Site-Selective Modification of Peptides and Proteins via Interception of Free-Radical-Mediated Dechalcogenation

Article abstract of DOI:10.1002/anie.202006260

The development of site-selective chemistry targeting the canonical amino acids enables the controlled installation of desired functionalities into native peptides and proteins. Such techniques facilitate the development of polypeptide conjugates to advance therapeutics, diagnostics, and fundamental science. We report a versatile and selective method to functionalize peptides and proteins through free-radical-mediated dechalcogenation. By exploiting phosphine-induced homolysis of the C?Se and C?S bonds of selenocysteine and cysteine, respectively, we demonstrate the site-selective installation of groups appended to a persistent radical trap. The reaction is rapid, operationally simple, and chemoselective. The resulting aminooxy linker is stable under a variety of conditions and selectively cleavable in the presence of a low-oxidation-state transition metal. We have explored the full scope of this reaction using complex peptide systems and a recombinantly expressed protein.

Full text of DOI:10.1002/anie.202006260

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Site-Selective Modification of Peptides and Proteins via
Interception of Free Radical-Mediated Dechalcogenation
Authors: Rhys Griffiths, Frances R. Smith, Jed E. Long, Huw Williams,
Robert Layfield, and Nicholas Mitchell
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202006260
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10.1002/anie.202006260
Angewandte Chemie International Edition
RESEARCH ARTICLE
Site-Selective Modification of Peptides and Proteins via
Interception of Free Radical-Mediated Dechalcogenation
Rhys Griffiths,[a] Frances R. Smith,[a] Jed E. Long,[a] Huw Williams,[b] Robert Layfield[c] and Nicholas J.
Mitchell*[a]
[a]
Dr Nicholas J. Mitchell, Mr Rhys Griffiths, Dr Frances R. Smith, Dr Jed E. Long
School of Chemistry
University of Nottingham
University Park, Nottingham, NG7 2RD, UK
*E-mail: nicholas.mitchell@nottingham.ac.uk
Dr Huw Williams
Biodiscovery Institute
University of Nottingham
[b]
[c]
University Park, Nottingham, NG7 2RD, UK
Prof. Robert Layfield
School of Life Sciences
University of Nottingham
University Park, Nottingham, NG7 2UH, UK
Supporting information for this article is given via a link at the end of the document.
Abstract: The development of site-selective chemistry, targeting the
canonical amino acids, enables the controlled installation of desired
functionality into native peptides and proteins. Such techniques
facilitate the development of polypeptide conjugates to advance
therapeutics, diagnostics and fundamental science. Herein, we report
a versatile and selective method to functionalize peptides and proteins
via the process of free radical-mediated dechalcogenation. By
exploiting phosphine-induced homolysis of the C-Se and C-S bonds
of selenocysteine and cysteine, respectively, we demonstrate the site-
selective installation of groups appended to a persistent radical trap.
The reaction is rapid, operationally simple, and chemoselective. The
resulting aminooxy linker is stable under a variety of conditions and
selectively cleavable in the presence of a low oxidation state transition
metal. We explore the full scope of this reaction using complex
peptide systems and a recombinantly expressed protein.
directed mutagenesis, reactions that target this residue have been
widely adopted throughout industry and academia.[6-7]
The ‘standard’ Cys-specific conjugation methods employ
electrophilic moieties such as a-halocarbonyl[7-8] and maleimide[7,
9]
groups. However, such components present chemoselectivity
and stability challenges, respectively. More recently, bromo-
maleimides,[10]
perfluoroaromatics,[11]
phosphonamidates,[12]
allenamides,[13] vinyl/alkynyl,[14] and acrylate groups[15] have been
investigated to selectively label Cys residues. Methods beyond
simple addition chemistry, such as thiol-ene/yne click reactions,[16]
metal-catalyzed[17] and metal-free arylation,[18] and oxidative
elimination to dehydroalanine (Dha) with subsequent Michael[19] or
radical addition,[20-22] have also been explored. Despite the broad
range of chemistry developed to target this residue, challenges
persist regarding reaction efficiency, chemoselectivity, tolerance,
operational simplicity, and conjugate stability. Thus, novel
methods are in high demand.
Ar
O
R
Introduction
X
N
Se
R
O
Cys-specific
chemistry
Sec-specific
chemistry
R
The diverse array of chemical functionality displayed by the
20 canonical amino acids presents both a challenge and an
opportunity for the site-selective functionalization of peptides and
proteins. A broad range of reactions have been reported to modify
the majority of the proteinogenic residues,[1-4] providing tools to
enable the study and manipulation of biological systems, and the
preparation of therapeutic and diagnostic agents. To be widely
applicable to peptide/protein bioconjugation such reactions must
be chemoselective, high yielding, rapid, and operationally simple.
The number of modified isoforms produced by a technique will be
dictated by both the chemoselectivity of the chemistry and the
relative abundance of the target amino acid. Reactions that select
the more abundant amino acids, such as lysine (Lys), which
accounts for approximately 6% of residues across the human
proteome, are liable to produce a mixture of isoforms which can
be challenging to purify.[2, 5] Conversely, the amino acid cysteine
(Cys) constitutes just 2% of our proteome. Due to this limited
presence, coupled with the enhanced nucleophilicity of the thiol
sidechain and ease of incorporation of non-native Cys via site-
S
N
N
O
S
R
O
R
NH
Se
Ar
S
O
S
Se
X = SH (Cys)/Se dimer (Sec)
Cys/Sec to
Dha
R
S
Michael
addition
Radical
addition
R
O
N
H
N
H
O
N
H
O
Dehydroalanine
(Dha)
Figure 1. Current approaches to selective peptide and protein modification at
selenocysteine (Sec) and cysteine (Cys) residues.
Selenocysteine (Sec) represents an alternative conjugation
handle to Cys. Known as the 21st proteinogenic amino acid,
biological expression of Sec is rare; only 25 selenoproteins have
1
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10.1002/anie.202006260
Angewandte Chemie International Edition
RESEARCH ARTICLE
been identified within the human proteome.[23] Incorporation of this
amino acid is facilitated by a dedicated suite of proteins and a Sec
Insertion Sequence Element (SECIS), a stem-loop RNA structure
that repurposes the opal codon (UGA) for Sec installation. Thus,
selenoproteins can be prepared using modified expression
techniques, albeit in lower yield than can be achieved with
standard recombinant expression.[24-27] The selenol moiety of the
Sec residue exhibits enhanced nucleophilicity over the thiol group
of Cys due to the increased polarizability of the selenium atom.[28]
With a low redox potential (-381 mV), Sec is readily oxidized, thus
its predominant role in nature is as a scavenger for damaging
oxidizing agents. Strategies to site-selectively functionalize Sec-
containing peptides and proteins include metal-catalyzed and
radical species will then abstract an H-atom from a thiol additive
to yield an Ala residue at this position. In the case of
deselenization, the alanyl radical can be trapped by a peroxide
salt (Oxone)[48-49] or O2[50] to install a hydroxyl group at the ligation
junction, and thus afford the amino acid serine (Ser) (fig. 2).
