Angewandte
Chemie
DOI: 10.1002/anie.201305876
Protein S-Sulfhydration
Detection of Protein S-Sulfhydration by a Tag-Switch Technique**
Dehui Zhang, Igor Macinkovic, Nelmi O. Devarie-Baez, Jia Pan, Chung-Min Park,
Kate S. Carroll, Milos R. Filipovic,* and Ming Xian*
Abstract: Protein S-sulfhydration (forming -S-SH adducts
from cysteine residues) is a newly defined oxidative posttranslational modification and plays an important role in H2Smediated signaling pathways. In this study we report the first
selective, “tag-switch” method which can directly label protein
S-sulfhydrated residues by forming stable thioether
conjugates. Furthermore we demonstrate that H2S
alone cannot lead to S-sulfhydration and that the
two possible physiological mechanisms include
reaction with protein sulfenic acids (P-SOH) or
the involvement of metal centers which would
facilitate the oxidation of H2S to HSC.
difficulties in developing selective detection methods for Ssulfhydration.[4]
So far two methods have been utilized in the detection of
S-sulfhydration (Scheme 1). The first method is a modified
biotin switch technique.[5a] It employs an alkylating agent S-
Hydrogen
sulfide (H2S) has been recently
classified as a critical cell-signaling molecule.[1]
Literature published in the past few years increasingly suggests that H2S is a mediator of many
physiological and/or pathological processes.[2]
Some of these effects are ascribed to the forma- Scheme 1. Current strategies for profiling protein S-sulfhydration.
tion of protein persulfides, or protein S-sulfhydration (i.e. conversion of cysteine residues -SH to
persulfides -S-SH). This has been defined as a new oxidative
methyl methanethiosulfonate (MMTS) to differentiate thiols
posttranslational modification (oxPTM).[3, 4] Formation of
and persulfides. Thiols (-SH) in proteins are first blocked by
MMTS. Persulfides (-S-SH) are believed to remain unreacted
persulfides is potentially significant because it provides
and be available for subsequent conjugation to N-[6-(bioa possible mechanism by which H2S alters the functions of
tinamido)hexyl]-3-(2-pyridyldithio)propionamide (biotina wide range of cellular proteins and enzymes.[5] To date, the
HPDP). Using this method, a large number of proteins
underlying mechanisms of S-sulfhydration mediated by H2S
were identified as targets for S-sulfhydration and the basal
are still unclear.[3, 4] A significant challenge is that the
sulfhydration level of some proteins was estimated to be as
persulfide group (-S-SH) shows reactivity akin to that of
high as 25 %. In the second method,[5c] it suggested that both
other sulfur species, especially thiols (-SH), which causes
-SH and -SSH units can be blocked by alkylating reagents like
iodoacetic acid (IAA). Then the persulfide adducts can be
reduced by dithiothreitol (DTT) to form free -SH groups, and
[*] Dr. D. Zhang,[+] Dr. N. O. Devarie-Baez, Dr. C.-M. Park, Prof. M. Xian
subsequently labeled with iodoacetamide-linked biotin
Department of Chemistry, Washington State University
(IAP).
Pullman, WA 99164 (USA)
From a chemistry perspective, both methods are problemE-mail: mxian@wsu.edu
atic. In Method 1, the underlying mechanism of selectivity of
I. Macinkovic,[+] Dr. M. R. Filipovic
MMTS for thiol versus persulfide is unclear. Studies have
Department of Chemistry and Pharmacy
University of Erlangen-Nuremberg
demonstrated that persulfides and thiols should have similar
Erlangen (Germany)
reactivity towards electrophiles such as MMTS.[4] In
E-mail: milos.filipovic@fau.de
Method 2, it is unclear how DTT reduction would distinguish
Dr. J. Pan, Prof. K. S. Carroll
persulfide modifications from other DTT-reducible residues,
Department of Chemistry, The Scripps Research Institute
such as disulfides and S-nitrosothiols.
Jupiter, FL 33458 (USA)
Therefore, the chemical foundations of current methods
+
[ ] These authors contributed equally to this work.
are questionable, which may lead to erroneous results.
[**] M.X. thanks the NSF-CAREER Program (0844931) and the American
Apparently more reliable methods for the detection of
Chemical Society (Teva USA Scholar Award). M.R.F. and I.M. thank
protein S-sulfhydration are needed. Having realized the
the University of Erlangen-Nuremberg within Emerging Field
very similar reactivity of both thiols and persulfides, we
Initiative (EFI-MRIC) for support. K.S.C. thanks NIH R01
proposed a tag-switch technique to detect S-sulfhydration.
GM102187.
Herein we report the development and application of this
Supporting information for this article is available on the WWW
method.
under http://dx.doi.org/10.1002/anie.201305876.
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Scheme 2. Proposed tag-switch technique for detecting S-sulfhydration.
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As illustrated in Scheme 2, we proposed that S-sulfhydration can be selectively detected by the tag-switch method (i.e.
using two reagents to label protein persulfides in two steps).
In the first step a SH-blocking reagent will be introduced and
it should tag both -SH and -SSH to form intermediate T. If an
appropriate tag is employed, the disulfide bonds in persulfide
adducts may show much enhanced reactivity to certain
nucleophiles relative to the reactivity of common disulfides
in proteins. Therefore we could introduce a tag-switching
reagent (containing both the nucleophile and a reporting
molecule such as biotin) to label only the persulfide adducts.
It should be noted that thiol adducts from the first step are
thioethers, which are not expected to react with the nucleophile.
A major challenge in this technology is whether the newly
generated disulfide linkage from persulfide moieties can
display a unique reactivity for a suitable nucleophile to an
extent that distinguishes them from common disulfides. SHblocking reagents are well known.[6] However, those fulfilling
the criteria for this assay are limited. For example, irreversible
thiol-blocking reagents such as maleimides and iodoacetamides displayed good selectivity and fast reactivity for
thiols.[6] If such reagents react with persulfides, alkyl disulfide
adducts are produced and their reactivity should not differ
from that of cysteine or glutathionylated protein disulfides.
Therefore, these reagents are not suitable for tag-switch. We
envisioned that a reagent, upon reaction with persulfides to
give a mixed aromatic disulfide linkage, could meet the
reactivity criteria. One potential candidate is methylsulfonyl
benzothiazole (MSBT), a thiol-blocking reagent recently
developed by our group.[7] We expected the disulfides
generated from MSBT and persulfides should be highly
activated and exert a unique reactivity with certain nucleophiles, in particular, enolates.[8]
With this idea in mind, we first tested the reaction
between MSBT and persulfide substrates. Since MSBT is
a very effective SH-blocking reagent[7] and persulfides
(-S-SH) are known to have very similar reactivity to thiols,[4]
we expected MSBT should effectively block persulfides.
However, it is known that small-molecule persulfides are
very unstable species.[9] We could not use purified/isolated
persulfides in the experiments. Instead we attempted several
approaches to generate persulfides in situ from precursors
like 1 (Scheme S1 in the Supporting Information) and used
the persulfide intermediates directly in MSBT blocking.
Indeed we obtained the desired product 3, although in low
yield (13 %). This result demonstrated that MSBT can react
with persulfides to form R-S-S-BT adducts. The major
product in the reaction was found to be polysulfides derived
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from persulfide 2. This should not be a concern in
the case of protein persulfides because polysulfide
formation is not expected to occur easily on
protein persulfides.
We next used a cysteine substrate 4 to screen
the appropriate nucleophile for the tag-switch step
(Table 1). The preparation of 4 is shown in
Scheme S2 in the Supporting Information. It
should be noted that R-S-S-BT products like 4
Table 1: Screening nucleophiles for the tag-switch step.
[a] Bn = Benzyl.
are quite stable. They do not react with potential nucleophilic
groups such as -OH and -NH2 (Scheme S3 in the Supporting
Information). We screened a series of carbon-based nucleophiles as potential candidates. As shown in Table 1, three
reagents (dimedone, malononitrile, and methyl cyanoacetate)
proved to be effective and the corresponding products 5 b, 5 e,
and 5 f were obtained in valuable yields. The reactions were
also found to be fast (completing within 20 min). Among
these candidates, methyl cyanoacetate (MCA; Table 1,
entry 6) was particularly attractive as the ester group could
allow easy installation of reporting molecules. Therefore
MCA was selected in subsequent studies.
Given the dramatic structure changes in protein persulfide substrates, we wondered whether MCA could effectively
react with different R-S-S-BT substrates. The reaction scope
was then studied using a series of cysteine-S-S-BT derivatives
(Scheme 3). Pleasingly, the reaction was found to be highly
effective. In all cases the substitution products were afforded
in good yields.
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Scheme 3. Scope of the reaction of MCA with R-S-S-BT derivatives.
Cbz = carbobenzyloxy.
