Oxidative deselenization of selenocysteine: Applications for programmed ligation at serine

Article abstract of DOI:10.1002/anie.201504639

Despite the unique chemical properties of selenocysteine (Sec), ligation at Sec is an under-utilized methodology for protein synthesis. We describe herein an unprecedented protocol for the conversion of Sec to serine (Ser) in a single, high-yielding step.

Full text of DOI:10.1002/anie.201504639

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Angewandte
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International Edition: DOI: 10.1002/anie.201504639
German Edition: DOI: 10.1002/ange.201504639
Protein Ligation
Oxidative Deselenization of Selenocysteine: Applications for
Programmed Ligation at Serine
Lara R. Malins, Nicholas J. Mitchell, Sheena McGowan, and Richard J. Payne*
Abstract: Despite the unique chemical properties of seleno-
cysteine (Sec), ligation at Sec is an under-utilized methodology
for protein synthesis. We describe herein an unprecedented
protocol for the conversion of Sec to serine (Ser) in a single,
high-yielding step. When coupled with ligation at Sec, this
transformation provides a new approach to programmed
ligations at Ser residues. This new reaction is compatible with
a wide range of functionality, including the presence of
unprotected amino acid side chains and appended glycans.
The utility of the methodology is demonstrated in the rapid
synthesis of complex glycopeptide fragments of the epithelial
glycoproteins MUC5AC and MUC4 and through the total
synthesis of the structured, cysteine (Cys)-free protein eglin C.
A major drawback of late-stage global desulfurization
protocols is the need to protect native and structurally
important Cys residues within the target sequence,[7] which
would be concomitantly desulfurized to Ala if left unpro-
tected. The recent report of peptide ligation at selenocysteine
(Sec)[8] coupled with selective deselenization (in the presence
of unprotected Cys residues) by treatment with tris-(2-
carboxyethyl)phosphine (TCEP) and dithiothreitol (DTT),[9]
provided, for the first time, a completely chemoselective
approach to Ala residues at the ligation junction (Sche-
me 1A). Mechanistically, the selective deselenization is
thought to proceed via a radical mechanism (through an
alanyl radical intermediate), taking advantage of the weak
nature of the carbon–selenium bond as well as the ability to
form selenium-centered radicals preferentially over sulfur-
centered radicals.[9] This key discovery laid the intellectual
framework for the development of ligation–deselenization
chemistry at unnatural selenol-derived amino acids,[10] includ-
ing in our laboratory at phenylalanine (Phe).[10b]
During the course of our investigations into the chemo-
selective deselenization of b-selenol Phe-containing peptides,
we observed the formation of minor hydroxylation byprod-
ucts consistent with the formation of diastereomeric b-
hydroxy Phe (Scheme 1B).[10b,11] Invoking a similar mecha-
nism to that proposed for the chemoselective deselenization
of Sec,[9] we presumed that this intriguing byproduct resulted
from reaction of a benzylic radical with dissolved oxygen in
the reaction media. Although an undesirable byproduct in the
context of ligation-deselenization chemistry at Phe, we
hypothesized that the development of an equivalent reaction
at Sec residues could provide a programmed approach to
access serine (Ser) residues at the ligation junction through
a ligation, followed by Sec to Ser transformation (Sche-
me 1C). It should be noted that other strategies for ligation at
Ser junctions have recently been explored, including the use
of thiol auxiliaries linked via the Ser side-chain,[12] ligation at
Ser through the rearrangement of O-acyl isopeptides[13] or
direct ligation with activated salicylaldehyde (SAL) esters.[14]
While these protocols enable ligation, there are limitations
associated with each, namely lengthy reaction times[12] and
a reliance on high substrate concentrations in non-aqueous
media.[12b,13,14] Recently, a procedure for the conversion of Cys
to Ser has been developed by Okamoto and Kajihara.[15]
Although this is a powerful transformation, the requirement
for multiple steps (including intermediary HPLC purifica-
tions) and the use of toxic reagents (e.g. CNBr)[15] to promote
the desired substitution are considerable drawbacks. Con-
current to this work, Bode and co-workers described an
effective ligation at Ser through application of an oxazetidine
