Palladium Oxidative Addition Complexes for Peptide and Protein Cross-linking

Article abstract of DOI:10.1021/jacs.8b00172

A new method for cysteine-lysine cross-linking in peptides and proteins using palladium oxidative addition complexes is presented. First, a biarylphosphine-supported palladium reagent is used to transfer an aryl group bearing an O-phenyl carbamate substituent to a cysteine residue. Next, this carbamate undergoes chemoselective acyl substitution by a proximal lysine to form a cross-link. The linkage so formed is stable toward acid, base, oxygen, and external thiol nucleophiles. This method was applied to cross-link cysteine with nearby lysines in sortase A?. Furthermore, we used this method for the intermolecular cross-linking between a peptide and a protein based on the p53-MDM2 interaction. These studies demonstrate the potential for palladium-mediated methods to serve as a platform for the development of future cross-linking techniques for peptides and proteins with natural amino acid residues.

Full text of DOI:10.1021/jacs.8b00172

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Article
Palladium Oxidative Addition Complexes for Peptide and Protein Crosslinking
Koji Kubota, Peng Dai, Bradley L. Pentelute, and Stephen L. Buchwald
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b00172 • Publication Date (Web): 06 Feb 2018
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Journal of the American Chemical Society
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Palladium Oxidative Addition Complexes for Peptide and Protein
Crosslinking
Koji Kubota,‡ Peng Dai,‡ Bradley L. Pentelute* and Stephen L. Buchwald*
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Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
Supporting Information Placeholder
ABSTRACT: A new method for cysteine-lysine crosslinking in peptides and proteins using palladium oxidative addition
complexes is presented. First, a biarylphosphine-supported palladium reagent is used to transfer an aryl group bearing an
O-phenyl carbamate substituent to a cysteine residue. Next, this carbamate undergoes chemoselective acyl substitution
by a proximal lysine to form a crosslink. The linkage so formed is stable towards acid, base, oxygen and external thiol nu-
cleophiles. This method was applied to crosslink cysteine with nearby lysines in sortase A*. Furthermore, we used this
method for the intermolecular crosslinking between a peptide and a protein based on the p53-MDM2 interaction. These
studies demonstrate the potential for palladium-mediated methods to serve as a platform for the development of future
crosslinking
techniques
for
peptides
and
proteins
with
natural
amino
acid
residues.
linking step and achieving chemo- and site-selectivity.
Therefore, we anticipated that judicious choice of the elec-
trophilic functional group would be required to achieve
the desired transformation with a high level of selectivity.
O-aryl carbamates are mild electrophiles, which
INTRODUCTION
Intra- and intermolecular crosslinking of pep-
tides and proteins have been recognized as an important
strategy to control protein conformations, enhance pro-
tein stability, study protein-protein interactions, and im-
prove pharmacological properties. For example, disulfide
bonds formed between two cysteine side chains serve as a
part of natural protein’s secondary or tertiary structures,
providing the essential framework for protein function
and/or stability.1 However, these naturally occurring
crosslinking processes are significantly limited. As a con-
sequence, chemists have developed numerous synthetic
crosslinking methodologies2 that can link specific side
chains in peptides3-7 and proteins8,9 for engineering their
structures and properties.
react with amines under basic conditions to form stable
urea adducts. Recently, Schultz and co-workers reported
an elegant new protein crosslinking method involving
incorporation of noncanonical amino acids with side
chains bearing an aryl carbamate.9 They demonstrated the
efficient crosslinking formation between the aryl carba-
mate and the proximal lysine residues within proteins.
Despite its utility, a limitation of this approach is that it
requires the incorporation of an unnatural amino acid into
each individual protein of interest.11 To compliment the
Schultz technique, we aimed to develop a direct method to
incorporate the O-aryl carbamate functional group at spe-
cific natural amino acid sites in unprotected peptides and
proteins to form protein crosslinked connections. Specifi-
cally, we envisioned using a palladium complex bearing a
phenyl carbamate group as a cysteine-lysine crosslinking
method at natural amino acid residues to form a linker
with stable S-aryl and urea attachments (Figure 1). In
analogy to our previous work,10a we expected that the SH
group of a cysteine would displace an appropriate leaving
group (X) on Pd. The ensuing reductive elimination would
provide the S-arylated intermediate. Subsequent nucleo-
philic attack by a proximal lysine residue would provide
the crosslinked peptide or protein (Figure 1a). In addition,
the same strategy could be used for intermolecular cross-
linking between two biomolecules (Figure 1b).
