0 min ;
b) t = 50 min and c) t = 180 min (pH = 6, 25 °C).
30 Figure S5. Conversion plot for the reaction between nitroso-probe 7d and methanesulfinic acid
(1:5) in D2O (pH = 6) at 25 °C (Table 3, entry 1).
Figure S6. First-order rate plot for the reaction between nitroso-probe 7d and methanesulfinic
acid (1:5) in D2O (pH = 6) at 25 °C (Table 3, entry 1).
31 Figure S7. First-order rate plots for Table 3, entries 2-4.
32 Figure S8. First-order rate plots for Table 3, entries 5-7.
33 Figure S9. LC-MS analysis for Table 4 – Reactivity of sulfinic acids with 7f (*absorbance from
buffer; §minor decomposition of 7f to 21d). HPLC traces show UV absorbance at 254 nm versus
time. Each peak affords a distinct mass, which is indicated alongside the corresponding structure. 34 Figure S10. 1H NMR of 7f (40 mM) in PBS-D2O solution (pH 7) at 10 min, 1, 2, 4, 8, and 24 h.
35 Figure S11. 1H NMR of 9e (40 mM) in PBS-D2O solution (pH 7) at 3 min, 1, 2, 4, 8, and 24 h.
36 Figure S12. Reactivity of the methanesulfonyl adduct 9e toward potentially reactive biological
species in aqueous media at pH 7. LC-MS trace of: a) 9e; b) 9e (1 equiv) with lysine (1 equiv)
after 1h; b) 9e (1 equiv) with cysteine (1 equiv) after 1h. HPLC traces show UV absorbance at
254 nm versus time. Each peak affords a distinct mass, which is indicated alongside the
corresponding structure.
37 Figure S13. LC-MS traces for Table 5 (Entries 1-4) – Reactivity of 7f toward potentially
reactive biological species in aqueous media at pH 7 (*absorbance from buffer; §minor
decomposition of 7f to 21d). HPLC traces show UV absorbance at 254 nm versus time. Each
peak affords a distinct mass, which is indicated alongside the corresponding structure.
38 Figure S14. Reactivity of 21b (see Scheme S3) toward potentially reactive biological species in
aqueous media at pH 7. LC-MS trace of: a) 21b; b) 21b (1 equiv) with lysine (1 equiv) after 1h;
b) 21b (1 equiv) with cysteine (1 equiv) after 1h. HPLC traces show UV absorbance at 254 nm
versus time. Each peak affords a distinct mass, which is indicated alongside the corresponding
structure.
39 Figure S15. Reactivity of 21c (see Scheme S3) toward potentially reactive biological species in
aqueous media at pH 7. LC-MS trace of: a) 21c; b) 21c (1 equiv) with lysine (1 equiv) after 1h;
b) 21c (1 equiv) with cysteine (1 equiv) after 1h. LC traces show UV absorbance at 254 nm
versus time. Each peak affords a distinct mass, which is indicated alongside the corresponding
structure.
40 Figure S16. LC-MS traces for Table 5 (Entries 5-9) – Reactivity of 7f toward potentially
reactive biological species in aqueous media at pH 7 (*absorbance from buffer; §minor
decomposition of 7f to 21d). Except entry 8, all LC-MS traces were obtained in negative mode.
HPLC traces show UV absorbance at 254 nm versus time. Each peak affords a distinct mass,
which is indicated alongside the corresponding structure.
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42