amino acid derivative in ketoacid–hydroxylamine ligation
T
he advent of native chemical ligation,[1] a method for the
mild and chemoselective condensation of two peptide frag-
ments to generate larger peptides and proteins, has enabled
access to numerous protein targets through chemical syn-
thesis.[2] The methodology involves the reaction of a peptide
bearing an N-terminal Cys residue with a C-terminal peptide
thioester in an initial transthioesterification reaction, fol-
lowed by a rapid S!N acyl shift to generate a native amide
bond in aqueous media and at neutral pH. The transformation
takes place in the presence of unprotected amino acid side
chains and is tolerant of additional functionality, including the
presence of a diverse array of post-translational modifica-
tions.[2,3]
Efforts focused on the extension of native chemical
ligation to include select non-Cys ligation junctions have
recently converged on ligation–desulfurization chemistry,[4] in
which an amino acid bearing a reactive thiol auxiliary
facilitates the ligation of two peptide fragments, with a sub-
sequent desulfurization[5] generating a native amino acid
residue. First demonstrated in the conversion of Cys to
alanine (Ala),[5a] synthetic access to thiol-derived proteino-
genic amino acids has recently facilitated the preparation of
a variety of targets via non-Cys ligation disconnections.[6]
[*] Dr. L. R. Malins,[+] Dr. N. J. Mitchell,[+] Prof. R. J. Payne
School of Chemistry, The University of Sydney
NSW 2006 (Australia)
E-mail: richard.payne@sydney.edu.au
Homepage: http://sydney.edu.au/science/chemistry/~payne/
index.html
Dr. S. McGowan
Department of Biochemistry and Molecular Biology
Monash University
Melbourne, VIC 3800 (Australia)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201504639.
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reaction was also tolerant of a wide range of pH
values (pH 4–7). To probe the reaction on
a preparative scale, we ligated selenopeptide
1 and thioester 2 (Ac-LYRANA-SR) in the
presence of excess 4-mercaptophenylacetic acid
(MPAA) to afford peptide 3 as the selenyl-
MPAA sulfide in 72% isolated yield (Sche-
me 2A). Upon treatment of peptide 3 with the
optimized Sec to Ser conditions (50 equiv TCEP,
50 equiv Oxone, aqueous buffer: 120 mm
Gn·HCl, 2 mm Na2HPO4, final concentration of
peptide 3 = 100 mm, pH 4.2–4.5), we were pleased
to observe rapid (t < 1 h) and clean conversion to
the desired Ser-containing product 4, which was
isolated in 90% yield following HPLC purifica-
tion, along with the formation of the TCEP-
derived phosphine selenide
5 (Scheme 2B).
Importantly, the identity and stereochemical
integrity of peptide
4 was confirmed by
1H NMR analysis through comparison with
authentic peptide standards bearing both d-
and l-Ser at the ligation junction (see Figure S45,
Supporting Information). It should also be noted
Scheme 1. Ligation–deselenization at A) Sec and B) b-selenol Phe. C) Proposed Sec-
mediated ligation followed by Sec to Ser conversion.
chemistry.[16] In this study, we describe the development of an
operationally simple Sec to Ser conversion under mild
conditions in a single post-ligation transformation. This
disclosure is an important addition to the currently available
tools for peptide and protein ligation and underscores the
unique chemical diversity offered by the strategic incorpo-
ration of Sec residues into target peptides and proteins.
Having attributed the minor hydroxylation pathway in our
b-selenol Phe work (Scheme 1B) to dissolved oxygen in the
reaction media, our initial examination of the proposed Sec to
Ser conversion began with the treatment of selenopeptides
with TCEP in oxygen-saturated buffer (see Figure S2, Sup-
porting Information). Under these conditions, we observed
the desired transformation together with Sec to Ala con-
version, akin to standard deselenization, as well as a number
of backbone cleavage byproducts. Difficulties in optimizing
the use of oxygen gas to maintain precise control of
stoichiometry prompted the exploration of an alternative
oxidant. Our focus turned to Oxone,[17] a commercially
available, bench stable and water-soluble oxidant. Impor-
tantly, there is also precedence for the use of Oxone in
peptide chemistry and the compatibility of the reagent with
proteinogenic amino acid side-chains.[18]
that this operationally simple oxidative deselenization proto-
col proceeded in an open-air flask without the need for
tedious solvent degassing.