The results shown above demonstrate the chemical
foundation of the tag-switch method. We then tested it in
protein samples. Gpx3, an established protein-S-SH model,[4]
was used in this experiment. Freshly prepared Gpx3 persulfide was treated with MSBT-A, a water-soluble MSBT
derivative,[7] followed by the addition of cyanoacetate. The
protein was then purified and analyzed by LC-MS. As shown
in Figure 1 B, cyanoacetate-labeled protein was clearly identified by MS. In the control (Figure 1 A, without MSBT-A),
we did not observe the peak for the cyanoacetate-labeled
protein. An oxidative byproduct (P-S-SO3H) was observed in
both samples and this is common for Gpx3 based on our
previous experience.[4]
We next tested the selectivity of tag-switch assay towards
different oxPTMs. A biotin-linked cyanoacetate (CN-biotin)
was prepared and used in this study. A relatively stable
sulfenic acid derivative of bovine serum albumin (BSA-SOH)
was prepared[10] and in its reactions with glutathione[10, 11] and
H2S the corresponding glutathionylated and S-sulfhydrated
derivatives were generated (Figure 2 A,B). Neither BSA-SH,
BSA-SOH, nor BSA-SSG gave positive signals in the tagswitch assay. Only in the case of BSA-SSH could biotinylated
product be pulled down by streptavidin agarose beads and
Angew. Chem. Int. Ed. 2013, 52, 1 – 8
Figure 1. MS analysis of the tag-switch assay with Gpx3-persulfide.
A) The control reaction between Gpx3 persulfide and ethyl cyanoacetate (without MSBT-A). B) The reaction between Gpx3 persulfide and
ethyl cyanoacetate (with MSBT-A).
detected by dot blot or ESI-TOF MS (Figure 2 C and
Figure S1 in the Supporting Information).
Although a few studies have suggested S-sulfhydration to
be a potential posttranslational modification mediated by H2S
that could regulate protein function,[5] there is no information,
to date, dealing with the mechanism(s) underlying its
formation. From a chemistry perspective, the direct reaction
of protein thiols (-SH) with H2S would not be possible.
However, the intermediate role of oxygen and metal centers
as well as the reactions with other posttranslational modifications of cysteine could be possible.
Based on the mechanistic studies for S-glutathionylation[3, 11] we addressed the following hypothetical reactions, as
potential paths for P-SSH/PSS generation under physiological conditions [Eq. (1)–(6)].
P-SH þ H2 S þ O2 ! P-S-SH þ H2 O2
ð1Þ
P-S-S-R þ H2 S ! P-S-SH þ RSH
ð2Þ
P-S-OH þ H2 S ! P-S-SH þ H2 O
ð3Þ
P-S-NO þ H2 S ! P-S-SH þ HNO
ð4Þ
P-SðHÞNOH þ H2 S ! P-S-SH þ NH2 OH
ð5Þ
P-SH þ HSC þ O2 ! P-S-SH þ HO2 C
ð6Þ
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If MCA is used to specifically label protein persulfide
derived R-S-S-BT moieties, it is critical to prove that MCA is
inert towards common disulfides. We thus carried out several
control experiments (Scheme S5 in the Supporting Information). We first examined the reactivity of MCA against Cys
disulfide 8 (Boc = tert-butyloxycarbonyl). Under the tagswitch reaction conditions the corresponding product was
not observed, even after hours. We also checked the reactivity
of MCA toward S-nitrosothiol 9, which represents another
well-known thiol modification in proteins. Again, no reaction
was observed. Finally a crossover experiment using both
R-S-BT 10 (derived from thiols) and R-S-S-BT 4 (derived
from persulfides) was tested. We only observed product 5 f
(from 4). The thiol-derived substrate 10 was unreactive and
could be fully recovered. These results suggested that the
proposed tag-switch method was selective for persulfides.
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We tested the reaction
pathways (1), (3), (5), and
(6) using glyceraldehyde
3-phosphate dehydrogenase
(GAPDH)
as
a model. Angelis salt
was used as a donor of
HNO[18] to form the
(hydroxyamino)sulfanyl
derivative. Formation of
P-SOH was induced by
reaction with H2O2. Proteins were also treated
with supraphysiological/
pharamcological concentrations of H2S alone and
H2S in combination with
rigorous
shaking
to
Figure 2. Testing the selectivity of the tag-switch assay. A) Schematic description of the tag-switch assay.
B) Preparative procedure for generating different oxPTMs of BSA. C) Percentage of the unbound protein after
increase the oxygenation
treatment with streptavidin agarose beads. Samples 1, 3, 5, and 7 are untreated samples of BSA-SH, BSA-SOH,
of the solution. As
BSA-SSG, and BSA-SSH, respectively. Samples 2, 4, 6, and 8 are BSA-SH, BSA-SOH, BSA-SSG and BSA-SSH,
a source of HSC we used
respectively, treated with tag-switch reagents. n = 3, *p < 0.001. Inset shows the dot blot detection of successful
a combination of waterbiotinylation.
soluble ferric porphyrin
and H2S.[19] Only when
H2S reacted with P-SOH or when HSC was generated with
The reaction shown in Equation (1) is only the sum of the
multiple reaction steps that could occur during the spontathe iron center was PS-SH detectable (Figure S2 in the
neous oxidation of H2S where HSC is a possible intermediSupporting Information). When BSA was used as a model of
the protein with intramolecular disulfide bonds, no S-sulfhyate[12] that would then lead to formation of S-sulfhydrated
dration was observed upon addition of H2S (data not shown),
protein by means of the reaction shown in Equation (6).
The reduction of a disulfide bond by H2S [Eq. (2)] is
confirming the low reducing power of free H2S. It is worth
thermodynamically unfavorable.[13] Based on the calculation
mentioning that an alternative mechanism could lead to
protein persulfide formation, such as the reaction with
of the bond energies of GSSG and GSSH, the bonding energy
polysulfides,[5g] although their physiological relevance
in the latter is roughly 18 kJ mol1 lower.[14] The reaction with
sulfenic acids [Eq. (3)] does occur, as demonstrated in
remains to be elucidated.
Figure 2 B. S-Nitrosothiols would react with H2S to give
As a proof of concept that the method can be applied to
more complex systems such as the intracellular environment,
HSNO, rather than to form HNO and the corresponding Swe attempted to label proteins by the tag-switch technique in
sulfhydrated protein as we previously demonstrated (with
cell extracts. Protein extracts from control and Jurkat cells
Drxn1G8 =+ 40 kJ mol1).[14] However, a recent computational
treated with 200 mm H2S (using Na2S as the equivalent) for
study pointed out that the surrounding of the SNO bond
could significantly affect the thermodynamic feasibility of the
30 min at 37 8C were labeled by the tag-switch method,
reaction shown in Equation (4), making the reaction possible
resolved by SDS-PAGE, transferred to a nitrocellulose memfor certain proteins that have positively charged amino acids
brane, and identified by anti-biotin horse radish peroxidase
in close proximity to the SNO bond.[15]
(HRP)-conjugated antibodies. Representative Western blots
(Figure 3 A–C) demonstrated that a small number of proteins
The reaction of nitroxyl (HNO), a redox sibling of NO
showed positive signals, confirming the existence of endogwith distinct signaling pathways,[16] with a protein thiol leads
enous S-sulfhydration. The treatment with Na2S increased the
to the formation of a (hydroxyamino)sulfanyl derivative. It is
that this (hydroxyamino)sulfanyl derivative then reacts with
signal intensity, but did not significantly affect the total
other thiols with elimination of hydroxylamine as shown in
number of S-sulfhydrated proteins. Importantly, when cell
Equation (5).[16]
lysates were treated with streptavidin agarose beads and
subsequently analyzed, no biotinylated proteins could be
Finally, a possible way to form S-sulfhydrated proteina is
detected, suggesting that all modified proteins could be pulled
the reaction of HSC with protein thiols, where initially PSSHC is
down and the method used for the further proteomic analysis
formed, which subsequently reacts quickly with O2 to give
(for example, see Figure S3A in the Supporting Information).
O2C and PSSH [Eq. (6)], as observed in the formation of SIt has been reported previously that GAPDH could be
glutathionylated proteins.[11] Formation of HSC under physioone of the major targets for S-sulfhydration.[5a,b] Indeed, when
logical conditions would require interaction with oxidized
metal centers, such as those in ferrioc heme porphyrins, which
we attempted to identify GAPDH with specific antibodies we
would be reduced forming HSC by means of inner-sphere
found that this protein was endogenously S-sulfhydrated,
electron transfer.[17]
although the signal was much stronger after the treatment
with H2S (Figure 3 C).
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The most prominent S-sulfhydration was detected on
a protein with a molecular weight of roughly 70 kDa. Using
antibodies specific for heat shock protein 70 (Hsp70), we
demonstrated that this protein is most likely Hsp70 (Figure 3 A). Hsp70 is of great pharmacological interest[20a] and
recent studies showed that it could serve as a redox sensor
through oxidation of its cysteines by sulfenylation (PSOH),[20b] which can also explain how Hsp70 could form
persulfides [Eq. (3)].