Successful trapping of the alanyl radical suggests potential scope
for the development of a novel bioconjugation strategy based on
free radical-mediated dechalcogenation. Previous research has
described the use of thiophosphonium salts to induce the
conversion of disulfides to thioethers[51] and also as a method to
deuterate Cys-containing peptides via desulfurization.[52] However,
to our knowledge, free radical-mediated dechalcogenation has not
been explored as a method to install groups of interest into
peptides and proteins. Interception of this pathway, using a
suitable functionalized trapping agent, would potentially be a
powerful addition to the synthetic methodology within this field.
metal-free
allenamidation.[30]
arylation,[29]
heteroarylation,
alkylation
and
Beyond applications in bioconjugation, both Cys and Sec
residues have been utilized to facilitate the chemical ligation of
peptide sequences. The technique of native chemical ligation
(NCL)[31-32] enables peptides bearing an N-terminal Cys residue to
be chemoselectively linked to sequences containing a C-terminal
thioester via a native amide bond. Further developments to this
powerful method[33] include the use of Sec-containing peptides[34-
36] and selenoester peptides to accelerate the ligation reaction.[37]
The development of diselenide-selenoester ligation (DSL)
employs both selenium-containing fragments to enable rapid,
additive-free peptide ligation.[38-43] Protocols that facilitate the post-
ligation conversion of internal Cys and Sec residues to alanine
(Ala), via desulfurization and deselenization respectively,[44-45]
permit peptide ligation at sites that contain this more abundant
residue. Further developments in this field include the utilization of
non-proteinogenic thiolated and selenolated residues, which,
when coupled with dechalcogenation,[46-47] grant access to a broad
range of ligation junctions, dramatically enhancing the scope of
the technique.
This work: Interception of dechalcogenation
using a persistent radical trap
N
TCEP
Mn(OAc)3
X
O
N
N
H
H
O
O
O
N
X = diselenide dimer (Sec)
X = SH (Cys)
• ‘Open flask’ reaction • 30 min - 4 hr reaction time • Tolerant to a range of
functionality Stereochemistry of the modified residue retained
•
•
Aminooxy linkage stable to pH, temperature, nucleophiles, UV, and
radicals • Selectively cleavable using Zn(0) in mildly acidic conditions
Figure 3. Trapping of the alanyl radical produced from free radical-mediated
deselenization/desulfurization at Sec/Cys residues using a TEMPO-based
persistent radical.
The persistent radical, TEMPO (2,2,6,6-tetramethyl-1-
piperidine-1-oxyl), would be an ideal trapping reagent in this
context due to the stability of the sterically shielded nitroxyl radical.
This reaction is likely to be chemoselective in the presence of
native proteinogenic chemical functionality and rapid, assuming it
can outcompete H-atom abstraction. Crucially, the generation and
trapping of a radical at the b-carbon of Sec or Cys, via the
described pathway, would maintain the integrity of the a-
stereocenter of the target residue. By applying this approach,
utilizing TEMPO derivatives that carry a broad range of desirable
moieties, we demonstrate the first example of site-selective
functionalization via trapping of free radical-mediated
dechalcogenation, representing an entirely novel method of
peptide and protein modification (fig. 3).
Se
HO
2
TCEP
N
H
N
Oxone/O2
H
O
O
Oxone/
O2
TCEP
R3P Se
CH2
-
Se PR3
N
H
N
H
O
O
Phosphoranyl radical
Alanyl radical
Figure 2. Proposed mechanism of free radical-mediated deselenization with
addition of Oxone or O2 to afford serine (Ser) at the site of deselenization.
Results and Discussion
It is proposed that the desulfurization and deselenization
pathways (mediated by a phosphine) proceed, in both cases, via
a free radical process. Desulfurization of Cys-containing peptides
requires the initial formation of a thiyl radical (usually via addition
of the initiator, VA-044) which adds into the phosphine (TCEP –
tris(2-carboxyethyl)phosphine) to form a phosphoranyl radical.
Sec undergoes this reaction spontaneously under ambient
conditions. Subsequent homolysis of the C-S/C-Se bond of the
phosphoranyl sulphide/selenide produces an alanyl radical (with
release of the phosphine sulfide/selenide). This intermediate
For our initial investigations into this concept, we synthesized the
small Sec-containing model peptide 1a (H-UAF-OMe) and
subjected it to standard deselenization conditions on an analytical
scale: 125 mM TCEP (50 molar eq.), 62.5 mM TEMPO (25 eq.) at
2.5 mM wrt the selenol monomer (1.25 mM wrt to the diselenide
dimer), in neutral buffer containing a high concentration of a
chaotropic salt (6 M Gdn•HCl, 0.1 M Na2HPO4, pH 6.5).
2
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10.1002/anie.202006260
Angewandte Chemie International Edition
RESEARCH ARTICLE
Table 1. Reaction exploration and optimization.
N
O
TCEP, TEMPO
+/- Mn(OAc)3,
Se
2
O
O
H
H
N
OMe
N
OMe
H2N
N
H2N
N
37 - 50 o
C
H
H
O
O
O
O
30 min - 6hrs
1a (2.5 - 0.2 mM wrt
selenol monomer)
2a
Entry
Peptide/
mM
Temp.
%
TCEP
TEMPO
eq.
Doping[b]
Additive
eq.
Full conversion
1a - 2a[c]
/ºC
37
37
37
37
37
50
50
50
50
50
50
~30
Co-solvent[a]
eq.
