Repurposing the Pummerer Rearrangement: Determination of Methionine Sulfoxides in Peptides

Article abstract of DOI:10.1002/cbic.201900463

The reversible oxidation of methionine residues in proteins has emerged as a biologically important post-translational modification. However, detection and quantitation of methionine sulfoxide in proteins is difficult. Our aim is to develop a method for specifically derivatizing methionine sulfoxide residues. We report a Pummerer rearrangement of methionine sulfoxide treated sequentially with trimethylsilyl chloride and then 2-mercaptoimidazole or pyridine-2-thiol to produce a dithioacetal product. This derivative is stable to standard mass spectrometry conditions, and its formation identified oxidized methionine residues. The scope and requirements of dithioacetal formation are reported for methionine sulfoxide and model substrates. The reaction intermediates have been investigated by computational techniques and by ¹³C NMR spectroscopy. These provide evidence for an α-chlorinated intermediate. The derivatization allows for detection and quantitation of methionine sulfoxide in proteins by mass spectrometry and potentially by immunochemical methods.

Full text of DOI:10.1002/cbic.201900463

Accepted Article
Title: Repurposing the Pummerer Rearrangement: Determination of
Methionine Sulfoxides in Peptides
Authors: Carolyn C Woodroofe, Rolf E Swenson, Joseph Ivanic,
Rodney L. Levine, and Sarah Monti
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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the VoR from the journal website shown below when it is published
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To be cited as: ChemBioChem 10.1002/cbic.201900463
Link to VoR: http://dx.doi.org/10.1002/cbic.201900463
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10.1002/cbic.201900463
ChemBioChem
Repurposing the Pummerer Rearrangement: Determination of Methionine Sulfoxides in Peptides
Carolyn C. Woodroofe*[a], Joseph Ivanic[b], Sarah Monti[c], Rodney L. Levine[c], and Rolf E. Swenson[a]
Abstract
The reversible oxidation of methionine residues in proteins has emerged as a biologically important
post-translational modification. However, detection and quantitation of methionine sulfoxide in
proteins is difficult. Our aim is to develop a method for specifically derivatizing methionine sulfoxide
residues. We report a Pummerer rearrangement of methionine sulfoxide treated sequentially with
trimethylsilyl chloride and then 2-mercaptoimidazole or pyridine-2-thiol to produce a dithioacetal
product. This derivative is stable to standard mass spectrometry conditions, and its formation identified
oxidized methionine residues. The scope and requirements of dithioacetal formation are reported for
methionine sulfoxide and model substrates. The reaction intermediates have been investigated by
computational techniques and by 13C NMR. These provide evidence for an alpha-chlorinated
intermediate. The derivatization allows for detection and quantitation of methionine sulfoxide in
proteins by mass spectrometry and potentially by immunochemical methods.
Table of Contents graphic
HN
N
TMSCl
SH
S
Cl
S
O
S
S
HN
N
Keywords: Protein modifications, reaction mechanism, Pummerer rearrangement, sulfoxides,
oxidoreductases
*Corresponding author: carolyn.woodroofe@nih.gov
Author Affiliations
[a]Imaging Probe Development Center, National Heart, Lung and Blood Institute, National Institutes of
Health, 9800 Medical Center Dr, Rockville MD 20850
[b] Advanced Biomedical Computational Sciences Group, Frederick National Laboratory for Cancer
Research operated by Leidos Biomedical Research, Inc., 1050 Boyles St, Frederick, MD 21702
[c] Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, 50
South Dr, Bethesda, MD 20892
1
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ChemBioChem
Introduction
Oxidative modification of proteins by reactive oxygen species had long been viewed as an inevitable,
negative by-product of aerobic metabolism. Over the past two decades, many investigators have
established that cells produce reactive oxygen species in a controlled fashion. These reactive oxygen
species mediate the reversible oxidative modification of specific proteins as an important mechanism of
cellular regulation.[1] Methionine in proteins is often thought to be a generic hydrophobic residue,
functionally replaceable with another hydrophobic residue such as valine or leucine. However, this
frequently is not the case because of the presence of sulfur in methionine. The methionine can be
oxidized to methionine sulfoxide, and all aerobic organisms contain methionine sulfoxide reductases
capable of reducing the sulfoxide back to the thioether.[2,3] Moreover, the cycle also constitutes a
reversible post-translational covalent modification analogous to phosphorylation that can regulate
cellular metabolism.[4,5,6] The reversible oxidative modification of methionine enables methionine
residues to provide a catalytically efficient antioxidant defense that scavenges reactive oxygen species.
Progress in identifying the proteins and pathways affected by methionine oxidation and reduction has
been slow because detection and quantitation of methionine sulfoxide in specific proteins is difficult. A
general antibody capable of specifically binding to any methionine sulfoxide residue would be very
useful, but such antibodies cannot be raised.[7,8] Mass spectrometry would seem an ideal method for
analysis, and various mass spectrometric methods have been proposed,[9] but they often suffer from
artifactual sulfoxide formation during sample preparation or analysis. A method for covalently and
specifically derivatizing methionine sulfoxide residues could circumvent these issues. Such a method
could be utilized in a variety of detection techniques, including one and two dimensional separations,
mass spectrometry, and fluorescence. Modified proteins or their proteolytic digests could also be
enriched by affinity techniques based on the covalent modification.
