Reactions of Sulfur-Containing Organic Compounds and Peptides in 1-Ethyl-3-methyl-imidazolium Acetate

Article abstract of DOI:10.1021/acs.joc.7b01272

The neat ionic liquid (IL) [C₂mim][OAc] is not just capable of dissolving thiol- and disulfide-containing compounds, but is able to chemically react with them without addition of any catalytic reagent. Through the analysis of four small organic molecules and a cysteine-containing peptide we could postulate a general reaction mechanism. Here, the imidazolium-carbenes preferentially react with the disulfide bond, but not thiol group. Moreover, the imidazole moiety was found to abstract the sulfur atom from the cysteine residue, providing an alternative way to transform Cys residues, which were artificially inserted into a peptide sequence in order to perform native chemical ligation (NCL) of two peptide fragments. Finally, the chemical reaction of [C₂mim][OAc] with a cysteine-containing biomolecules can be tuned or even suppressed through the addition of at least 30% of water to the reaction mixture.

Full text of DOI:10.1021/acs.joc.7b01272

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Article
Reactions of sulfur-containing organic compounds
and peptides in 1-ethyl-3-methyl-imidazolium acetate
Andreas C. Baumruck, Daniel Tietze, Annegret Stark, and Alesia A. Tietze
J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 22 Jun 2017
Downloaded from http://pubs.acs.org on June 23, 2017
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Reactions of sulfur-containing organic compounds and pep-
tides in 1-ethyl-3-methyl-imidazolium acetate
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†
‡
†
∥
Andreas C. Baumruck , Daniel Tietze , Annegret Stark and Alesia A. Tietze*
†
Clemens-Schöpf Institute of Organic Chemistry and Biochemistry, Darmstadt University of Technology, Alarich-Weiss Str.
4, 64287 Darmstadt, Germany
‡
Eduard-Zintl-Institute of Inorganic and Physical Chemistry, Darmstadt Univeristy of Technology, Alarich-Weiss-Str. 4, D-
6
4287
∥
Chemical Engineering, University of KwaZulu-Natal, 238 Mazisi Kunene Rd, Glenwood, Durban, 4001, South Africa
ABSTRACT: The neat ionic liquid (IL) [C mim][OAc] is not just capable of dissolving thiol- and disulfide-
2
containing compounds, but is able to chemically react with them without addition of any catalytic reagent.
Through the analysis of four small organic molecules and a cysteine-containing peptide we could postulate a gen-
eral reaction mechanism. Here, the imidazolium-carbenes preferentially react with the disulfide bond, but not
thiol group. Moreover, the imidazole moiety was found to abstract the sulfur atom from the cysteine residue,
providing an alternative way to transform Cys residues, which were artificially inserted into a peptide sequence in
order to perform native chemical ligation (NCL) of two peptide fragments. Finally, the chemical reaction of
[
C mim][OAc] with a cysteine-containing biomolecules can be tuned or even suppressed through the addition of at
2
least 30 % of water to the reaction mixture.
1. Introduction
Ionic liquids (ILs), consist entirely of ionic species and offer a vast variety of physical and chemical
properties primarily by the possibility of tailoring their structure, i.e. exchanging or modifying the cation
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1ꢀ3
or anion. During the last decades, ILs attracted great attention as organic solvents, in reactions such
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as cycloaddition, esterification, Wittig and nucleophilic substitution reactions, for the dissolution of
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cellulose, for electrolytes, for lithiumꢀion batteries and other industrial applications. On the other
hand, they participate as reactants in various reaction processes such as organic catalysis and oxidaꢀ
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tive addition.
Both, the kinetics and thermodynamics of a reaction are dependent on the type of
ionic liquid chosen, and even the reaction mechanism can be influenced. However, very little presciꢀ
ence currently exists on this topic.
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In our previous studies we successfully applied ILs as solvents for reactions of bioactive peptides conꢀ
taining a high number of cysteine moieties in their sequence (> 4), e.g. in oxidative folding and native
14ꢀ16
chemical ligation.
The advantageous in the use of IL was the good (up to 15 mM) solubility of hyꢀ
drophobic peptides and selective formation of thermodynamically stable conformation (so called “native
fold”) of peptides during oxidative folding. Further reports describe the advantages of using ILs for reꢀ
actions involving biological macromolecules, i.e. enzyme catalysis, because of their structureꢀstabilizing
14,17,18
capabilities or good solubilizing properties for hydrophobic peptides.
However, the possibility of
chemical modifications through a proposed carbeneꢀbased chemical reaction are not fully understood
yet and greatly limits their advantageous applicability for reactions involving biomolecules. This inforꢀ
mation is tremendously important for the knowledgeꢀbased application of ILs in a wide range of reacꢀ
tions involving biomolecules.
