Self-liganded Suzuki-Miyaura coupling for site-selective protein PEGylation

Article abstract of DOI:10.1002/anie.201208626

Building with PEGs: PEG-boronic acids, in the presence of simple Pd sources, are capable of acting as direct and effective Suzuki reagents in 70-98 % yield. When combined with non-natural amino acids, they allow efficient and direct, site-selective PEGylation of proteins at predetermined positions under biologically compatible conditions without the need for exogenous ligands.

Full text of DOI:10.1002/anie.201208626

Angewandte
Chemie
DOI: 10.1002/anie.201208626
Protein PEGylation
Self-Liganded Suzuki–Miyaura Coupling for Site-Selective Protein
PEGylation**
Anaꢀlle Dumas, Christopher D. Spicer, Zhanghua Gao, Tsuyoshi Takehana, Yuya A. Lin,
Tohru Yasukohchi, and Benjamin G. Davis*
The discovery and development of palladium-catalyzed
carbon–carbon bond formation has revolutionized modern
organic chemistry in recent decades. The versatility and
robustness of these processes have enabled major progress in
total synthesis, materials development, and medicinal chemis-
try.[1] Currently, a large variety of nucleophiles and electro-
philes can be efficiently coupled, and part of the efforts to
increase the utility of these reactions are now directed
towards the development of simpler, more active, and
economic catalytic systems that work under mild conditions.
Owing to their broad functional-group tolerance and low
associated toxicity, as well as their chemical and biological
compatibility, Pd-catalyzed reactions are now attractive
approaches for protein modification.[2] These processes are
particularly interesting, as a variety of non-natural amino
acids can be genetically incorporated into peptides and
proteins,[3] thus providing specific chemical handles for
a number of post-translational manipulations, including
palladium-mediated reactions.[2b] Recent contributions from
our group and others report robust and highly efficient
Suzuki–Miyaura[2a–c,e] and Sonogashira[2d] cross-coupling on
protein substrates bearing a halide or alkyne moiety, respec-
tively, under ambient conditions both in vitro[2a,b] and on cell
surfaces.[2e]
As part of our continuing interest in developing chemical
tools for the mild and specific post-translational modification
of proteins,[4] we investigated the use of the Suzuki–Miyaura
coupling to site-selectively attach polyethylene glycol (PEG)
chains to proteins. Protein PEGylation is a widespread
approach to improve the stability and pharmacokinetics of
protein drugs by reducing clearance rates, while providing
a steric shield from proteolytic enzymes and immune system
recognition.[5] However, reduced biological activity is often
observed for PEG-conjugated proteins.[6] This effect is
attributed to steric crowding resulting from the low positional
control achieved with traditional PEGylation chemistry;
commonly used PEG-derivatives react with the side chains
of natural amino acids, such as lysines or cysteines, or protein
termini and disulfide bonds.[5b,7] When more than one reactive
site is present on the protein, these approaches lead to
heterogenous mixtures of potentially less-/in-active conju-
gates that are difficult to separate.[5a,8] The possibility of site-
selectively PEGylating proteins could potentially overcome
this problem but, to date, only a few examples use the PEG-
derivatization of genetically-encoded non-natural amino
acids. These include Sonogashira coupling at homopro-
pargyl-glycine,[2d] and triazole formation[6a] or Staudinger
phosphite reaction[9] at p-azidophenylalanine. The two
former examples involve metal catalysts and required exog-
enous ligands and consequent optimization.
Air- and moisture-stable Pd-ligands provide convenient
systems that do not require inert atmosphere or degassed
solvents.[2a] Nonetheless, even more direct “ligandless” (self-
liganded or lacking an added ligand) methods are of
significant interest.[10] This would provide a more straightfor-
ward method that is particularly attractive in biotechnological
applications to be conducted on scale such as PEGylation. Pd-
nanoparticle-based catalysts and metal catalysts stabilized by
polymers such as polyethylene glycol (PEG) have been
reported.[11] Taking advantage of these potential stabilizing
properties of PEG, we considered the possibility of direct
palladium-catalyzed PEGylation of halogenated amino acids
with PEG–boronic acid derivatives in the absence of addi-
tional ligand.