Our groups have been interested in applying
transition-metal-mediated cross-coupling strategies to-
wards the modification of complex biomolecules.7e,7f,10
Recently, we reported that aryldialkylphosphine-based
palladium oxidative addition complexes are able to medi-
ate cysteine- or lysine-selective arylations.7f,10a Further-
more, bis-palladium complexes have been applied to pre-
pare stapled peptides through the construction of intra-
molecular cysteine-cysteine and lysine-lysine linkages.7e,7f
In order to expand the generality of transition-metal-
mediated intra- and intermolecular protein crosslinking
technology, we envisioned the use of a palladium oxidative
addition complex bearing an appropriate electrophilic
functional group. This bifunctional reagent could react
with unprotected peptides or proteins through palladium-
mediated cysteine-arylation,10a after which the bound elec-
trophilic group could react with a proximal nucleophilic
residue to furnish the desired crosslink. The main chal-
lenges associated with this strategy include avoiding hy-
drolysis of the bioconjugation handle prior to the cross-
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a Intramolecular Cys-Lys protein crosslinking with palladium
Scheme 1. Synthesis of the Palladium Oxidative Addi-
Lys bioconjugation
O
handle
1
2
3
4
5
6
7
8
tion Complex 1, which Bears a Phenyl Carbamate.
Lys
PhO
H
H
H2N
O
L
N
N
OMe
Cys
SH
OPh
NH
NH2
tBu
tBu
Pd Ar
Br
NH
(1.1 equiv)
tBuBrettPhos
O
O
X
S
P
OPh
S
Protein or
polypeptide
i
Pr
NH
Pd
Protein or
polypeptide
–HOPh
CH2TMS
CH2TMS
MeO
(1.1 equiv)
iPr
Protein or
Br
Pd
polypeptide
Storable
phenyl carbamate
transfer reagent
cyclohexane
rt, 16 h
1
iPr
92% yield
b Intermolecular Cys-Lys crosslinking with palladium
L
9
Interacting
protein
We first examined the use of 1 for the intramo-
Pd Ar
Interacting
protein
10
11
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X
lecular cysteine-lysine crosslinking in a model peptide 2,
which also contained a variety of other nucleophilic ami-
no acid residues such as tyrosine, serine, arginine, histi-
dine and glutamine which possess nucleophilic groups on
their side chains (Figure 2). In the presence of 1 (11 µM, 1.1
equiv) in 50 mM pH 8.5 HEPES buffer in water/DMSO
(95:5) solvent at 37° C for 24 hours,14 the substrate 2 (10
µM) was converted to the desired intramolecular cross-
linked product 3. The reaction outcome was characterized
by liquid chromatography-ESI-QTOF mass spectrometry
(LC-MS). We observed the complete disappearance of 2,
accompanied by the appearance of a new major peak cor-
responding to the loss of phenol and formation of a urea
crosslinked product 3 (Figure 2). A small amount of the
in-situ hydrolysis cysteine-arylation product 4 was detect-
ed.
C
C
C
S
H2N
S
SH
OPh
O
HN
–HOPh
Peptide
binder
Arylated
binder
N
O
N
H
H
Lys bioconjugation
handle
Figure 1. Strategy for palladium-mediated peptide and pro-
tein crosslinking.
