Palladium-Mediated Direct Disulfide Bond Formation in Proteins Containing S-Acetamidomethyl-cysteine under Aqueous Conditions

Article abstract of DOI:10.1002/anie.201900988

One of the applied synthetic strategies for correct disulfide bond formation relies on the use of orthogonal Cys protecting groups. This approach requires purification before and after the deprotection steps, which prolongs the entire synthetic process and lowers the yield of the reaction. A major challenge in using this approach is to be able to apply one-pot synthesis under mild conditions and aqueous media. In this study, we report the development of an approach for rapid disulfide bond formation by employing palladium chemistry and S-acetamidomethyl-cysteine [Cys(Acm)]. Oxidation of Cys(Acm) to the corresponding disulfide bond is achieved within minutes in a one-pot operation by applying palladium and diethyldithiocarbamate. The utility of this reaction was demonstrated by the synthesis of the peptide oxytocin and the first total chemical synthesis of the protein thioredoxin-1. Our investigation revealed a critical role of the Acm protecting group in the disulfide bond formation, apparently due to the generation of a disulfiram in the reaction pathway, which significantly assists the oxidation step.

Full text of DOI:10.1002/anie.201900988

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Accepted Article
Title: Palladium Mediated Direct Disulfide Bond Formation in Proteins
Containing S-Acetamidomethyl-Cysteine under Aqueous
Conditions
Authors: Ashraf Brik, Shay Laps, Hao Sun, and Guy Kamnesky
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201900988
Angew. Chem. 10.1002/ange.201900988
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10.1002/anie.201900988
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Palladium Mediated Direct Disulfide Bond Formation in Proteins
Containing S-Acetamidomethyl-Cysteine under Aqueous
Conditions
Shay Laps†, Hao Sun†, Guy Kamnesky and Ashraf Brik*
Abstract: One of the applied synthetic strategies for correct disulfide
this approach has enabled the chemical syntheses of various
bond formation relies on the use of orthogonal Cys protecting groups. interesting peptides and proteins (i.e. insulin),[2, 5c, 6, 9] the harsh
Such an approach requires purification before and after the
deprotection steps, which prolongs the entire synthetic process and
lowers the yield of the reaction. A major challenge in using this
approach is to be able to apply one-pot synthesis under mild
conditions and aqueous media. In this study, we report on the
development of rapid disulfide bond formation employing palladium
chemistry and S-acetamidomethyl-cysteine (Cys(Acm)). The
oxidation of Cys(Acm) to the corresponding disulfide bond was
achieved within minutes and one-pot operation by applying
palladium and diethyldithiocarbamate. The utility of this chemistry
were exemplified in the synthesis of oxytocin peptide and in the first
total chemical synthesis of Thioredoxin-1 protein. Our investigation
revealed a critical role of the Acm protecting group in the disulfide
bond formation apparently due to the generation of disulfiram in the
reaction pathway, which significantly assists the oxidation step.
removal conditions of the Cys PGs reduce significantly the
10]
efficiency of this approach.[8,
For example, a widely used
method to convert two Cys(Acm) residues to the corresponding
disulfide is based on iodine oxidation in acetic acid,[11] however,
over-oxidation of the thiol functionality to sulfonic acid could
occur as well as other side reactions have been reported when
Tyr, Trp and Met are present. [12]
Herein we report on the development of a new approach
for rapid disulfide bond formation employing Pd chemistry and
Cys(Acm) and its utility in various systems. The oxidation of
Cys(Acm) to the corresponding disulfide bond was achieved in
one-pot by applying Pd and diethyldithiocarbamate (DTC) in a
highly efficient manner. We also report on initial mechanistic
aspects of this transformation, which revealed an important role
of the Acm PG in this process.
We have recently discovered unprecedented reactivity of
PdII complexes for the efficient deprotection of several important
side chain PGs.[13] In particular, we have reported the use of
PdCl2 under NCL conditions for the rapid Cys(Acm) deprotection.