19ꢀ22
The in situ formation of Nꢀheterocyclic carbenes (NHC)
in imidazoliumꢀbased ILs (e.g. 1ꢀethylꢀ3ꢀ
methylꢀimidazolium acetate) has long been suspected. NHCs in [C mim][OAc] were detected in the gas
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phase by infrared spectroscopy and mass spectrometry. A few reports suggest the presence of
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NHC in the neat IL.
However, so far, no direct spectroscopic observations of carbenes in ionic liqꢀ
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uids were reported.
Several chemical reactions were used as an indirect indication of formation or presence of carbenes in
C mim][OAc]. In 2011, Gurau et al. detected an acetic acid – acetate complex formed by chemisorpꢀ
[
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tion of carbon dioxide in [C mim][OAc] with the help of Xꢀray crystallography. Rodriguez et al. was
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able to detect 1ꢀethylꢀ3ꢀmethylimidazoleꢀ2ꢀthione as a result of chemical reaction of the neat
[
C mim][OAc] with elemental sulfur. Moreover, the authors indicated the inhibition of product formation
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in 50% IL/water solution. One early literature example of a carbeneꢀmediated reaction carried out in
related 4ꢀ and 5ꢀmethylthiazoliumꢀbased ionic liquids, albeit in the presence of small quantities of trimeꢀ
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thylamine, was reported by Davis and Forrester. Similarly, 1,3ꢀdialkylimidazoliumꢀbased ionic liquids
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were activated by adding auxiliary base.
In 2011, Kelemen et al. found that the formation of the
carbene takes place in situ if the anion of the ionic liquid is sufficiently basic, e.g. in acetateꢀbased ionic
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liquids.
The process of imidazolꢀ2ꢀylidene formation in imidazoliumꢀbased ILs is reported to occur by a simple
24,25,33
deprotonation of the carbon atom inꢀbetween the two nitrogens of the imidazolium ring.
For 1,3ꢀ
dialkylꢀsubstituted imidazolium ILs Hollóczki et al. reported that the equilibrium between the ion pair
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and the corresponding hydrogenꢀbonded complex of the free acid and NHC can be shifted towards
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NHC formation in the presence of the relatively basic anion of the IL.
The hypothesis about the presence of trace amounts of highly reactive carbenes in the neat ILs with
relatively basic anions might pave the way for a general facile use of in situ formed NHCs in catalytic
reactions. Additionally, simple protonation of these carbenes (e.g. by addition of water) results in a
switchable reactive solvent system, allowing the physicoꢀchemical properties to be tuned by the strucꢀ
ture of the imidazolium cation.
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In biomolecules of different origin, e.g. neuropeptides, hormones, enzymes and growth factors, disulꢀ
fideꢀbonds play a crucial role in folding, bioactivity, function and stabilization of their threeꢀdimensional
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structure. In general, disulfideꢀbond formation can be manipulated by its formation and reformation.
This makes this area of chemistry both interesting and essential to a number of biochemical transforꢀ
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mations. It is well known that phosphorous nucleophiles are able to react with disulfide bonds by a
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SN2ꢀmechanism.
Banerjee et al. achieved disulfideꢀbond decomposition by a combination of terꢀ
tiary phosphines and the IL 1ꢀpropylꢀ3ꢀmethylimidazolium bromide ([C mim]Br) that serves as catalytic
3
system, which facilitates the thiolalkoxyꢀphosphonium cation and thiolate anion formation. A subseꢀ
quent reaction with alkyl halides and activate alkenes leads to the formation of unsymmetrical sulfides
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and 1,4ꢀaddition products.
This report, in combination with the knowledge about similar nucleophilic properties of phosphines and
imidazolꢀ2ꢀylidenes inspired our studies to prove whether [C mim][OAc] is capable to react with selectꢀ
2
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ed sulfurꢀcontaining compounds, such as disulfides or thiols.
Selected organic compounds were
meant to serve as model substances. Additionally, a cysteine bearing peptide, derived from the influꢀ
enza virus B proton channel (residues 22ꢀ35) was analyzed whether [C mim][OAc] can chemically reꢀ
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act with the thiol group of the cysteine side chain. This is especially of importance with respect to our
recent approach which introduced [C mim][OAc] as a highly promising reaction medium for the native
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chemical ligation (NCL) of highly hydrophobic peptides. Thus, we purse to identify reaction products
of the IL with the organic model compounds and with the cysteine side chain of the peptide fragment
unveiling a general reaction mechanism. Additionally, we could show, that the addition of more than
30% of water to the IL was necessary to suppress the reaction with the peptide. Consequently, we
found conditions where [C mim][OAc] can be used as a solvent only and is not involved in chemical
2
transformations as a reactant. Furthermore, one can think of making use of carbene chemistry using
ILs as in situ carbene generators for reactions without the use of additional reagent, and using ILs as
an alternative solvent for highly hydrophobic peptides and their transformations, i.e. NCL.
