Enhanced aqueous Suzuki-Miyaura coupling allows site-specific polypeptide 18F-labeling

Article abstract of DOI:10.1021/ja4049114

The excesses of reagents used in protein chemistry are often incompatible with the reduced or even inverse stoichiometries used for efficient radiolabeling. Analysis and screening of aqueous Pd(0) ligand systems has revealed the importance of a guanidine core and the discovery of 1,1-dimethylguanidine as an enhanced ligand for aqueous Suzuki-Miyaura cross-coupling. This novel Pd catalyst system has now allowed the labeling of small molecules, peptides, and proteins with the fluorine-18 prosthetic [ ¹⁸F]4-fluorophenylboronic acid. These findings now enable site-specific protein ¹⁸F-labeling under biologically compatible conditions using a metal-triggered reaction.

Full text of DOI:10.1021/ja4049114

Communication
pubs.acs.org/JACS
Enhanced Aqueous Suzuki−Miyaura Coupling Allows Site-Specific
18
Polypeptide F‑Labeling
Zhanghua Gao, Veronique Gouverneur, and Benjamin G. Davis*
́
Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, U.K.
S
* Supporting Information
Using a “tag-and-modify” approach26 in our research in
ABSTRACT: The excesses of reagents used in protein
chemistry are often incompatible with the reduced or even
inverse stoichiometries used for efficient radiolabeling.
Analysis and screening of aqueous Pd(0) ligand systems
has revealed the importance of a guanidine core and the
discovery of 1,1-dimethylguanidine as an enhanced ligand
for aqueous Suzuki−Miyaura cross-coupling. This novel
Pd catalyst system has now allowed the labeling of small
molecules, peptides, and proteins with the fluorine-18
prosthetic [18F]4-fluorophenylboronic acid. These findings
now enable site-specific protein 18F-labeling under bio-
logically compatible conditions using a metal-triggered
reaction.
protein post-translational modification, we have demonstrated
efficient Suzuki−Miyaura coupling (SMC) on proteins under
biologically compatible conditions.27−31 Suitable aryl iodide-
containing “tag” residues may be site-specifically introduced into
proteins either by chemical modification27 or in a genetically
encoded manner28,29,31 with great site flexibility, making this a
useful strategy for metal-mediated protein-labeling. The
efficiency, directness, and chemoselectivity as well as the low
toxicity of palladium29 encouraged us to expand the application
scope of SMC protein modification into highly challenging site-
specific 18F-protein-labeling. Here we describe the development
of an enhanced aqueous Pd ligand system that now enables Pd-
mediated protein and peptide 18F-labeling.
Due to the metabolic stability32 of aryl fluorides as well as the
structural simplicity, we designed 4-fluorophenylboronic acid (1)
as a relevant “minimal” boronic acid prosthetic for such labeling.
Despite the power of the SMC reaction, to our knowledge, 1 has
not previously been used as an 18F-prosthetic; a successful SMC
between [19F]1 and a model protein was disclosed in our
previous research.27 Using a phosphine-free, water-soluble
ligand, 2-amino-4,6-dihydroxy-pyrimidine (ADHP, L1), com-
plete coupling of 2 (0.03 mM) was achieved by using a 50-fold
excess of (L1)2Pd(OAc)2 and a 500-fold excess of 1. (L1)2Pd-
(OAc)2 has also recently been successfully used in small-
molecule 18F-labeling, catalyzing the SMC of [18F]2-bromo-4-
fluorophenol and phenylboronic acid in quantitative yield.33
Despite these encouraging precedents, SMC coupling of an 18F-
prosthetic, such as 1, presents particular and striking challenges.
In no-carrier-added 18F-radiochemistry, the radioactive material
is generally the least abundant component in a reaction (and the
key species on which RCY is based). Such labeling with a
nonabundant reagent (perhaps even under reverse stoichiom-
etry) is not addressed well by standard protein chemistry
methods; this has largely prevented effective bioconjugations in
which neither moiety can be used in great excess. Limitations in
current 18F-labeling methods include elevated temperature,17
multistep processes,15−23 and the use of organic solvent.12,18
Additionally, the half-life of 18F, although longer than for other
PET isotopes, still encourages short reaction times (<1 h
typically). Moreover, protein substrates often place strong limits
on concentrations. However, we considered that the apparent
efficiency and utility of the SMC might uniquely tackle these
combined constraints of rapid aqueous reaction under low
concentrations with nonabundant reagent.