We began our studies by treating model selenopeptides
(on analytical scales) with excess TCEP (50 equiv) and Oxone
(50 equiv) at a variety of concentrations and pH values (see
Supporting Information, Figure S3–S5). We found that opti-
mal conditions involved first solubilizing the peptide in
denaturing buffer (6m Gn·HCl, 0.1m Na2HPO4, 5 mm with
respect to the selenopeptide) followed by dilution with H2O
to give a final peptide concentration of 100 mm before the
simultaneous addition of TCEP (50 equiv) and Oxone
(50 equiv). At final concentrations of selenopeptide greater
than 100 mm, we observed some deselenization to yield the
corresponding Ala residue, while reactions also proceeded
smoothly at peptide concentrations lower than 100 mm. The
Scheme 2. A) Ligation of selenopeptide 1 and thioester 2 and Sec to
Ser conversion. B) Crude analytical HPLC trace (t=1 h) of Sec to Ser
conversion of peptide 3 to afford product 4 (0 to 50% MeCN over
30 min, l=230 nm).
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Table 1: Scope of the Sec to Ser conversion.
Intrigued by the mechanistic considerations of this new
transformation, we prepared model dipeptide dimer 6 bearing
an unnatural N-terminal b-methyl l-Sec analogue (Scheme 3,
see Supporting Information for synthetic details), with R-
configuration at the b-position, to enable investigation of the
stereochemical outcome at this site upon reaction with TCEP
and Oxone. Treatment of 6 under the optimized Sec to Ser
conditions revealed a complete loss of stereochemical integ-
rity at the b-position to afford 7 as a 1:1 mixture of the l-Thr
and l-allo-Thr products. The loss of stereochemistry at the b-
position (but not the a-position) is consistent with an alkyl
radical intermediate rather than the more common selenium
oxidation-nucleophilic displacement or oxidation-elimination
pathways. In addition, formation of phosphine selenide 5 (see
Scheme 2) is consistent with the radical-based mechanisms
proposed in the phopshine-mediated desulfurization of Cys[5b]
and deselenization of Sec.[9]
Entry USPZYS
Thioester Ligation product/ Sec to Ser Product/
(X)
Yield [%][a]
Yield [%][a]
1
2
3
4
5
6
7
8
9
1: Z=G
1: Z=G
1: Z=G
1: Z=G
1: Z=G
2: X=A
3: 72
4: 90
8: X=G 16: 93
9: X=M 17: 77
10: X=F 18: 93
11: X=L 19: 76
24: 83
25: 97[b,c]
26: 93
27: quant.[b]
28: 93
12: Z=K 10: X=F 20: 73
13: Z=H 2: X=A
14: Z=W 2: X=A
15: Z=C 2: X=A
21: 74
22: 97
23: 85
29: 95
30: 80
31: 80[d]
[a] Yields of isolated product. [b] Additional TCEP (10 equiv) and Oxone
(10 equiv) added at t=1 h. [c] Product of the Sec to Ser conversion
possessed an oxidized Met residue. Reduction to the native peptide
product was achieved in quant. yield under the following conditions:
NH4I (20 equiv), dimethyl sulfide (20 equiv), TFA, 08C, 30 min.[19]
[d] Additional TCEP (50 equiv) and Oxone (50 equiv) added at t=1 h;
product isolated along with 20% of a product corresponding to Sec to
Ala conversion.
Scheme 3. Synthesis of dipeptide 6 to probe the Sec to Ser mecha-
nism.
To probe the scope of the new reaction, we next prepared
a number of selenopeptide substrates by ligation chemistry
between model thioesters 2, 8–11 and model selenopeptides 1,
12–15 (see Supporting Information and Table 1), bearing
a range of functionality and oxidation prone amino acids (e.g.
Met, Phe, Trp, His, Lys and Cys). In all cases, Sec-mediated
ligations proceeded in less than 18 h to afford the desired
products (16–23), as the selenyl-MPAA sulfide adducts (e.g.