2. Results and Discussion
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ILs were reported to be able to promote disulfideꢀbond formation on air. However, it was observed
that ILs not only build a network around biomolecules and therefore support the formation of thermodyꢀ
namically stable disulfideꢀbond connectivity, but chemically interact with them. We supposed Cysꢀ
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residues to be involved in this reaction. On the other hand, the cleavage of sulfurꢀsulfur bonds was reꢀ
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ported by phosphorous nucleophiles following a carbene mechanism.
Moreover, the combination of
IL and triphenylphosphin (PPh ) was observed to serve as a catalyst which is able to cleave diphenyl
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disulfides facilitating the formation of a thiolalkoxyꢀphosphonium cation and a thiolate anion. So far, in
situ generated NHCs formed by the neat IL were not considered to be responsible for these processes.
Therefore, we decided to analyze if the neat IL might react with the sulfurꢀcontaining compounds withꢀ
out the need of auxiliary catalytic compounds.
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Scheme 1. Reaction processes of sulfurꢀcontaining compounds with [C mim][OAc]
The reaction mechanism outlined above (Scheme 1) is based on the analysis of the reaction of the
small organic molecules, diphenyl disulfide (1), dibenzyl disulfide (3) and their reduced forms thiopheꢀ
nol (2) and benzyl mercaptan (4) with [C mim][OAc] (5). Here, the carbeneꢀfacilitated reaction between
2
sulfurꢀcontaining molecules and the ionic liquid [C mim][OAc] is based on the deprotonation of the C2
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atom of the imidazolium ring by the IL’s anion acetate, formation of the singlet carbene and subsequent
nucleophilic substitution resulting in the intermediate imidazoliumꢀthio adduct a, which is converted in a
consecutive reaction to yield products b and c (Scheme 1). The molecules which contain the thiolꢀ
groups must be oxidized first to be able to react with the neat IL.
Compounds 1ꢀ4 were chosen as organic model molecules because of their simple structure in comꢀ
parison to complex biomolecules, such as peptides to interpret potential interactions between biomoleꢀ
cules and ionic liquids. After the underlying reaction mechanism of the IL with the organic compounds
was understood we used a Cysꢀcontaining model peptide, in order to follow up the reaction of the bioꢀ
molecule.
Complete dissolution of substances 1ꢀ4 occurred within 5 min by treatment in an ultrasonic bath and
reaction monitoring was commenced immediately after dissolution. Reaction products were analyzed
by liquid chromatography–mass spectrometry (LCꢀMS), electrospray ionization (ESI) MS and NMR
spectroscopy (Fig. 1, Fig. 2). Reactions were carried at room temperature and in the dark to exclude
39
hemolysis of the sulfurꢀsulfur bond.
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a) Reaction of diphenyl disulfide (1) and thiophenol (2) in [C mim][OAc] (5)
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During the reaction of diphenyl disulfide (1) (t = 19.39 min) in [C mim][OAc] (5) (t =1.37 min) (Fig.1 A),
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three different peaks appeared with retention times (t ) of 4.49, 10.79 and 12.39 min. According to refꢀ
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erence chromatogram the peak, eluting at t 10.79 min was assigned to thiophenol (2) (the reduced
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form of 1), which is formed only in small amounts, being at a constant concentration during 2 h and
disappearing after that time, indicating the equilibrium reaction between the reduced and oxidized
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forms of 1 (Fig. 1A). Already at the starting point of the reaction the intense peak at t 4.39 min was
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observed corresponding to the main product. According to NMR (Fig. 1C, Fig. S1A), ESIꢀMS (Fig. 1D)
spectra and thinꢀlayer chromatography results (Fig. S1), the product a (1a) was the main product
formed during the reaction of 1 in 5 (Scheme 1). The experimental isotopic pattern of product 1a was in
a good agreement with the calculated isotopic pattern (Fig. 1D). The concentration of product 1a
reached its maximum already at 5 min and stays constant for 12 h, since in the reaction mixture the
initial compound 1 is still present to form this product 1a. After 12 h compound 1 is completely transꢀ
formed to product 1a, which concentration then started to decrease in the ensuing time, indicating the
instability of product 1a in the ionic liquid. Thus, two new products 1e, 1f appeared as intense peaks in
the chromatogram at t between 2 – 3 min (Fig. S3).
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The additional peak at 12.39 min (Fig. 1A) was highly volatile during the work up procedure and difficult
to obtain from organic solvents by vacuum evaporation. However, we were able to isolate product 1d
for NMR analysis by direct extraction with cyclohexaneꢀd12 from the reaction mixture after 72 h. To our
surprise, we identified thioanisole as product 1d and confirmed this finding by comparison with the reꢀ
spective reference chromatogram and NMR data (Fig. 1A, E, Fig. S1B). Consequently, this indicates a
+
second sideꢀreaction between diphenyl disulfide (1) and the ionic liquid cation, in which [C mim] was
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obviously capable to methylate the thiol group of thiophenol. Interestingly, only the methylated form 1d,
but not the ethylated form was found as a product in this reaction (Fig. 1E, Fig. S1B). An efficient reacꢀ
tion of thiol methylation was described by Xie et al., applying [C mim]Cl as a catalyst using dimethyl
4
40
carbonate as a source of the methyl group. With respect to our data, [C mim][OAc] seemed to perꢀ
2
form methylation of aromatic thiols, i.e. thiophenol, without the need of an additional reactant.