This step was also followed by NCL coupled with desulfurization
Disulfide bridges are often evolutionarily conserved motifs,
which play an important role in the stabilization of the native
conformation of various peptides and proteins as well as in the
catalysis of known enzymes.[1] Therefore, when attempting the
synthesis of these macromolecules, the correct connectivities of
the disulfide bonds must be formed in order to obtain functional
molecules.[2] Chemical protein synthesis [3] is a useful method to
prepare complex protein analogues in high homogeneity and
workable quantities.[4] Nevertheless, the generation of proteins
bearing disulfide bonds remains a challenging task despite
success in interesting cases (e.g. erythropoietin).[1,2, 5]
Two main synthetic strategies are often applied for the
formation of disulfide bridges in peptides and proteins.[1, 6] The
first one relies on subjecting the reduced peptide to freely
oxidative folding conditions using buffer containing redox
reagents (e.g. Cys/Cystine) to form the correct connectivity
under thermodynamic control.[5a, 5b, 5d, 7] The main limitations of
this approach are the slow rate of the reaction (hours-days) and
the requirement for careful optimize of the desired ratio between
the redox reagents for each system.[6, 8]
in a one-pot manner as demonstrated in the total chemical
13e, 13h]
synthesis of various proteins.[13a,
In order to recover
efficiently the peptide, we used excess dithiothreitol (DTT) to
scavenge the free and the bound Pd to the peptide side chains.
[13a, 13e, 13h] However, DTT is a reducing agent,[14] which cannot be
used when in situ oxidation of Cys residues is desired. Therefore,
finding an appropriate Pd scavenger as an alternative to DTT is
the essential step in performing one-pot Cys deprotection
coupled with disulfide formation.
The second approach is based on
a regioselective
disulfide bond formation via stepwise oxidation by employing
various orthogonal Cys protecting groups (PGs) such as the S-
acetamidomethyl (Acm) and the tert-butyl (t-butyl).[5c, 8] Although
[*]
[†]
S. Laps,† Dr. H. Sun,† Dr. G. Kamnesky, Prof. A. Brik
Schulich Faculty of Chemistry
Technion-Israel Institute of Technology
Haifa, 3200008 (Israel)
E-mail: abrik@technion.ac.il
These authors contributed equally to this work.
Figure 1. Analytical HPLC and mass analysis of the model peptide 1 treated
with 10 equiv PdCl2 at 37 °C, pH 7 in 6 M Gn·HCl followed by quenching with
Supporting information for this article is given via a link at the end of
the document.
various scavengers. (A) After
5 min treatment with PdCl2 and 25 min
incubation with 25 equiv DTT: the main peak corresponds to unmasked
peptide 1, with the observed mass 1241.5 ± 0.1 Da (calcd 1241.4 Da,
average isotopes). The following tests were done with peptide 1 after 5 min
treatment with PdCl2 and 25 min incubation with 25 equiv: (B) DMG. (C) EDTA.
(D) KI. (E) DTC: the main peak corresponds to unmasked peptide 1, with the
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observed mass 1241.5 ± 0.1 Da (calcd 1241.4 Da, average isotopes). Peak a
corresponds to the dimerized peptide 1 after deprotection, with the observed
mass 2480.6 ± 0.1 Da (calcd 2481.0 Da, average isotopes).
followed by quenching for 25 min with: (D) 25 equiv of DTC, and (E) 100 equiv
of DTC. Peak b corresponds to the reduced unmasked oxytocin with the
observed mass 1108.3 ± 0.1 Da (calcd 1109.2 Da, average isotopes). Peak c
corresponds to the oxidized unmasked oxytocin with the observed mass
1106.2 ± 0.1 Da (calcd 1107.2 Da, average isotopes).
For this purpose we prepared the model peptide
1
(LYRAGC(Acm)LYRAG) and treated with 10 equiv PdCl2 for 5
min for Acm removal[13a] followed by incubation with various
We then attempted to examine the use of Pd and DTC in
one-pot Acm removal and Cys oxidation. We chose as a model
system the natural peptide-hormone oxytocin that consists of 9
potential Pd chelators yet cannot reduce disulfide bonds. This
13e, 13h]
list includes (1) DTT as a reference reaction[13a,
(2)
amino acids (AAs) and two native Cys that forms a disulfide
dimethylglyoxime (DMG),[15] (3) ethylenediaminetetraacetic acid
(EDTA),[16] (4) potassium iodide (KI),[17] and (5) DTC.[18] The
reactions were analyzed using HPLC and Mass analysis in
which we found that the peptide was not recovered upon
incubation for 25 min with DMG, EDTA or KI (Figure 1B, C &D),
probably due to the inability to release the thiolate group from Pd.
However, we were pleased to observe quantitative recovery of
our model peptide when using DTC as a scavenger similar to
DTT (Figure 1A&E).
[19]
bridge for its active structure.