Herein, we report PEGylation of amino acids, peptides,
and proteins through palladium-catalyzed Suzuki–Miyaura
coupling of 2 kDa and 20 kDa monomethoxy PEG (mPEG)
phenylboronic acids (mPEG2k-PBA and mPEG20k-PBA,
respectively). An evaluation of pyrimidine- or guanidine-
based palladium complexes and comparison with PEG
reagents alone reveals that the latter can act as potent self-
liganding agents for the stabilization of metal species, thus
allowing complete conversion using water-soluble palladium
salts in the absence of external ligands. The resulting reaction
system combines the advantages of reaction biocompatibility
for protein modification with the possibility of combining
a highly simple, single-component, economic, and environ-
mentally friendly catalyst with a self-liganding coupling
partner. We believe this shows the first example of ligandless
metal-mediated ligation on a protein surface.
[*] Dr. A. Dumas, C. D. Spicer, Dr. Z. Gao, Y. A. Lin, Prof. B. G. Davis
Department of Chemistry, University of Oxford, Chemistry Research
Laboratory, Mansfield Road, Oxford OX13TA (UK)
E-mail: ben.davis@chem.ox.ac.uk
Dr. T. Takehana, T. Yasukohchi
NOF Corporation (Japan)
[**] We thank the Swiss National Science Foundation (A.D.), UCB-
Celltech (C.D.S.), EPSRC (Z.G., Y.A.L.), and CRUK (Z.G.) for
funding, and Profs V. Gouverneur and C. J. Schofield for helpful
discussions. B.G.D. is a Royal Society Wolfson Merit Award
recipient.
Our initial study focused on the PEGylation of N-Boc-4-
iodo-l-phenylalanine (1; Boc-pIPhe) with mPEG20k-PBA
(2) by Suzuki–Miyaura cross-coupling in 20 mm phosphate
buffer, pH 8.0. The palladium catalyst system first tested used
an additional ligand 2-amino-4,6-dihydroxy-pyrimidine (L1),
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201208626.
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which is known to be an efficient system for Suzuki–Miyaura
cross-couplings on iodinated amino acids,[2a] proteins,[2b,c] and
on cell surfaces[2e] with small molecule boronic acids. Surpris-
ingly, in the presence of catalyst (5 mol%) and PEG20k-PBA
(1.5 equiv), no reaction was observed (Table 1, entry 1), even
increased level of PEG reagent proved beneficial with lower
molecular weight PEG (2kDa); the coupling of BocpIPhe
with mPEG2k-PBA (3) and the palladium catalysts (5 mol%)
involving L2, L3, and L4 showed 85–93% conversion under
these conditions (Table 1, entries 8–10), whereas L1 showed
only low activity, even after prolonged reaction times
(Table 1, entry 7).
This unusual and intriguing PEG reagent dependency led
us to explore its role further. Strikingly, when a Pd source
(K2PdCl4) was used alone without the addition of an external
ligand, the aryl halide was > 90% coupled in one hour with
both mPEG2k-PBA (3) and mPEG20k-PBA (2) in the
presence of 5 and 20 mol% of the metal, respectively
(Table 1, entries 11 and 12). To elucidate the possible role
played by the polymer reagent in promoting this efficient
coupling,[11e,11f,11 h] the reaction was investigated with 4-(N-
methylamino carbonyl)-phenyl boronic acid (6), which is
similar to the mPEG-PBA core found in 2 and 3, but lacking
the PEG chain (Table 2). Although L1 showed moderate
efficiency with this deactivated boronic acid under these
Table 1: Suzuki–Miyaura coupling of PEG-bound phenyl boronic acids
with Boc-pIPhe in aqueous solutions with pyrimidine– and guanidine–
palladium complexes or under ligandless conditions.
Entry PEG [kDa]
L
PEG [equiv] Pd loading [mol%] Yield [%][a]
1
2
3
4
5
6
7
8
20
20
20
20
20
20
2
2
2
2
20
2
L1 1.5
L2 1.5
L3 1.5
L4 1.5
L4 1.5
5
5
5
0
68
63
69
64
90
2
92
85
93
92
93
Table 2: Suzuki–Miyaura coupling of 4-(N-methylaminocarbonyl)-phenyl
boronic acid with BocpIPhe in aqueous solution with pyrimidine– and
guanidine–palladium complexes or under ligandless conditions with or
without PEG.[a]
5
20
20
5
5
5
5
20
5
L4
L1
L2
L3
L4
3
3
3
3
3
3
3
9
10
11
12
[b]
–
[b]
–
[a] Yields determined by 1H NMR spectroscopy. [b] K2PdCl4 was used as
the palladium source.