During our investigation we observed that not only diphenyl disulfide (1) but also the reduced form thiꢀ
ophenol (2) was able to react with the ionic liquid forming the same products 1a/2a and 1d/2d. Moreoꢀ
ver, we found that thiophenol is partially oxidized and diphenyl disulfide partially reduced in
[
C mim][OAc], unveiling that both forms exist in an equilibrium. Nevertheless, the formation of product
2
a is faster in diphenyl disulfide (1) than in thiophenol (2). This observation corresponds to our postulatꢀ
ed mechanism in Scheme 1, where the thiolꢀcompound must first be oxidized before it is attacked by
an in situ formed imidazoliumꢀcarbene. In both reactions, the initial compounds diphenyl disulfide (1)
and thiophenol (2) fully disappeared within 12 h, yielding the main products 1a/2a and reaching its
maximum concentration at 12 h. The second product 1d/2d appears after 2 h of reaction time and
reaches its maximum concentration at 24 h.
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Figure 1. HPLC elution profiles of the reaction of diphenyl disulfide 1 (A) and thiophenol 2 (B) in [C mim][OAc] (molar ratio
1
1:50, 123 mM of 1 or 2). (C) H NMR spectrum and assignment of product 1a. (D) ESIꢀMS spectrum and comparison of experꢀ
1
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imental and calculated isotopic patterns for product 1a. (E) C NMR spectrum of product 1d in cyclohexaneꢀd12. HPLC condiꢀ
tions: 20 % ꢀ 55 % eluent B in 15 min followed by a gradient of 55 % ꢀ 95% eluent B for 7 min at a flow rate 1 mL/min and
detection wavelength at 240 nm.
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(
b) Reaction of dibenzyl disulfide (3) and benzyl mercaptan (4) in [C mim][OAc] (5)
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Figure 2. HPLC elution profiles of reaction of dibenzyl disulfide 3 (A) and benzyl mercaptan 4 (B) in [C
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mim][OAc] (molar ratio
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:50, 123 mM of 3 or 4). HPLC conditions: 20 % ꢀ 55 % eluent B in 15 min followed by a gradient of 55 % ꢀ 95% eluent B for 7
min at flow rate 1 mL/min and detection wavelength at 240 nm. ESIꢀMS spectrum of products 3ꢀ4a (C) and 3ꢀ4b (E), and
1
comparison of their experimental and calculated isotopic patterns. (D) H NMR spectrum and assignment of the products 3ꢀ4a
(
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D) and 3ꢀ4b (F) in D O.
For the reaction of dibenzyl disulfide (3) and benzyl mercaptan (4) in [C mim][OAc] (5) the formation of
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the products aꢀc, according to the Scheme 1, was monitored by HPLC. The product a (3a and 4a), elutꢀ
ing at t 5.31 min, was already formed at the beginning of reaction (5 min) as indicated by a small peak
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in the chromatogram (Fig. 2A, B). Its concentration constantly increased until 2 h and then began to
decrease completely disappearing after 48 h. This product was isolated and identified by ESIꢀMS and
NMR analysis (Fig. 2C, D, Fig. S2C). Also product b (3b and 4b), eluting at t 2.82min, and product c
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(
3c and 4c), eluting at t 18.44min, were already present immediately after the reaction was started
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(
Fig. 2A, B, E,F, Fig. S2D). The concentration of both products 3b and 4b slightly increased during the
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5
5
5
5
5
6
reaction time, while the concentration of 3c and 4c remained constant. Interestingly, no benzyl mercapꢀ
tan (4) was observed during the reaction of 3 in [C mim][OAc], in comparison to diphenyl disulfide (1)
2
(
Fig. 1A, Fig.2A). Moreover, for the reaction of 4 in [C mim][OAc] the initial compound was completely
2
expended already at the beginning of the reaction yielding its oxidized form dibenzyl disulfide (3), indiꢀ
cating a much faster oxidation of benzyl mercaptan (4) in comparison to thiophenol (2). Furthermore,
this is an evidence that the reduce compounds indeed oxidize first to be able to react with the imidazꢀ
oleꢀcarbene. The products 3b/4b were isolated and assigned according to ESIꢀMS and solution NMR
analysis (Fig. 2E, F) and 3c/4c according to reference elution of dibenzyl sulfide (Fig. 2A).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In summary, the obtained data for compounds 1ꢀ4 clearly support our initial hypothesis (Scheme 1) that
constantly formed carbenes in [C mim][OAc] are capable to participate in nucleophilic substitution of
2
disulfides forming product a. For diphenyl disulfide/thiophenol product 1a/2a was formed first and then
subsequently transformed to products 1e and 1f. Moreover, [C mim][OAc] seemed to perform methylaꢀ
2
tion of aromatic thiols, i.e. thiophenol, by forming product 1d without the need of an additional reactant.