50
50
50
25
25
50
50
10
10
50
50
50
1
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
0.2
20
20
20
20
0[d]
20
20
20
20
20
20
20
25
25
5
0
2
2
2
2
0
2
2
0
0
2
2
-
-
-
-
-
-
-
-
6 hrs
2
3 hrs
3
4 hrs
4
5
4 hrs
5
5
4 hrs
6
5
4 hrs
7
5
2 hrs
8
2
2 hrs
9
2
2 (Mn(OAc)3)
4 (Mn(OAc)3)
2 (Mn(OAc)3)
0.05 (Eosin Y)[f]
90 mins
60 mins
30 mins
2 hrs
10[e]
11
12
2
5
5
[a] 20% methanol in ligation buffer (6 M Gdn•HCl, 0.1 M Na2HPO4, pH 7.0); degassing of the solution was not required; [b] Addition of TCEP and TEMPO at stated
eq. at 15 and 45 mins - entry 11 doped at 5 and 15 mins; [c] Reaction reached 100% conversion to 2a by the stated time, as determined by analytical HPLC; [d]
100% ligation buffer (6 M Gdn•HCl, 0.1 M Na2HPO4, pH 7.0); [e] Conditions selected as the optimal balance between rate and stoichiometry; [f] Sample irradiated
under blue LEDs, temp. measured as approximately 30 ºC.
B
TCEP concentration
A
Peptide Concentration
C
TEMPO concentration
100
100
100
80
60
40
20
0
80
60
40
20
0
80
60
40
20
0
25 eq. TEMPO
5 mM UAF-OMe
100 eq. TCEP
10 eq. TEMPO
5 eq. TEMPO
2 eq. TEMPO
2.5 mM UAF-OMe
1 mM UAF-OMe
0.1 mM UAF-OMe
50 eq. TCEP
25 eq. TCEP
5 eq. TCEP
0
2
4
6
8
10 12 14 16
0
2
4
6
8
10 12 14 16
0
2
4
6
8
10 12 14 16
Time (hours)
Time (hours)
Time (hours)
E TCEP:TEMPO:Mn(OAc)3 optimization
D
Comparison of initiators
100
100
80
60
40
20
0
80
2 eq. Cu(OAc)2
10:2:0
60
2 eq. VA-044
10:2:2
2 eq. Mn(OAc)3
40
25:4:2
0.05 eq Eosin Y
No Initiator
50:2:4
20
0
0.0
0.5
1.0
1.5
2.0
0.0
0.5
1.0
1.5
2.0
Time (hours)
Time (hours)
Figure 4. Data plots illustrating the effect of several variables on reaction rate (1a - 2a); plots generated from integration of the desired product peak (2a) relative to
the starting peptide (1a) by analytical HPLC; see SI figs S7 - S16 for further details regarding reaction conditions.
3
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10.1002/anie.202006260
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RESEARCH ARTICLE
A co-solvent (20% methanol) was included to facilitate dissolution
of the reagents and the reaction run at 37 ºC. Full conversion to
2b was complete within 6 hours (table 1, entry 1; reaction
monitored by analytical HPLC). Gratifyingly, the undesired Ala by-
product, produced via quenching of the alanyl radical by H-atom
abstraction from a suitable donor, was not observed. The
persistent TEMPO radical is able to outcompete this process to
yield quantitative conversion to the desired conjugate. To enhance
the rate of conjugation several variables were then explored,
including; temperature, pH, stoichiometry of reagents, co-solvent,
and the use of various additives (see table 1, fig. 1 and SI for
details). The reaction was observed to proceed at a slightly slower
rate if the excess of TCEP was dropped to 25 eq. and failed to
reach completion over 16 hrs with 5 eq. (fig. 4B). Doping the
solution with TCEP and TEMPO twice over the first hour pushed
the reaction to completion within 3 hours (entry 2). Doping also
enabled us to lower the excess of TEMPO to 5 eq. without
dramatically decreasing the rate of reaction (entry 3). Running the
reaction without a co-solvent did not affect the rate (entry 5).
Raising the temperature to 50 ºC further accelerated the reaction
eliminating the need for doping, allowing us to employ a more
acceptable overall stoichiometry of TEMPO (entry 6). Doping
under these conditions dropped the conversion rate to 2 hours
(entry 7) and allowed us to again reduce the excess of TCEP and
TEMPO, to 10 and 2 eq. respectively (entry 8).
To further accelerate the rate of the reaction, we next
explored the introduction of several additives to facilitate
production of the alanyl radical. Cu(OAc)2, Mn(OAc)3, the radical
initiator VA-044, and the photosensitive dye, Eosin Y, were all
evaluated (fig. 4D). Each additive successfully enhanced the rate
to afford complete conversion within 2 hrs. Mn(OAc)3 gave the
fastest conversion; 4 eq. of Mn(OAc)3 enabled us to decrease the
excess of TEMPO to 2 eq. with complete conversion observed in
under 60 minutes, without doping (entry 10). However, doping with
2 eq. of Mn(OAc)3 did allow us to push the reaction to completion
within 30 minutes (entry 11). Additionally, the presence of an
additive accelerated the reaction at lower concentrations of
peptide 1a; a catalytic amount of the dye Eosin Y (5 mol%), with
doping under blue LEDs, afforded full conversion at 0.2 mM over
2 hours (entry 12). The need for a balance between the rate of the
reaction and a reasonable stoichiometry of TEMPO, which will
carry the desired group for conjugation, led us to conclude that
entry 10 represented the optimal set of conditions to take forward
(referred to hereafter as protocol A, see table S1). These
conditions were repeated on a larger scale (8 mg of model peptide
1a) and the product purified via HPLC to afford the desired
conjugate 2a in excellent yield (83%, table 2, entry 13).
TEMPO (3 - 7, fig. 5), using mild, well-established chemistry in
high yield (see SI for details).
O
H
H
N
N
O
N
O
O
=
S
OH
4 - Propargyl
O
H
HO
O
N
N
OMe
O
5 - Fluorescein
NH
3
3 - Tetra-EG
O
OH
O
N
NH
H
HN
H
O
H
N
N
7 - Gemcitabine
H
F
F
OH
S
O
6 - Biotin
Figure 5. TEMPO-based persistent radical traps 3 - 7.
Tetraethylene glycol (tetra-EG) polymer (3) was included to
enable the attachment of a group to modulate the stability of a
polypeptide; propargyl (4) and biotin (6) groups were explored to
enable the isolation of tagged peptides/proteins; a fluorophore
(fluorescein, 5) was included for imaging, and a cytotoxic drug
(gemcitabine, 7) used to demonstrate protein-drug conjugation
(via a stable, non-cleavable linker, thus relying on lysosomal
degradation of the conjugate for drug release). Each trap was then
conjugated to model peptide 1a using protocol A to demonstrate
the tolerance of the technique. The reactions all proceeded to
completion within 1 hour to afford the desired product in good -
excellent isolated yields (67 - 83%, products 8 - 12, table 2).
50 eq. TCEP,
N
Se
2 eq. TEMPO/
O
2
O
O
H
3-7
H
N
OMe
N
OMe
H2N
N
H2N
N
H
4 eq. Mn(OAc)3,
20% DMSO in
ligation buffer,
50 oC, 60 min
H
O
O
O
O
1a (2.5 mM wrt the
selenol monomer)
2a, 8 - 12
Table 2. Isolated yields of peptide conjugates 2a, 8 - 12 from model peptide 1a.
