A convenient catalyst for aqueous and protein Suzuki-Miyaura cross-coupling

Article abstract of DOI:10.1021/ja907150m

(Figure Presented) A phosphine-free palladium catalyst for aqueous Suzuki-Miyaura cross-coupling is presented. The catalyst is active enough to mediate hindered, ortho-substituted biaryl couplings but mild enough for use on peptides and proteins. The Suzuki-Miyaura couplings on protein substrates are the first to proceed in useful conversions. Notably, hydrophobic aryl and vinyl groups can be transferred to the protein surface without the aid of organic solvent since the aryl- and vinylboronic acids used in the coupling are water-soluble as borate salts. The convenience and activity of this catalyst prompts use in both general synthesis and bioconjugation.

Full text of DOI:10.1021/ja907150m

Published on Web 10/23/2009
A Convenient Catalyst for Aqueous and Protein Suzuki-Miyaura
Cross-Coupling
Justin M. Chalker, Charlotte S. C. Wood, and Benjamin G. Davis*
Chemistry Research Laboratory, Department of Chemistry, UniVersity of Oxford, 12 Mansfield Road,
Oxford OX1 3TA, U.K.
Received August 24, 2009; E-mail: ben.davis@chem.ox.ac.uk
Table 1. Model Cross-Couplings with Pd-pyrimidine Catalysta
The utility of palladium-catalyzed cross-coupling in organic
synthesis is indisputable.1 With broad functional group tolerance
and the advent of aqueous cross-coupling,2 it is not surprising these
reactions have been explored in bioconjugation. In particular, cross-
coupling on peptides3 and proteins4 has received much attention
because of the prospect of selective formation of a nonlabile linkage
at a residue functionally orthogonal to natural amino acids.5 Such
modified proteins may then find use in a variety of biochemical
and therapeutic contexts.6 Cross-coupling as a method for protein
modification is particularly attractive since a variety of non-natural
amino acid coupling partners can be genetically incorporated into
peptides and proteins.4,7 Nonetheless, efficient cross-coupling on
proteins remains an outstanding problem and efforts to date have
suffered from low conversion4c or required denaturing conditions.4d
These reports prompt the need for readily available, active catalysts
that mediate cross-coupling under benign conditions suitable for
proteins: buffered aqueous media at ambient temperature.
As part of our continued interest in adapting the tools of organic
chemistry to synthetic biology,8 we considered worthwhile an
exploration of Suzuki-Miyaura cross-coupling9 on peptides and
proteins. We restricted ourselves to phosphine-free catalysts that
are commercially available. Phosphines are susceptible to oxida-
tion,10 and it is convenient to have a system that does not require
an inert atmosphere or degassed solvents. Moreover, phosphine
ligands are often expensive and their price can exceed that of the
palladium salt.
Exploratory work11 on model systems led us to 2-amino-4,6-
dihydroxypyrimidine, a ligand used in Sonogashira cross-couplings
in organic solvents.12 We found that the sodium salt of this ligand
forms a complex with Pd(OAc)2 that is freely soluble in water
(Table 1). We were pleased to find that this system catalyzed
Suzuki-Miyaura cross-couplings in good yields on amino acid and
peptide models (Table 1). The couplings were run at 37 °C in
buffered water. No organic solvent was used and no effort was
made to exclude oxygen.13 The amino acid substrates in Table 1
were selected because they can be incorporated into proteins
genetically (entries 1-4) or chemically (entries 5-7).4,7 No reaction
was observed under these conditions with 4-chlorophenylalanine
(entry 3). Entry 6 indicates that C-terminal cysteine derivatives may
inhibit this catalytic system, but internal alkyl-cysteines are tolerated
and high yields were observed with the peptide in entry 7 containing
the amino acid p-iodobenzyl cysteine (Pic). Free cysteine, however,
is detrimental, and no cross-coupling was observed in interference
experiments where reduced glutathione was added at the same
loading as palladium.11
a For preliminary models and catalysts, see Supporting Information.
borates at the serine of the active site.14 The protein was therefore
irreversibly inhibited by sulfonylation at S221 with phenylmeth-