In contrast, for dibenzyl disulfide/benzyl mercaptan we observed two additional products b and c. Most
likely, the additional CH2ꢀgroup between the phenyl ring and [C mim]S in product 3a/4a allows for the
2
-
nucleophilic attack of the RS intermediate at this position. Moreover, thiophenol (2) and benzyl merꢀ
captane (4) dimerized first forming diphenyl disulfide (1) and dibenzyl disulfide (3) before they were
reacting with the NHC of [C mim][OAc]. Thus, we concluded that the carbene itself is able to “break”
2
the disulfide bond, without the need of further catalytic compounds. Consequently, this type of reaction
must be taken into consideration when discussing increased m/z peaks of proteins derived from IL mixꢀ
tures, or the highly selective oxidative folding (disulfide bond formation) to a single thermodynamically
14
stable isomer in ILs. Finally, the chemical reactions observed in this study are clearly indicative for the
presence of a small carbene fraction in neat [C mim][OAc].
2
(
c) Reaction of model peptide CWTIGHLNQIKRGI in [C mim][OAc] (5)
2
With respect to the chemical reaction of [C mim][OAc] (5) with the disulfide bonds of the small organꢀ
2
ic compounds 1 and 3 and their reduced forms, compounds 2 and 4, a small cysteine containing pepꢀ
tide with the sequence CWTIGHLNQIKRGI (6) was investigated.
From the results discussed above, one would expect for this reaction the disulfide bond formation, folꢀ
lowed by nucleophilic substitution and scission of the SꢀS bond by the in situ formed NHC carbene from
IL according to Scheme 1. The reaction was monitored by RPꢀHPLC and all peaks were characterized
by LCꢀMS (Fig. 3). Product peaks were isolated and reinjected to prove their stability. The LCꢀMS analꢀ
ysis was performed for two reaction times, at 1 h and 24 h (Fig. S4). The reaction products are depictꢀ
ed in Fig. 3A. Directly after dissolving the peptide (6) in [C mim][OAc] (5), an intensive peak was obꢀ
2
served (t 16.52 min) in the RPꢀHPLC chromatogram (Fig. 3B), indicating peptide oxidation and forꢀ
R
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mation of the peptide dimer (IGRKIQNLHGITWCCWTIGHLNQIKRGI, 6d) through a disulfide bond
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(Fig. S4A, Fig. S6). Disulfide bond formation typically occurs through peptide oxidation in the presence
13
of oxygen from the air. Within 2 h, this peak completely disappeared, while three other main peaks at
tR 7.99 and 9.24 and 10.71 min built up reaching their maximum concentration at 2 h. According to the
LCꢀMS analysis and observations made for the small organic model compounds in this study the peak
at t 7.99 min corresponded to the reaction product a (6a) of Scheme 1, indicating the chemical reacꢀ
R
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
7
8
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3
4
5
6
7
8
9
0
tion of peptide (6) with 5 (Fig. S4A, Fig. S6). The peak at t 9.24 min had a corresponding m/z, which
R
was 32 m/z higher than for the nonꢀaltered peptide (6) and most likely corresponded to peptide Cysꢀ
sulfone (product 6e) (Fig. 3B, Fig. S4A, Fig. S6). The peak at t 10.71 min increased until 24 h and was
R
assigned to product 6f (Fig. S4B, Fig. S6), assuming further oxidation of product 6a by forming the sulꢀ
fone product. After 2 h product 6a (t 7.99 min) continuously decreased and in parallel peaks at tR
R
12.44/12.79 min were observed. The analysis of the isotopic pattern of this product indicated the desulꢀ
furization of the Cysꢀresidue of the peptide yielding product AWTIGHLNQIKRGI (6g), where Cys was
transformed to Ala (Fig. S4B, Fig. S6). Interestingly, our data suggest that the desulfurization process
observed herein might proceed via a very similar mechanism as reported for the wellꢀdescribed reacꢀ
41
tion of desulfurization by tris(2ꢀcarboxyethyl)phosphine (TCEP). So far, [C mim][OAc] had not been
2
described to be involved in such kind of reactions and further investigations are needed whether this
reaction is an alternative procedure for the desulfurization of cysteine residues, which were artificially
inserted into a peptide sequence for NCL purposes.
Product 6a could not be isolated most likely being unstable upon the workꢀup procedure. Instead, from
the respective HPLC peak we isolated the desulfurized peptide 6e and product 6h, which was the oxiꢀ
dized form of 6c (Fig. S5). Conclusively, the Cysꢀcontaining peptide underwent the same reactions in
neat [C mim][OAc] as observed for the small organic model compounds. But unlike for the model comꢀ
2
pounds peptide products 6a and 6c were not stable and further oxidized yielding products 6f and 6h
(
Fig. 3) confirming the reactions outlined in Scheme 1.