RESULTS AND DISCUSSION
The palladium oxidative addition complex 1 bear-
ing a phenyl carbamate can be prepared from phenyl N-
(4-bromophenyl)carbamate in high yield, using our pre-
viously reported protocol with tBuBrettPhos as the ligand
(Scheme 1).10a,12 This phenyl carbamate transfer reagent is
a solid that is stable in the air, and may be stored at 4°C
for at least 5 months without detectable decomposition.13
H
a
N
O
HO
HO
NH
NH2
HS
S
HO
HO
O
50 mM HEPES
pH 8.5
H2N
tBuBrettPhos
CONH2
4
4
OPh
AcHN
NH2
R
S
Q
F
Y
K
H
D
A
G G
C
CONH2
R
S
Q
F
Y
K
H
D
A
G
C
G
AcHN
NH2
+
Pd
Br
NH
S
+
H2O:DMSO (95:5)
37 °C, 24 h
NH2
NH2
NH
NH
2
2
3
3
HN
N
HN
N
H
H
O
O
N
N
2, 10 µM
1, 11 µM
(1.1 equiv)
Crosslinked peptide
Hydrolysis product
3
4
b
t = 0 h
t = 24 h
2
3
4
time [min]
time [min]
Figure 2. Selective intramolecular peptide crosslinking using the palladium oxidative addition complex 1. (a) Palladium
complex 1-mediated intramolecular crosslinking of model peptide 2. (b) LC-MS analysis of starting material and the reac-
tion mixture. The mass spectra for peptide 2 and 3 are shown as insets.
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carbamate derived from cysteine S-arylation was observed
1
2
3
4
5
6
7
8
As confirmation of the chemoselectivity of this
reaction, we mutated the lysine in peptide 2 to leucine
(Figure 3). Under identical conditions as above, crosslink-
ing was not observed. Instead, despite the presence of
numerous nucleophilic residues, only hydrolysis of the
a
by LC-MS analysis. This suggests crosslinking occurs se-
lectively between cysteine and lysine residues in peptide 2
using 1
.
H2N
HO
HO
HO
HS
S
HO
O
50 mM HEPES
pH 8.5
tBuBrettPhos
CONH2
9
OPh
R
S
Q
F
Y
L
H
D
A
G
C
G
AcHN
NH2
+
NH
Pd
Br
AcHN
NH2
R
S
Q
F
Y
L
H
D
A
G G
C
CONH2
10
11
12
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H2O:DMSO (95:5)
37 °C, 24 h
NH2
NH2
NH
NH
2
3
2
HN
N
3
HN
N
H
O
H
O
N
N
5
1, 11 µM
(1.1 equiv)
Hydrolysis product
10 µM
6
b
t = 0 h
t = 24 h
5
6
time [min]
time [min]
Figure 3. Chemoselectivity of palladium complex 1-mediated peptide crosslinking is confirmed by mutation study. (a)
Even in the presence of nucleophilic residues, no crosslink was formed in lysine-t0-leucine mutated model peptide 5 us-
ing the palladium complex 1, indicating crosslinking occurs between cysteine and lysine residues. (b) LC-MS analysis of
starting material and the reaction mixture. The mass spectra for peptide 5 and 6 are shown as insets.
a
O
tBuBrettPhos
H
We next varied the distance between the lysine
and cysteine residues to adjust the size of the newly
formed macrocycle. Analogs of peptide 2, in which the
relative position of cysteine and lysine residues ranged
from i,i+1 to i,i+11, were prepared. When treated with the
palladium complex 1 under the standard conditions,
good-excellent yields of product were observed in all cas-
es (Figure 4).
OPh
N
O
Pd
Br
NH
NH2
NH
HS
S
Size
1, 11 µM
(1.1 equiv)
Size
4
4
Peptide analogs
of 2
Peptide analogs
of 2
50 mM HEPES
pH 8.5
H2O:DMSO (95:5)
Position i,i+ j
Position i,i+ j
37 °C, 24 h
b
100
50
0
1 2 3 4 5 6 7 8 9 10 11
j
Figure 4. Palladium oxidative addition complex 1 enables
highly efficient cysteine-lysine crosslinking to form mac-
rocycles with different sizes. (a) Tuning the size of the
macrocycles. (b) Yield summary for the macrocyclization
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scan. Yield of the desired products were determined by
Page 4 of 9
was accompanied by the appearance of a new peak corre-
sponding to a mass of 19,331.7 Da, consistent with the
formation of an S-aryl and urea linkage to produce the
desired product 8. To confirm the crosslinking site, the
reaction mixture was subjected to trypsin and the result-
ant mixture was analysed by the LC-MS/MS (See the Sup-
porting Information for details). The MS/MS fragmenta-
tion of the products (Figure S2) indicated that crosslinks
had occurred between Cys(184) and either Lys(162) or
Lys(198), in approximately equal proportion.