As we suggested in Figure S2 in the Supporting Information and in Equation (6), metal-assisted generation of P-SSH
could be the predominant way for forming P-SSH, but also
a source of its artificial formation. Indeed when Jurkat cell
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Figure 3. Detection of protein S-sulfhydration in cell lysates by the tagswitch assay. A) Jurkat cell lysates of the control (lane 1) and H2Streated cells (lane 2) (200 mm Na2S, 30 min, 37 8C) analyzed by the tagswitch assay. In parallel the same cell extracts were tested by Western
blot analysis for the presence of Hsp70, which appeared at exactly the
same position as the strongest S-sulfhydrated band. B) Quantification
of the S-sulfhydration levels in the control and H2S-treated cells based
on the intensity of the band at 70 kDa. C) Detection of GAPDH as
a standard S-sulfhydrated protein.
lysates used in the experiments shown in Figure 3 were
additionally exposed to 200 mm H2S, much stronger overall
sulfhydration was detected than when living cells were treated
(Figure S3B in the Supporting Information).
Finally, in a preliminary set of experiments we tried to
adapt the tag-switch technique for the in situ labeling of
methanol- or paraformaldehyde-fixed cells. Human umbilical
vein cells (HUVECs), previously exposed to 100 mm Na2S for
30 min or to 2 mm propargylglycine, a cystathionine gamma
lyase inhibitor,[14] for 2 h, were fixed with ice-cold methanol.
Free thiols were blocked with MSBT-A and the protein Ssulfhydration (protein persulfides) tagged with CN-biotin.
Finally, the cells were exposed to fluorescein-labeled streptavidin. A similar protocol was used with cells treated with Na2S
or 2-ketobutyric acid (inhibitor of mercaptopiruvate S-transferase, a mitochondrial enzyme for H2S production), but with
the initial difference that they were fixed with paraformaldehyde.
As shown in Figure 4 A,B, detectable S-sulfhydrated
proteins increased in HUVECs treated with H2S. Partial
inhibition, relative to the control, was achieved by treatment
with propargylglycine but it was almost completely abolished
by the use of 2-ketobutyric acid, implying the essential role of
mitochondrially produced H2S, as suggested previously.[21]
Almost complete absence of the signal in 2-ketobutyric acid
treated cells also confirms that the nonselective binding of the
fluorescent probe, incomplete blocking of free thiols, and/or
unselective background fluorescence are not contributors of
the main fluorescence signal. In addition, the pretreatment of
the fixed cells with dimedone did not affect the detection of
intracellular persulfides (Figure S4 in the Supporting Information). These data suggest that the tag-switch assay is
selective for P-S-SH in the presence of P-S-OH. It should be
noted that the reactivity of sulfenic acids toward carbon
nucleophiles like cyanoacetate may change depending on the
protein enviroment. However, even if certain P-S-OH groups
would react with cyanoacetate, samples could always be
pretreated with dimedone to remove the false signals from PS-OH, as we previously demonstrated that dimedone reacts
with P-S-OH but not with P-S-SH.[4]
Higher magnification microscopy (100 ) gave us some
indication of the intracellular distribution of the signal
(Figure 4 D). The perinuclear localization of the signal is
indicative of localization in the mitochondrion and/or endoplasmic reticulum (ER). Since the signal in the cells was less
diffused with methanol fixation we used it for co-localization
studies. Mitochondrial and ER trackers proved that most of
the detected persulfide signal is localized within these two
organelles, within the ER predominantly (Figure S5 in the
Supporting Information).
Taken together, these data offer a new, selective method
for the detection of protein S-sulfhydration. Our results
demonstrate that carbon-based nucleophiles such as cyanoacetate do not react with common disulfides in proteins, but
with highly chemically activated disulfide species. Some
mechanistic insight into physiological mechanisms for the
formation of protein S-sulfhydration is presented, suggesting
that the metal-center-assisted oxidation of H2S could be
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Communications
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[2] a) K. Abe, H. Kimura, J. Neurosci. 1996, 16, 1066 – 1071; b) W.
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Redox Signaling 2011, 13, 1649 – 1657; c) J. M. Fukuto, C. H.
c) G. Yang, L. Wu, B. Jiang, W. Yang, J. Qi, K. Cao, Q. Meng,
Switzer, K. M. Miranda, D. A. Wink, Annu. Rev. Pharmacol.
A. K. Mustafa, W. Mu, S. Zhang, S. H. Snyder, R. Wang, Science
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Steppan, S. M. Jung, A. K. Bhunia, V. M. Barodka, F. K. Gazi,
Inorg. Chem. 2010, 49, 1017 – 1026.
R. K. Barrow, R. Wang, L. M. Amzel, D. E. Berkowitz, S. H.
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Snyder, Circ. Res. 2011, 109, 1259 – 1268; e) J. W. Elrod, J. W.
Calvert, J. Morrison, J. E. Doeller, D. W. Kraus, L. Tao, X. Jiao,
Ford, N. Paolocci, T. Katori, C. G. Tocchetti, D. Mancardi, D. D.
6
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.
www.angewandte.org
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
These are not the final page numbers!
Angew. Chem. Int. Ed. 2013, 52, 1 – 8
Angewandte
Chemie
Angew. Chem. Int. Ed. 2013, 52, 1 – 8
Srinivasan, C. A. Dickey, J. E. Gestwicki, Chem. Biol. 2012, 19,
1391 – 1399.
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Biochem. 2009, 146, 623 – 626; b) K. Mdis, K. Coletta, K.
Erdlyi, A. Papapetropoulos, C. Szabo, FASEB J. 2013, 27, 601 –
611.
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Thomas, M. G. Espey, K. N. Houk, J. M. Fukuto, D. A. Wink, J.
Am. Chem. Soc. 2005, 127, 722 – 731.
[19] J. L. Miljkovic, I. Kenkell, I. Ivanovic-Burmazovic, M. R.
Filipovic, Angew. Chem. 2013, DOI: 10.1002/ange.201305669;
Angew. Chem. Int. Ed. 2013, DOI: 10.1002/anie.201305669.
[20] a) T. Liu, C. K. Daniels, S. Cao, Pharmacol. Ther. 2012, 136, 354 –
374; b) Y. Miyata, J. N. Rauch, U. K. Jinwal, A. D. Thompson, S.
These are not the final page numbers!
.
Angewandte
Communications
Communications
Protein S-Sulfhydration
D. Zhang, I. Macinkovic,
N. O. Devarie-Baez, J. Pan, C.-M. Park,
K. S. Carroll, M. R. Filipovic,*
M. Xian*
&&&&—&&&&
8
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Ü
Detection of Protein S-Sulfhydration by
a Tag-Switch Technique
www.angewandte.org
Selective detection: The first selective
tag-switch method can be used to directly
label protein persulfide units (sites of Ssulfhydration) in the form of stable
thioether conjugates. It is thought that
H2S alone cannot lead to S-sulfhydration
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
These are not the final page numbers!
and that the two possible physiological
mechanisms include reaction with protein sulfenic acids (P-SOH) and the
involvement of metal centers which
would facilitate the oxidation of H2S to
HSC.
Angew. Chem. Int. Ed. 2013, 52, 1 – 8
Supporting Information
Wiley-VCH 2013
69451 Weinheim, Germany
Detection of Protein S-Sulfhydration by a Tag-Switch Technique**
Dehui Zhang, Igor Macinkovic, Nelmi O. Devarie-Baez, Jia Pan, Chung-Min Park,
Kate S. Carroll, Milos R. Filipovic,* and Ming Xian*
anie_201305876_sm_miscellaneous_information.pdf
Materials: Reagents and solvents were of the highest grade available. Reagent grade solvents were used for either
chromatography or extraction without further purification before use. Dichloromethane (DCM) and tetrahydrofuran
(THF) were directly used from a solvent purifier (Pure Solv, Innovative Technology, Inc.). Partially protected
amino acids were purchased from Advanced ChemTech and used directly. 2-mercaptobenzothiazole, 2,2’dibenzothiazolyl disulfide, malononitrile, methyl-2-cyanoacetate, and 1,3-cyclopentadione were purchased from
TCI America and used directly. D-(+)-biotin was purchased from Acros and used directly.
Buffers was prepared with nano-pure water, stirred with Chelex-100 resins to remove traces of heavy
metals and kept above the resins until used. Sodium sulfide (Na2S) was purchased as anhydrous, opened and stored
in glove box (<2 ppm O2 and <1 ppm H2O). 100 mM stock solutions of sodium sulfide were prepared as described
previously.14 Fe3+(P) water-soluble porphyrin was a kind gift from Dr Norbert Jux (Department of Chemistry and
Pharmacy, University of Erlangen-Nuremberg). HUVE cells (Human umbilical vein endothelial cells) were
obtained from Promo Cell (Heidelberg, Germany). Jurkat cells (T-lymphoblastic cells) were a kind gift from
professor Martin Herrmann (Medizinische Klinik 3, Rheumatologie und Immunologie, Erlangen, Germany).