The Pummerer rearrangement is a reaction specific to sulfoxides. Sulfoxides can be O-alkylated or
acylated with a strong electrophile or other acid, forming an adduct that then eliminates to give rise to a
thionium ion. The thionium ion may undergo a variety of reactions: in the most common incarnation of
the Pummerer rearrangement, an acetate ion or similar weak to moderate nucleophile adds to generate
an alpha-substituted sulfide as the product, essentially transferring the oxidation from the original sulfur
atom to the adjacent carbon. Attack of water yields a hemithioacetal, which can decompose to the
corresponding aldehyde and thiol. Other nucleophiles have included carboxylic acids, alcohols, phenyl
rings, indoles, phenols, anilines and amides.[10,11] Intramolecular Pummerer-like rearrangements have
recently been reviewed.[12] Sulfur nucleophiles, while known, are somewhat underrepresented in
comparison, with just a few examples of conversion of a sulfoxide to a dithioacetal.[13,14] We now report
the application of Pummerer rearrangement using thiol nucleophiles to selectively transform oxidized
methionine residues from sulfoxides into dithioacetals.
Results and Discussion
An attempt to quantitate methionine oxidation via the Pummerer rearrangement was reported in 1972
by Lunder.[15] However, we have been unable to reproduce his results, and there are no published
2
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10.1002/cbic.201900463
ChemBioChem
papers that utilized this protocol. Classical Pummerer protocols were often carried out in acetic acid or
trifluoroacetic acid with acetic anhydride or trifluoroacetic anhydride as initiators.[11] These harsh
conditions usually covalently modify or degrade proteins. Trimethylchlorosilane[16] (TMSCl) was an
intriguing alternative to acetic anhydride or trifluoroacetic anhydride, particularly as TMSCl has been
used as a cleavage and deprotection reagent in peptide synthesis[17] and would be expected to cause
minimal protein decomposition. In addition, TMSCl is often used for transient protection of sensitive
groups, as it readily forms adducts with hydroxyls or amines that are spontaneously removed during
aqueous workup.[18,19] We considered that TMSCl could transiently protect alcohols, amines, or other
sensitive side chains during the activation step, an effect that should simultaneously increase solubility
in organic solvents. Subsequent interception of the thionium ion or other reactive intermediate[20] with
an appropriate thiol could give rise to a dithioacetal adduct, as shown in Scheme 1. Dithioacetals are
generally quite stable to all but forcing conditions,[21] including mass spectrometry, and a methionine-
HN
N
TMSCl
SH
HN
N
S
S+
S
S
O
Scheme 1. Proposed labeling approach
based dithioacetal should be readily differentiated from a methionine thioether by MS or other
analytical methods. Such an approach could offer the ability to both monitor total sulfoxide levels and
determine the regiospecificity of oxidation; i.e. which specific methionine residues within the protein of
interest were in the sulfoxide oxidation state.
We chose the commercially available Fmoc-Met[O]-OH (1) as our initial test substrate, as the Fmoc
group is both acid-stable and presents a useful UV handle for LC analysis studies. Initial reaction of 1
with TMSCl in THF was promising, as a mass of 370 corresponding to a putative thionium intermediate
was observed by LCMS. Upon treatment with 2-mercaptopyridine (2d, Table 1) robust signals with m/z
481 were observed, consistent with the desired adduct. With this preliminary result in hand, we set out
to explore the scope and limitation of the reaction of 1 to form dithioacetal adducts.
An initial screen of small molecule thiols for adduct formation, with a focus on thiol-substituted
heterocycles, suggested that the scope of effective nucleophiles was relatively narrow (Table 1). Most
tested thiols formed adducts of 1 in very modest amounts, even when the structure was closely related
to a thiol that formed the corresponding dithioacetal adduct in good yield. 4-Mercaptopyridine (2e) for
example, yielded far less adduct than 2d, and 2-mercaptopyrimidine (2i) yielded very little adduct. 2-
Mercaptoimidazole (2a), in contrast, displayed adduct formation similar to that of 2d. Closer analysis
suggested the formation of multiple regioisomeric products for most nucleophiles, presumably owing to
formation of the thionium ion from either carbon adjacent to the unsymmetrical sulfoxide (Scheme 2).
3
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10.1002/cbic.201900463
ChemBioChem
R'
S
1. TMSCl
2. R'SH
S
O
R
S
+
R'
S
R
S
R
4a-k
5a-k
3a-k
5a-k
Table 1. Thiol nucleophilesa
Thiol name
Thiol structure
Fmoc-Met
Adduct (3+4)b
82
DMSO Adduct (5)c
70
2a
2b
2-mercapto imidazole
3-Carboxy-2-
mercaptopyridine
6
6.1
2c
3-Carboxy-6-
mercaptopyridine
48
81
19
78
2d
2-Mercaptopyridine
4-Mercaptopyridine
12
4
56
9
2e
2f
6-Amino-2-mercapto
benzothiazole
2g
2h
2-mercapto benzimidazole
Cysteamine
37
42
-
5.2
2i
2-mercapto pyrimidine
Thiourea
7
21
-
2j
2k
21
15
Thiazolidine
46
4
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10.1002/cbic.201900463
ChemBioChem
aReactions were performed at 0.25 mmol scale (Fmoc-Met(O)-OH, 1) following the General Procedure
for Dithioacetal Formation or 1 mmol scale (DMSO), following the General Procedure for Condensation
of DMSO with Thiol Nucleophiles. bR = Fmoc-alanine; estimated percent yield of adducts are reported
based on integration of the 280 nm absorbance trace between 3 min and 5 min. No correction was
made for the contribution of the nucleophile to the absorbance of 3+4. cR = H; isolated percent yield
HN
O
S
HN
S
N
S
1. TMSCl
HN
N
S
S
OH
2.