Our strategy was to make the
peptide synthetically with two Cys(Acm) and test the Acm
removal followed by quenching with DTC and performing in situ
oxidation step. After preparing the oxytocin peptide with the
Cys(Acm) using Fmoc-SPPS we performed Acm deprotection
using 10 equiv of PdCl2 for 5 min[13a] followed by quenching the
reaction with 25 equiv DTC for 25 min at pH 7 and 37 °C
(Supporting Information, Figure S4B). The reaction was then
monitored by HPLC-MS analysis, which indicated the formation
of two products including the oxidized oxytocin (15%) and
reduced form (85%). After overnight incubation under these
conditions the oxidation reaction was completed without the
requirement of isolation and oxidation. A control experiment with
DTT as the scavenger clearly showed no oxidation even after
overnight treatment (Supporting Information, Figure S4D).
At this stage we wondered if changes in the reaction
conditions could affect the oxidation rate. Therefore, we initially
focused our attention in examining the effect of pH (6-8), which
did not show major effect (Supporting Information, Figure S5).
We then decided to examine the effect of the DTC amount on
the rate of disulfide bond formation. We treated the protected
oxytocin with 10 equiv PdCl2 followed by quenching the
reactions with increasing amount of DTC up to 100 equiv.
Interestingly, the rate of the oxidation was significantly
accelerated with increasing the DTC amount. In this case, the
oxidation was completed within 30 min when 100 equiv of DTC
was used in comparison to requirement for overnight treatment
with 25 equiv DTC (Figure 2).
To test the applicability of our new strategy for the total
chemical synthesis of proteins containing native disulfide bonds,
we chose as a target the E. coli thioredoxin-1 (Trx-1) enzyme.
The sequence of Trx-1 consists of 109 AAs and bears two Cys
residues in positions 33 and 36, which form a disulfide bond as
part of its catalytic machinery.[20] Trx-1 acts as thiol disulfide
oxidoreductase and catalyzes the reduction of disulfides in
various proteins.[20b] Trx-1 is an interesting challenge in terms of
synthesis due to the fact that it contains two native Cys in non-
strategical positions for NCL. Therefore, the native Ala in
position 57 has to be mutated into Cys in order to perform NCL.
Subsequently, this Cys must be converted back to Ala after the
NCL step via desulfurization.[21] Thus, the two native Cys
residues have to be protected with the Acm PG to keep them
intact during the desulfurization step.[22] We envisioned here the
use
of
our
new
approach
to
enable
one-pot
ligation/desulfurization steps[23] followed by Acm removal and
disulfide bond formation without the need of multiple purification
steps and the loss of material.
Figure 2. Optimization of one-pot Acm removal and disulfide bond formation in
oxytocin. (A) Schematic presentation of Acm removal via PdCl2 followed by
treatment with DTC. (B) The effect of DTC amount on disulfide formation after
25 min. (C) Analytical HPLC and mass analysis of purified protected oxytocin,
peak a with the observed mass 1150.3 ± 0.1 Da (calcd 1151.2 Da, average
isotopes). The reaction of protected oxytocin with 10 equiv PdCl2 for 5 min
We divided the Trx-1 sequence into two segments, Trx-
1(1–56)-thioester (Trx-1-N) and Cys-Trx-1(58–109) (Trx-1-C).
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Trx-1-C was synthesized on a Rink amide resin, while Trx-1-N
was synthesized on the diaminobenzoic acid (Dbz) linker
after 30 min of incubation at 37 °C only 10% oxidized oxytocin
was obtained compared to full conversion when the two Cys
residues were masked with the Acm (Figure 4B). The oxidation
was completed only after overnight. These findings triggered us
to further investigate the necessity of the Acm for enhancing the
oxidation reaction. To examine this, we prepared another
oxytocin variant bearing unprotected N-terminal Cys while Cys6
was protected with the Acm. The peptide was treated under the
same conditions where 80% oxidation was observed after 30
min and completed within 2 h (Figure 4C). Together these
results indicated that the deprotection step of the Acm under our
conditions plays an important role in the oxidation step. Notably,
it has been proposed that disulfiram (DSF)- the oxidized form of
DTC, is capable to induce glutathione (GSH) oxidation probably
via disulfide bonds exchange.[28] In a different study, a complex
[24]
(Supporting Information, Figure S7-8). Subsequently, we ligated
Trx-1-C and Trx-1-N using 4-mercaptophenylacetic acid
(MPAA) and tris(2-carboxyethyl)phosphine (TCEP) as additives
in 6 M Gn·HCl, pH 7.3 at 37 °C.[25] The ligation was completed
within 10 h and the product was dialyzed against 6 M Gn·HCl to
enable one-pot desulfurization, which completed within 2 h
(Figure A-C, Supporting Information, Figure S9). The reaction
mixture was then dialyzed again, treated with 10 equiv PdCl2 for
5 min and quenched with 100 equiv DTC for 25 min to afford the
full length Trx-1 protein with the disulfide bond between Cys33
and Cys36 in one-pot and 37% overall isolated yield (Figure 3D).