Instrumentation. NMR spectra were recorded on a Varian Vx 300 NMR spectrometer and are reported in parts per
million (ppm) on the ! scale relative to residual CHCl3 (! 7.25 or ! 77.0) and DMSO-d6 (! 2.49 or ! 39.5) for 1H or
13
C. NMR experiments were performed at room temperature. All reported melting points for solid materials were
measured by Fisher-Johns melting point apparatus and not corrected. FT-IR spectra were recorded on a Thermo
Scientific Nicolet iS10 (Thin film) and reported in units of cm-1. Mass spectra were recorded using an electrospray
ionization mass spectrometry (ESI, Thermo Finnigan LCQ Advantage). Mass data were reported in units of m/z for
[M+H]+ or [M+Na]+.
Chromatography. The progress of the reactions was monitored by analytical thin layer chromatography (VWR,
TLC 60 F254 plates). Plates were visualized first with UV (254 nm) and then illuminated by CAM stain (2.5 g of
ammonium molybdate tetrahydrate and 1 g of cerium ammonium sulfate in a solution of 10% sulfuric acid in
water), KMnO4 solution (1.5 g of KMnO4, 10 g of K2CO3, and 1.25 mL of 10% NaOH), or ninhydrin solution
(0.3 % ninhydrin in a solution of 3 % acetic acid in ethanol). Flash column chromatography was performed using
silica gel (230-400 mesh). The solvent compositions for all separations are on a volume/volume (v/v) basis.
Scheme S1. The reaction between MSBT and small molecule persulfides
Reaction
between
MSBT
and
persulfides
(the
preparation
of
3):
To
a
solution
of
S-(4-
trifluoromethylbenzothioacidic)-N-Ac-penicillamine-NHBu 1 (73.0 mg, 0.163 mmol) in dry THF (12 mL) was
added MSBT (139 mg, 0.652 mmol) under argon atmosphere. Then a solution of benzyl amine (52.4 mg, 0.489
mmol) was added dropwise into the mixture at rt. The resulting mixture was stirred overnight and the solvent was
removed under vacuum. The crude material was subjected to flash column chromatography (2% MeOH in DCM)
1
to afford the desired product 3 as a brownish solid in 13 % yield (9 mg). mp 167-168 oC; 1H NMR (300 MHz,
CDCl3) ! 8.04 (s, 1H), 7.78 (t, J = 8.9 Hz, 2H), 7.42 (t, J = 7.7 Hz, 1H), 7.32 (t, J = 7.5 Hz, 1H), 6.90 (d, J = 8.9 Hz,
1H), 4.87 (d, J = 9.1 Hz, 1H), 3.43 (m, 1H), 3.27 – 3.09 (m, 1H), 2.02 (s, 3H), 1.52 (m, 5H), 1.39 (m, 5H), 0.89 (t, J
= 7.2 Hz, 3H);
13
C NMR (75 MHz, CDCl3) ! 170.4, 168.9, 153.9, 136.1, 126.6, 125.1, 121.9, 121.5, 58.7, 55.5,
39.7, 31.6, 31.2, 25.2, 23.6, 20.5, 14.0; IR (thin film, cm-1) 3273, 3080, 2951, 2864, 1683, 1634, 1564, 1455, 1424,
1380, 1363, 1002, 749, 721; MS (ESI) m/z calcd for C18H25N3NaO2S3 [M+Na]+ 434.0, found 434.1.
Synthesis of R-S-S-BT compounds
Preparation of compound 4: To a solution of 2,2’-dibenzothiazoyl disulfide (523 mg, 1.57 mmol) in THF (50
mL) was added Ac-Cys-NHBn (330 mg, 1.30 mmol) in CHCl3. The reaction mixture was stirred for 48 h at rt then
concentrated. The resulting residue was subjected to flash column chromatography (2% MeOH in DCM) to give
the desired product 4 as a solid (80% yield). mp 181-182 oC; 1H NMR (300 MHz, CDCl3) ! 8.22 (t, J = 5.2 Hz,
1H), 7.80 – 7.70 (m, 1H), 7.41 (dt, J = 7.3, 3.1 Hz, 1H), 7.36 – 7.20 (m, 7H), 6.99 (d, J = 7.5 Hz, 1H), 4.84 (td, J =
7.7, 4.7 Hz, 1H), 4.68 – 4.37 (m, 2H), 3.51 (dd, J = 14.2, 4.7 Hz, 1H), 3.11 (dd, J = 14.2, 8.0 Hz, 1H), 1.98 (s, 3H);
13
C NMR (75 MHz, CD3OD/CDCl3) ! 171.6, 169.9, 154.2, 137.6, 135.8, 128.7, 127.7, 127.6, 126.6, 125.1, 121.9,
121.4, 52.4, 43.7, 41.5, 22.6; FT-IR (thin film, cm-1) 3293.1, 3268.6, 3060.2, 3023.4, 1618.5, 1544.2, 1454.3,
1421.6, 1004.6, 735.1; MS (ESI) m/z calcd for C19H19N3NaO2S3 [M+Na]+ 440.1, found 440.1.
Scheme S2. Preparation of activated-disulfide model substrates
General Procedure for compounds 6. To a solution of 2,2’-dibenzothiazoyl disulfide (1.2 mmol) in THF (50 mL)
was added a solution of cysteine derivative (1 mmol) in CHCl3. The reaction mixture was stirred for 48 h at room
temperature and then concentrated. The resulting residue was subjected to flash column chromatography to give the
desired product.
Compound 6a was obtained as a white solid (70% yield). mp 135-136 oC; 1H NMR (300 MHz, CDCl3/CD3OD) !
8.36 – 8.17 (m, 2H), 7.94 – 7.83 (m, 1H), 7.85 – 7.75 (m, 1H), 5.31 (dd, J = 7.4, 4.7 Hz, 1H), 4.18 (s, 3H), 3.92 (dd,
J = 14.1, 4.8 Hz, 1H), 3.80 (dd, J = 14.1, 7.4 Hz, 1H), 2.47 (s, 3H); 13C NMR (75 MHz, CDCl3/CD3OD) ! 171.6,
170.6, 154.5, 135.6, 126.6, 125.0, 121.8, 121.3, 52.7, 51.9, 40.8, 22.2; FT-IR (thin film, cm-1) 3288.9, 3061.0,
2
2920.6, 1745.2, 1733.0, 1649.3, 1545.3, 1467.8, 1337.3, 1241.4, 1002.8, 747.8; MS (ESI) m/z calcd for
C13H14N2NaO3S3 [M+Na]+ 365.0, found 365.0.
Compound 6b was obtained as a white solid (77% yield). mp 128-129 oC; 1H NMR (300 MHz, CDCl3) ! 7.68-7.76
(m, 4H), 7.26-7.53 (m, 6H), 5.05-5.08 (m, 1H), 3.69 (s, 3H), 3.56 (d, J= 4.8 Hz, 2H); 13C NMR (75 MHz, CDCl3) !
170.7, 167.4, 154.6, 136.1, 133.7, 132.2, 129.0, 128.9, 127.5, 126.7, 125.1, 122.4, 121.6, 53.2, 52.4, 41.7; FT-IR
(thin film, cm-1) 3361.3, 2954.4, 2926.1, 1741.0, 1641.1, 1516.1, 1462.0, 1210.0, 1003.9, 764.4; MS (ESI) m/z
calcd for C18H16N2NaO3S3 [M+Na]+ 427.0, found 427.1.
Compound 6c was obtained as a white solid (75% yield). mp 174-175 oC; 1H NMR (300 MHz, CDCl3/CD3OD) !
8.47 (d, J = 7.8 Hz, 2H), 8.24 – 7.96 (m, 3H), 7.86 (q, J = 7.0 Hz, 4H), 5.44 (s, 1H), 5.32 (s, 1H), 4.35 (s, 3H), 4.17
– 4.02 (m, 1H), 3.93 (m, 1H), 3.73 (m, 1H), 3.58 (m, 1H), 2.58 (s, 3H);
13
C NMR (75 MHz, CDCl3/CD3OD) !
171.9, 171.8, 170.1, 154.6, 136.5, 129.2, 128.5, 126.8, 126.6, 125.0, 121.9, 121.4, 54.4, 54.3, 51.8, 40.7, 37.9, 37.8,
22.4, 22.3; FT-IR (thin film, cm-1) 3301.2, 3260.4, 3068.3, 3027.6, 1732.1, 1670.6, 1642.2, 1527.8, 1425.7, 1000.7,
743.2; MS (ESI) m/z calcd for C22H23N3NaO4S3 [M+Na]+ 512.1, found 512.2.