OH
FmocHN
FmocHN
O
N
HS
O
4a
1
OH
FmocHN
TMSCl
3a
O
2a
Cl
TMSO
S+
TMSO-
Cl
Cl-
S+
S
Cl-
S+
OH
FmocHN
insoluble
OH
OH
FmocHN
FmocHN
O
OH
8
O
FmocHN
O
S+
O
O
9a
10a
11a,
soluble
2a
+
Cl
S
S+
S
TMSO-
Cl-
2a
H
Cl
OH
OH
OH
OH
FmocHN
FmocHN
FmocHN
FmocHN
O
O
O
7
9b
10b
11b,
soluble
Scheme 2. Mechanism of dithioacetal formation.
Adducts of C3 produced two distinct diastereomers. The dithioacetal adducts 3a and 4a, formed from
reaction of 1 with 2a, eluted as a single slightly asymmetric peak in most cases, and this nucleophile was
therefore used in reaction optimization screenings. While some conversion was observed with a single
equivalent of 2a, the best yields were obtained with the addition of 4 equivalents or more. Estimated
conversions (LCMS) of 1 to 3 + 4 for all nucleophiles are listed in Table 1. The formation of 2a
dithioacetal adduct was also examined for simpler systems, using DMSO and methyl phenyl sulfoxide as
substrates, to ensure a single product (Scheme 3). The expected adducts 5a and 6a were formed in 70%
and 64% isolated yield after preparative HPLC. The thiol nucleophiles previously examined via HPLC
analysis of their reaction with 1 were screened against a DMSO substrate in order to enable full
characterization and yield analysis of the products 5b-k. The results are shown in Table 1.
5
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10.1002/cbic.201900463
ChemBioChem
1. TMSCl
2. R'SH
O
S
S
S
R
R
R'
5a-k
R = CH
R = Ph
3 6a
Scheme 3. Model system Pummerer rearrangement
The reaction was most effective when conducted in two steps: an initial activation step in ethereal
solvent with 0.2-0.33 M TMSCl followed by condensation with a thiol, typically 2-mercaptoimidazole.
The initial activation appeared to be a multi-step process itself, as premature addition of the thiol
nucleophile resulted in the production of significant amounts of reduced Fmoc-Met-OH (7), presumably
due to thiol attack on a sulfur-electrophile species. However, allowing the reaction mixture to progress
Table 2. Effect of solvent on Fmoc-Methionine Sulfoxide Pummerer Reactiona
HN
O
S
HN
S
N
S
1. TMSCl
HN
N
S
S
OH
2.
OH
FmocHN
FmocHN
O
N
HS
O
4a
1
OH
FmocHN
3a
O
Solvent
Dithioacetal Thioether Starting
Adduct
7
Material
1
3a+4ab
Acetic acid
Chloroform
Dichloromethane
Diglyme
Dioxane
Dimethoxyethane
Triglyme
THF
Ethyl Acetate
Acetonitrile
Dimethylacetamide
Dimethylformamide
NMP
17
7
6
58
41
43
1
3
6
3
3
8
31
32
90
54
19
94
-
-
-
-
-
-
-
-
-
-
63
3
37
79
-
83
82
71
71
79
62
43
-
-
-
-
-
Toluene
Trifluoroethanol
aReaction as shown in Scheme 2; percent yields of each species are based on LCMS analysis. A 1 mmol-
portion of TMSCl at ambient temperature was added to a suspension of 0.25 mmol Fmoc-Met(O)-OH (1)
in 3 mL of the indicated solvent and the mixture was stirred for a minimum of 16 h, followed by the
6
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10.1002/cbic.201900463
ChemBioChem
b
addition of 2a and stirring for a further 24 h. Adduct percent yields are reported as the sum of mixed
isomers.
from a turbid and opaque mixture in the ethereal solvent to a translucent solution prior to addition of 2-
mercaptoimidazole delivered consistent LCMS yields of approximately 80% dithioacetal adduct 3a and
4a, as judged by absorbance at 280 nm. Reactions were analyzed at 280 nm in order to maximize the
Fmoc absorbance signal while minimizing the imidazole contribution to dithioacetal absorbance.
The reaction was strongly dependent on solvent, as shown in Table 2, with ethereal solvents
consistently producing optimal yields. Less polar solvents such as dichloromethane or chloroform were
significantly less effective, while EtOAc and acetonitrile were moderately effective. Dimethylformamide,
N-methylpyrrolidone and dimethylacetamide yielded varying amounts of reduction to the thioether 7.
Acidic solvents such as trifluoroethanol or acetic acid tended to favor reduction to 7 over formation of
3a and 4a. In the case of trifluoroethanol, it is possible that the well-precedented mechanism of alcohol
oxidation by activated sulfoxide dominates,[22] which would support the high efficiency of reduction to 7.
Mixed-phase systems such as 1:1 water:THF or water:DCM were not effective.
A variety of activating agents were screened for effectiveness. Silyl chlorides were found to be generally
effective, although increased bulk around the silane corresponded to a requirement for extended
reaction times (Table 3). Indeed, TBDPSCl failed to generate any 3a or 4a products. Several traditional
Pummerer electrophiles proved to be ineffective in the conversion to 3a and 4a, as acetic or
trifluoroacetic anhydrides yielded no adduct (Table S1). Interestingly, chloride appears to be required
for dithioacetal formation. While it is perhaps unsurprising that the strongly reactive TMSBr does not
yield a great deal of desired products,[23,24] or that the less reactive TMS-imidazole and TMS-
polyphosphate are ineffective, it is telling that the addition of LiCl to the TMS-polyphosphate reaction is
sufficient to “rescue” this reaction and produce a significant amount of dithioacetal adduct. LiBr and LiI
were not effective in this context, perhaps owing to the in situ generation of TMSBr or TMSI.[25] Of the
non-silyl based activating agents tested, only those containing chloride were even modestly effective
(Table S1). The Pummerer-like reaction of thioethers with N-chlorosuccinimide is known,[26] and
treatment of 1 with NCS followed by addition of 2a gave rise to a moderate yield of 3a + 4a as well. The
addition of base was not productive as smaller amounts tended to promote thioether 7 formation and
larger amounts suppressed Pummerer-like reactions altogether. Lower temperature promoted the side
reduction to 7, while modestly heating the reaction to 40 oC or 60 oC accelerated the activation for
adduct formation, with an optimal activation time of 2-4 h. Isolated yields of the product mixture 3a
and 4a using 1 as a substrate were identical (77%) for the reaction with activation at RT (20 h) or 40 oC (3
h). The dithioacetal adduct of the terminal methyl group 3a was isolated and characterized. However,
the adducts 4a of the internal C3 methylene were inseparable chromatographically and appeared to be
less stable to prolonged exposure to aqueous TFA solution. This runs counter to other dithioacetals,
which are typically stable to the mild acidic conditions of preparative HPLC[21], and could be largely due
to the relatively electron-poor nature of 2a, which is in resonance with a thiourea form.