16-MPAA) and/or the corresponding symmetrical diselenide
dimers (e.g. 16-dimer), in high isolated yields (72–97%, see
Table 1 and Supporting information for details). Ligation
products were next treated with TCEP and Oxone under the
optimized conditions. Gratifyingly, reactions proceeded in
high isolated yields in all cases (80%–quant.) to afford the
target Ser-containing peptides (24–31, see Table 1). Both the
MPAA ligation adducts and the diselenide dimers were found
to be suitable substrates for the Sec to Ser conversion (see
Supporting Information for details). In most cases, reactions
were complete in less than 1 h after a single dose of TCEP and
Oxone. Due to the competing inactivation of TCEP through
oxidation by Oxone, however, some substrates required an
additional dose (10 equiv TCEP and 10 equiv Oxone) to
reach completion (see entries 3 and 5, Table 1).
dimethyl sulfide in TFA (trifluoroacetic acid), robust reduc-
tion conditions described by Hackenberger (see Supporting
Information).[19] The presence of a Cys residue in the target
peptide sequence (entry 9) reduced the rate of the Sec to Ser
transformation, which reached only 50% conversion after the
first dose of TCEP and Oxone. However, a second dose of
TCEP (50 equiv) and Oxone (50 equiv) enabled complete
consumption of the ligation product to yield the target Sec to
Ser product 31 in 80% yield together with 20% conversion to
the corresponding Ala peptide, as determined by HPLC-MS
analysis. Given that a model peptide bearing a Cys residue at
the ligation junction (in the absence of Sec) was completely
inert to the oxidation conditions (see Figure S23, Supporting
Information), we hypothesized that conversion of Sec to Ala
in the presence of Cys might be proceeding through a standard
deselenization pathway (see Scheme 1A), whereby the puta-
tive alanyl radical intermediate is appropriately positioned
for rapid intramolecular H-abstraction from the neighboring
Cys thiol moiety. An investigation into the effect of the
relative positioning of Sec and Cys within a target peptide on
the outcome of the Sec to Ser transformation further supports
the likelihood of an intramolecular H-abstraction event. For
four model polypeptides with differential spacing between the
Sec and Cys residues (spaced 4, 9, 15 or 27 amino acids apart,
see Supporting Information for details), treatments with
TCEP and Oxone led to increasing amounts of Ala byproduct
The transformation was found to be compatible with
peptides bearing internal Lys, His and Trp residues (entries 6–
8), which proceeded without unwanted side-chain oxidation.
Unsurprisingly, Sec to Ser conversion of ligation product 17-
MPAA, bearing an internal Met residue, led to concomitant
oxidation of the Met thioether side-chain to the correspond-
ing sulfoxide. However, facile reduction to the native Met
side-chain was achieved in quantitative yield using NH4I-
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formation (relative to the target Ser product) as the internal
Cys residue was placed closer along the peptide backbone to
the Sec residue. Interestingly, peptides with Cys residues in
close proximity to Sec also resulted in lower overall con-
versions of the selenopeptide starting material, suggesting
that the intramolecular proximity of an H-atom donor may
also inhibit the rate of the transformation. Conversely, the
lack of observed Ala formation or significant effect on
reaction rate in an intermolecular competition study between
a Sec-containing model peptide and a Cys analogue (see
Figure S24, Supporting Information) suggests that the corre-
sponding intermolecular process is unlikely.
containing a native Ser residue at the ligation junction, in an
excellent 83% isolated yield.
MUC4 thioester 36 and selenopeptide 37, each decorated
with three copies of the Thr-linked T antigen disaccharide,
were next synthesized and ligated. The reaction was complete
in 24 h to afford glycopeptide 38, bearing a selenyl-MPAA
sulfide, in an excellent 86% isolated yield. Upon two simple
treatments with Oxone and TCEP, we were pleased to
observe the clean conversion of peptide 38 to the correspond-
ing MUC4 32-mer 39 (Scheme 4B), comprised of two MUC4
VNTRs and six copies of the Thr-derived T antigen (72%
isolated yield).