Compound 6d was obtained as a white solid (72% yield). mp 145-146 oC; 1H NMR (300 MHz, CDCl3) ! 7.88 (d, J
= 8.1 Hz, 1H), 7.77 (d, J = 7.9 Hz, 1H), 7.55 – 7.26 (m, 8H), 5.58 (d, J = 7.5 Hz, 1H), 5.11 (s, 2H), 4.99 – 4.83 (m,
1H), 4.47 – 4.26 (m, 1H), 3.72 (s, 3H), 3.42 (m, 2H), 1.40 (d, J = 7.1 Hz, 3H); 13C NMR (75 MHz, CDCl3) ! 172.6,
170.9, 170.3, 156.2, 154.9, 136.4, 136.1, 128.8, 128.4, 128.3, 126.7, 125.2, 122.5, 121.5, 67.3, 53.2, 52.1, 50.7,
41.2, 18.8; FT-IR (thin film, cm-1) 3269.0, 3093.1, 2987.5, 1736.2, 1646.3, 1683.1, 1531.9, 1258.1, 1000.7, 755.5;
MS (ESI) m/z calcd for C22H23N3NaO5S3 [M+Na]+ 528.1, found 528.1.
Compound 6e was obtained as a white solid (89% yield). mp 100-101 oC; The NMR spectra are reported for a
dynamic equilibrium between two rotamers: 1H NMR (300 MHz, CDCl3) ! 7.96 – 7.67 (m, 3H), 7.34 (m, 7H), 5.16
3
(s, 2H), 4.83 (m, 1H), 4.38 (m, 1H), 3.82 – 3.23 (m, 7H), 2.41 – 1.83 (m, 4H); 13C NMR (75 MHz, CDCl3) ! 172.6,
171.9, 170.3, 156.1, 155.1, 136.6, 136.1, 128.7, 128.3, 128.2, 126.6, 125.1, 122.4, 121.5, 67.7, 61.0, 60.7, 53.1,
52.2, 51.7, 47.9, 47.3, 41.4, 31.4, 28.5, 24.8; FT-IR (thin film, cm-1) 3329.9, 2974.3, 2945.7, 1740.3, 1703.4,
1695.3, 1654.5, 1519.6, 1417.5, 1352.1, 1205.0, 1111.0, 1090.6, 1000.7, 763.7; MS (ESI) m/z calcd for
C24H25N3NaO5S3 [M+Na]+ 554.1, found 554.1.
Compound 6f was obtained as a white solid (79% yield). mp 160-161 oC; 1H NMR (300 MHz, CDCl3) ! 7.76 (d, J
= 8.0 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.59 (d, J = 7.7 Hz, 1H), 7.45 – 7.27 (m, 2H), 7.17 (m, 8.7 Hz, 6H), 5.00 –
4.75 (m, 2H), 3.68 (s, 3H), 3.43 (dd, J = 14.1, 6.0 Hz, 1H), 3.19 (dd, J = 14.3, 5.8 Hz, 2H), 3.07 (dd, J = 13.9, 7.0
Hz, 1H), 1.92 (s, 3H); 13C NMR (75 MHz, CDCl3) ! 171.6, 170.7, 169.8, 154.1, 136.4, 129.5, 128.8, 127.3, 126.7,
125.2, 122.2, 121.5, 54.0, 52.7, 52.6, 42.7, 37.8, 23.3; FT-IR (thin film, cm-1) 3288.6, 3059.9, 2926.8, 1736.4,
1642.1, 1537.3, 1458.7, 1376.6, 1214.1, 1008.0, 754.7; MS (ESI) m/z calcd for C22H23N3NaO4S3 [M+Na]+ 512.1,
found 512.1.
The reaction between 4 and MCA: To a round bottom flask containing compound 4 (93.7 mg, 0.224 mmol) was
added 7 mL of THF. The solution was stirred for 5 min and then 7 mL of NaPi buffer (20 mM, pH 7,4) was added
into the flask. The mixture was a homogeneous solution. MCA (2 eq, 44.5 mg, 0.449 mmol) was added at rt and the
reaction was found to complete within 20 min (monitored by TLC). The aqueous mixture was quenched with 1N
HCl (16 mL) and extracted with EtOAc (10 mL " 3). The combined organic layers were dried and concentrated.
Flash column chromatography afforded the desired product 5f (as 1:1 diastereomers) in 98 % as colorless sticky oil.
1
H NMR (300 MHz, CDCl3) ! 7.42 (s, 1H), 7.36 – 7.16 (m, 5H), 7.12 – 6.89 (m, 1H), 4.93 – 4.74 (m, 1H), 4.58 (d,
J = 8.3 Hz, 1H), 4.47 – 4.28 (m, 2H), 3.82 (s, 3H), 3.29 – 2.98 (m, 2H), 1.95 (s, 3H); 13C NMR (75 MHz, CDCl3) !
165.1, 164.7, 137.6, 128.9, 127.9, 127.8, 114.4, 114.2, 54.7, 54.6, 52.1, 52.1, 43.9, 36.2, 36.1, 34.9, 34.7, 23.2; FTIR (thin film, cm-1) 3280.8, 3072.4, 3035.6, 2953.9, 2925.3, 2247.0, 1746.5, 1635.7, 1523.7, 1458.3, 1433.8,
1368.4, 1311.2, 1229.5, 1025.2, 702.4; MS (ESI) m/z calcd for C16H19N3NaO4S [M+Na]+ 372.1, found 372.1.
Scheme S3. Stability of R-S-S-BT toward potential biological nucleophiles
4
General procedure. To a solution of 6b (dissolved in 1:1 THF/NaPi (pH 7.4)) was added 5 equiv of corresponding
nucleophiles (L-Lysine, L-Serine, and L-methionine). The reaction mixture was stirred for 5 h at room temperature.
The reaction was monitored by TLC. We found that the starting material 6b was always fully recovered with no
reactions.
Scheme S4. The reaction of R-S-S-BT substrates with carbon nucleophiles
General procedure. To a stirring solution of R-S-S-BT substrate (0.2 mmol) in THF (8 mL) and NaPi (20 mM, 8
mL) was added each carbon nucleophiles (0.4 mmol). The reaction mixture was then stirred for 20 min at room
temperature and quenched with 1N HCl (16 mL). The solution was extracted with ethyl acetate (10 mL " 3). The
combined organic layers were washed with brine, dried over with anhydrous MgSO4, and concentrated. The
resulting residue was subjected to flash column chromatography to isolate the possible products or recovered
starting materials.
Compound 5b: 1H NMR (300 MHz, CDCl3) ! 8.30 (t, J = 6.0 Hz, 1H), 7.76 (d, J = 6.9 Hz, 1H), 7.35 – 7.20 (m,
5H), 4.44 (m, 4H), 3.12 (dd, J = 13.6, 4.4 Hz, 1H), 2.43 (m, 5H), 2.02 (s, 3H), 1.09 (s, 6H); 13C NMR (75 MHz,
CDCl3) ! 191.8, 171.9, 171.4, 137.7, 128.6, 127.4, 127.4, 104.5, 53.1, 43.5, 37.7, 31.5, 29.7, 28.2, 22.6; FT-IR (thin
film, cm-1) 3268.5, 3066.1, 3029.5, 2952.4, 2923.4, 2854.0, 1645.1, 1543.0, 1475.5, 1369.9, 1264.3, 1144.9, 1025.5,
1009.9, 731.3; MS (ESI) m/z calcd for C20H26N2NaO4S [M+Na]+ 413.2, found 413.4.
5
Compound 5e: 1H NMR (300 MHz, DMSO-d6) ! 8.73 (t, J = 6.0 Hz, 1H), 8.51 (d, J = 8.4 Hz, 1H), 7.90 (s, 1H),
7.32 – 7.23 (m, 5H), 4.73 (m, 1H), 4.29 – 4.25 (m, 2H), 3.32 – 3.21 (m, 2H), 1.91 – 1.81 (m, 3H);13C NMR (75
MHz, CDCl3/MeOH-d4) ! 171.7, 169.4, 137.4, 128.8, 127.9, 127.7, 111, 110.9, 51.9, 43.9, 36.1, 29.9, 22.9; FT-IR
(thin film, cm-1) 3276.7, 3064.2, 3031.6, 2917.1, 2851.8, 2202.0, 2157.1, 2112.1, 1638.1, 1523.7, 1450.2, 1368.4,
1241.8, 698.3; MS (ESI) m/z calcd for C15H16N4O2S [M-H]- 351.1, found 351.5.
Compound 7a was obtained as a thick oil (as 1:1 diastereomers, 93% yield). 1H NMR (300 MHz, CDCl3) ! 6.38 (s,
1H), 4.90 (q, J = 5.9 Hz, 1H), 4.43 (d, J = 27.1 Hz, 1H), 3.87 (s, 3H), 3.81 (s, 3H), 3.53 – 3.31 (m, 1H), 3.32 – 3.15
(m, 1H), 2.07 (s, 3H); 13C NMR (75 MHz, CDCl3) ! 170.7, 170.5, 164.6, 164.3, 114.1, 113.9, 54.6, 53.3, 51.8, 51.7,
36.1, 35.9, 34.7, 23.3; FT-IR (thin film, cm-1) 3374.8, 3280.8, 2963.9, 2917.1, 2843.6, 2247.0, 1740.5, 1654.5,
1531.9, 1429.7, 1368.4, 1213.2, 1008.9; MS (ESI) m/z calcd for C10H14N2NaO5S [M+Na]+ 297.1, found 297.1.