7
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ChemBioChem
Table 3. Silane-Based Pummerer Activation of Fmoc-Met(O)-OH (1)a
HN
O
S
HN
S
1. R1R2R3
HN
N
S
SiX
N
S
S
OH
2.
OH
FmocHN
FmocHN
O
N
HS
O
4a
1
OH
3a
FmocHN
O
R1
R2
R3
X
Reaction Adduct
Thioether
(7)
2±0.4
44±2.6
11±6.9
56±1.8
Starting
Material (1)
-
-
82±10.5
38±2.5
Time
16-20h
16h
(3a+4a)
82±0.8
8±0.5
-
-
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Cl
Br
Imidazole 16h
16h
PPb
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Allyl
Vinyl
Et
iPropyl Cl
tButyl
Ph
Pfp
CH2Cl
Cl
Me
Et
Cl
Cl
Cl
20h
40h
40h
40h
64h
24h
40h
40h
16h
20h
64h
64h
77±1.6
77±3.2
73±0.6
51±0.6
45±6
75±0.1
79±0.85
65±2.7
85±4.9
78±3.5
35±4.5
-
4±1.9
4±0.3
6±4.5
34±4.9
31±6
3±0.7
4.5±1.5
5±2.1
2±0
-
-
-
-
-
-
-
-
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
-
-
-
CH2CHSiMe2Clc Me
3±1.4
39±8.2
55±4.1
Et
tButyl
Et
Ph
Ph
24±5.8
aActivation was carried out by stirring for a minimum of 16 h at RT after addition of 1 mmol of a silyl
activating agent R1R2R3SiX to 0.25 mmol of 1 in 3 mL of dioxane. Upon clarification of the reaction
solution, 2a was added and the reaction was stirred for an additional 24 h before sampling. Yields are
based on LCMS analysis and are an average of at least 2 runs. Adduct yields are reported as the sum of
mixed isomers. bPolyphosphate cEntry refers to 1,2-bis(chlorodimethylsilyl)ethane.
We propose that the mechanism of dithioacetal formation involves a fast initial reaction of the sulfoxide
1 with TMSCl to generate the adduct 8 shown in Scheme 2, which could then either first undergo
chloride exchange to form species 9 or lose TMSOH directly to form thionium ion 10. The dependence
of dithioacetal 3a+4a formation on the presence of a halide ion, preferably a chloride ion, suggests that
the active species is either the chloride-thionium ion pair 10 or quite possibly the alpha-chloro thioether
11. The latter species has been invoked previously,[20] and Jung et al observed incorporation of a
chloride ion into an aromatic ring under Pummerer rearrangement conditions.[27] Both of these
examples used the highly reactive thionyl chloride.
To empirically examine the nature of the active intermediate, we carried out the reaction on 1 that had
been 13C -labeled at the methyl group of the side chain and monitored the reaction by 13C NMR
8
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ChemBioChem
spectroscopy. The parent sulfoxide 13C -enriched resonances appeared at 38.86 and 38.78 ppm
presumably owing to diastereomeric oxidation of the parent 7. Treatment with excess TMSCl in
deuterated THF for 2 h at 40 oC yielded roughly equivalent new signals at 50.6, 13.5 and 14.1 ppm. This
result is consistent with formation of alpha-chlorinated thioethers 11a+11b as a statistical mixture either
at the 13C -enriched methyl (50.6 ppm) or at the interior C3 methylene (13.50, 14.1 ppm). A trace of
presumed thioether 7 was also observed at 15.3 ppm. Subsequent condensation of the reaction with
excess 2a saw these peaks migrate to 40.2, 13.8, and 14.5 ppm, respectively; consistent with formation
of one primary (3a) and two secondary (diastereomeric, 4a) dithioacetals. A similar experiment was
carried out using unlabeled DMSO as a substrate. The initial 13C NMR showed a single signal at 42.0
ppm, which gave rise to a split signal at 53.00/52.99 ppm as well as a signal at 15.0 ppm after 20 h of
TMSCl treatment. Condensation with 2a resulted in peaks at 42.7 and 15.4 ppm. 13C NMR spectra for
both model systems are shown in Figure S1.
To shed light on the mechanisms and energetic profiles of the synthesis processes described above, we
performed quantum chemistry calculations on the Pummerer rearrangement of DMSO using the Density
Functional Theory (DFT) method with the hybrid meta-GGA M11 functional. All reported results
represent the M11/cc-pVTZ level of theory (see Methods for full details).