Circular dichroism (CD) spectroscopy analysis of the purified
oxidized Trx-1 exhibited the expected CD signature of the folded
protein[26] (Figure 3E). In addition, we confirmed the catalytic
activity of the synthetic Trx-1 using established protocol for Trx-1
activity assay[27] (Figure 3F).
Figure 3. Total chemical synthesis of Trx-1. (A) Schematic presentation of the
Trx-1 synthesis. (B) Trx-1 sequence. (C) Analytical HPLC and mass analysis
of purified Trx-1(Acm)2 with the observed mass 11909.6 ± 0.4 Da (calcd
11911.5 Da, average isotopes) after one-pot ligation and desulfurization. (D)
Analysis of purified full length oxidized Trx-1 with the observed mass 11765.9
± 0.3 Da (calcd 11767.5 Da, average isotopes). (E) CD analysis of the Trx-1
final product. (F) Activity assay of the reduction of bovine insulin catalyzed by
the chemically synthesized Trx-1 with and without DTT. The turbidity of the
reaction mixture analyzed via plotting the absorbance at 650 nm versus time.
Figure 4. The effect of Acm on the Pd/DTC mediated disulfide bonds
formation: (A) Schematic presentation of the reaction of different analogues of
oxytocin with PdCl2 followed by treatment with DTC in 6 M Gn·HCl, pH 7.5,
37 °C. Incubation of oxytocin analogues with 10 equiv PdCl2 for 5 min followed
by quenching with 100 equiv DTC for additional 25 min: (B) the case of
unprotected oxytocin; peak a corresponds to the oxidized oxytocin with the
observed mass 1106.2 ± 0.1 Da (calcd 1107.2 Da, average isotopes). Peak b
corresponds to the reduced and unmasked oxytocin with the observed mass
1108.3 ± 0.1 Da (calcd 1109.2 Da, average isotopes). (C) The case of
oxytocin analogue bearing one Cys(Acm). (D) The case of oxytocin bearing
two Cys(Acm).
At this stage of our study we were puzzled about the effect
of DTC combined with Pd on the rate of oxidation and were
curious to examine if the enhancement of the oxidation reaction
could occur in the case of the free and reduced Cys residues.
Therefore, we synthesized the oxytocin peptide with unprotected
and reduced Cys residues. Subsequently, the peptide was
exposed to 10 equiv of Pd and 100 equiv of DTC and the
reaction was monitored by analytical HPLC-MS. To our surprise,
of CuII and DTC as a ligand was reported to promote GSH
oxidation possibly via a redox cycle of the copper.[29] Although
the exact mechanism is unclear, the generation of DSF through
a redox cycle of CuII(DTC)4 to CuI(DTC)2 has been proposed.[29]
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These previous findings prompted us to examine whether in
our reaction DSF could also be a key element in the oxidation
step, which could be formed via a redox cycle involving Pd. To
examine this assumption we first exposed the unprotected
oxytocin to our optimized conditions in the presence of
commercially available DSF (2 equiv). A complete oxidation was
observed within 5 min compared to overnight treatment in the
absence of DSF. When this peptide was incubated with DSF
only and without Pd and DTC, the same results were obtained.
In addition, when oxytocin bearing the two Cys(Acm) was
treated with 10 equiv of PdCl2, 25 equiv of DTC and 2 equiv of
DSF we observed Cys deprotection and oxidation in situ within
total 10 min, compared to 30 min when using 100 equiv DTC
and Pd. These results support possible formation of DSF during
the Acm deprotection step and a critical role of DSF in the rapid
disulfide bond formation. Notably, external addition of
acetamidomethyl alcohol (2 equiv), which is one of the products
of the deprotection step, [13a] to the reaction mixture had no effect
on the oxidation rate. To further support the requirement of
Cys(Acm) for the generation of DSF assisted by Pd, we treated
unprotected oxytocin peptide with external peptide containing
Cys(Acm) in the presence of PdCl2 and DTC, which led to a
complete disulfide bond formation within 1.5 h compared to
overnight treatment in the absence of Cys(Acm) (Supporting
Information, Figure S14).
addition of 25 equiv DTC and 2 equiv DSF for another 5 min to
furnish the desired oxidized product (Supporting Information,
S23).