1
2
3
4
5
6
7
8
LC-MS. See the Supporting Information for the details.
A high degree of stability of the linker and the
macrocyclic peptide is key for any practical crosslinking
methodology. Thus, we investigated the stability of the
cysteine-lysine crosslinked peptide 3 under a number of
conditions (Figure 5). When conjugate 3 (100 µM) was
subjected to basic (500 µM K2CO3)15, acidic (10 mM HCl)
or oxidative (open air) conditions, LC-MS analysis showed
that no degradation had occurred after 24 hours. Addi-
tionally, no reaction of 3 was observed in the presence of
the external nucleophile glutathione (500 µM) in Tris
buffer (100 mM) at pH 7.5 over the same period of time.
9
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O
tBuBrettPhos
OPh
Pd
NH
O
Br
1, 11 µM
(1.1 equiv)
NH
N
H
a
b
50 mM HEPES
pH 8.5
H2O/DMSO (95:5)
S
Basic conditions: K2CO3, rt
Acidic conditions: HCl, rt
37 °C, 24 h
3
3
t = 0 h
t = 0 h
t = 0 h
SrtA* (7), 10 µM
Crosslinked SrtA* (8)
After
crosslinking
Before
crosslinking
3
3
t = 24 h
t = 24 h
time [min]
time [min]
c
d
Nucleophile: glutathione, pH 7.5, rt
Oxidative conditions: air, 37 °C
Deconvoluted mass
Deconvoluted mass
t = 0 h
t = 0 h
3
3
Figure 6. SrtA* (7) was crosslinked intramolecularly by
the palladium complex 1. The deconvoluted mass spectra
for the whole protein peak from LC-MS analysis of the
starting material and crude reaction mixture are shown.
t = 24 h
t = 24 h
3
3
Our method was also used to form an intermo-
lecular crosslinking between a peptide and an interacting
protein. MDM2 protein (9) is known to bind to the pep-
tide pDIQ with high affinity (Figure 7a).17ƒ We carried out
the crosslinking process in two separate steps. An ad-
vantage of this approach is that the product of the first
step can be stored and used over a period of time. First,
pDIQ analog 10 was prepared with the leucine residue
mutated to cysteine (Figure 7a). To incorporate a phenyl
carbamate group into the peptide substrate 10, we carried
out the arylation reaction of 10 (50 µM) with the palladi-
um oxidative addition complex 1 (55 µM, 1.1 equivalents,
relative to 10) with 100 mM pH 7.5 phosphate buffer in
water/DMSO (95:5) co-solvent at room temperature for 10
minutes. This yielded the desired arylation product 11
with complete conversion of 10 (Figure 7b). The product
11 was purified by a reverse phase HPLC to provide the
pure product. Notably, even though it contains a free N-
terminal amine, 11 does not undergo macrocyclization in
acidic media during HPLC purification and can be stored
in water at 500 µM in the freezer for at least three
months.
time [min]
time [min]
Figure 5. Chemical stability of the intramolecular cross-
linked peptide 3 (100 µM) under (a) basic (500 µM K2CO3)
condition; (b) acidic (10 mM HCl) condition; (c) oxidative
conditions (air) in Tris buffer (100 mM, pH 7.5) and (d) in the
presence of glutathione (500 µM) in Tris buffer (100 mM, pH
7.5). LC-MS analysis indicated that little or no decomposi-
tion had occurred after 24 hours.