We first investigated the initial step, reaction of DMSO with TMSCl, in order to elucidate: (i) the highest
energy barriers, or “bottlenecks,” that must be overcome and (ii) which active species product, chloride-
thionium ion pair or alpha-chloro thioether, is produced. Geometry optimizations and subsequent
vibrational frequency calculations (to confirm true minima and transition states) were performed in gas
and tetrahydrofuran (THF) solvent phases. Figure 1 illustrates the full computed energy profile for this
initial activation process. Note that gas-phase structures are shown where transition states have been
definitively mapped to their corresponding neighboring minima. The activation process is seen to
proceed through three intermediates, and thus four distinct transition states. The final energetically
most stable product is the alpha-chloro thioether. This computed mechanism affirms that shown in
Scheme 2; however, important details are garnered from the quantum chemistry results. One of these is
that the rate-limiting step, or bottle-neck, is the proton transfer, shown as TS2 in Figure 1, with
computed barriers of 37 (gas) and 38 (THF) kcal/mol (relative to separated reactants DMSO and TMSCl).
The best experimental thioacetal yields were obtained with aprotic ethereal solvents (Table 2) which
cannot participate directly in proton exchange. While ethereal solvents may participate in hydrogen
bonding, thereby slightly lowering the energy of the proton transfer transition state, stabilization of
minima is also likely. The slight increase in energy barrier (one kcal/mol) when going from gas to THF-
solvent phase suggests that the hydrogen bonding capabilities of the solvent do not appreciably
facilitate the proton transfer step.
9
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10.1002/cbic.201900463
ChemBioChem
Figure 1. Computed reaction mechanism and energy profiles describing the initial activation process
leading to formation of alpha-chlorinated thioether: DMSO + TMSCl → MeSCH2Cl + TMSOH. Calculations
were performed using the M11/cc-pVTZ level of theory in gas (black lines and text, structures shown)
and THF solvent (blue lines and italic text) phases. All energies (kcal/mol) are relative to separated
reactants DMSO + TMSCl. The main points to note are: (i) the pathway to thioether formation
progresses through four distinct transition states and three intermediates, (ii) the proton transfer step is
predicted to be rate-limiting, having computed barriers of 37 (gas) and 38 (THF) kcal/mol, and (iii) as
shown at far right, upon formation of TMSOH and the chloride-thionium ion pair MeS(+)CH2--Cl(−), the
isolated latter species should convert to the much more stable alpha-chlorinated thioether MeSCH2Cl.
Selected bond lengths (Å) are also shown.
Thus, the only real way to go from TMSO-S(+)Me2 to TMSOH + MeS(+)=CH2 is via intramolecular proton
transfer, as exemplified by TS2. We also find that for the portion of the activation process leading to
TMSOH---MeS(+)=CH2 (I2), solvent effects (THF) are negligible for four of the stationary points, including
the rate-limiting proton transfer step, but stabilize the other species (I1a, TS1b, I1b) by 5 to 12 kcal/mol.
The other key point is that upon formation of the chloride-thionium ion pair, conversion to the
substantially more stable alpha-chloro thioether (MeSCH2Cl) should readily occur. The computed gas-
phase barrier of 34 kcal/mol is lowered to 18 kcal/mol when modeled in THF solvent. This step is
illustrated at far right of Figure 1 where TMSOH was considered a spectator species. We speculate that if
the proton transfer energy barrier can be overcome, then formation of alpha-chloro thioether
(MeSCH2Cl) product is almost certain. Since the separated products TMSOH + MeSCH2Cl are computed
to be more stable than the separated reactants by 17 (gas) and 14 (THF) kcal/mol, the complete
exothermic activation process is highly unlikely to be reversible because the backwards proton transfer
energy barrier is over 50 kcal/mol. These quantum chemistry results nicely account for the experimental
observations, particularly the slowness of reaction and irreversibility. They also indicate that the alpha-
chloro thioether is produced, rather than ion pair, in line with the 13C NMR results.
10
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Next, we determined reaction energies in THF solvent for the final step, formation of thioacetals: RSH +
MeSCH2Cl → RSCH2SMe + HCl. We investigated the four thiols 2-mercaptoimidazole (2a), 2-
mercaptopyridine (2d), 4-mercaptopyridine (2e), and 2-mercaptopyrimidine (2i). Full potential energy
surface (PES) scans were performed using the cc-pVDZ basis sets to discover lowest-energy structures
for separated reactants and products. Their geometries were then optimized and vibrational frequencies
computed using the cc-pVTZ basis sets. The results are shown in Figure 2 where electronic reaction
energies (ΔE), lying between −6.5 and −5.3 kcal/mol, indicate favorable thioacetal formation. However,
free energies of reaction (ΔG298) are less exothermic, with products computed to be only slightly more
stable than reactants by 3.8 to 2.4 kcal/mol. There are many complex factors that can shift these
reaction energies up/down by a few kcal/mol, and thus cause experimental yields to vary significantly as
seen in Table 1. These factors include reaction conditions, explicit solvent interactions, and reactant-
reactant/product-product complex formations. As such, all we can really surmise from the computed
results is that thioacetal formation is barely exothermic and that actual experimental yields may vary
due to other factors that are difficult to model with kcal/mol accuracy.
Figure 2. Computed structures and energetics for conversions of four thiols to dithioacetals: RSH +
MeSCH2Cl → RSCH2SMe + HCl. The M11(THF)/cc-pVTZ level of theory was used, electronic (ΔE) and free
(ΔG298) energies of reaction are given in kcal/mol.
Having investigated the optimal conditions for dithioacetal formation in small molecules, we turned our
attention to using the optimized method for labeling methionine sulfoxide residues in peptides.