Given our interest in exploiting the over-expression and
aberrant glycosylation of mucin epithelial proteins for the
purpose of developing novel synthetic cancer vaccine candi-
dates,[20] we were next interested in utilizing the Sec to Ser
transformation to rapidly access multiple copies of the serine-
rich variable number tandem repeat (VNTR) sequences of
the MUC5AC and MUC4 glycoproteins. Each target peptide
would bear a defined glycosylation pattern, consisting of
multiple copies of the disaccharide (T) or monosaccharide
(TN) tumor-associated carbohydrate antigens (TACAs). To
this end, we first synthesized, via divergent Fmoc-SPPS, the
target MUC5AC thioester 32 and MUC5AC selenopeptide
33, both of which are derived from the eight amino acid
MUC5AC VNTR sequence and bear two copies each of the
Thr-derived TN antigen (see Scheme 4 and Supporting
Information). Peptides 32 and 33 were then ligated to afford
selenyl-MPAA sulfide 34 after 38 h and in 65% isolated yield.
Ligation product 34 was subjected to the optimized Sec to Ser
conditions to afford peptide 35, a MUC5AC VNTR dimer,
To probe the utility of the new transformation in the
synthesis of a protein target, we finally embarked upon the
synthesis of eglin C, a structurally ordered serine protease
inhibitor derived from the leech Hirudo medicinalis.[21]
Recombinantly, the N-acetylated protein is produced,[22]
which is functionally equivalent to the native protein. As
a Cys-free protein, eglin C maintains a three-dimensional
structure through a network of electrostatic interactions
rather than disulfide bridges and is highly resistant to
denaturation.[21] The lack of Cys residues in this 70-amino
acid target, however, also precludes its construction using
traditional native chemical ligation methods. There are also
no centrally placed Ala residues to facilitate a ligation-
desulfurization-based assembly. As such, only Cys mutants of
eglin C, in which the native Ser residue at position 41 has been
replaced by a Cys residue (S41C), have been accessible using
ligation chemistry.[23] Taking advantage of the centrally
located Ser-41, we envisaged the first ligation-based approach
to the native sequence using a tandem Sec ligation–Sec to Ser
Scheme 4. A) Synthesis of MUC5AC VNTR dimer, a=6m Gn·HCl, 0.1m Na2HPO4, 5 mm conc. of peptide 33, 200 mm MPAA, pH 7.2, 38 h,
b=TCEP (3”50 equiv), Oxone (3”50 equiv), aqueous buffer (120 mm Gn·HCl, 2 mm Na2HPO4), 100 mm conc. of peptide 34, RT, 6 h; B) Synthesis
of MUC4 VNTR dimer, c=6m Gn·HCl, 0.1m Na2HPO4, 5 mm conc. of peptide 37, 200 mm MPAA, pH 7.2, 24 h, d=TCEP (2”50 equiv), Oxone
(2”50 equiv), aqueous buffer (120 mm Gn·HCl, 2 mm Na2HPO4), 100 mm conc. of peptide 38, RT, 3.5 h; C) Synthesis of eglin C, e=6m Gn·HCl,
0.1m Na2HPO4, 200 mm MPAA, 70 mm TCEP, 210 mm TCEP=Se, 50 mm sodium ascorbate, 308C, pH 6.5, 30 h, f=TCEP (5”50 equiv), Oxone
(5”50 equiv), 6m Gn·HCl, 0.1m Na2HPO4, 100 mm conc. of peptide 42, pH 4.8–4.9, RT, 6 h; R=4-mercaptophenylacetic acid.
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conversion. Accordingly, we first prepared thioester 40
(eglin C 1–40) and Sec-containing peptide 41 (eglin C 41–
70) using Fmoc-SPPS (Scheme 4C). Adoption of Sec-ligation
conditions recently reported by Melnyk and co-workers,[24]
which utilize a more reducing environment (denaturing buffer
in the presence of 200 mm MPAA, 70 mm TCEP, 210 mm
thews for assistance with circular dichroism spectroscopy, and
Dr. Nick Proschogo for technical support.