This palladium-mediated methodology was next
applied to the intramolecular crosslinking within a pro-
tein (Figure 6). Engineered sortase A (SrtA*, 7) containing
one cysteine and 18 lysine residues was chosen,16 in order
to investigate the site-selectivity of the crosslinking. The
X-ray crystal structure (PDB ID 2KID) of wild type SrtA
shows that Cys(184) is close to Lys(134), Lys(162) and
Lys(198) (Figure S1). Thus, we would expect that cross-
linking between cysteine and these residues would be
most probable. The reaction of SrtA* (7) with 1 under the
standard reaction conditions was conducted and the re-
sulting reaction mixture was analysed by LC-MS. As ex-
pected, the disappearance of the peak corresponding to
the SrtA* (7) with a mass of 19,214.5 Da was observed. This
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The crosslinking reaction between the conjugate
a
1
2
3
4
5
6
7
8
11 and MDM2 (9) was carried out. MDM2 (9) contains one
cysteine and 9 lysine residues (Figure 8). The reaction of 9
(5 µM) and 10 equivalents of 11 (50 µM) in pH 8.5 HEPES
buffer produced the mono-crosslinked product 12 (Figure
8a). In the deconvoluted mass spectra from LC-MS analy-
sis, the original mass peak corresponding to 9 at 14,568.0
Da entirely disappeared and a single new peak at 16,236.7
Da emerged, consistent with the loss of phenol and for-
mation of a urea crosslink in the desired product 12. No-
tably, multiply crosslinked products were not observed.
The LC-MS analysis also showed that the conjugate 11
underwent intramolecular cyclization between the phenyl
carbamate and its terminal amine to form the corre-
sponding macrocyclic peptide. We note that although we
only see product from the addition of one equivalent of 11
to 9, we cannot say whether only one crosslinked species
is formed. To demonstrate the observed selectivity in the
crosslinking reaction of MDM2 (9) is not derived from the
competing cyclization of 11, we added an additional 10
equivalents of 11 to the reaction mixture and waited for
another 24 hours. Only a trace amount of the double-
crosslinked product was observed (Figure S3), highlight-
ing the crosslinking reaction is primarily mediated by the
protein-peptide interaction.
MDM2 (9)
W
W
S
H
Q
E
F
L
T
E
L
pDIQ
S
CONH2
pDIQ
H2N
Leu11 mutated
to Cys
W
W
S
H
Q
E
F
L
T
9
C
S
E
10
11
12
13
14
15
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Crystal stracture
of MDM2-pDIQ
complex
H2N
CONH2
HS
Cysteine-mutated
pDIQ analog 10
b
O
tBuBrettPhos
Pd
Br
OPh
W
W
S
H
NH
Q
E
F
L
H
N
1, 55 µM
(1.1 equiv)
T
O
C
S
E
10
50 µM
OPh
11
CONH2
H2N
S
100 mM phosphate
pH 7.5, H2O/DMSO (95:5)
rt, 10 min
Given the efficient crosslinking reaction between
11
9 and 11, we wondered whether the reaction was MDM2
(9) selective if carried out in a mixture of proteins (Figure
8b). To this end, 10 equivalents of the arylated pDIQ ana-
log 11 (50 µM) and a mixture of three proteins, MDM2 (9,
5 µM), the C-terminal domain of HIV-1 capsid assembly
polyprotein (CCA, 13, 5 µM), which contains 6 lysines, and
SrtA* (7, 5 µM), which contains 18 lysines, were incubat-
ed. Analysis by LC-MS indicated that the mono-
crosslinked MDM2 (12) had been formed (94% conversion
determined by LC-MS) leaving CCA (13) and SrtA* (7)
unmodified (Figure 8b).
time [min]
Figure 7. Preparation of MDM2 binding peptide analog 11
bearing a phenyl carbamate. (a) X-ray crystal structure of
MDM2-pDIQ complex (PDB ID: 3JZS) and design of pDIQ
analog 10 for cysteine arylation. (b) The palladium-
mediated cysteine arylation of 10. LC-MS analysis showed
full conversion of 10 to form the desired product 11.