Conversion of sulfoxides to dithioacetals offers a stable mass tag to differentiate oxidized methionine
residues definitively from the thioether state. We chose the peptide met-enkephalin (H-Tyr-Gly-Gly-
Phe-Met-OH, 12) for our study. The peptide was oxidized with NaIO4 to the sulfoxide form 13. The
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sulfoxide-containing peptide 13 was then activated for a minimum of 2 days in 10% v/v TMSCl in
dioxane, followed by addition of solid 2-mercaptoimidazole and stirring for at least 24 h. In the case of
the oxidized peptide, more than 70% of peptide-associated absorbance at 220 nm corresponded to a
mass of 672, consistent with the expected dithioacetal adduct 14a + 15a. Approximately 17% of the
signal corresponded to reduction to methionine thioether form 12. In a control reaction using the
parent peptide, > 90% of 220 nm absorbance corresponded to a mass of 574 consistent with the starting
peptide 12 (Figure S2.) Approximately 6% of peptide absorbance corresponded to dithioacetal adducts
14a + 15a, presumably formed after air oxidation of 12 to 13. The use of an inert atmosphere was found
to be useful in suppressing background dithioacetal formation. The requirement for ethereal solvents
may not be compatible with the solubilty of some proteins. However, prior fragmentation with trypsin,
pepsin, or other proteases commonly employed in protein chemistry may enable Pummerer-based
dithioacetal formation of the resulting fragments. In addition, TMSCl is expected to transiently protect
many polar groups, such that minimal initial solubility may be sufficient to ultimately enable reaction.
Although total conversion of the peptide will be less than indicated by the absorbance percentage,
owing to contribution of the thioimidazole substituent at 220 nm, our protocol presents clearly
differentiable results between otherwise-identical peptides containing either sulfoxide or thioether
functionality.
Conclusions
We have demonstrated that the Pummerer rearrangement can be used to covalently label oxidized
methionine side chains with good efficiency. We have described the use of silyl chlorides, especially
TMSCl, to convert alkyl sulfoxides into intermediate alpha-chlorinated thioethers, which subsequently
undergo chlorine displacement by thiol nucleophiles. The efficacy of a variety of thiol nucleophiles has
been investigated, and 2-mercaptoimidazole and 2-mercaptopyridine proved to be most effective.
Ethereal solvents were shown to be optimal for dithioacetal formation. The alpha-chlorinated
intermediates were investigated by computation and by 13C NMR spectroscopy. Finally, we have
demonstrated the use of our optimized conditions to selectively label a peptide containing an oxidized
methionine residue while the corresponding non-oxidized peptide remained essentially unlabeled. It
should be possible to raise an antibody specific for the dithioacetal derivative. Such an antibody could
be used to detect and quantitate methionine sulfoxide in proteins by immunochemical methods or for
affinity purification of derivatized peptides prior to mass spectrometric sequencing. We anticipate that
this approach can be used to identify peptides with oxidized methionine residues by tagging them with a
thiol nucleophile functionalized with mass or other reporter groups.
Experimental Section
General
Reagents were purchased from commercial sources and used without further purification. Fmoc-
methionine 13C -methyl was purchased from Cambridge Isotope Laboratories. Met-Enkephalin was
obtained from Chempep Inc. NMR spectra were obtained on a 400 MHz Varian NMR and processed
using MestReNova software. LCMS data for small molecules were acquired on an Agilent Technologies
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1290 Infinity HPLC system using a 6130 quadrupole LC/MS detector and a Poroshell 120 SB-C18 2.7 um
column (4.6 x 50 mm). Peptides were analyzed using an Agilent 1200 series HPLC system with an
LC/MSC Trap XCT detector and a Zorbax 300SB-C18 3.5 um column (4.6 x 50 mm). Preparative HPLC
chromatography was performed on a Shimadzu system using a 30 mm x 150 mm Zorbax SB C18 column
(Agilent) or a 19mm x 150 mm Xbridge C4 column (Waters). Flash chromatography was performed on a
Teledyne Isco Combiflash Rf+ instrument using hexane:ethyl acetate gradients. HRMS data was acquired
on a Waters XEVO G2-XS QTOF running MassLynx version 4.1.
Quantum chemistry. The computations in this study were executed using the GAMESS[28,29] package and
molecular structures were illustrated using MacMolPlt.[30] We used the density functional theory (DFT)[31]
method with the hybrid meta-GGA M11 functional.[32] Calculations were performed in gas and
tetrahydrofuran (THF) solvent phases, and the latter was accomplished using the Polarizable Continuum
Model (PCM)[33,34,35,36] (with a high density of tesserae: NTSALL = 960 in $TECAV). Geometries were
optimized (maximum Cartesian gradient < 10−4 Hartree/Bohr) using analytic gradients and Hessians were
computed seminumerically (double differences) using analytic gradients. The cc-pVDZ basis sets[36,37] were
used to probe potential energy surfaces for locations of lowest-energy conformations and transition state
structures (having exactly one imaginary frequency). Refined optimized geometries and subsequent
Hessians were computed using the cc-pVTZ basis sets[36,37] and minimum energy paths connecting
transition states to corresponding reactant/product minima were determined using the second-order
intrinsic reaction coordinate (IRC) method of Gonzalez and Schlegel.[38] All data presented and discussed
in the manuscript represent the M11/cc-pVTZ level of theory. Structures and energies (electronic (E) and
free (G298)) of all stationary points described are given in the Supporting Information.
Synthesis
General Procedure for Dithioacetal Formation: TMSCl 127 uL (108 mg, 1 mmol) was added to a stirred
solution of sulfoxide (0.25 mmol) in 3 mL of dioxane. The reaction was stirred for at least 24 h at RT, at
which point the thiol nucleophile (1.04 mmol) was added. The reaction was stirred for an additional 24
h at RT. Neutralization with 1 M triethylammonium bicarbonate solution was followed by either
preparative HPLC or extraction and flash chromatography purification to afford the pure material.