Entry
L
Yield [%][b]
1
2
3
4
5
6
L1
L2
L4
34
>95
93
20
86
1
after 2 h at 378C, as determined by H NMR spectroscopy
[c]
(see the Supporting Information). Other nitrogen-based
ligands containing the same or similar structural motifs were
therefore investigated, including 2-(N,N-dimethyl)-amino-
4,6-dihydroxypyrimidine (L2), 1,1-dimethyl guanidine (L3)
and 1,1,3,3-tetramethyl guanidine (L4).[2d,12] With catalytic
systems involving L2–L4, 63–69% of the aryl halide was
consumed in one hour under the same conditions (Table 1,
entries 2–4). Importantly, increasing the palladium loading
did not improve the conversion (Table 1, entry 5), and the full
consumption of the PEG–boronic acid was identified as the
–
mPEG 2000[c]
mPEG 2000, D[c,d]
>95
[a] Reaction conditions: 6 (3 equiv)/BocpIPhe, Pd (5 mol%), 1 h, 378C.
[b] Yields determined by 1H NMR spectroscopy. [c] K2PdCl4 was used as
the palladium source. [d] mPEG2000 pre-incubated with K2PdCl4 at 378C
for 15 min.
conditions (Table 2, entry 1), the coupling with BocpIPhe
showed over 93% conversion in one hour with catalysts based
on L2 and L4 (Table 2, entry 2–3). However, in clear contrast
to the PEG–boronic acid derivatives, only low (20%)
conversion was observed in this case in the absence of
ligand: palladium black precipitated from the solution, also in
contrast to the PEG-based systems. Consistent with this
critical role of PEG in coordinating Pd, the addition of
monomethoxy polyethylene glycol (mPEG2000) to the reac-
tion mixture allowed 86% conversion under the same
conditions, with little palladium precipitate observed. Finally,
pre-incubation of the metal with the polymer for 15 min at
378C before the addition of the other reagents eliminated
1
limiting factor by H NMR analysis. This arose mainly from
the formation of the homocoupled product, by reduction of
the PdII precatalyst to the active Pd0 species in situ,[13] or by
phenyl-ring oxidation as a result of palladium-catalyzed
hydroxylation of the boronic acid in the presence of O2.[14]
The addition of agents such as sodium ascorbate or sodium
formate, to reduce the precatalyst did not lead to any
significant improvement in yield (see the Supporting Infor-
mation).
Increased equivalents of PEG-reagent boronic acid to
BocpIPhe led to an improved conversion of the halide in one
hour at 378C (90%; Table 1, entry 6). Analogously, this
2
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metal precipitation entirely and led to full conversion after
one hour (Table 2, entries 4–6). Together these results suggest
that, in the case of the PEG boronic acids, the interaction of
K2PdCl4 with the PEG side chains of mPEG2k-PBA and
mPEG20k-PBA is likely to stabilize the relevant catalytic
species and provide a robust system to promote Suzuki–
Miyaura coupling under ligandless/self-liganded conditions,
without the addition of external ligands.[10a,11b–f,h]
These encouraging results on a model amino acid system
(BocpIPhe) prompted us to investigate these coupling con-
ditions to PEGylate two different non-natural, iodinated
amino acids, p-iodo-benzylcysteine (Pic)[2a] and p-iodo-phe-
nylalanine (pIPhe), in the context of two differing protein
structural motifs/folds, 3-layer-a/b-Rossman-fold protein sub-
tilisin from Bacillus lentus (SBL) and all-b-helix protein 275–
276 from Nostoc punctiforme (Npb),[15] respectively.
The non-natural amino acid pIPhe was genetically instal-
led at predetermined site 69 of Npb, using amber stop codon
suppression;[16] this site was chosen based on accessibility
analysis as observed from the crystal structure.[15] His-tagged
Npb-69pIPhe was expressed in E. coli cells with high fidelity
and with full pIPhe occupancy, and then purified using Ni-
NTA affinity chromatography. Pic was chemically installed[2a]
at predetermined site 156 of SBL to create SBL-156Pic;
autolytic degradation was controlled through the addition of
PMSF.[2a,17]
Suzuki–Miyaura PEGylation reactions were tested on
these two model proteins by incubation with mPEG2k-PBA
and Pd with either the pyrimidine and guanidine ligands (L1–
L4) or under ligandless conditions. The physical properties of
the PEG chains have a non-linear effect on the Stokes radius
of the proteins to which they are attached, and cause
PEGylated proteins to migrate significantly more slowly in
polyacrylamide gels than proteins of the same total molecular
weight, thus providing an effective means to resolve them
from the native protein on SDS-PAGE.[18] The reactions were
therefore analyzed by SDS-PAGE, and, after Coomassie
staining, quantified by gel densitometry. Gratifyingly, after
incubation at 378C with mPEG2k-PBA and palladium
catalyst, both proteins demonstrated a higher molecular
weight species by electrophoresis, which corresponded to
the PEGylated mutants mPEG2k-Np or mPEG2k-SBL
(Figure 1). In both cases, no PEGylated product was observed
in the absence of palladium catalyst or PEG–boronic acid.