eptides and proteins have been increasingly applied as
Psubstrates for positron emission tomography (PET) tracers,
but such tracers remain difficult to create.1−6 Their application
requires practical methods to incorporate positron emission
nuclides (such as 18F, 11C, 68Ga). The most common
radionuclide used in PET imaging is 18F. The relatively long
half-life (t1/2 = 109 min) allows for chemical synthesis, and a low
positron energy offers superior imaging resolution.7 Protein-
labeling using prosthetic 18F-synthons has been developed and
applied to a variety of substrates, generally targeting either
lysine8−11 or cysteine12−14 residues. Some variants employ
heterobifunctional cross-linkers to Lys/Cys, allowing a two-step
process combining other modification chemistries15−21 or even
complexation.22,23 However, these protein-labeling techniques
can result in nonspecific labeling, which may affect the biological
activity and typically generate mixtures of differently labeled
proteins as product. Site-specific labeling of proteins is highly
desirable because a homogeneous product allows unambiguous
biophysical and activity-based characterization, leading to
reproducibility in labeling and imaging. It also allows labeling
to be precisely targeted to functionally benign sites in a given
protein substrate; this is of particular relevance to proper
therapeutic protein evaluation. In particular, site-specific
installation of unnatural amino acids can provide diverse
chemical functionality (a “tag”) for subsequent reaction
(“modification”) that will allow protein-labeling. A few
approaches have been developed; these include the use of
expressed protein ligation to install an aminoxy group labeled by
oxime chemistry24 (used at the C-terminus) and the use of an
unnatural alkynyl amino acid “tag” modified by triazole chemistry
(in low radiochemical yield (RCY)).25
Received: May 16, 2013
© XXXX American Chemical Society
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To probe the limits of the SMC, and so test its possible utility
under such stringent conditions, we explored key parameters.
The impact of boronic acid concentration ([1]) was tested first
using L1 as the ligand, and a model 3-layer-α/β-Rossman-fold
protein subtilisin from Bacillus lentus (SBL) containing the
haloaromatic amino acid Pic27 SBL-156ArI (2) as the substrate
(Scheme 1). The reaction was conducted under key constraints:
(38%) and L4 (<15%). Moreover, variation of boronic acid (Ph,
p-CN, p-Me, p-MeO, o,p-diOMe, furyl; Table S3) confirmed
generality (65−90% yield) of these low substrate conditions
toward other substrates.
To test this enhanced catalytic system in a lowered or reversed
stoichiometry ([[18F]1] < [ArI]) more relevant to radiochemical
conditions, the model unnatural amino acid aryl iodide p-iodo-L-
phenylalanine residue (Boc-pIPhe, 3) was subjected to SMC
with 1 (0.5 equiv to 3) at low concentrations ([3] = 0.10 mM, [1]
= 0.05 mM) (Scheme 2). Notably, optimal reactions required
Scheme 1. Suzuki−Miyaura Coupling of 1 with Model
Proteins SBL-ArI (2) under Constrained Conditions,
Identifying the Guanidine Catalyst “Core” and More Effective
a
Ligand System L3
Scheme 2. SMC of 1 with Model Haloaromatic Small
Biomolecules under Low or Reversed Stoichiometry
low Pd loading of 0.1 mM (2 equiv to 1). Higher catalyst
amounts led to decreased yield, possibly due to the
decomposition of 1 or the opening of another Pd-mediated
reaction manifold (see SI for more details.) Thus, 75% SMC
yield was obtained with only 0.10 mM (L3)2Pd(OAc)2 catalyst
loading (Scheme 2), and >70% yields were obtained from all
three enhanced ligands under these conditions (see SI). Based on
the above, enhanced ligand L3 was selected for more complex
peptide and protein substrates.
To further evaluate the utility of SMC coupling, a decamer
peptide (Ala-pIPhe-Ala-Val-Asn-Thr-Ala-Asn-Ser-Thr, 4) con-
taining haloaromatic unnatural residue p-iodo-L-phenylalanine
was designed, constructed, and tested. Peptide 4 contains the
“tag” amino acid pIPhe inserted between two alanine residues in
an N-terminal cap, followed by a VNTANST sequence as the C-
terminal. VNTANST, namely the Comprehensive Carcinoma
Homing Peptide (CCHP), has been suggested as a broad cancer-
targeting mini-peptide.37 Upon reoptimization with fixed
temperature and reaction time (37 °C, 30 min), the quantitative
SMC conversion (>95%) of 4 (0.1 mM) was achieved with 1.0
mM 1 and [Pd] no lower than 0.3 mM. Even at lower boronic
acid concentration (0.2 mM, 2 equiv), 70% conversion was
possible ([Pd] = 1.0 mM). To explore positional dependency in a
peptide, we also tested a variant of the CCHP in which the “tag”
amino acid pIPhe was found directly at the N-terminus (pIPhe-
Val-Asn-Thr-Ala-Asn-Ser-Thr, 5); >95% conversion was also
possible.
a
Pd source added as precatalyst from Pd(II)OAc2 (see SI for details).