S-(((1H-imidazol-2-yl)thio)methyl)-N-(((9H-fluoren-9-yl)methoxy)carbonyl)-L-homocysteine (3a):
White amorphous solid. 1H NMR (CD3OD) δ 7.80 (dd, J = 7.6, 3.1 Hz, 2H), 7.67 (t, J = 7.6 Hz, 2H), 7.62 (d, J
= 2.7 Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H), 7.35 – 7.26 (m, 2H), 4.46 – 4.27 (m, 5H), 4.24 (d, J = 7.2 Hz, 1H),
3.37 – 3.27 (m, 2H), 2.84 (s, 1H), 2.76 (q, J = 6.8, 6.1 Hz, 1H), 2.22 – 2.14 (m, 1H), 1.99 (s, 1H). 13C
NMR{1H} (CD3CN + D2O): δ 174.6, 157.6, 144.5, 141.8, 138.8, 128.5, 127.9, 125.9, 122.2, 120.7, 67.3,
+
53.4, 47.6, 39.8, 31.2, 28.1. HRMS: Calculated for C23H24N3O4S2 470.1208; found 470.1206
General Procedure for Condensation of DMSO with Thiol Nucleophiles: TMSCl 127 uL (108 mg, 1
mmol) was added to a stirred solution of dimethyl sulfoxide (19.5 mg, 17.7 uL, 0.25 mmol) in 3 mL of
dioxane. The reaction was stirred for 24 h at RT, and the thiol nucleophile 2 (1.04 mmol) was then
added. The reaction was stirred for an additional 24 h at RT. Neutralization with 1 M triethylammonium
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bicarbonate solution was followed by either preparative HPLC or extraction and flash chromatography
purification to afford the pure material.
2-(((Methylthio)methyl)thio)-1H-imidazole (5a). 28.2 mg white solid, 70% 1H NMR (CD3OD): δ 7.62 (s, 2
H); 4.33 (s, 2 H); 2.27 (s, 3 H). 13C{1H} NMR (CD3OD): δ 140.3, 123.2, 43.2, 15.2. HRMS (ESI/QTOF) m/z:
[M+H]+ Calculated for C5H9N2S2 161.0207; Found 161.0207.
2-(((Methylthio)methyl)thio)nicotinic acid (5b). 3.2 mg white solid, 6.1%. 1H NMR (CD3CN): δ 8.54 (ddd,
J = 4.8, 1.9, 0.6 Hz, 1H), 8.23 (ddd, J = 7.8, 1.8, 0.5 Hz, 1H), 7.18 (dd, J = 7.8, 4.8 Hz, 1H), 4.25 (s, 2H), 2.15
(s, 3H). 13C{1H} NMR (CD3CN): δ 168.3, 161.3, 153.1, 140.5, 124.9, 120.3, 35.8, 15.9. HRMS (ESI/QTOF)
m/z: [M+H]+ Calculated for C8H10NO2S2 216.0153; Found 216.0152
6-(((methylthio)methyl)thio)nicotinic acid (5c). 10.3 mg white solid, 19%. 1H NMR (CD3CN): δ 8.96 (dd, J
= 2.2, 0.9 Hz, 1H), 8.08 (dd, J = 8.4, 2.2 Hz, 1H), 7.35 (dd, J = 8.4, 0.9 Hz, 1H), 4.39 (s, 2H), 2.21 (s,
3H).13C{1H} NMR (CD3CN) δ 166.6, 164.7, 151.6, 138.1, 123.3, 122.6, 36.1, 15.7. HRMS (ESI/QTOF) m/z:
[M+H]+ Calculated for C8H10NO2S2 216.0153; Found 216.0152
2-(((Methylthio)methyl)thio)pyridine (5d).[39] Colorless liquid, 33.3 mg, 78%. 1H NMR (CDCl3): δ 8.46
(ddd, J = 4.9, 1.9, 1.0 Hz, 1H), 7.51 (ddd, J = 8.1, 7.4, 1.9 Hz, 1H), 7.22 (dt, J = 8.0, 1.0 Hz, 1H), 7.02 (ddd, J
= 7.3, 4.9, 1.1 Hz, 1H), 4.37 (s, 2H), 2.25 (s, 3H).13C{1H} NMR (CDCl3): δ 157.9, 149.7, 136.3, 122.8, 120.1,
35.9, 15.7. HRMS (ESI/QTOF) m/z: [M+H]+ Calculated for C7H10NS2 172.0255; Found 172.0254.
4-(((Methylthio)methyl)thio)pyridine (5e): Colorless liquid, 23.9 mg, 56%. 1H NMR (CDCl3): δ 8.45 (s,
2H), 7.25 – 7.14 (m, 2H), 4.09 (s, 2H), 2.27 (s, 3H). 13C{1H} NMR (CD3CN) δ 161.0; 142.4, 123.6; 37.0,
15.7. HRMS (ESI/QTOF) m/z: [M+H]+ Calculated for C7H10NS2 172.0255; Found 172.0253
1
2-(((Methylthio)methyl)thio)benzo[d]thiazol-6-amine (5f). Yellowish solid, 5.4 mg, 9%. H NMR (CDCl3):
δ 7.68 (dt, J = 8.7, 0.5 Hz, 1H), 7.03 (dt, J = 2.4, 0.5 Hz, 1H), 6.79 (ddd, J = 8.7, 2.3, 0.4 Hz, 1H), 4.42 (s,
2H), 3.79 (s, 2H), 2.29 (s, 3H). 13C{1H} NMR (CDCl3): δ 160.3, 146.8, 144.2, 137.5, 122.6, 115.5, 105.7,
39.9, 15.9. HRMS (ESI/QTOF) m/z: [M+H]+ Calculated for C9H11N2S3 243.0084; Found 243.0082.