ylsulfonylfluoride (PMSF) to avoid complications during the cross-
coupling.
As a first attempt, phenylboronic acid was used as the cross-
coupling partner (Table 2, entry 1). Gratifyingly, the coupling was
complete within 30 min at 37 °C, as monitored by LC-MS. While
an excess of palladium (50 equiv) and boronic acid (500 equiv)
was used to ensure rapid coupling, these reagent loadings are still
lower than the thousands of equivalents often employed in protein
modification.15 In control experiments, only unreacted starting
material was observed when 1 was treated with palladium or
phenylboronic acid alone. No reaction was observed under the
coupling conditions when the protein did not contain an arylio-
dide.11
These promising results prompted validation of this catalytic
system on a protein substrate. A model protein (1, Table 2) was
obtained by chemically installing Pic at the single cysteine of
subtilisin Bacillus lentus (SBL) mutant S156C.11 SBL is a serine
protease, and boronic acids are inhibitors of this protein, forming
A variety of aryl- and vinylboronic acids could also be coupled
to the protein (Table 2). It is noteworthy that no organic solvent is
used in these reactions. The borate confers water-solubility to an
otherwise insoluble substrate and allows efficient conjugation of a
9
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10.1021/ja907150m CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
Table 2. Suzuki-Miyaura Cross-Coupling on a Protein Surface
Table 3. Pd-Pyrimidine Catalyst in Aqueous Biaryl Synthesisa
a For more examples, TON/TOF analysis, and related control experi-
ments, see Supporting Information.
Acknowledgment. We thank the Rhodes Trust for funding and
the National Science Foundation for a Graduate Research Fellow-
ship (J.M.C.). Mitul Patel is acknowledged for technical assistance.
Supporting Information Available: Full experimental details and
compound characterization. This material is available free of charge
via the Internet at http://pubs.acs.org.
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variety of hydrophobic aryl groups and lipids (entries 1-8). Finally,
cross-coupling was used as a route to a synthetic glycoprotein16
(Entry 9).
While our main goal was to validate Suzuki-Miyaura cross-
coupling in protein modification, we are not insensitive to general
synthesis. Indeed, the catalyst mediates efficient coupling of a
variety of aryliodides and arylbromides. Selected examples are
shown in Table 3. Microwave irradiation was used for rapid
coupling, though conventional heating suffices in most cases.
The catalyst is functionally tolerant, and phenols, anilines,
thiophenes, and pyridines were synthesized in excellent yield.
Hindered, ortho-substituted biaryls were also readily accessible
(Table 3). These congested couplings are a common testing
ground for catalyst activity.17 Cross-coupling with this catalyst
is also scalable. A 50-g coupling of 4-bromobenzoic acid and
phenylboronic acid was carried out in a beaker of water, open
to air, using only 0.01 mol % Pd. The product crystallizes directly
from the mixture and is isolated in excellent yield (97%).11 No
organic solvent is needed in the reaction or workup, an important
feature in the context of green chemistry.18
In conclusion, we have disclosed a convenient, easily pre-
pared11 catalyst for aqueous Suzuki-Miyaura cross-coupling.
The ligand is commercially available and far cheaper than water-
soluble phosphines typically used in aqueous cross-coupling. The
catalyst is mild enough to promote cross-coupling on proteins
and sufficiently active to mediate hindered biaryl coupling. The
cross-couplings reported on protein substrates are the first to
proceed to completion and vindicate extensive effort to geneti-
cally incorporate cross-coupling partners into proteins. Currently,
we are investigating the use of this catalyst in other Pd-mediated
transformations in water. We are also exploring the scope of
Suzuki-Miyaura cross-coupling in bioconjugation and applica-
tions in fluorogenic protein labeling. Progress to this end will
be reported in due course.
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arylboronic acid is also capable of this reduction to the active catalyst.
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