Entry
13
Product
Trap
Yield
83%
72%
80%
69%
78%
67%
2a
8
TEMPO
14
Tetra-EG (3)
Propargyl (4)
Fluorescein (5)
Biotin (6)
15
9
Based on the proposed mechanism of dechalcogenation
under the described conditions, it is assumed that epimerization
of the a-carbon at the target residue should not occur. To confirm
this, model peptide 1b was prepared carrying the D-isomer of Sec
(H-D-UAF-OMe) and subjected to protocol A to yield conjugate
16
10
11
12
17
18
Gemcitabine (7)
1
2b. Comparison of the H NMR spectra for epimers 2a and 2b
clearly highlights that the signal shifts observed for 2b are not
present in the NMR for 2a and vice versa (fig. S36). Therefore, it
can be concluded that the integrity of the stereochemistry at the
target residue remains intact throughout the conjugation process.
Confident that we had fully optimized the method, a range of
TEMPO traps, carrying potentially desirable functionality, were
then prepared from either 4-amino-, carboxy-, oxo-, or hydroxy-
Although Sec provides a useful bioconjugation handle,
especially when employing peptide ligation to access large
peptides or small proteins, Cys is the target residue of choice for
researchers wishing to modify recombinantly expressed proteins.
Therefore, to test the potential of our reaction to label Cys-
containing peptides, the model H-CAF-OMe (13) was prepared
and conjugation reactions with TEMPO trialed to afford conjugate
4
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10.1002/anie.202006260
Angewandte Chemie International Edition
RESEARCH ARTICLE
14 (table S13). Although it was gratifying to confirm that the
procedure could be applied to trap the alanyl radical produced via
desulfurization of Cys, it was observed that the standard protocol
A did not give an optimal rate for this model. It was also noted that
use of Eosin Y as a catalytic initiator resulted in production of an
unidentified impurity which co-eluted with the product. Therefore,
the stoichiometry of TCEP, TEMPO and Mn(OAc)3 was again
explored to determine the ideal conditions. It was observed that 1
mM peptide, 50 eq. TCEP, 2 eq. TEMPO, 5 eq. Mn(OAc)3, 50 ºC
in 20% co-solvent in ligation buffer resulted in full conversion to 14
over 2 hours (referred to hereafter as protocol B). The model
peptide Ac-CWHISKEY-NH2 (15) was prepared to test the
efficiency and chemoselectivity of the conjugation at Cys against
more diverse proteinogenic chemical functionality. The isolated
yields obtained on model 15 using protocol B with traps 3 - 7 were
comparable to those of the Sec model (16 - 20, 71 - 82% yield,
table 3). No by-products were observed from side-reactions with
diselenide (DPDS) within 60 seconds indicated that the ligation
had reached completion; this precipitate was extracted with
hexane and a sample of the solution was removed for analysis.
The crude ligation solution was partitioned into five equal volumes
and the required equivalents of the reagents added from stock
solutions; TCEP (0.625 M stock solution), TEMPO trap (3 - 7) (0.1
M stock solution) and Mn(OAc)3 (0.1 M stock solution). The
reactions were diluted with buffer and DMSO (up to 20% co-
solvent) to give the final concentrations as described by
conjugation protocol A: 2.5 mM peptide wrt selenol monomer,
125 mM TCEP (50 eq.), 5 mM TEMPO (2 eq.), 10 mM Mn(OAc)3
(4 eq.). The conjugations were heated at 50 ºC for 1 hour; once
the starting material was shown to be consumed by analytical
HPLC, the crude reactions were purified by preparative HPLC to
yield the desired products 27 - 31 in high yield (69 – 80%) in one-
pot over two steps (fig. 6).
After demonstrating that Cys can be modified under the
the nucleophilic (Lys) or aromatic (His) residues within this peptide. conjugation conditions containing Mn(OAc)3 as an additive
Quenching of the alanyl radical via H-atom abstraction from the
thiol groups of the Cys-containing peptides in solution was a
concern for this model. However, the Ala by-product was not
observed under the optimized conditions. Furthermore, to ensure
that the reaction did not result in the oxidation of methionine (Met)
residues, model peptide H-UWIMKY-NH2 (21) was synthesized
and subjected to protocol A conditions to afford conjugate 22 in
good yield (68%, fig. S64) with no detectable oxidation of the Met
residue.
(products 16 - 20, table 3), we were intrigued to explore the
selective modification of Sec in the presence of Cys. To confirm
that an internal Cys present in the sequence would not interfere
with the modification of Sec, the model peptide H-UHISCY-NH2
(25) was synthesised and the one-pot ligation-conjugation
attempted with selenoester 23 under conditions similar to
protocol A, but with omission of Mn(OAc)3, run at 37 ºC (50 eq.
TCEP, 5 eq. TEMPO at a final peptide concentration of 2.5 mM –
labelled as protocol C). The reaction was monitored via HPLC
and observed to reach completion over 4 hours. The reaction
mixture was purified to yield the desired product 32 in an excellent
yield (90%) over the two steps, with no apparent interference from
the internal Cys residue (fig. 6). This ligation-conjugation protocol
was successfully repeated with these peptide models using the
propargyl-TEMPO (4) and biotin-TEMPO (6) traps to yield
products 33 and 34 in 83% and 71% respectively. No conjugation
or desulfurization at the internal Cys residue was detected under
these conditions. Crucially, the presence of the internal thiol (an
excellent H-atom source) within the peptide sequence did not
quench the conjugation reaction at the Sec residue.
To demonstrate the application of our method to a ligation-
conjugation protocol using N-terminal Cys residues with a
selenoester peptide, we synthesized the model peptide H-
CHISKY-NH2 (26) and submitted it to a ligation-conjugation
protocol with selenoester 23. However, post-ligation the internal
Cys residue (the intended site of conjugation) reacts with any
excess of the selenoester starting peptide to give an undesired
pendent selenoester. This by-product is not observed using an N-
terminal Sec residue for ligation as the low redox potential of this
residue leads to rapid oxidation to the diselenide product.
Treatment with hydroxylamine results in successful hydrolysis of
the selenoester by-product to yield the desired thiol, however, use
of this additive prevents direct conjugation using the crude solution.
We therefore switched to a two-pot protocol for model peptide 26.