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a
1
2
3
4
5
6
7
8
9
O
Mono-crosslinked
MDM2 (12)
W
W
S
H
Q
50 mM HEPES
pH 8.5
NH
E
F
L
H
N
MDM2 (7)
N
T
O
C
H
S
+
E
H2O/glycerol
(91:9)
37 °C, 24 h
OPh
H2N
CONH2
S
S
11
50 µM
(10 equiv)
Mono-crosslinked
Deconvoluted mass
MDM2 (9), 5 µM
MDM2 (12)
10
11
12
13
14
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b
O
NH
N
H
CCA (13)
SrtA* (7)
S
W
W
H
S
Q
E
F
Mono-
crosslinkd
MDM2
(12)
50 mM HEPES
L
H
T
pH 8.5
O
N
C
MDM2 (9), 5 µM
Mono-crosslinked
S
E
+
MDM2 (12)
OPh
H2O/glycerol
(91:9)
37 °C, 24 h
H2N
CONH2
S
+
11
50 µM
(10 equiv)
Unreacted
CCA (13)
MDM2 (9)
+
Unreacted
SrtA* (7)
Deconvoluted mass
CCA (13), 5 µM SrtA* (7), 5 µM
Figure 8. Protein-selective intermolecular crosslinking between a peptide and an interacting protein. (a) Intermolecular
crosslinking of MDM2 (9) with the arylated pDIQ binder analog 11. The deconvoluted mass spectra for the whole protein
peak from LC-MS analysis of the crude reaction mixture showed no multiple crosslinked products. (b) Protein-selective
intermolecular crosslinking of MDM2 (9) in a protein mixture. Only MDM2 (9) was modified as indicated by the LC-MS
analysis.
In order to probe the relationship between bind-
ing affinity of the substrate peptide and the rate of inter-
molecular crosslinking with MDM2 (9), a second arylated
MDM2 binder analog 14 was prepared (See the Support-
ing Information for the details) and its reaction with
MDM2 (9) was examined (Figure 9). Peptides 11 and 14
were derived from pDIQ (IC50 = 8 nM) and pDI6N (IC50
=
400 nM) respectively, which have different binding affini-
ties toward 9.17 Indeed, the more tightly binding pDIQ
analog 11 provided a higher conversion at every time point
(1, 3, 6 and 9 hours) compared to that of the arylated
pDI6N analog 14 (Figure 9).
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a
Supporting Information
1
2
3
Experimental procedures, data and computational details.
This material is available free of charge via the Internet at
http://pubs.acs.org.
O
50 mM HEPES
pH 8.5
4
5
6
7
NH
N
11 or 14
50 µM
(10 equiv)
H
+
AUTHOR INFORMATION
Corresponding Author
H2O/glycerol
(91:9)
37 °C, 24 h
S
Mono-
crosslinked
MDM2 (12)
8
9
sbuchwal@mit.edu
blp@mit.edu
MDM2 (9), 5 µM
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11
12
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14
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W
W
N
W
H
S
H
A
Q
L
E
Q
Author Contributions
E
F
F
L
H
N
H
N
T
T
O
C
O
C
‡These authors contributed equally.
S
E
S
L
OPh
OPh
H2N
S
CONH2 H2N
CONH2
S
ACKNOWLEDGMENT
11 derived from pDIQ
14 derived from pDI6N
Financial support for this work was provided by the National
Institutes of Health (R01GM46059 and R01GM110535). MIT
has patents on some of the ligands and precatalysts used in
this work from which S.L.B. and former co-workers receive
royalty payments. We gratefully acknowledge Sigma-Aldrich
for the gift of RuPhos and BrettPhos. K.K. gratefully
acknowledge support from the Japan Society for the Promo-
tion of Science. We thank Dr. Ekaterina Vinogradova for
performing related initial experiments. We thank Dr. Mette
Ishoey and Dr. Faycal Touti for assistance in protein expres-
sion. We thank Richard Liu, Dr. Nicholas White, and Dr.
Andy Thomas (MIT) for assistance on the preparation of this
manuscript.
b
100
50
0
0
2
4
6
8
10
time [h]
REFERENCES
(1) Fass, D. Annu. Rev. Biophys. 2012, 41, 63–79.
Figure 9. The efficiency of intermolecular crosslinking of
MDM2 is mediated by peptide binding affinity. (a) MDM2
(9) was reacted with two analogs 11 and 14 derived from
two MDM2 binding peptides of different affinities. (b)
Summary of MDM2 conversion at different time points.