Compound 7b was obtained as a thick oil (as 1:1 diastereomers, 99% yield). 1H NMR (300 MHz, CDCl3) ! 7.91 –
7.74 (m, 2H), 7.59 – 7.36 (m, 3H), 7.18 – 7.02 (m, 1H), 5.08 (s, 1H), 4.59 – 4.35 (m, 1H), 3.82 (s, 6H), 3.54 (ddd, J
= 14.9, 10.0, 4.7 Hz, 1H), 3.34 (ddd, J = 14.2, 9.2, 6.0 Hz, 1H); 13C NMR (75 MHz, CDCl3) ! 170.8, 167.5, 164.6,
164.4, 133.4, 132.4, 128.9, 127.5, 114.0, 113.9, 54.6, 53.5, 53.4, 52.2, 52.2, 36.1, 35.9, 34.8, 34.6; FT-IR (thin film,
cm-1) 3329.9, 2953.9, 2921.2, 2855.8, 2242.9, 1740.8, 1642.2, 1519.6, 1486.9, 1429.7, 1213.2, 1017.0, 710.6; MS
(ESI) m/z calcd for C15H16N2NaO5S [M+Na]+ 359.1, found 359.1.
Compound 7c was obtained as a yellowish solid (as 1:1 diastereomers, 99% yield). mp 113-114 oC; 1H NMR (300
MHz, CDCl3) ! 7.37 – 7.04 (m, 6H), 6.53 – 6.30 (m, 1H), 4.87 – 4.65 (m, 2H), 4.55 – 4.28 (m, 1H), 3.77 (s, 3H),
3.67 (s, 3H), 3.36 – 2.97 (m, 4H), 1.89 (s, 3H); 13C NMR (75 MHz, CDCl3) ! 171.7, 170.7, 170.1, 169.9, 164.7,
6
136.5, 129.6, 129.5, 128.8, 127.2, 114.3, 54.6, 54.6, 54.5, 53.3, 52.1, 52.0, 38.2, 36.2, 35.9, 34.6, 34.5, 23.3; FT-IR
(thin film, cm-1) 3287.4, 3034.3, 2910.5, 2251.4, 1746.0, 1730.8, 1664.2, 1545.0, 1431.1, 1367.4, 1298.2, 1032.3,
754.2; MS (ESI) m/z calcd for C19H23N3NaO6S [M+Na]+ 444.1, found 444.2.
Compound 7d was obtained as a thick oil (as 1:1 diastereomers, 96% yield): 1H NMR (300 MHz, CDCl3) ! 7.61 –
6.98 (m, 6H), 5.60 (s, 1H), 5.10 (s, 2H), 4.87 (s, 1H), 4.67 – 4.42 (m, 1H), 4.32 (s, 1H), 3.81 (s, 3H), 3.76 (s, 3H),
3.47 – 3.11 (m, 2H), 1.38 (d, J = 6.9 Hz, 3H); 13C NMR (75 MHz, CDCl3) ! 172.9, 170.4, 170.3, 164.7, 164.7,
156.3, 136.4, 128.8, 128.4, 128.3, 114.4, 114.1, 67.3, 67.3, 54.6, 54.6, 53.3, 53.3, 52.0, 50.8, 36.2, 35.9, 34.6, 34.4,
18.6, 18.4; FT-IR spectra (thin film, cm-1) 3313.5, 2953.9, 2921.2, 1851.8, 2247.0, 1740.3, 1738.5, 1670.8, 1515.6,
1429.7, 1319.4, 1209.1, 1017.0, 735.1, 702.4; MS (ESI) m/z calcd for C19H23N3NaO7S [M+Na]+ 460.1, found 460.2.
Compound 7e was obtained as a thick oil (as 1:1 diastereomers, 92% yield): The NMR spectra are reported for a
dynamic equilibrium between two rotamers: 1H NMR (300 MHz, CDCl3) ! 7.79 – 7.11 (m, 6H), 5.13 (s, 2H), 4.98
– 4.52 (m, 2H), 4.49 – 4.23 (m, 1H), 3.93 – 3.65 (m, 6H), 3.60 – 3.27 (m, 3H), 3.13 (s, 1H), 2.29 – 1.87 (m, 4H);
13
C NMR (75 MHz, CDCl3) ! 172.8, 172.3, 170.3, 164.9, 164.7, 156.2, 155.2, 136.6, 128.7, 128.3, 128.2, 128.1,
114.2, 67.6, 61.0, 60.7, 54.5, 53.3, 53.22, 52.1, 51.9, 51.2, 51.1, 47.8, 47.2, 36.3, 35.7, 35.0, 34.7, 29.9, 28.9, 24.8,
23.9; FT-IR (thin film, cm-1) 3317.1, 2962.1, 2947.5, 2851.8, 2242.9, 1748.5, 1679.0, 1519.6, 1454.3, 1433.8,
1405.2, 1352.1, 1209.1, 111.0, 739.2; MS (ESI) m/z calcd for C21H25N3NaO7S [M+Na]+ 486.1, found 486.1.
Compound 7f was obtained as a thick oil (as 1:1 diastereomers, 99% yield): 1H NMR (300 MHz, CDCl3) ! 7.33 –
7.15 (m, 4H), 7.18 – 7.02 (m, 2H), 6.88 – 6.65 (m, 1H), 4.89 – 4.68 (m, 2H), 4.61 (d, J = 6.5 Hz, 1H), 3.82 (s, 3H),
3.70 (s, 3H), 3.26 – 2.93 (m, 4H), 1.95 (s, 3H); 13C NMR (75 MHz, CDCl3) ! 171.6, 171.6, 170.9, 169.9, 165.2,
164.8, 135.9, 129.4, 128.8, 128.8, 127.4, 127.4, 114.6, 114.4, 54.7, 54.6, 53.9, 52.7, 51.9, 37.8, 37.8, 36.3, 36.3,
7
34.8, 34.5, 23.2; FT-IR (thin film, cm-1) 3289.0, 3060.2, 3027.5, 2958.0, 2940.3, 2851.8, 2247.0, 1744.4, 1650.4,
1519.6, 1429.7, 1972.5, 1245.9, 1209.1, 1017.0, 743.2; MS (ESI) m/z calcd for C19H23N3NaO6S [M+Na]+ 444.1,
found 444.2.
Scheme S5. Control experiments
Scheme S6. Synthesis of biotinylated #-cyano ester (CN-biotin)
Compound S8. To a suspension of biotin S7 (4.06 mmol, 990 mg) in anhydrous DMF (60 mL) was added 1.1
equiv of N-hydroxysuccinimide (4.46 mmol, 514 mg). EDC$HCl (4.87 mmol, 933 mg) was then added and the
reaction was stirred for 24 hours (at this time the solution turned clear). The solvent was then removed under
reduced pressure to provide a white solid. The solid was washed thoroughly with anhydrous methanol, filtered and
dried to provide S8 as a white solid (1.104 g, 80% yield). The product was used directly in the next step without
any further purification.
Compound S9. To a solution of compound S8 (3.24 mmol, 1.104 g) in anhydrous DMF (66 mL) was added 2aminoethanol (4.86 mmol, 0.29 mL) dropwise. Then, triethylamine (6.48 mmol, 0.9 mL) was added and the
reaction mixture was stirred overnight. The solvent was removed under reduced pressure and the resulting residue
8
was purified by flash column chromatography (gradient elution: 50:1 DCM/MeOH to 7:1 DCM/MeOH). The
product S9 was used directly in the next step.
CN-Biotin. To a solution of compound S9 (3.24 mmol) in DMF (50 mL) was added cyanoacetic acid (3.89 mmol,
331 mg). DCC (4.2 mmol, 867 mg) was then added followed by DMAP (0.32 mmol, 40 mg,). The reaction mixture
was stirred overnight and the solvent was removed under reduced pressure to give the crude product. Flash column
chromatography (gradient elution: 50:1 DCM/MeOH to 7:1 DCM/MeOH) afforded the final product as a white
solid (804 mg). Yield: 70% for two steps. mp 132-134 oC; 1H NMR (300 MHz, DMSO-d6) ! 7.96 (t, J = 5.3 Hz,
1H), 6.43 (s, 1H), 6.37 (s, 1H), 4.30 (dd, J = 7.7, 4.8 Hz, 1H), 4.11 (m, 3H), 3.97 (s, 2H), 3.36 – 3.23 (m, 2H), 3.16
– 3.03 (m, 1H), 2.82 (dd, J = 12.4, 5.0 Hz, 1H), 2.57 (m, 1H), 2.06 (t, J = 7.3 Hz, 2H), 1.68 – 1.37 (m, 4H), 1.29 (m,
2H);
13
C NMR (75 MHz, DMSO-d6) ! 172.3, 164.3, 162.6, 114.9, 64.4, 60.9, 59.1, 55.3, 39.7, 37.1, 34.9, 28.1,
27.9, 25.0, 24.5. FT-IR (thin film, cm-1) 3270.6, 3079.7, 2931.3, 2264.3, 2200.7, 1742.4, 1696.7, 1549.2, 1264.7,
1032.1, 726.8. MS (ESI) m/z, calcd for C15H22N4NaO4S [M+Na]+ 377.1, found 377.1.