Ligation of the two peptides was followed by extraction of DPDS,
addition of hydroxylamine, and purification of the ligated product
to afford the desired peptide 35 in 77% yield (fig. S85). When
applying protocol B (optimized for model 15; Ac-CWHISKEY-
NH2) to the purified ligation product using propargyl-TEMPO 4, it
was found that a slightly higher excess of Mn(OAc)3 (10 eq.) at 2.5
mM wrt the starting peptide was required to successfully modify
an internal Cys residue (labelled protocol D - table S1).
N
50 eq. TCEP,
2 eq. 3-7
SH
O
O
O
O
NH2
W H I S K E Y
16 - 20
NH2
W H I S K E Y
N
N
H
H
5 eq. Mn(OAc)3,
20% DMSO in
ligation buffer,
50 oC, 2 hrs
O
15 (1 mM)
Table 3. Isolated yields of peptide conjugates 16 - 20 from model peptide 15.
Entry
19
Product
16
Trap
Yield
80%
82%
71%
73%
80%
Tetra-EG (3)
Propargyl (4)
Fluorescein (5)
Biotin (6)
20
17
21
18
22
19
23
20
Gemcitabine (7)
The chemoselectivity demonstrated using these model
peptides was encouraging, however, to ensure selectivity in larger,
more complex peptides, and to demonstrate a successful one-pot
ligation-functionalization protocol, the selenoester peptide Ac-
YEPLA-SePh (23) and selenopeptide H-UHISKY-NH2 (24) were
synthesized. Together, these models contain the majority of the
chemical functionality present in larger protein systems, including;
a nucleophilic amine (Lys), carboxylic acid (glutamic acid, Glu),
aromatic groups (tyrosine, Tyr, and His), a primary alcohol (Ser),
a primary amide (C-terminus) and an aliphatic, sterically bulky
group (leucine, Leu). Peptide ligation of 24 via DSL[38] proceeded
in buffer (6 M Gdn•HCl, 0.1 M Na2HPO4, pH 6.5) at 2.5 mM wrt to
the diselenide dimer (5 mM wrt the selenol monomer) with a slight
excess of the selenoester 23 (1.05 eq.). Precipitation of diphenyl
5
This article is protected by copyright. All rights reserved.

10.1002/anie.202006260
Angewandte Chemie International Edition
RESEARCH ARTICLE
O
1) Ligation; 6M Gdn•HCl, 0.1 M
Na2HPO4, pH 6.5-7.0
Ac
SePh
Y E P L A
N
O
24 X = Se dimer, Z = Lys
25 X = Se dimer, Z = Cys
26 X = SH, Z = Lys
X
23 5.25 mM
O
+
NH2
H I S Z Y
NH2
H I S Z Y
H2N
N
H
Ac
Y E P L A
O
O
2) Conjugation; TCEP,
Mn(OAc)3, 20% DMSO in
ligation buffer
24 - 26 5.0 mM wrt selenol/
thiol monomer
27 - 34
(48 - 90% yield)
2 - 5 eq
3 - 7
N
O
Protocol A; X = Se dimer; Z = Lys; Protocol C; X = Se dimer; Z = Cys; Protocol D; X = SH; Z = Lys
O
S
NH
H
O
O
HN
H
H
HN
O
OMe
O
HN
N
O
N
O
N
O
N
O
O
O
O
NH2
H I S K Y
NH2
NH2
H I S K Y
H I S K Y
N
Ac
Y E P L A
N
N
H
Ac
Ac
Y E P L A
Y E P L A
H
H
O
O
O
HO
27 78% X = Se dimer
O
28 80% X = Se dimer
29 76% X = Se dimer
52% X = SH[a]
OH
HO
S
H
O
N
O
O
HN
N
OH
H
O
N
N
O
F
F
O
N
O
N
O
O
O
NH2
NH2
H I S K Y
H I S K Y
N
N
H
Ac
Ac
Y E P L A
Y E P L A
H
O
O
31 78% X = Se dimer
48% X = SH[a]
NH
H
30 69% X = Se dimer
HN
H
H
O
HN
N
S
O
N
O
N
O
N
O
SH
SH
SH
O
O
O
NH2
H I S C Y
NH2
H I S C Y
NH2
H I S C Y
N
H
Ac
Y E P L A
N
H
Ac
N
Y E P L A
Ac
Y E P L A
H
O
O
O
32 90% X = Se dimer
34 71% X = Se dimer
33 83% X = Se dimer
Figure 6; Ligation-conjugation reactions; Additive-free ligation conditions: 5 mM wrt selenol monomer (2.5 mM wrt diselenide dimer), and Cys-peptide, 1.05 eq.
selenoester; Conjugation protocol A; 2.5 mM peptide (wrt selenol monomer), 50 eq. TCEP, 2 eq. TEMPO trap, 4 eq. Mn(OAc)3, 50 ºC, 1 hour; Protocol C; 2.5
mM peptide (wrt selenol monomer), 50 eq. TCEP, 5 eq. TEMPO trap, 37 ºC, 4 hours; Protocol D; 2.5 mM peptide, 50 eq. TCEP, 2 eq. TEMPO trap, 10 eq.
Mn(OAc)3, 50 ºC, 4 hours; [a] Overall yield over 2 steps.
peptide 37 was successfully isolated in 64% yield (fig. S92), thus
demonstrating that alternative moieties can be selectively installed
at Sec and Cys residues by tuning the reaction conditions.
These conditions afforded the desired conjugate 28 in 81%
conversion (by HPLC) over 4 hours. The conjugation was
repeated on an isolatable scale using peptide 35 with propargyl-
TEMPO 4 and gemcitabine-TEMPO 7 to yield the desired
conjugates (28 and 31) over the same time course in 67% and
62% isolated yield, respectively (52% and 48% over two steps).
Our investigation of one-pot ligation-conjugation using
model peptide 25 (H-UHISCY-NH2) demonstrates that we can
selectively modify a Sec residue in the presence of Cys. To further
explore this selectivity, peptide 25 was subjected to the mild
conjugation protocol C (i.e. no Mn(OAc)3) using the propargyl-
TEMPO trap 4 and purified to give mono-modified peptide 36 in
68% yield (fig. S90). This peptide was then submitted to the
standard protocol A with the tetra-PEG trap 3 and the di-modified
The stability of polypeptide conjugates is a vital aspect of
any bioconjugation method. It is crucial, for instance, that protein-
drug conjugates do not prematurely release their cytotoxic cargo
before reaching their target site. Conversely, applications within
proteomics and protein profiling require selectively cleavable
linkers to release isolated protein material for analysis. To
investigate the stability of our aminooxy linker, TEMPO-peptide
conjugates 2a and 38, prepared from starting peptides 1a (H-UAF-
OMe) and 24 (H-UHISKY-NH2), were explored over 16 hrs at
varying pH (2 - 9), elevated temperature, exposure to UV radiation,
and in the presence of additives, including; VA-044 (a radical
6
This article is protected by copyright. All rights reserved.