2-(((Methylthio)methyl)thio)-1H-benzo[d]imidazole (5g): White solid, 22 mg, 42%. 1H NMR (CD3OD): δ
7.48 (br s, 2 H); 7.18-7.23 (m, 2 H); 4.40 (s, 2 H); 2.25 (s, 3 H). 13C{1H} NMR (CD3OD): δ 150.5, 124.1,
123.7, 111.0, 39.8, 15.4. HRMS (ESI/QTOF) m/z: [M+H]+ Calculated for C9H11N2S2 211.0364; Found
211.0364
1
2-(((Methylthio)methyl)thio)pyrimidine (5i): White solid, 9.0 mg, 21%. H NMR (CD3CN): δ 8.56 (d, 2 H,
J = 4.9); 7.12(t, 1 H, J = 4.9); 4.33 (s, 2 H); 2.21 (s, 3 H). 13C{1H} NMR (CD3OD) δ 172.6, 158.9, 118.5, 37.3,
15.6. HRMS (ESI/QTOF) m/z: [M+H]+ Calculated for C6H9N2S2 173.0207; Found 173.0200.
2-(((Methylthio)methyl)thio)-4,5-dihydrothiazole (5k). Colorless liquid, 20.6 mg, 46%. 1H NMR (CDCl3):
δ 4.36 – 4.14 (m, 4H), 3.41 (t, J = 8.0 Hz, 2H), 2.24 (s, 3H). 13C{1H} NMR (CDCl3): δ 165.1, 64.4, 38.2, 35.7,
15.9. HRMS (ESI/QTOF) m/z: [M+H]+ Calculated for C5H10NS3 179.9975; Found 179.9981.
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2-(((Phenylthio)methyl)thio)-1H-imidazole (6a). TMSCl 127 uL (108 mg, 1 mmol) was added to a stirred
solution of methyl phenyl sulfoxide (35 mg, 0.25 mmol) in 3 mL of dioxane. The reaction was stirred for
24 h at RT, then 2-mercaptoimidazole (104 mg, 1.04 mmol) was added. The reaction was stirred for an
additional 24 h at RT. Neutralization with 1 M triethylammonium bicarbonate solution was followed by
preparative HPLC (5% MeCN 2 min, then ramp to 50% MeCN over 13 min), concentration of the
1
appropriate fractions, and lyophilization to afford 30 mg (54%) of the pure material as a white solid. H
NMR (CD3CN): δ 7.43-7.46 (m, 2 H); 7.28-7.27 (m, 5 H); 4.70 (s, 2 H). 13C{1H} NMR (CD3OD): δ 140.0,
134.3, 132.3, 130.7, 129.2, 123.4, 42.4. HRMS (ESI/QTOF) m/z: [M+H]+ Calculated for C10H11N2S2
223.0364; Found 223.0369.
13C -Methyl Fmoc-methionine sulfoxide. To a stirred solution of 13C -methyl Fmoc-methionine (10.3 mg,
27.7 umol) in 4 mL of 50% aqueous MeOH was added sodium periodate (7.1 mg, 1.2 equiv), and the
reaction was stirred for 16 h at RT. Saturated ammonium chloride solution was added and the aqueous
layer was extracted with 3 x 4 mL DCM. The combined organic layers were dried over Na2SO4 and
evaporated to yield 13C -methyl Fmoc-methionine oxide (white solid, 10.7 mg, quantitative).
13C NMR Experiment
13C -Methyl Fmoc-methionine oxide (10.7 mg, 27.7 umol) was dissolved in THF-d8 (1 mL) and the 13C
NMR spectrum was acquired. TMSCl (50 uL, 13.9 equiv) was added, and the reaction was heated to 40˚
C. After 2 h, the reaction was cooled and the 13C NMR spectrum was acquired. After returning the NMR
sample to the reaction, mercaptoimidazole (39.8 mg, 14 equiv) was added and the reaction was stirred
at RT for 24 h. The 13C NMR spectrum of the reaction was then acquired once again.
Met-Enkephalin Sulfoxide (13). Met-Enkephalin (12, 20 mg, 35 umol) was dissolved in 1 mL of
deionized water, and sodium periodate (9.0 mg, 42 umol) was added. The reaction was stirred
overnight, then neutralized with AcOH. The desired oxidized peptide was purified by preparative HPLC
on a Waters C4 column using a gradient of 10->45% MeCN (0.05% TFA) in water (0.05% TFA). 13.3 mg of
pure material was obtained after lyophilization.
Pummerer Reaction of Met-Enkephalin. Met-enkephalin sulfoxide (13, 0.6 mg, 1 umol) and met-
enkephalin (12, 0.6 mg, 1 umol) were each suspended in 0.1 mL of a solution of TMSCl in dioxane (10%
v/v) that had been degassed by passing Ar through for 30 sec. The suspension was stirred for 2 days at
RT, at which point 2 mg of 2-mercaptoimidazole were added to each vial. The reaction was stirred
further, and samples were withdrawn and diluted in H2O/MeCN prior to LCMS analysis. Integration of
the 220 nm trace was performed between 4 and 6 min and peaks were assigned based on mass analysis.
Supporting Information
Computational details and NMR spectra can be accessed free of charge via the Internet at wiley.com.
Acknowledgements
We are grateful to Burchelle N. Blackman for performing high-resolution mass spectrometry. This
research was supported by the NIH Intramural Research Program and NHLBI. S.M. and R.L.L were
15
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supported by the Intramural Research Program of the National Heart, Lung and Blood Institute (Grant
ZIA HL000225). J.I. was supported with federal funds from the National Cancer Institute, National
Institutes of Health, under Contract No. HHSN 261200800001E. The content of this publication does not
necessarily reflect the views or policies of the Department of Health and Human Services.
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