(2) The selected reviews on synthetic polypeptide crosslinking strate-
gies, see: (a) Koniev, O.; Wagner, A. Chem. Soc. Rev. 2015, 44, 5495–
5551; (b) Kluger, R.; Alagic, A. Bioorg. Chem. 2004, 32, 451–472.
(3) For the selected examples of peptide crosslinking by olefin me-
tathesis, see: (a) Wang, D.; Chen, K.; Kulp, J. L.; Arora, P. S. J. Am.
Chem. Soc. 2006, 128, 9248–9256. (b) Speltz, T, E.; Fanning, S. W.;
Mayne, C. G.; Fowler, C.; Tajkhorshid, E.; Greene, G. L.; Moore, T. W.
Angew. Chem. Int. Ed. 2016, 55, 4252–4255.
(4) For the selected examples of peptide crosslinking by alkylation,
see: (a) Muppidi, A.; Doi, K.; Edwardraja, S.; Drake, E.; Gulick, A.;
Wang, H.; Lin, Q. J. Am. Chem. Soc. 2012, 134, 14734–14737. (b) Jo H.;
Meinhardt, N.; Wu Y.; Kulkarni, S.; Hu, Z.; Low, K. E.; Davies, P. L.;
DeGrado, W. F.; Greenbaum, D. C. J. Am. Chem. Soc. 2012, 134, 17704–
17713. (c) Wang, Y.; Chou, D. Angew. Chem. Int. Ed. 2015, 54, 10931–
10934. (d) Zambaldo, C.; Luo, X.; Mehta, A. P.; Schultz, P. G. J. Am.
Chem. Soc. 2017, 139, 11646–11649.
CONCLUSION
In summary, we have developed a new protocol
for peptide and protein crosslinking at natural amino acid
residues using palladium oxidative addition complexes.
This method involves the palladium-mediated arylation of
cysteine residues to incorporate an O-phenyl carbamate
group into peptides or proteins, followed by acyl substitu-
tion by proximal lysine residues. Compared to other cys-
teine-lysine crosslinking reagents such as N-
(5) For the selected examples of peptide crosslinking by cycloaddi-
tion, see: (a) Scrima, M.; Le Chevalier-Isaad, A.; Rovero, P.; Papini, A.
M.; Chorev, M.; D’Ursi, A. M. Eur. J. Org. Chem. 2010, 2010, 446–457.
(b) Lau, Y.; de Andrade, P.; Quah, S. –T.; Rossmann, M.; Laraia, L.;
Skold, N.; Sum, T.; Rowling, P.; Joseph, T.; Verma, C.; Hyvonen, M.;
Itzhaki, L.; Venkitaraman, A.; Brown, C.; Lane, D.; Spring, D. Chem.
Sci. 2014, 5, 1804–1809.
(6) For the selected examples of peptide crosslinking by lactamiza-
tion, see: (a) Taylor, J. W. Biopolymers 2002, 66, 49–75. (b) de Araujo,
A. D.; Hoang, H. N.; Kok, W. N.; Diness, F.; Gupta, P.; Hill, T. A.; Driv-
er, R. W.; Prince, D. A.; Liras, S.; Fairlie, D. P. Angew. Chem. Int. Ed.
2014, 53, 6965–6969.
hydroxysuccinimide(NHS)-maleimide,
NHS-
pyridyldithiol or NHS-haloacetyl crosslinkers, the palla-
dium oxidative complex 1 shows potential advantages in
high linker stability and minimal hydrolysis side reactions.
The method was successfully applied to intramolecular
crosslinking of SrtA* (7) and intermolecular crosslinking
between interacting MDM2 (9) and pDIQ analog 11 with
excellent protein-selectivity. These studies demonstrate
the potential for palladium oxidative addition complexes
to serve as new reagents for protein crosslinking.
(7) For peptide crosslinking by arylation, see: (a) Spokoyny, A. M.;
Zou, Y.; Ling, J. J.; Yu, H.; Lin, Y. S.; Pentelute, B. L. J. Am. Chem. Soc.