10.1002/anie.202006260
Angewandte Chemie International Edition
RESEARCH ARTICLE
initiator) and glutathione (see tables S15 and S16 for details). The
conjugate was found to be remarkably stable under the conditions
explored. However, the aminooxy linker is labile in the presence
of a low oxidation state transition metal in mildly acidic conditions.
Introduction of 10 eq. of Zn(0) in 10% acetic acid resulted in
quantitative and clean degradation of the linker to leave a Ser
residue at the site of conjugation. This protocol was applied to
purified conjugate 28 with successful and exceptionally clean
release of the propargyl trap to afford 39 in high yield (92%) (fig.
S103). To evaluate this method as a potential technique to
achieve peptide ligation at a Ser residue, we investigated a one-
pot DSL-TEMPO conjugation-reductive cleavage protocol.
Selenoester model 23 and diselenide model 21 were ligated via
standard DSL conditions. Following hexane extraction of the
precipitated DPDS, conjugation protocol A was carried out using
TEMPO to afford conjugate 40. To effect reductive cleavage of the
aminooxy linkage of the conjugate in the crude mixture at 1 mM
wrt 40, 0.5 M Zn(0) was required in 10 vol.% AcOH (fig. 7). The
cleavage was complete after 16 hr and the desired peptide (41),
bearing a Ser residue at the ligation site, was isolated in good yield
(74%). This protocol is thus comparable to the previously reported
Oxone-TCEP method for programmable ligation at Ser (fig.
S111).[48-49]
following hexane extraction of the precipitated DPDS, the
propargyl-TEMPO trap 4 was conjugated using protocol A. The
desired conjugate 44 was isolated in a 42% one-pot yield.
Se
2
ZEGFR-2 (30-58)
OH
O
H2N
29
O
43 5.0 mM
wrt selenol monomer
SePh
H
ZEGFR–1 (1-28)
+
42 6.0 mM
3) Conjugation protocol A:
2.5 mM ligation product,
50 eq.TCEP, 4 eq. Mn(OAc)3,
20% DMSO in ligation buffer,
50 oC, 60 mins
1) Ligation: 6M
Gdn•HCl, 0.1 M
Na2HPO4, pH 6.5, 5 mins
2) Hexane extraction
O
O
N
O
4 2 eq.
N
O
O
ZEGFR-2 (30-58)
OH
H
ZEGFR-1 (1-28)
N
H
29
O
44 42% one-pot yield
Se
VDNKFNKEMW–AAWEEIRNLP–NLNGWQMTAF-IASLVDDP
SQ–SANLLAEAKK-LNDAQAPK
2
NH2
W I M K Y
H2N
O
O
21 5.0 mM wrt selenol
Figure 8. One-pot ligation-conjugation of ZEGFR:1907 affibody (native sequence
shown): DSL ligation of fragments 42 and 43 followed by conjugation of the
propargyl 4 trap using protocol A afforded peptide conjugate 44.
Ac
SePh
Y E P L A
+
monomer
23 6 mM
3) Conjugation protocol A:
2.5 mM ligation product,
50 eq.TCEP, 2 eq. TEMPO
4 eq. Mn(OAc)3, 20% DMSO
in ligation buffer, 50 ºC, 60
mins
1) Ligation: 6M
Gdn•HCl, 0.1 M
Na2HPO4, pH 6.5, 5 mins
2) Hexane extraction
To demonstrate our conjugation method on a biologically
expressed protein sample, a K48C mutant of ubiquitin (45) was
prepared via recombinant expression in E. coli.[54] It was observed
that modification of Cys within a protein required slightly higher eq.
than present in protocol D. Therefore, at
1 mM protein
N
concentration, the propargyl-TEMPO trap 4 was conjugated using
100 mM TCEP, 5 mM 4, and 20 mM Mn(OAc)3 at 50 ºC for 2 hours
(protocol E). The product was purified by preparative HPLC and
desired conjugate 46 isolated in 62% yield (fig. 9).
O
O
NH2
W I M K Y
N
Ac
Y E P L A
H
O
40 (not isolated)
Protocol E:
100 mM TCEP,
20 mM Mn(OAc)3,
20% DMSO in
ligation buffer,
50 oC, 2 hrs
4) Reductive cleavage:
O
1 mM 40, 0.5 M Zn(0), 10
SH
N
vol% AcOH, 50 oC, 16 hrs
O
OH
O
NH2
W I M K Y
O
N
N
Ac
Y E P L A
H
O
O
46 62% isolated yield
45 1 mM
41 74% isolated yield
4 5 mM
MQIFVKTLTG–KTITLEVESS–DTIDNVKSKI–QDKEGIPPDQ–QRLI
FAGCQL–EDGRTLSDYN–IQKESTLHLV–LRLRGG
Figure 7. One-pot ligation at Ser; DSL ligation of model fragments 23 and 21,
followed by conjugation of TEMPO and reductive cleavage, affords the desired
peptide bearing Ser at the ligation junction.
Figure 9. Conjugation of propargyl-TEMPO trap 4 to recombinantly expressed
Ub 45 (K48C; sequence shown)[54] to yield conjugate 46 (Ub represented in
figure by PDB entry 2L00).
Finally, we applied our conjugation reaction to two larger,
more complex polypeptide systems. To fully demonstrate the
scope of the one-pot ligation-conjugation protocol, the affibody,
ZEGFR:1907 (a 58 mer peptide derived from immunoglobin-binding
protein A)[53] was synthesized as two fragments; selenoester 42
(amino acids 1-28) and diselenide 43 (amino acids 29-58). These
two peptides were ligated under standard DSL conditions and,
Purified Ub conjugate 46 and the K48C mutant 45 were re-
dissolved in ligation buffer and folded via dialysis into 25 mM
Na2HPO4, 100 mM NaCl, pH 6.9. The samples were analysed via
1H NMR and assigned by comparison to the published data.[55]
Both conjugate 46 and K48C mutant 45 showed extended NH
7
This article is protected by copyright. All rights reserved.

10.1002/anie.202006260
Angewandte Chemie International Edition
RESEARCH ARTICLE
regions (6.5 - 9.5 ppm) and upshifted methyl groups indicating
formation of the native tertiary structure (figs S121 - S123). A
comparison of the chemical shift values of the NH signals for 45
and 46 shows very little deviation across the majority of the
sequence (fig. S120). As expected, those residues flanking
modified position 48 do experience minor perturbations in
chemical shift due to the installed moiety.