0.05 mM protein at fixed temperature (37 °C) and short reaction
time (30 min) in the absence of any organic solvents. This
revealed a striking dependency of coupling yield (judged with
respect to the protein) upon boronic acid concentration to the
extent that, when [1] was reduced from 10 mM to 2 mM, protein
conversion decreased from 72% to 5%.
We then looked for a more active Pd catalyst system. The
dimethylated analogue of L1, 2-dimethylamino-4,6-dihydrox-
ypyrimidine (L2), has recently been reported as a variant ligand
for Pd(0)-mediated reactions on proteins.34 Although structural
information on the new aqueous Pd catalyst systems does not yet
exist, our developmental studies30 have suggested that it is the
central guanidine moiety that coordinates Pd. To this end we also
considered “minimal” ligand structures, dimethylguanidine (L3)
and tetramethylguanidine (L4). Importantly, both are widely
available and water-soluble.35,36 Moreover, their lack of toxicity
opens up future possibilities for in vivo application. We were
pleased to find all three ligand variants, L2, L3, and L4, afforded
increased catalytic activity at lower boronic acid concentration.
With [1] = 1.0 mM, all three ligands offered >60% protein
conversion, conditions under which essentially no coupling
product was observed from L1; this emphasized the critical need
for novel ligands in such low concentrations and excesses. When
[1] was further decreased to 0.5 mM (only 10 equiv to protein),
L3 stood out by affording a 58% conversion, followed by L2
With these promising small-molecule and peptide reactions in
hand, a final non-radioactive (cold) optimization of protein SMC
B
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Journal of the American Chemical Society
Communication
was carried out at the very low boronic acid:protein ratio of 2. As
for all of the reactions thus described, only reaction times of 30
min were considered to be of realistic utility in analogous hot
chemistry given the half-life of 18F (109 min). By varying the pH,
buffer, concentration of Pd catalyst, and concentration of protein,
reaction was seen under these unprecedentedly stringent
conditions; at pH 8, with 2 mM (L3)2Pd(OAc)2 and 0.2 mM
[SBL-ArI], 10% conversion of protein was obtained. Although
low in synthetic terms, this first observable Pd-mediated protein
F-labeling under such very dilute and time-restricted conditions
paves the way for detectable (and so useful) protein 18F-
radiolabeling.
The radiochemical synthesis (Scheme 3a) of our chosen, novel
18F-prosthetic reagent [18F]1 exploited [18F]4-fluoroiodoben-
zene ([18F]7) as a known 18F-intermediate.38−41 Thus, di(4-
iodophenyl)iodonium triflate (6) was treated with Kryptofix
2.2.2-potassium [18F]fluoride to give [18F]7.38−42 Since a free
boronic acid was of high importance to execute the desired SMC
rapidly, tetrahydroxydiborane (8) was employed to allow direct
C−B(OH)2 formation, thereby avoiding protecting group
manipulation. Despite an array of reported possible conditions
for borylations,43−45 KOAc and DMSO were found to be critical
to success as base and solvent, as originally noted by Miyaura in
“cold” borylations using bis(pinacolato)diboron46 (see SI for
more details). Thus, [18F]7 was converted to [18F]1 by treatment
with 8 in DMSO at 90 °C in the presence of Pd(dppf)Cl2 and
potassium acetate. The two-step radiosynthesis gave excellent
radiochemical purities (RCPs) at each step (RCP >95%) and fair
yields, giving an overall 5−10% decay-corrected RCY (specific
activity 9.7 GBq/μmol; 3.1−14.4 GBq/μmol, n = 3). After solid-
phase extraction purification, the prosthetic reagent [18F]1 was
concentrated and reformulated in aqueous-only pH 8 phosphate
buffer for ready “hot” SMC. It is important to note that the
boronic acid moiety valuably allowed flexible use of [18F]1 in
both organic and aqueous solvents. In addition, to extend
accessibility of our method, we also successfully developed one-
pot methods that allowed direct access to [18F]1 from 6 (see SI).