Mass analysis of tag-switch assay with Gpx3-persulfide: A freshly prepared Gpx3 protein persulfide solution4
(20 µM final concentration) was incubated with (or without) MSBT-A (20 mM final concentration) respectively,
followed by the addition of ethyl cyanoacetate (20 mM final concentration) at rt for 1 hour. The protein was then
purified through a P-30 spin column. Sample aliquots were then generated and analyzed by LC%MS.
Evaluation of the tag switch assay: Preparation of bovine serum albumin (BSA) sulfenic acid (BSA-SOH) was
done following the published protocol.[10] Briefly, 0.98 mM BSA was reduced with 5 mM 2-mercaptoethanol and
dialysed over night. Samples were additionally degassed with argon. BSA was then incubated with 10 mM H2O2
(15 min, 37 °C) and the reaction was stopped by the addition of bovine heart catalase (1221 U, Sigma Aldrich,
USA). Formation of the sulfenic acid was confirmed by treating the freshly prepared BSA-SOH with 50 mM
dimedone and detecting the dimedone-labeled peptide (after trypsin digestion) by LC-ESI-TOF-MS.
Glutathionylated samples (BSA-SSG) were made by incubation of BSA-SOH with 2 mM glutathione, and Ssulfhydrated samples (BSA-SSH) by incubation with 2 mM H2S (15 min, room temperature). Each sample was
desalted on biospin column, treated with 10 mM MSBTA (15 min, 37 °C) and 2 mM CN-biotin (with desalting on
biospin column after each step). Prepared samples were then analyzed by ultra-high resolution ESI-TOF-MS
(maXis, Bruker Daltonics). One part of the samples was mixed with streptavidin agarose resin (Thermo Fischer
Scientific), incubated for 2 h at room temperature and the concentration of remaining proteins in the supernatant
quantified by RotiR Nanoquant (Karl Roth, Karlsruhe, Germany). Resins were washed, the bound proteins eluted
following the manufacturer’s instruction and the absorbance at 280 nm measured. In a separate experiment, 10 µL
of BSA-SH, BSA-SOH, BSA-SSG and BSA-SSH (treated with tag switch assay) were loaded on nitrocellulose
membrane. After drying, membrane was blocked with 5 % non-fat milk for 1 h at room temperature, and incubated
2 h with HRP-labelled mouse monoclonal anti-biotin antibody (Sigma Aldrich, St. Louis, MO) according to the
manufacture recommendation. Signal was visualized using Pierce ECL reagent (Thermo Scientific, IL, USA) and
exposing membrane to x-ray films.
Mechanistic studies of protein S-sulfhydration: 2 mg/mL GAPDH (from rabbit muscles, Sigma Aldrich, USA)
was incubated with either 100 µM H2S, 100 µM H2S with rigorous vortexing, 100 µM Angeli’s salt and 100 µM
H2O2 for 30 min at rom temperature. GAPDH was also treated with 100 µM Angeli’s salt and 100 µM H2O2 for 15
9
min followed by additional 15 min with 100 µM H2S. Additionally, 2 mg/mL GAPDH was mixed with 100 µM
H2S and 20 µM water-soluble iron prophyrin for 30 min. Samples were then desalted in biospin columns and
further labeled by tag switch assay.
Cell culture: Jurkat cells were cultured in RPMI supplemented with 10% FCS, 1% nonessential amino acid mix
and 1% Streptomycin-Penicillin at 37 °C, and 5% CO2. Human umbilical vein endothelial cells (HUVECs, passage
2-3) were cultured in 35-mm &-Dishes (ibidi, Martinsried, Germany) in endothelial cell growth medium 2
(PromoCell GmbH) at 37 °C and 5% CO2.
Jurkat cell extracts: JURKAT cells (5 x 106) were incubated without or with 200 µM Na2S (30 min). Cells were
centrifuged and lysed in HEN buffer (250 mM HEPES, 50 mM NaCl, 1mM EDTA, 0.1 mM neocuproine, 1 %
protease inhibitors, pH 7.4) containing 1 % NP40 detergent using ultrasonicator. Total cell lysates were then split
into two parts, one kept as it was and the other one was additionally mixed with 200 µM Na2S and vortexed for 30
min.
Tag-switch assay for S-sulfhydration: Protein samples were treated with 50 mM MSBT-A (2.5% SDS, 1 h, 37
°C) and desalted on biospin columns (BioRad). Proteins were then treated with CN-biotin (1h, 37 °C). Prior the
electrophoresis, samples were additionally desalted, mixed with 5 x Laemmli buffer, and then resolved on 8% or
10% SDS polyacrylamide gel. Proteins were transferred on nitrocellulose membrane using semi dry system (Bio
Rad, USA), blocked with 5% non-fat milk for 1 h at room temperature, and incubated over night with HRP-labelled
mouse monoclonal anti-biotin antibody (Sigma Aldrich, St. Louis, MO) according to the manufacture
recommendation. Signal was visualized using Pierce ECL reagent (Thermo Scientific, IL, USA) and exposing
membrane to x-ray films.
Pull-down assay: Biotinylated whole cell lysates were additionally incubated with streptavidin agarose resins (1-3
mg of biotin/mL binding capacity) for 30 min at room temperature with constant mixing and unbound proteins
analysed by SDS-PAGE followed by Western Blot.
MeOH fixation and S-Sulfhydration detection: HUVECs were grown in ibidi dishes and then treated with 100
µM Na2S for 30 min, 2 mM PG (propargylglycine, CSE inhibitor) for 2 h or pre-treated with 2 mM PG (2 h) and
then exposed to Na2S for 30 min. After the treatment cells were fixed with ice cold MeOH and kept at -20 °C for 15
min. Cells were washed with 80 % MeOH/20 % HEN buffer (250 mM HEPES, 50 mM NaCl, 1mM EDTA, 0.1
mM neocuproine) (v/v) and then exposed to 50 mM MSBT-A over night in the same solvent (MeOH/HEN buffer,
80/20). After thorough washing (5 x 10 min) the cells were exposed to 2 mM CN-biotin for 2 h in the same solvent.
Finally, cells were incubated 1 h with fluorescein-labelled streptavidin (Thermo Fischer Scientific, IL, USA) in
PBS.
Paraformaldehyde fixation and S-sulfhydration: HUVECs were grown in ibidi dishes and treated with 100 µM
Na2S (30 min), and 2 mM 2-ketobutiric acid (2 h). Cells were fixed with 4 % paraformaldehyde for 20 min. After
washing with PBS cells were exposed to PBS containing 1 % Triton X-100 (v/w) for 2 h to permeabilize the cells
and increase availability of thiols to MSBT-A. 50 mM MSBTA was made in the same solvent and cells kept in this
solution at room temperature over night. Cells were then washed (5 x 10 min) with cold PBS and treated with 2
10
mM CN-biotin in PBS for 2 h. This was followed by incubation with fluorescein-labelled streptavidin (Thermo
Fischer Scientific, IL, USA) for 1 h.
Colocalization studies: For visualization of endoplasmic reticulum HUVECs were transfected with CellLightTM
ER-GFP following the instruction of the manufacturer (Molecular Probes, Invitrogen, Germany). Mitochondria
were visualized by treating the cell with 500 nM MitoTrackerR Red CMXRos (Molecular Probes, Invitrogen) for 45
min prior the fixation. Cells were treated with 100 µM Na2S (30 min), washed and fixed with ice cold methanol for
20 min at -20 °C. Cell permabilization was performed with 5 min exposure to ice cold acetone. After that cells
were washed and treated with 50 mM MSBT-A, then 5 mM CN-biotin and finally with DyLightTM405 conjugated
streptavidin (Thermo Fischer Scientific, IL, USA), 45 min at 37 °C for each step. Between each step samples were
washed extensively (5 x 5 min).
Evaluation of the in situ detection of S-sulfhydrated proteins: Paraformaldehyde fixed HUVEC cells were
exposed to either CN-biotin alone or the whole tag switch assay. Additionally, after fixation and permeabliziation
HUVECs were pre-incubated with 50 mM dimedone and protein persulfides labelled by tag switch assay. This was
followed by incubation with fluorescein-labelled streptavidin (Thermo Fischer Scientific, IL, USA) for 1 h. Nuclei
were stained with DAPI.
Fluorescent microscopy: Fluorescent microscopy was carried out using Carl Zeiss Axiovert 40 CLF inverted
microscope, equipped with monochromatic RGB CoolLed light source (Andover, UK) and monochromatic
AxioCam Icm1 camera. All experiments were performed at least in triplicate. Images were post-processed in
ImageJ software (NIH, USA).