In summary we have established the use of Pd chemistry,
for first time, in the context of disulfide bridge formation within
peptides and proteins under mild and aqueous conditions. Our
results revealed unprecedented enhanced oxidation of
Cys(Acm) residues to the corresponding disulfide mediated by
Pd and DTC in a highly efficient manner and was found to be
compatible with one-pot total chemical synthesis of proteins as
exemplified with Trx-1. In this process, the generation of DSF
appears to be the key reagent for the rapid disulfide bond
formation. Our study also shows the first utilization of DSF as an
efficient and mild reagent for rapid disulfide bond formation in
peptide and protein synthesis under aqueous media. This
approach has the potential to be further expanded to generate
multiple disulfide bonds in peptides and proteins in
regioselective manner and one-pot operation
a
Based on these observations we propose a mechanism in
which after the rapid Acm deprotection step the bound Pd to the
unprotected thiolate undergoes complexation with the added
DTC. Such a complex could contain Pd and a DTC ligand on
each Cys of the unprotected peptide. Indeed, we were able to
isolate such an intermediate in the case of Trx-1 and
characterize it by mass spectrometry, which matches the mass
of the Trx-1 in addition to two Pd and two DTC ligands (3,
Scheme 1). Moreover, this isolated intermediate in presence of
excess Pd and DTC was converted to the oxidized Trx-1 product
within 30 min (Supporting Information, Figure S12). Therefore,
we also propose that such an intermediate in presence of
excess DTC collapses rapidly to the free peptide (4, Scheme 1)
and generate DSF, similar to the CuII case, in yet unclear
mechanism.[29] Subsequently, the free Cys react with DSF to
facilitate rapid disulfide bond formation (Scheme 1), which is the
most stable disulfide bond of which DTC cannot further reduce.
Due to the quantitative appearance of the free thiols
immediately after adding the DTC to the bound Pd-peptide (4,
Scheme 1) as we detected by HPLC-MS analysis (Supporting
Information, Figure S11), it seems that the generation of DSF
after the removal of the Acm is a rate-limiting step. Hence, the
additional 25 min that was required to obtain the product is likely
the time, which is needed to generate enough DSF for the rapid
oxidation step. We also observed a clear correlation between
the amount of DSF and the oxidation rate, as the addition of 1
equiv of DSF led to a relatively slower oxidation of oxytocin,
which completed within 45 min compared to 5 min with 2 equiv.
This is also consistent with our study on the oxytocin peptide
bearing one Cys(Acm) where the oxidation step was slower and
required 2 h compared to 30 min in the case of oxytocin with two
Cys(Acm), (Figure 4). Based on these results we developed an
optimized protocol in which Cys(Acm) deprotection is performing
by treatment with 10 equiv PdCl2 for 5 min followed by the
Scheme 1. Proposed mechanism for disulfide bond formation via Pd
mediated Acm removal and DSF formation.
in light of our recent findings regarding the on demand
deprotection of various Cys PGs with Pd.[13h] These exciting
directions and others are currently under investigation in our
laboratory.
Acknowledgements
A. Brik holds The Jordan and Irene Tark Academic Chair. H. S.
is supported in part at the Technion by a Technion-Guangdong
Fellowship.
Keywords: desulfurization • disulfide bonds • palladium • protein
synthesis • S-acetamidomethyl-cysteine
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10.1002/anie.201900988
Angewandte Chemie International Edition
COMMUNICATION
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COMMUNICATION
S-Acetamidomethyl protects and
connects: The chemistry of palladium
and diethyldithiocarbamate was
applied for the efficient and rapid
conversion of S-Acetamidomethyl-
Cysteine to the corresponding
Shay Laps†, Hao Sun†, Guy Kamnesky,
Ashraf Brik*
Page No. – Page No.
Palladium Mediated Direct Disulfide
Bond Formation in Proteins
disulfide, in which disulfiram is
suggested to play a key role in this
process. This approach enabled one-
pot synthesis of disulfide bond
contaiting pepide/protein under mild
and fully aquous conditions.
Containing S-Acetamidomethyl-
Cysteine under Aqueous Conditions
This article is protected by copyright. All rights reserved.