Keywords: deselenization · glycopeptides · ligation ·
native chemical ligation · peptides
How to cite: Angew. Chem. Int. Ed. 2015, 54, 12716–12721
Angew. Chem. 2015, 127, 12907–12912
=
TCEP Se and 50 mm sodium ascorbate), afforded the
MPAA-ligation product 42 in 36% isolated yield. Initial
subjection of peptide 42 to the Sec to Ser transformation
resulted in substantial peptide cleavage byproducts as well as
products consistent with Sec to Ala conversion and off target
oxidation. However, upon carrying out the reaction entirely
in denaturing buffer (6m Gn·HCl/0.1m Na2HPO4, pH 4.8–
4.9), we were pleased to observe clean conversion of peptide
42 to the target Ser-containing product 43 upon treatment
with TCEP and Oxone (5 ” 50 equiv each). We hypothesize
that in the case of eglin C, high concentrations of chaotropic
salts serve to disrupt sequence-specific electrostatic interac-
tions and expose the internal Sec residue, which appears to be
a critical factor for efficient oxidative deselenization trans-
formations. Following Sec to Ser conversion, the full-length
protein was isolated in 66% yield. Interrogation of the
structure of 43 using circular dichroism revealed structural
elements consistent with an authentic sample of recombinant
eglin C (see Figure S42 and S43, Supporting Information). In
addition, the synthetic protein exhibited inhibitory activity
against both cathepsin G and elastase, albeit less than that
measured for the recombinant protein (see Figure S44,
Supporting Information). Future work in our laboratory will
further probe the utility of the oxidative deselenization
reaction for the synthesis of folded protein targets.
In summary, we have disclosed an unprecedented and
mechanistically distinct oxidative deselenization reaction for
the conversion of Sec to Ser in a single-step within unpro-
tected peptides and proteins. The high-yielding and opera-
tionally simple protocol enables programmed ligations at Ser
junctions in aqueous media (with and without chaotropic
salts), under mild conditions. We have demonstrated the
compatibility of the transformation at a range of pH values
and probed the remarkable chemoselectivity of the trans-
formation in the presence of potentially reactive proteino-
genic amino acid side chains. Extension of the methodology to
MUC5AC and MUC4 VNTR glycopeptide targets and in the
total synthesis of the Cys-free protein eglin C further confirms
the compatibility of the reaction protocol with complex
(glyco)peptide and protein systems and highlights the utility
of the method as a complement to native chemical ligation for
the construction of Cys-free protein targets. We further
envisage that the mild reaction conditions and wide functional
group tolerance may render this novel transformation gen-
erally applicable for the synthesis of functionalized small
molecules. Studies of this nature are currently underway in
our laboratory.
[1] P. E. Dawson, T. W. Muir, I. Clark-Lewis, S. B. H. Kent, Science
1994, 266, 776 – 779.
[2] S. B. Kent, Chem. Soc. Rev. 2009, 38, 338 – 351.
[3] a) D. P. Gamblin, E. M. Scanlan, B. G. Davis, Chem. Rev. 2009,
109, 131 – 163; b) R. J. Payne, C. H. Wong, Chem. Commun.
2010, 46, 21 – 43; c) L. Raibaut, N. Ollivier, O. Melnyk, Chem.
Soc. Rev. 2012, 41, 7001 – 7015.
[4] a) H. Rohde, O. Seitz, Biopolymers 2010, 94, 551 – 559; b) P. E.
Dawson, Isr. J. Chem. 2011, 51, 862 – 867; c) C. P. R. Hacken-
berger, D. Schwarzer, Angew. Chem. Int. Ed. 2008, 47, 10030 –
10074; Angew. Chem. 2008, 120, 10182 – 10228; d) L. R. Malins,
R. J. Payne, Curr. Opin. Chem. Biol. 2014, 22, 70 – 78.
[5] a) L. Z. Yan, P. E. Dawson, J. Am. Chem. Soc. 2001, 123, 526 –
533; b) Q. Wan, S. J. Danishefsky, Angew. Chem. Int. Ed. 2007,
46, 9248 – 9252; Angew. Chem. 2007, 119, 9408 – 9412.
[6] a) D. Crich, A. Banerjee, J. Am. Chem. Soc. 2007, 129, 10064 –
10065; b) J. Chen, Q. Wan, Y. Yuan, J. L. Zhu, S. J. Danishefsky,
Angew. Chem. Int. Ed. 2008, 47, 8521 – 8524; Angew. Chem.
2008, 120, 8649 – 8652; c) C. Haase, H. Rohde, O. Seitz, Angew.