11
12
13
Figure S1. Evaluation of the tag switch assay. A) Preparation of bovine serum albumin (BSA) sulfenic acid
(BSA-SOH) was done following the published protocol.[10] Formation of the sulfenic acid was confirmed by
treating the freshly prepared BSA-SOH with 50 mM dimedone and detecting the dimedone-labeled peptide (after
trypsin digestion) by LC-ESI-TOF-MS. Original MS data from DataAnalysis software shown in A) represent the
measured, deconvoluted spectrum of GLVLIAFSQYLQQ34CPFDEHVK peptide. Spectrum bellow is simulated
isotopic distribution for the peptide labeled with dimedone. The third spectrum represents the simulation of the
isotopic distribution of the peptide only, and the last spectrum is the simulation of isotopic distribution for the
peptide containing cysteine in the form of sulfenic acid. B) Isotopic distribution of the measured (upper spectrum)
and simulated (lower spectrum) of dimedone-labeled GLVLIAFSQYLQQ34CPFDEHVK confirming that the used
synthetic protocol indeed gives mainly BSA-SOH. C) UV-vis detection of the proteins eluted from streptavidin
agarose resins. BSA-SH (black line), BSA-SOH (blue line), BSA-SSG (red line) and BSA-SSH (green line) were
treated with tag switch assay and mixed with streptavidin agarose resins. Resins were washed and the eluted
proteins analyzed by UV-vis spectroscopy for the presence of the proteins (280 nm). D) ESI-TOF MS analysis of
the BSA-SOH, BSA-SSG and BSA-SSH treated with tag switch assay. Figure shows re-drown deconvoluted MS
spectra obtained using DataAnalysis software (Bruker Daltonics). Untreated BSA served as a control giving the
peak at m/z 66430±2 Da. BSA-SOH sample gave only the peak at m/z 66462±2 Da which we assigned to BSASO2H (mass change is 32 when compared to the control spectrum). BSA-SO2H was inevitable end product after
treating the samples of BSA-SOH for few hours with all the reagents of tag switch assay. The same peak could be
seen in BSA-SSG and BSA-SSH samples. However, BSA-SSG sample clearly shows the peak at m/z 66735±2 Da
(addition of a glutathione moiety) while the BSA-SSH sample has a peak at m/z 66782±2 Da, which is assigned to
the BSA labeled with CN-biotin. ~50 % yield of the BSA-SSH is in good agreement with the data obtained by the
analysis of the proteins bound after exposure to streptavidin agarose resins (Figure 2). The peak at m/z 66728 could
be the decomposition product of BSA-CN biotin derivative, suggesting the loss of NC(O)N moiety of biotin
structure.
14
Figure S2. Possible reaction pathways for S-sulfhydration. A) Coomassie blue-staining for the protein load
(left) and Western blot of S-sulfhydrated GAPDH. B) Quantification of the S-sulfhydration yield, compared to the
control. GAPDH (2 mg/mL) was used as a model protein. Untreated sample served as a control. Samples were
treated as marked in the figure.
15
Figure S3. Tag-switch assay for protein S-sulfhydration. Jurkat cells (5 x 106) were incubated without or with
200 µM Na2S (30 min) and then centrifuged and lysed in HEN buffer (250 mM HEPES, 50 mM NaCl, 1mM
EDTA, 0.1 mM neocuproine, 10 % protease inhibitors, pH 7.4) containing 1 % NP40 detergent. Total cell lysates
were then split into two parts, one kept as it was and the other one was additionally mixed with 200 µM Na2S and
vortexed for 30 min. A) Western blot of the whole cell lysate before (1) and after (2) it has been mixed with
streptavidin agarose resins. B) Original, unprocessed scan of the nitrocellulose membrane. Inset: Coomasie blue
staining for the protein load. C) Quantification of the S-sulfhydration based on the intensity of the 70 kDa band. 1:
control, 2: H2S-treated cells, 1 + H2S: control cell lysates treated with H2S, 2 + H2S: cell lysates from H2S-treated
cells additionally treated with 200 µM H2S.
16
DAPI
PSSH
Overlay
Cells treated with
CN-biotin only
Cells treated with
tag-switch assay
Cells pre-treated
with dimedone
and then with
tag-switch assay
Figure S4. Evaluation of the in situ method for the intracellular detection of protein persulfides.
Paraformaldehyde fixed HUVEC cells (treated with 200 &M Na2S) were exposed to either CN-biotin alone or the
whole tag switch assay. Additionally, after fixation and permeablization HUVECs were pre-incubated with 50 mM
dimedone (to block all intracellular sulfenic acids) and protein persulfides labelled by tag switch assay.
Biotinylated proteins were visualized with fluorescein-labelled streptavidin (Thermo Fischer Scientific, IL, USA)
for 1 h. Nuclei were stained with DAPI.
17
ER tracker
P-SSH
Overlay
Mito tracker
P-SSH
Overlay
Figure S5. Colocalization of intracellular persulfides with endoplasmic reticulum and mitochondria. For
visualization of endoplasmic reticulum HUVECs were transfected with CellLightTM ER-GFP (green fluorescence)
following the instruction of the manufacturer (Molecular Probes, Invitrogen, Germany). Mitochondria were
visualized by treating the cell with 500 nM MitoTrackerR Red CMXRos (red fluorescence, Molecular Probes,
Invitrogen) for 45 min prior the fixation. Cells were treated with 100 µM Na2S (30 min), washed and fixed with ice
cold methanol for 20 min at -20 °C. Cell permabilization was performed with 5 min exposure to ice cold acetone.
After that cells were washed and treated with 50 mM MSBT-A, then 5 mM CN-biotin and finally with
DyLightTM405 conjugated streptavidin (blue fluorescence, Thermo Fischer Scientific, IL, USA), 45 min at 37 °C
for each step. For better presentation pictured were artificially colored green and red.
18
300MHz 1H NMR for compou
und 3 in CDCl3 (ppm
m)
75
5MHz 13C NMR fo
or compound 3 in CDCl3/MeOH-d4 (pppm)
O
S
S
NHBn
NH
HAc
S
N
4
300MHz 1H NMR for compound 4 in CDCl3 (ppm)
O
S
S
NHBn
NHAc
S
N
4
75
5MHz 13C NMR fo
or compound 4 in CDCl
C
ppm)
3/MeOH-d4 (p
300MHz 1H NM
MR for compound 6a in CDCl3/MeOH
H-d4 (ppm)
75M
MHz 13C NMR for compound 6a in CDCl3/MeOH-d4 (pppm)
300
0MHz 1H NMR forr compound 6b in CDCl3 (ppm)
75MHz 13C NM
MR for compound 6b in CDCl3 (ppm)
30
00MHz 1H NMR fo
or compound 6c in CDCl3/MeOH-d4 (ppm)
75MHz 13C NM
MR for compound 6c in CDCl3/MeOH
H-d4 (ppm)
30
00MHz 1H NMR fo
or compound 6d in
n CDCl3 (ppm)
75MHz 13C NMR
R for compound 6d
d in CDCl3 (ppm)
300MHz 1H NM
MR for compound 6e in CDCl3 (ppm)
75MHz 13C NM
MR for compound
d 6e in CDCl3 (ppm
m)
300MHz 1H NMR for compound
d 6f in CDCl3 (ppm
m)
75MHz 13C NMR
R for compound 6ff in CDCl3 (ppm)
30
00MHz 1H NMR for compound 5b in
n CDCl3 (ppm)
75MHz 13C NM
MR for compound 5b
b in CDCl3/MeOH
H-d4 (ppm)
300MHz 1H NMR for comp
pound 5e in DMSO
O-d6 (ppm)
75MH
Hz 13C NMR for compound 5e in CDC
Cl3/MeOH-d4 (ppm
m)
300MHz 1H NMR
R for compound 5ff in CDCl3 (ppm)
75MHz 13C NMR
R for compound 5ff in CDCl3 (ppm)
300MHz 1H NMR for compound 7a in CDCl3 (ppm)
75MHz 13C NMR for compou
und 7a in CDCl3 (pppm)
300MHz 1H NM
MR for compound 7b in CDCl3 (ppm
m)
75MHz 13C NM
MR for compound 7b in CDCl3 (ppm
m)
300MHz 1H NMR for compound
d 7c in CDCl3 (ppm
m)
75MHz 13C NMR for compound 7c in CDCl3 (ppm)
300MHz 1H NMR for compoun
nd 7d in CDCl3 (pppm)
75MHz 13C NM
MR for compound 7d in CDCl3 (ppm)
300MHz 1H NMR
R for compound 7e in CDCl3 (ppm)
75MHz 13C NMR for compound 7e in
n CDCl3 (ppm)
NC
O
MeO
O
Ph
CO2Me
S
H
N
N
NHAc
O
7f
300MHz 1H NMR
R for compound 7ff in CDCl3 (ppm)
NC
O
MeO
Ph
CO2Me
S
H
N
NHAc
O
7f
75MHz 13C NMR
N
for compound
d 7f in CDCl3 (ppm
m)
300MHz 1H NMR fo
or compound CN-biiotin in DMSO-d6 (ppm)
75MH
Hz 13C NMR for co
ompound CN-biotiin in DMSO-d6 (pppm)