Keywords: Conjugation
•
Cysteine
•
Deselenization
•
Desulfurization • Selenocysteine • Peptides • Radicals
References
[1]
[2]
[3]
E. A. Hoyt, P. M. Cal, B. L. Oliveira, G. J. Bernardes, Nat. Rev.
Chem. 2019, 3, 147-171.
O. Boutureira, G. J. L. Bernardes, Chem. Rev. 2015, 115, 2174-
2195.
J. N. deGruyter, L. R. Malins, P. S. Baran, Biochemistry 2017, 56,
3863-3873.
Conclusion
[4]
[5]
O. Koniev, A. Wagner, Chem. Soc. Rev. 2015, 44, 5495-5551.
N. Krall, F. P. Da Cruz, O. Boutureira, G. J. Bernardes, Nat. Chem.
2016, 8, 103.
In summary, we have developed a novel approach to site-
selective polypeptide modification via trapping of free radical-
mediated dechalcogenation. The reaction is operationally very
simple (carried out on the bench in an open vessel, with no
requirement to degas the solutions), and tolerant to a diverse
range of moieties appended to a persistent radical trap. Crucially,
we demonstrate that the stereochemistry of the a-center of the
modified Sec or Cys residue is retained during conjugation.
Reagent stoichiometry has been optimized throughout (protocols
A - E, see table S1) to demonstrate rapid labelling within 30 - 60
minutes for Sec residues and slightly longer for Cys (2 - 4 hrs).
The reaction is efficient down to a concentration of 100 µM (over
16 hrs, Fig 4A), and selective for Sec in the presence of Cys. The
aminooxy linker of the conjugate is stable under the conditions
explored and can be controllably degraded in mild acid with the
addition of Zn(0) with exceptionally clean release. The method
affords good - excellent yields on simple model systems and this
efficiency translates well onto larger and more complex peptides
and proteins carrying a wealth of chemical diversity. While
conjugation to an internal Cys residue within a recombinantly
expressed protein (ubiquitin) required a higher excess of the
reagents relative to the peptide models, this increase does not
translate into a significant quantity of material at the scale
appropriate for protein chemistry. In addition, the functionalised
TEMPO-based traps can be synthesised on the gram-scale in high
yield, and the TCEP and Mn(OAc)3 reagents are relatively
[6]
[7]
S. B. Gunnoo, A. Madder, ChemBioChem 2016, 17, 529-553.
J. M. Chalker, G. J. Bernardes, Y. A. Lin, B. G. Davis, Chem. Asian
J. 2009, 4, 630-640.
D. R. Goddard, L. Michaelis, J. Biol. Chem. 1935, 112, 361-371.
G. T. Hermanson, Bioconjugate techniques, Academic press,
2013.
M. E. Smith, F. F. Schumacher, C. P. Ryan, L. M. Tedaldi, D.
Papaioannou, G. Waksman, S. Caddick, J. R. Baker, J. Am.
Chem. Soc. 2010, 132, 1960-1965.
A. M. Spokoyny, Y. Zou, J. J. Ling, H. Yu, Y.-S. Lin, B. L.
Pentelute, J. Am. Chem. Soc. 2013, 135, 5946-5949.
M. A. Kasper, M. Glanz, A. Stengl, M. Penkert, S. Klenk, T. Sauer,
D. Schumacher, J. Helma, E. Krause, M. C. Cardoso, Angew.
Chem. Int. Ed. 2019, 58, 11625-11630.
[8]
[9]
[10]
[11]
[12]
[13]
[14]
A. Abbas, B. Xing, T. P. Loh, Angew. Chem. Int. Ed. 2014, 53,
7491-7494.
M. J. Matos, C. D. Navo, T. Hakala, X. Ferhati, A. Guerreiro, D.
Hartmann, B. Bernardim, K. L. Saar, I. Compañón, F. Corzana,
Angew. Chem. Int. Ed. 2019, 58, 6640-6644.
B. Bernardim, P. M. Cal, M. J. Matos, B. L. Oliveira, N. Martínez-
Sáez, I. S. Albuquerque, E. Perkins, F. Corzana, A. C. Burtoloso,
G. Jiménez-Osés, Nat. Commun. 2016, 7, 1-9.
C. E. Hoyle, C. N. Bowman, Angew. Chem. Int. Ed. 2010, 49,
1540-1573.
E. V. Vinogradova, C. Zhang, A. M. Spokoyny, B. L. Pentelute, S.
L. Buchwald, Nature 2015, 526, 687-691.
C. Zhang, E. V. Vinogradova, A. M. Spokoyny, S. L. Buchwald, B.
L. Pentelute, Angew. Chem. Int. Ed. 2019, 58, 4810-4839.
G. J. Bernardes, J. M. Chalker, J. C. Errey, B. G. Davis, J. Am.
Chem. Soc. 2008, 130, 5052-5053.
T. H. Wright, B. J. Bower, J. M. Chalker, G. J. Bernardes, R.
Wiewiora, W.-L. Ng, R. Raj, S. Faulkner, M. R. J. Vallée, A.
Phanumartwiwath, O. D. Coleman, M.-L. Thézénas, M. Khan, S. R.
G. Galan, L. Lercher, M. W. Schombs, S. Gerstberger, M. E. Palm-
Espling, A. J. Baldwin, B. M. Kessler, T. D. Claridge, S.
Mohammed, B. G. Davis, Science 2016, 354, aag1465.
A. Yang, S. Ha, J. Ahn, R. Kim, S. Kim, Y. Lee, J. Kim, D. Söll, H.-
Y. Lee, H.-S. Park, Science 2016, 354, 623-626.
J. Dadová, S. R. Galan, B. G. Davis, Curr. Opin. Chem. Biol. 2018,
46, 71-81.
H. J. Reich, R. J. Hondal, ACS Chem. Biol. 2016, 11, 821-841.
E. S. Arnér, H. Sarioglu, F. Lottspeich, A. Holmgren, A. Böck, J.
Mol. Biol. 1999, 292, 1003-1016.
J. Liu, Q. Chen, S. Rozovsky, J. Am. Chem. Soc. 2017, 139, 3430-
3437.
J. Liu, F. Zheng, R. Cheng, S. Li, S. Rozovsky, Q. Wang, L. Wang,
J. Am. Chem. Soc. 2018, 140, 8807-8816.
J. Liu, R. Cheng, S. Rozovsky, Curr. Opin. Chem. Biol. 2018, 46,
41-47.