Similar to the observations made at the amino acid level, little
or no reaction was observed with L1 as a catalyst ligand,
whereas L2–L4 gave the PEGylated product in 25–55% yield
for Npb-69pIPhe (Figure 1B and Table 3) and 70–80% yield
for SBL-156Pic (Figure 1C and Table 3). The reaction of
mPEG20k-PBA with Npb-69pIPhe showed similar results,
yielding 40–70% PEGylated protein mPEG20k-Np after
incubation with catalysts based on L2–L4 or in the absence of
additional ligands (see the Supporting Information). As can
be seen in Table 3, the ligandless conditions resulted in the
highest conversions with Npb-69pIPhe and conversions that
were among the highest obtained for SBL-156Pic.
cation of reaction, especially when conducted in a site-
selective manner, as demonstrated here. After palladium
scavenging using 3-mercaptopropionic acid,[2b] MALDI-TOF-
MS analysis of the reaction mixtures confirmed the presence
of the desired PEGylated products at a m/z 2300–2400 higher
than for the native protein (Figure 1D,E). Together these
results confirmed the ability of not only guanidine-based
catalyst systems, but also the direct ligandless PEG system
presented here to promote Suzuki–Miyaura coupling as a new
strategy for site-selective PEGylation of proteins.
The overall conversion with these bulky polymer reagents
was not as high on the protein surface as with some small-
molecule modifications,[2a,b] but compare well with other
PEGylation reactions with genetically incorporated non-
natural amino acids (which show 42–85% conversions to
PEGylated product).[2d,6a,9] Protein “mapping” through com-
bined tryptic digest and MS/MSMS was performed on the
material contained in the lower-molecular-weight electro-
phoretic region after reactions with mPEG2k-PBA or
mPEG20k-PBA using either [L42Pd(OAc)2] or ligandless
conditions. These revealed a complete loss of iodide (Phe
instead of the expected pIPhe at the site of modification in the
relevant peptide). This suggests that, in the protein environ-
ment, although the initial oxidative addition is essentially
complete, subsequent dehalogenation competes with trans-
metalation, thus preventing full conversion into the coupled
product. The steric bulk from the interaction between the
protein and the PEG derivative may indeed slow down the
transmetalation and result in possible dehalogenation. This
pathway may potentially be diminished by conducting the
conjugation at less sterically hindered amino acid residues or
through the use of different, potentially more active boron
species, such as boronic esters or triolborates.[20]
ICP-OES analysis performed on the protein mixture
obtained from the reaction of Npb-69pIPhe and mPEG2k-
PBA under ligandless conditions showed the presence of ca.
1800 ppm palladium. Although this is below the indicative
pharmaceutical permitted exposure, given the low doses of
PEGylated proteins administered,[21] appropriate scavenging
resins may help further remove this residual metal on an
applied biotechnological scale. However, to the best of our
knowledge, no extensive toxicological testing has revealed
significant toxicity of palladium in humans to date,[21] and
recent contributions show minimal cellular toxicity of suitably
complexed palladium catalysts.[2e]
In summary, a novel way to PEGylate halogenated amino
acids and proteins through the Suzuki–Miyaura coupling of
PEG–boronic acid derivatives was demonstrated. The PEG–
boronic acid derivatives themselves were shown to be
sufficient to stabilize the reactive palladium-species and
promote the coupling using a water-soluble PdII salt in the
absence of external ligands. These ligandless conditions
enabled full conversion of the iodinated amino acid in one
hour at 378C. This system was then applied to site-selectively
PEGylate proteins at a genetically-encoded pIPhe or chemi-
cally-installed Pic. Although guanidine-based catalysts also
promoted Suzuki–Miyaura coupling on proteins, ligandless
conditions provided among the highest conversions for the
PEGylation of proteins. Dehalogenation, leading to full
These results were also confirmed by mass spectrometry.