2013, 135, 5946–5949. (b) Brown, S.; Smith, A. J. Am. Chem. Soc. 2015,
ASSOCIATED CONTENT
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137, 4034–4037. (c) Kalhor-Monfared, S.; Jafari, M. R.; Patterson, J. T.;
Kitov, P. I.; Dwyer, J. J.; Nuss, J. M.; Derda, R. Chem. Sci. 2016, 7, 3785–
3790. (d) Lautrette, G.; Touti, F.; Geun Lee, H.; Dai, P.; Pentelute, B. L.
J. Am. Chem. Soc. 2016, 138, 8340–8343. (e) Rojas, A. J.; Zhang, C.;
Vinogradova, E. V.; Buchwald, N.; Reilly, J.; Pentelute, B. L.; Buch-
wald, S. L. Chem. Sci. 2017, 8, 4157–4263. (f) Geun Lee, H.; Lautrette,
G.; Pentelute, B. L.; Buchwald, S. L. Angew. Chem. Int. Ed. 2017, 56,
3177–3181.
1
2
3
4
5
6
(8) The selected examples of protein crosslinking, see: (a) Xiang, Z.;
Ren, H.; Hu, Y. S.; Coin, I.; Wei, J.; Cang, H.; Wang, L. Nat. Methods
2013, 10, 885–888. (b) Furman, J. L.; Kang, M.; Choi, S.; Cao, Y.; Wold,
E. D.; Sun, S. B.; Smider, V. V.; Schultz, P. G.; Kim, C. H. J. Am. Chem.
Soc. 2014, 136, 8411–8417. (c) Chen, X. –H.; Xiang, Z.; Hu, Y. S.; Lacey,
V. K.; Cang, H.; Wang, L. ACS Chem. Biol. 2014, 9, 1956–1961. (d)
Hoppmann, C.; Lacey V. K.; Louie, G. V.; Wei, J.; Noel, J. P.; Wang, L.
Angew. Chem. Int. Ed. 2014, 53, 3932–3936.
7
8
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(9) Xuan, W.; Shao, S.; Schultz, P. G. Angew. Chem. Int. Ed. 2017, 56,
5096–5100.
(10) (a) Vinogradova, E. V.; Zhang, C.; Spokoyny A. M.; Pentelute, B.
L.; Buchwald, S. L. Nature 2015, 526, 687–691. (b) Zhao, W.; Geun Lee,
H.; Buchwald, S. L.; Hooker, J. M. J. Am. Chem. Soc. 2017, 139, 7152–
7155. (c) Rojas, A. J.; Pentelute, B. L.; Buchwald, S. L. Org. Lett. 2017,
19, 4263–4266.
(11) (a) Xiao, H.; Schultz, P. G. Cold Spring Harbor Perspect Biol. 2016,
8, a023945. (b) Liu, C. C.; Schultz, P. G. Annu. Rev. Biochem. 2010, 79,
413–444.
(12) McAtee, J. R.; Martin, S. E. S.; Ahneman, D. T.; Johnson, K. A.;
Watson, D. A. Angew. Chem. Int. Ed. 2012, 51, 3663–3667.
(13) After aging the palladium reagent 1 for 5 months at 4°C in a freez-
er, we performed the crosslinking of peptide 2 using this batch and
quantitatively obtained the desired product 3.
(14) The full optimization data is described in the Supporting Infor-
mation.
(15) In contrast, a significant amount of degradation of maleimide-
cysteine conjugate was observed under basic conditions in our previ-
ous study (ref. 10a).
(16) The SrtA* used here contains P94S/D160N/K196T triple
mutation, which promotes enhanced catalytic efficiency, see: Chen,
I.; Dorr, B. M.; Liu, D. R. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 11399–
11404.
(17) Jason, P.; Zhenyu, L.; Agnieszka, K.; Baozong, L.; Said, S.; Wayne,
G.; Ernst, S. J. Biol. Chem. 2010, 285, 2174–2183.
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OMe
tBu
tBu
O
O
P
1
2
3
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6
OPh
HN
iPr
NH
Pd
HN
MeO
iPr
Br
SH
iPr
S
New Pd reagent for crosslinking
1. Cysteine arylation
2. Urea bond formation with
lysine residues
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Unprotected
peptides and proteins
Intra- and intermolecular
crosslinking
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