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1
2
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8
9
1
1
1
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2
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2
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3
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6
0
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0
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0
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2
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6
7
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9
0
1
2
3
4
5
6
7
8
9
0
2
Figure 3. Reaction of Cysꢀcontaining peptide in [C mim][OAc] and products formed during the reaction
d)The effect of water on product formation during reaction of peptide 6 in 5
From the observations made for the chemical transformation of the small organic model compounds
and the Cysꢀcontaining peptide fragment induced by small fractions of NHCs in neat [C mim][OAc] we
2
concluded that the addition of water might prevent NHC formation. Since, peptide 6 was water soluble,
we investigated if water can prevent the reactions observed in [C mim][OAc]. Indeed, HPLC analysis
2
revealed that at water:IL ratios ranging from 10 to 30 % (v/v) the reaction with imidazoleꢀcarbene was
slowed down and fewer products were formed in comparison to reaction in the neat IL (Fig. 3B, Fig. 4).
2
Figure 4. The effect of water on product formation during reaction of peptide 6 in [C mim][OAc] 5
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Over time, the concentration of dimerized peptide 6d increased and reached its maximum at 2 h stayꢀ
ing at a constant concentration for the rest of the monitored reaction time (24 h) when the water conꢀ
tent was 20 or 30 % (Fig. 4B, C), respectively. At a water content of 10% product 6a was formed after
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
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2
2
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2
2
2
2
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3
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3
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4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
h, whereas at 20 % water only small traces of product 6a were observed in the HPLC chromatogram
after 24 h (Fig. 4B). Also, only small amounts of the oxidation products 6e and 6f were formed within
the whole reaction process (Fig. 4B). In contrast, at water concentration of at least 30 % (Fig. 4C)
product 6a was not observed within 24 h of reaction time, but minimal concentrations of products 6h
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
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8
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0
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0
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2
3
4
5
6
7
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0
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2
3
4
5
6
7
8
9
0
and 6f. So far, our results support previous findings, which described the inhibition of carbene forꢀ
42,43
mation (in reaction with elemental sulfur) with water at concentrations higher than 50 %
due to the
26, 44
disruption of interaction between the anion and the cation of the IL through water.
As a result, water reduces the reactivity of imidazoliumꢀcarbenes towards sulfurꢀrich peptides and small
41,42
amounts of carbene present in neat IL can be protonated or even decomposed in water.
ing explains the absence of product 6a in the RPꢀHPLC chromatogram for water contents ≥ 30 %
Fig. 4).
This findꢀ
(
3. Conclusions
In this work we demonstrated, that the ionic liquid [C mim][OAc] is not just capable of dissolving thiolꢀ
2
and disulfideꢀcontaining model compounds, such as diphenyl disulfide, dibenzyl disulfide, thiophenol
and benzyl mercaptan, but also are able to chemically react with them without addition of any catalytic
reagents. Due to the anion’s basicity (acetate) the equilibrium shifts towards the deprotonated C2
atom of the imidazolium cation allowing for a reaction with thiolꢀ or disulfideꢀ containing comꢀ
pound. Timeꢀresolved RPꢀHPLC analysis indicated the formation of a various products. By ESIꢀMS,
NMR spectroscopy and TLC analysis, we could unambiguously identify and characterize all formed
products. Consequently, our data fully supports the reaction scheme as outlined in Scheme 1 hypotheꢀ
sizing that the imidazoliumꢀcarbene can react with a disulfide bond, but not with a thiol group. In turn,
thiols first must be oxidized in order to be able to form products a, b and c (Scheme 1). Further, the
structure of the disulfide played an important role. In order to allow the formation of products b and c
+
from a, the thiol group must have an adjacent methylene group, allowing [C mim]S to cleave off. If
2
there is no adjacent methylene group, the reaction stops after the formation of product a. However,
several other products were formed, namely thioanisole (1d/2d), in the reaction of 1/2 in 5 indicating
that 5 is capable to methylate the thiolꢀgroup without the help of additional methylation reagents.
Moreover, we could show that a cysteineꢀcontaining peptide results into similar products as observed
for the organic model compounds in [C mim][OAc] which also had to be oxidized first to react with imꢀ
2
+
idazoleꢀcarbenes forming product a (Fig. 3A). Additionally, the spontaneous elimination of [C mim]S
2
from the peptide leads to the desulfurization of the respective Cys residue which might provide an alꢀ
ternative way for Cys transformation e.g. after NCL of two peptide fragments.
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Despite the interesting ability of neat [C mim][OAc] to react with sulfurꢀcontaining molecules, there are
2
1
2
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4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
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3
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3
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3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
some reactions, where IL is preferred to promote its features as a solvent, i.e. NCL where such reactiviꢀ
ty is undesired. Thus, the addition of at least 30 % of water to [C mim][OAc] was sufficient to suppress
2
any chemical reaction of the IL with the peptide.
Finally, our results allow for making use of a tailored combination of carbene chemistry in the field of
organic and biochemical reactions, with the beneficial use of the environmental friendly ILs, but also
overcomes the problematic use of ILs in the presence of cystineꢀrich peptides and proteins.