The heterogeneity of PEG reagents[19] prevents precise
analysis by ESI-MS, but MALDI-MS provides a good indi-
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consumption of the iodi-
nated amino acid, was
revealed as the major side
reaction by tryptic protein
mapping. However, up to
70%
conversions
into
PEGylated protein were
obtained upon coupling
under these ligandless con-
ditions.
Using the simplest pal-
ladium species, this reaction
shows the first example of
self-liganded palladium-cat-
alyzed coupling on protein
surfaces and has been
applied here to an impor-
tant, biotechnologically rel-
evant transformation to
allow an operationally sim-
plified process. It is impor-
À
tant to note that the C C
linkage created here is one
with much greater stability
than those typically cur-
rently used for PEG-attach-
ment to proteins, an issue
and/or problem of primary
importance in some PEGy-
lated therapeutics.[7] More-
over, this ability to site-
selectively install PEG,
combined with strategies
for accessing highly pure
PEG,[19] now opens up intri-
guing possibilities on pro-
teins for both crosslinking
moieties
with
PEG
(“hetero-PEG”) and in pre-
cisely “seeding” the helical
[19]
properties of PEG.
When combined with the
recently discovered atypical
switch-like behavior of Pd
in biological systems[2e]
exciting
possibilities
emerge for the application
of this type of unified
ligand–reagent–metal
system in contexts where
multiple reagents would be
difficult to apply, such as in
in vivo reactions or in large-
scale biotechnological ap-
plications.
Figure 1. Suzuki–Miyaura PEGylation of Npb-69pIPhe and SBL-156 Pic with mPEG2k-PBA. A) Reaction
scheme. B and C) Coomassie-stained SDS-PAGE gel of the reaction of Npb-69pIPhe (B) and SBL-156Pic (C),
showing the formation of PEGylated proteins (mPEG-Prot.); lanes 1–5: reaction mixtures involving L1–L4 and
ligandless conditions, lane 6: reaction in the absence of palladium catalyst, lane 7: reaction in the absence of
mPEG2k-PBA. D and E) MALDI-TOF analysis of the unreacted proteins, or reaction mixture catalyzed by
[L42Pd(OAc)2] (lane 4 in B and C), K2PdCl4 (lane 5 in B and C) or run in the absence of palladium (lane 6 in B
and C) for Npb-69pIPhe (D) and SBL-156Pic (E). (1000 equiv mPEG2k-PBA, 40 (Npb-69pIPhe) or 10 (SBL-
156Pic) equiv Pd catalyst, 378C, 2 h.) [a] K2PdCl4 was used as the palladium source.
4
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Miyaura, A. Suzuki, Chem. Rev. 1995, 95, 2457 – 2483; e) Y.
Table 3: Yield of the Suzuki–Miyaura PEGylation of Npb-69pIPhe and
SBL-156Pic with mPEG2k-PBA, as determined by gel densitometry.
Nakama, O. Yoshida, M. Yoda, K. Araki, Y. Sawada, J.
Nakamura, S. Xu, K. Miura, H. Maki, H. Arimoto, J. Med.
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Npb-69pIPhe
yield [%]
SBL-156Pic
yield [%]
ligand
ligand
L1
L2
L3
L4
0
25
40
55
60
L1
L2
L3
L4
0
70
80
70
70
[a]
[a]
–
–
[a] K2PdCl4 was used as the palladium source.
[2] a) J. M. Chalker, C. S. C. Wood, B. G. Davis, J. Am. Chem. Soc.
2009, 131, 16346 – 16347; b) C. D. Spicer, B. G. Davis, Chem.
Commun. 2011, 47, 1698 – 1700; c) Y. S. Wang, W. K. Russell,
Z. Y. Wang, W. Wan, L. E. Dodd, P. J. Pai, D. H. Russell, W. S. R.
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Edwardraja, Q. Lin, J. Am. Chem. Soc. 2011, 133, 15316 – 15319;
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134, 800 – 803; f) J. Li, P. R. Chen, ChemBioChem 2012, 13,
1728 – 1731.
Experimental Section
General method for Suzuki–Miyaura coupling of Boc-pIPhe: mPEG-
PBA (1.5–3 equiv) diluted with phosphate buffer (200 mm, pH 8),
water, and D2O were added to a solution of N-Boc-4-iodo-l-
phenylalanine in phosphate buffer (20 mm, pH 8). Palladium catalyst
stock solution (10 mm; 0.05 or 0.2 equiv) was then added and the
reaction was mixed and shaken at 378C. After 1 h, the reaction was
analyzed by NMR spectroscopy.