Chem. Int. Ed. 2008, 47, 6807 – 6810; Angew. Chem. 2008, 120,
6912 – 6915; d) Z. Harpaz, P. Siman, K. S. A. Kumar, A. Brik,
ChemBioChem 2010, 11, 1232 – 1235; e) R. L. Yang, K. K.
Pasunooti, F. P. Li, X. W. Liu, C. F. Liu, J. Am. Chem. Soc.
2009, 131, 13592 – 13593; f) K. S. Ajish Kumar, M. Haj-Yahya, D.
Olschewski, H. A. Lashuel, A. Brik, Angew. Chem. Int. Ed. 2009,
48, 8090 – 8094; Angew. Chem. 2009, 121, 8234 – 8238; g) J. Chen,
P. Wang, J. L. Zhu, Q. Wan, S. J. Danishefsky, Tetrahedron 2010,
66, 2277 – 2283; h) L. R. Malins, K. M. Cergol, R. J. Payne,
ChemBioChem 2013, 14, 559 – 563; i) R. E. Thompson, B. Chan,
L. Radom, K. A. Jolliffe, R. J. Payne, Angew. Chem. Int. Ed.
2013, 52, 9723 – 9727; Angew. Chem. 2013, 125, 9905 – 9909;
j) L. R. Malins, K. M. Cergol, R. J. Payne, Chem. Sci. 2014, 5,
260 – 266; k) S. Y. Shang, Z. P. Tan, S. W. Dong, S. J. Danishefsky,
J. Am. Chem. Soc. 2011, 133, 10784 – 10786; l) K. M. Cergol,
R. E. Thompson, L. R. Malins, P. Turner, R. J. Payne, Org. Lett.
2014, 16, 290 – 293; m) P. Siman, S. V. Karthikeyan, A. Brik, Org.
Lett. 2012, 14, 1520 – 1523; n) Z. P. Tan, S. Y. Shang, S. J.
Danishefsky, Angew. Chem. Int. Ed. 2010, 49, 9500 – 9503;
Angew. Chem. 2010, 122, 9690 – 9693; o) L. R. Malins, R. J.
Payne, Aust. J. Chem. 2015, 68, 521 – 537; p) L. R. Malins, A. M.
Giltrap, L. J. Dowman, R. J. Payne, Org. Lett. 2015, 17, 2070 –
2073; q) K. Sato, K. Kitakaze, T. Nakamura, N. Naruse, K.
Aihara, A. Shigenaga, T. Inokuma, D. Tsuji, K. Itoh, A. Otaka,
Chem. Commun. 2015, 51, 9946 – 9948.
[7] To the best of our knowledge, selective desulfurization reactions
have only been reported for ligation–desulfurization at b-thiol
Asp residues (see reference [6i]).
[8] a) R. Quaderer, A. Sewing, D. Hilvert, Helv. Chim. Acta 2001,
84, 1197 – 1206; b) R. J. Hondal, B. L. Nilsson, R. T. Raines, J.
Am. Chem. Soc. 2001, 123, 5140 – 5141; c) M. D. Gieselman, L.
Xie, W. A. van der Donk, Org. Lett. 2001, 3, 1331 – 1334.
[9] N. Metanis, E. Keinan, P. E. Dawson, Angew. Chem. Int. Ed.
2010, 49, 7049 – 7053; Angew. Chem. 2010, 122, 7203 – 7207.
[10] a) S. D. Townsend, Z. Tan, S. Dong, S. Shang, J. A. Brailsford,
S. J. Danishefsky, J. Am. Chem. Soc. 2012, 134, 3912 – 3916;
b) L. R. Malins, R. J. Payne, Org. Lett. 2012, 14, 3142 – 3145;
Acknowledgements
We acknowledge an Australian Research Council Discovery
Project for funding (DP130101984), Prof. Jacqueline Mat-
12720 www.angewandte.org
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Angewandte
Chemie
c) L. R. Malins, N. J. Mitchell, R. J. Payne, J. Pept. Sci. 2014, 20,
64 – 77.
Bode, Angew. Chem. Int. Ed. 2012, 51, 5114 – 5118; Angew.