Our study has a major impact on further IL application for sulfurꢀcontaining compounds, e.g. peptides
and proteins or small organic molecules. To investigate the border case of basicity of anion in ionic liqꢀ
uid to be able to deprotonate IL cation, ILs with anions of different basicity, according to Hofmeister
0
1
2
3
4
5
6
7
8
9
0
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2
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4
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0
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7
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6
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6
7
8
9
0
45,46
series, will be studied.
Optimal reaction conditions (choice of IL, water amount, temperature, reacꢀ
tant concentration) and selectivity for the formation of a single reaction product for specific applications
will be determined in future work.
4. Experimental Section
General experimental: For the reaction diphenyl disulfide 1 (39.3 mg), thiophenol 2 (19.8mg, 1.83
l), dibezyl disulfide 3 (44.4 mg) and benzyl mercaptan 4 (22.3 mg, 2.11ꢁl) were dissolved in 1.5 ml of
ꢁ
[
C mim][OAc] (molar ratio 1:50, 123 mM) respectively. The reaction mixtures were leaved to react while
2
agitating at room temperature until the reaction was completed. For reaction monitoring of compounds
ꢀ4 in [C mim][OAc] aliquots of the mixtures (5 ꢁl) were diluted with 250 ꢁl acetonitrile with 0.1% TFA
1
2
and 5 ꢁl of the resulting solution was injected for analytical RPꢀHPLC. The reaction was monitored at
h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h and 72 h. Products 1a/2a, 3a/4a and 3b/4b were isolated by
0
column chromatography, applying a mixture of acetonitile/water (9:1) as a mobile phase. After lyophiꢀ
lization, a light yellow, viscous liquid was obtained (product 1ꢀ2a: 31.6 mg (yield: 80%), product 3ꢀ4a:
11.2 mg (yield: 26%)). The fraction, containing the product 3ꢀ4b was evaporated under reduced presꢀ
sure and finally 15.4 mg (yield: 60%) of a dark orange liquid was obtained.
Synthesis of peptide CWTIGHLNQIKRGI (6)
Peptide 6 was synthesized on an automated peptide synthesizer (Liberty, CEM) according to a
standard Fmocꢀprotocol on AmphiSpheres RAM resin (0.37 mmol/g loading size). Coupling reactions
(
15 min, double coupling) were performed using Fmocꢀamino acids (4 equiv.), activated with 1ꢀ
[bis(dimethylamino)methylene]ꢀ1Hꢀ1,2,3ꢀtriazolo[4,5ꢀb]pyridinium 3 oxide hexafluorophosphate (HATU,
.9 equiv.) and NꢀethylꢀNꢀ(propanꢀ2ꢀyl)propanꢀ2ꢀamine (DIEA, 8 equiv.) in N,Nꢀdimethylformamide
DMF) under microwave irradiation (50 °C, 35W). Fmocꢀprotecting group was removed by treating the
3
(
peptideꢀresin with 20% piperidine in DMF under microwave irradiation (50 °C, 35W), twice. All deproꢀ
tection and coupling steps were followed by intensive washings using DMF and dichloromethane
(
DCM).
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Peptide cleavage and deprotection was accomplished with a cleavage cocktail of a 90 % TFA, 5 %
water, 2.5 % TIPS and 2.5 % anisole for 3 h at room temperature. The crude peptides were precipitatꢀ
ed in cold diethyl ether, centrifuged and washed with diethyl ether. The crude peptide was purified by
preparative RPꢀHPLC using a C18 column (MultoKrom 100ꢀ5, 10 nm, 250 x 20 mm,), eluent A: water
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
0.1% TFA) and eluent B: acetonitrile (0.1% TFA). Preparative HPLC conditions: 20% ꢀ 80% acetoniꢀ
trile in 40 min using a flow rate of 10 ml/min.
0
1
2
3
4
5
6
7
8
9
0
1
2
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6
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0
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2
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6
7
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0
Peptide CWTIGHLNQIKRGI (6) reaction in [C mim][OAc] (5)
2
For the reaction 0,825 mg of peptide 6 were dissolved in 43 ꢁl of [C mim][OAc] (molar ratio 1:500,
2
12mM). The reaction mixture was leaved to react while agitating at room temperature until the reaction
was completed.
The effect of the water content on the reaction progress was determined for the peptide BM2(22ꢀ35).
A series containing different amounts of water (10 %, 20 % and 30 % (v/v)) were prepared and the reꢀ
action progress was followed by RPꢀHPLC at 0 min, 30 min, 1 h, 2 h, 4 h, 8 h and 24 h.