L. R. Malins, N. J. Mitchell, R. J. Payne, J. Pept. Sci. 2014, 20, 64-
77.
D. T. Cohen, C. Zhang, B. L. Pentelute, S. L. Buchwald, J. Am.
Chem. Soc. 2015, 137, 9784-9787.
[15]
[16]
[17]
[18]
[19]
[20]
inexpensive. Beyond direct modification,
a DSL-conjugation
protocol has also been described to enable the application of this
methodology to the one-pot chemical synthesis and modification
of large peptides and small proteins. Furthermore, we have
exploited the lability of the aminooxy linker to low oxidation state
[21]
[22]
[23]
[24]
transition metals to develop
a one-pot ligation-conjugation-
reductive cleavage protocol that allows peptide ligation at Ser
residues.
Due to the tolerance, efficiency, and operational simplicity of
the described protocols, coupled with the stability of the aminooxy
linker and option of controllable degradation, this method
[25]
[26]
[27]
[28]
[29]
[30]
[31]
represents
a versatile new approach to polypeptide-small
molecule conjugation for researchers working across diverse
scientific themes.
L. Pedzisa, X. Li, C. Rader, W. R. Roush, Org. Biomol. Chem.
2016, 14, 5141-5147.
P. E. Dawson, T. W. Muir, I. Clark-Lewis, S. Kent, Science 1994,
266, 776-779.
[32]
[33]
S. B. Kent, Chem. Soc. Rev. 2009, 38, 338-351.
V. Agouridas, O. a. El Mahdi, V. Diemer, M. Cargoët, J.-C. M.
Monbaliu, O. Melnyk, Chem. Rev. 2019, 119, 7328-7443.
R. J. Hondal, B. L. Nilsson, R. T. Raines, J. Am. Chem. Soc. 2001,
123, 5140-5141.
M. D. Gieselman, L. Xie, W. A. van der Donk, Org. Lett. 2001, 3,
1331-1334.
R. Quaderer, A. Sewing, D. Hilvert, Helv. Chim. Acta 2001, 84,
1197-1206.
Acknowledgements
This work was supported by funding from the Engineering and
[34]
[35]
[36]
[37]
Physical
Sciences
Research
Council
(EP/S028323/1,
EP/S017739/1) and from the University of Nottingham (PhD
studentship for RG, EPR upgrade funded by the University of
Nottingham Propulsion Futures Beacon).
T. Durek, P. F. Alewood, Angew. Chem. Int. Ed. 2011, 50, 12042-
12045.
8
This article is protected by copyright. All rights reserved.

10.1002/anie.202006260
Angewandte Chemie International Edition
RESEARCH ARTICLE
[38]
N. J. Mitchell, L. R. Malins, X. Liu, R. E. Thompson, B. Chan, L.
Radom, R. J. Payne, J. Am. Chem. Soc. 2015, 137, 14011-14014.
N. J. Mitchell, J. Sayers, S. S. Kulkarni, D. Clayton, A. M. Goldys,
J. Ripoll-Rozada, P. J. B. Pereira, B. Chan, L. Radom, R. J.
Payne, Chem 2017, 2, 703-715.
[39]
[40]
J. Sayers, P. M. Karpati, N. J. Mitchell, A. M. Goldys, S. M. Kwong,
N. Firth, B. Chan, R. J. Payne, J. Am. Chem. Soc. 2018, 140,
13327-13334.
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
X. Wang, L. Corcilius, B. Premdjee, R. J. Payne, J. Org. Chem.
2019, 85, 1567-1578.
T. S. Chisholm, S. S. Kulkarni, K. R. Hossain, F. Cornelius, R. J.
Clarke, R. J. Payne, J. Am. Chem. Soc. 2019, 142, 1090-1100.
S. S. Kulkarni, E. E. Watson, B. Premdjee, K. W. Conde-Frieboes,
R. J. Payne, Nat. Protoc. 2019, 14, 2229-2257.
Q. Wan, S. J. Danishefsky, Angew. Chem. Int. Ed. 2007, 46, 9248-
9252.
N. Metanis, E. Keinan, P. E. Dawson, Angew. Chem. Int. Ed. 2010,
49, 7049-7053.
S. S. Kulkarni, J. Sayers, B. Premdjee, R. J. Payne, Nat. Rev.
Chem. 2018, 2, 1-17.
A. C. Conibear, E. E. Watson, R. J. Payne, C. F. Becker, Chem.
Soc. Rev. 2018, 47, 9046-9068.
L. R. Malins, N. J. Mitchell, S. McGowan, R. J. Payne, Angew.
Chem. Int. Ed. 2015, 54, 12716-12721.
N. J. Mitchell, S. S. Kulkarni, L. R. Malins, S. Wang, R. J. Payne,
Chem. Eur. J. 2017, 23, 946-952.
S. Dery, P. S. Reddy, L. Dery, R. Mousa, R. N. Dardashti, N.
Metanis, Chem. Sci. 2015, 6, 6207-6212.
G. J. Bernardes, E. J. Grayson, S. Thompson, J. M. Chalker, J. C.
Errey, F. El Oualid, T. D. Claridge, B. G. Davis, Angew. Chem. Int.
Ed. 2008, 47, 2244-2247.
[52]
[53]
K. Jin, T. Li, H. Y. Chow, H. Liu, X. Li, Angew. Chem. Int. Ed.
2017, 56, 14607-14611.
M. Friedman, A. Orlova, E. Johansson, T. L. Eriksson, I. Höidén-
Guthenberg, V. Tolmachev, F. Y. Nilsson, S. Ståhl, J. Mol. Biol.
2008, 376, 1388-1402.
[54]
[55]
T. P. Garner, J. Strachan, E. C. Shedden, J. E. Long, J. R. Cavey,
B. Shaw, R. Layfield, M. S. Searle, Biochemistry 2011, 50, 9076-
9087.
Biological Magnetic Resonance Data Bank (BMRDB); entries 68
and 4769; http://www.bmrb.wisc.edu/.
9
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Angewandte Chemie International Edition
RESEARCH ARTICLE
Entry for the Table of Contents
X
N
H
A radical approach to bioconjugation: Functionalization of peptides and proteins via
trapping of peptide radical species is described. This novel and versatile strategy exploits the
process of desulfurization and deselenization of cysteine and selenocystine residues to enable
the site-selective installation of a broad range of groups appended to a persistent radical trap.
O
X = diselenide dimer (Sec)
X = SH (Cys)
O
N
@mitchell_chem; @NottsChemistry
N
O
TCEP
Mn(III)
N
H
O
10
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