Chem. 2012, 124, 5204 – 5208.
[11] Dawson and co-workers also observed a small amount of Sec to
Ser conversion in their unoptimized deselenization conditions
(see reference [9]).
[12] a) M. Y. Lutsky, N. Nepomniaschiy, A. Brik, Chem. Commun.
2008, 1229 – 1231; b) H. Hojo, C. Ozawa, H. Katayama, A. Ueki,
Y. Nakahara, Y. Nakahara, Angew. Chem. Int. Ed. 2010, 49,
5318 – 5321; Angew. Chem. 2010, 122, 5446 – 5449.
[13] M. El Khatib, M. Elagawany, F. Jabeen, E. Todadze, O.
Bolꢀshakov, A. Oliferenko, L. Khelashvili, S. A. el-Feky, A.
Asiri, A. R. Katritzky, Org. Biomol. Chem. 2012, 10, 4836 – 4838.
[14] a) Y. Zhang, C. Xu, H. Y. Lam, C. L. Lee, X. Li, Proc. Natl.
Acad. Sci. USA 2013, 110, 6657 – 6662; b) X. Li, H. Y. Lam, Y.
Zhang, C. K. Chan, Org. Lett. 2010, 12, 1724 – 1727.
[19] C. P. Hackenberger, Org. Biomol. Chem. 2006, 4, 2291 – 2295.
[20] a) D. M. McDonald, B. L. Wilkinson, L. Corcilius, M. Thaysen-
Andersen, S. N. Byrne, R. J. Payne, Chem. Commun. 2014, 50,
10273 – 10276; b) B. L. Wilkinson, S. Day, R. Chapman, S.
Perrier, V. Apostolopoulos, R. J. Payne, Chem. Eur. J. 2012, 18,
16540 – 16548; c) B. L. Wilkinson, S. Day, L. R. Malins, V.
Apostolopoulos, R. J. Payne, Angew. Chem. Int. Ed. 2011, 50,
1635 – 1639; Angew. Chem. 2011, 123, 1673 – 1677.
[21] a) U. Seemüller, M. Meier, K. Ohlsson, H. P. Muller, H. Fritz,
Hoppe-Seyler’s Z. Physiol. Chem. 1977, 358, 1105 – 1107; b) U.
Seemüller, M. Eulitz, H. Fritz, A. Strobl, Hoppe-Seyler’s Z.
Physiol. Chem. 1980, 361, 1841 – 1846.
[22] H. Rink, M. Liersch, P. Sieber, F. Meyer, Nucleic Acids Res. 1984,
12, 6369 – 6387.
[23] W. Y. Lu, M. A. Starovasnik, J. J. Dwyer, A. A. Kossiakoff, S. B.
Kent, W. Lu, Biochemistry 2000, 39, 3575 – 3584.
[24] N. Ollivier, A. Blanpain, E. Boll, L. Raibaut, H. Drobecq, O.
Melnyk, Org. Lett. 2014, 16, 4032 – 4035.
[15] R. Okamoto, Y. Kajihara, Angew. Chem. Int. Ed. 2008, 47, 5402 –
5406; Angew. Chem. 2008, 120, 5482 – 5486.
[16] I. Pusterla, J. W. Bode, Nature Chem. 2015, 7, 668 – 672.
[17] R. J. Kennedy, A. M. Stock, J. Org. Chem. 1960, 25, 1901 – 1906.
[18] a) L. Ju, A. R. Lippert, J. W. Bode, J. Am. Chem. Soc. 2008, 130,
4253 – 4255; b) J. W. Bode, R. M. Fox, K. D. Baucom, Angew.
Chem. Int. Ed. 2006, 45, 1248 – 1252; Angew. Chem. 2006, 118,
1270 – 1274; c) A. O. Ogunkoya, V. R. Pattabiraman, J. W. Bode,
Angew. Chem. Int. Ed. 2012, 51, 9693 – 9697; Angew. Chem. 2012,
124, 9831 – 9835; d) V. R. Pattabiraman, A. O. Ogunkoya, J. W.
Received: May 22, 2015
Revised: July 8, 2015
Published online: September 1, 2015
Angew. Chem. Int. Ed. 2015, 54, 12716 –12721
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 12721