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[4] J. M. Chalker, G. J. L. Bernardes, B. G. Davis, Acc. Chem. Res.
2011, 44, 730 – 741.
General method for Suzuki–Miyaura protein PEGylation: A
solution of Na2HPO4 (5 mL, 100 mgmLÀ1
, 3.5 mmol) containing
mPEG-PBA (1000 equiv) was added to a solution of pIPhe- or Pic-
tagged protein (20 mL, 0.3–0.5 mgmLÀ1) in phosphate-buffered saline
(PBS, pH 8.0). Stock palladium catalyst (10 mm, ꢀ 10–40 equiv) was
then added and the mixture was shaken at 378C for 2 h. The reactions
were analyzed by SDS-PAGE on 12% Bis–Tris polyacrylamide gel
with MOPS running buffer. Control reactions with either Pd catalyst
or PEG–boronic acid excluded were diluted with water to maintain
concentrations.
[5] a) S. N. S. Alconcel, A. S. Baas, H. D. Maynard, Polym. Chem.
ˇ
2011, 2, 1442 – 1448; b) S. Jevsevar, M. Kunstelj, V. G. Porekar,
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applications (Ed.: F. Veronese), Birkhꢂuser, Basel, 2009, pp. 11 –
31.
[6] a) A. Deiters, T. A. Cropp, D. Summerer, M. Mukherji, P. G.
Schultz, Bioorg. Med. Chem. Lett. 2004, 14, 5743 – 5745; b) A. J.
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MALDI-MS: Prior to analysis, palladium was scavenged from the
reaction mixtures with 3-mercaptopropionic acid at 378C for
30 min.[2b] The reaction mixture (1 mL) was diluted with water
(3 mL) and mixed 1:1 with matrix (sinapic acid matrix 10 mgmLÀ1 in
4:6 acetonitrile/H2O+0.1% TFA); 1 mL was applied to the plate.
“In gel” tryptic digestions: 3 mL of SDS loading buffer was added
to 10 mL of reaction mixture and the samples were heated to 958C for
10 min. The reactions were then run on SDS-PAGE on 12% Bis–Tris
polyacrylamide gel with MOPS running buffer. Proteins were
revealed with Instant Blue stain and then extensively washed
(water). Bands were then excised from the gel and washed
(NH4HCO3 in Milli-Q water/HPLC grade MeCN ꢀ 2 then MeCN),
dried, treated with DTT at 378C for 30 min, washed as above, treated
with iodoacetamide solution for 60 min in the dark, washed, and
dried. Sequencing Grade Modified Trypsin solution (1 mg, Promega)
was added and the digest was incubated at 378C overnight. 1 mL of
formic acid was added to quench, and the supernatant containing the
peptides transferred to a new tube. 50 mL of extraction buffer (20 mL
formic acid in 10 mL Milli-Q water and 10 mL HPLC grade
acetonitrile) was then added and the mixture was incubated for
30 min, and the supernatant was pooled with the existing supernatant.
The mixtures were then dried and dissolved in water (10 mL). The
samples were analyzed by MS/MS-MS.
[7] a) S. Balan, J.-W. Choi, A. Godwin, I. Teo, C. M. Laborde, S.
Heidelberger, M. Zloh, S. Shaunak, S. Brocchini, Bioconjugate
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PEGylation, basic science and biological applications (Ed.: F.
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Received: October 26, 2012
Published online: && &&, &&&&
Keywords: catalysis · PEGylation · proteins ·
.
Suzuki-Miyaura coupling
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.
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6
www.angewandte.org
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2013, 52, 1 – 7
These are not the final page numbers!

Angewandte
Chemie
Communications
Protein PEGylation
A. Dumas, C. D. Spicer, Z. Gao,
T. Takehana, Y. A. Lin, T. Yasukohchi,
B. G. Davis*
&&& — &&&
Self-Liganded Suzuki–Miyaura Coupling
for Site-Selective Protein PEGylation
Building with PEGs: PEG–boronic acids,
in the presence of simple Pd sources, are
capable of acting as direct and effective
Suzuki reagents in 70–98% yield. When
combined with non-natural amino acids,
they allow efficient and direct, site-selec-
tive PEGylation of proteins at predeter-
mined positions under biologically com-
patible conditions without the need for
exogenous ligands.
Angew. Chem. Int. Ed. 2013, 52, 1 – 7
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
7
These are not the final page numbers!