Reversed-phase high performance liquid chromatography (RP-HPLC): Reactions were moniꢀ
tored by analytical RPꢀHPLC on a Prontosil 120ꢀ5ꢀC8ꢀSH 5.0 ꢁm analytical column. The gradient eluꢀ
tion system was 0.1% TFA in water (eluent A) and 0.1% TFA in acetonitrile (eluent B). Organic comꢀ
pounds 1ꢀ4 were eluted with a gradient of 20 % ꢀ 55 % eluent B in 15 min followed by 55 % ꢀ 95 % eluꢀ
ent B in 22 min and a flow rate of 1 mL/min. The peptide BM2(22ꢀ35) was eluted with a gradient of
20 % ꢀ 30 % eluent B in 20 min and a flow rate of 1 mL/min. The detector was operated at 240 nm for
organic compounds 1ꢀ4 and at 214nm for the peptide BM2(22ꢀ35).
Thin-layer chromatography (TLC): Thin layer chromatography was performed on silica gel 60 F
254 coated glass plates. Two solvent systems were applied for each compound TLC nꢀhexane or nꢀ
hexane/ethyl acetate (59:1, v/v) and acetonitrile/water (9:1, v/v). The R values are summarized in Table
f
1.
NMR spectroscopy: Solution NMR experiments were performed on a Bruker Avance III spectromeꢀ
ter or on a Bruker DRX 500 at proton frequencies of 300 MHz or 500 MHz, respectively. The products
1a, 3a, 3b were dissolved in D O and 1d in cyclohexaneꢀd12.
2
1
1
a/2a: H NMR (300 MHz, D O) δ = 7.7 (d, J = 2.2 Hz, 1ꢀH), 7.6 (d, J = 2.2 Hz, 1ꢀH), 7.4 (m, 3ꢀH, Ar),
2
13
7
.3 (m, 2ꢀH, Ar), 4.3 (q, J = 7.3 Hz, 2ꢀH), 3.8 (s, 3ꢀH), 1.3 (t, J = 7.3 Hz, 3ꢀH) ppm. C NMR (D O) δ =
2
130.3, 130.0, 129.2, 128.6, 125.6, 123.5, 45.17, 36.1, 14.3 ppm.
1
1d/2d: H NMR (500 MHz, cyclohexaneꢀd ) δ 7.88 – 7.80 (m, 4ꢀH, Ar), 7.70 – 7.67 (m,1ꢀH, Ar), 3.04
12
13
(
s, 3ꢀH) ppm. C NMR (cyclohexaneꢀd ) δ = 139.8, 129.0, 127.5, 125.3, 16.1 ppm.
12
1
3
a/4a: H NMR (300 MHz, D O) δ = 7.5 (d, J = 2.1 Hz, 1ꢀH), 7.5 (d, J = 2.2 Hz, 1ꢀH), 7.3 – 7.2 (m, 3ꢀ
2
H), 7.1 – 7.0 (m, 2ꢀH), 4.1 (s, 2ꢀH), 4.0 (q, J = 7.3 Hz, 2ꢀH), 3.6 (s, 3ꢀH), 1.2 (t, J = 7.4 Hz, 3ꢀH) ppm.
13
C NMR (D O) δ = 136.0, 129.2, 128.7, 128.5, 125.2, 123.0, 44.6, 39.8, 35.5, 14.2 ppm.
2
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1
3
b/4b: H NMR (300 MHz, D O) δ = 7.0 (d, J = 2.3 Hz, 1ꢀH), 7.0 (d, J = 2.3 Hz, 1ꢀH), 4.0 (q, J = 7.3
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3
Hz, 2ꢀH), 3.5 (s, 3ꢀH), 1.2 (t, J = 7.3 Hz, 3ꢀH) ppm. C NMR (D O) δ = 119.8, 117.8, 43.0, 34.7,
2
13.6 ppm.
LC-MS: The LCꢀMS of the reaction of peptide CWTIGHLNQIKRGI (6) with [C mim][OAc] (5) was perꢀ
2
formed using a gradient of 20 – 30 % eluent B in 20 min and a flow rate of 0.2 mL/min. The gradient
elution system was 0.1% TFA in water (eluent A) and 0.1% TFA in acetonitrile (eluent B).
0
1
2
3
4
5
6
7
8
9
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9
0
Characterization of starting materials and products, including retention times and response factors, is
given in the Supporting Information along with selected chromatograms.
ASSOCIATED CONTENT
Supporting Information
NMR spectra, LCꢀMS analysis, RPꢀHPLC chromatograms, MS spectra. (PDF). This material is availaꢀ
ble free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*
Eꢀmail: a.tietze@biochemieꢀtud.de
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The Liebig Fellowship of the Fonds der Chemischen Industrie and the LOEWE iNAPO funded by the
Ministry of Higher Education, Research and the Arts (HMWK) of the Hessen state are gratefully
acknowledged (A.A.T). The authors thank the MSꢀService team of the Chemistry Department, especialꢀ
ly C. Rudolph and Dr. A. Schiesser (TU Darmstadt). We thank Diana Imhof (University of Bonn) and
members of the COST Action CM1206 Exchange on Ionic Liquids for useful scientific discussions.
REFERENCES
(1)
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