18F-SMC was first demonstrated by the labeling of two small
molecules, 3 and 4-iodobenzoic acid (9) (Scheme 3b). Thus, 3 or
9 (0.1 mM) was mixed with [18F]1 and 1 mM (L3)2Pd(OAc)2
under low concentration conditions in pH 8 buffer and shaken at
37 °C for 30 min to give RCY = 87% or 83%, respectively. To our
knowledge, these are the lowest substrate concentrations used in
Pd-catalyzed 18F chemistry;47,48 as a result, purification was
greatly simplified due to the very low quantity of remaining
reagents, starting material, and potential side products present in
the final product mixture (<5 nmol).
Scheme 3. (a) Radiosynthesis of [18F]1 and (b) Its Use in the
Direct 18F-Labeling of Small Molecules, Peptide, and Protein;
(c) HPLC (Radio or UV/Vis Detection) of Labeled Protein
14
18F-SMC also proved successful on the Carcinoma Homing
VNTANST peptide 4 and with the peptide variant 5 bearing a
direct N-terminal tag; reaction of [18F]1 under optimized
conditions ([4] or [5] = 0.1−0.4 mM, [(L3)2Pd(OAc)2] = 2.0
mM, 37 °C, 30 min) gave 18F-labeled peptide 12 in 33% RCY
(RCP >95%, n = 9) and 13 in 48% RCY (RCP >95%, n = 4).
Finally, protein-labeling was carried out by incubating the
reaction mixture (SBL-ArI (2), reformulated [18F]1 (10−20
MBq), and (L3)2Pd(OAc)2 in pH 8 phosphate buffer) at 37 °C
for just 30 min. Palladium catalyst was scavenged by 3-
mercaptopropionic acid28 before the protein product 14 was
purified by size-exclusion chromatography down to levels as low
as 1 ppm Pd (as measured by ICP-OES, see SI). Even under
these conditions, detection of radiolabeled protein was
confirmed by HPLC (Scheme 3c) with a RCY of ∼2−5% from
boronic acid 1 (decay-corrected);49 addition of 2 equiv of carrier
[19F]1 did not improve the yield.
This first Pd-catalyzed incorporation of 18F into a protein using
a direct method is an encouraging first step in a new strategy in
18F-protein-labeing and is another, rare, metal-mediated example
(see SI). The 18F[1] aryl boronic acid may also find use in other
powerful synthetic methods, and these are being explored
currently. Further improvement of the RCY would help the
clinical application of this methodology. Nevertheless, despite
the low yield, the current research has demonstrated the power of
Pd-catalyzed cross-coupling reactions under extreme conditions
(biomacromolecules, biocompatible aqueous context, low
concentrations and excesses, and short time frames dictated by
isotope half-life) to allow detectable 18F-protein-labeling. This
was enabled by the discovery of the enhanced, readily available
Pd ligand L3. It is an effective Pd ligand for aqueous Suzuki−
Miyaura coupling at low substrate concentrations (0.10−0.20
mM), allowing “hot SMC” coupling of small molecules and
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Journal of the American Chemical Society
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peptides in radiochemical yields of up to 87%. This discovery
further expands the application scope of aqueous Pd catalysis to
radiobiology.
(23) Laverman, P.; McBride, W. J.; Sharkey, R. M.; Eek, A.; Joosten, L.;
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ASSOCIATED CONTENT
* Supporting Information
■
S
Details of experimental procedures of nonradioactive reaction
optimization, 18F-radiolabeling, and preparation of aqueous Pd
catalysts and substrates. This material is available free of charge
via the Internet at http://pubs.acs.org.
(24) Flavell, R. R.; Kothari, P.; Bar-Dagan, M.; Synan, M.;
Vallabhajosula, S.; Friedman, J. M.; Muir, T. W.; Ceccarini, G. J. Am.
Chem. Soc. 2008, 130, 9106−9112.
(25) Boutureira, O.; D’Hooge, F.; Fernandez-Gonzalez, M.; Bernardes,
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AUTHOR INFORMATION
Corresponding Author
ben.davis@chem.ox.ac.uk
■
(26) Chalker, J. M.; Bernardes, G. J. L.; Davis, B. G. Acc. Chem. Res.
2011, 44, 730−741.
(27) Chalker, J. M.; Wood, C. S. C.; Davis, B. G. J. Am. Chem. Soc. 2009,
131, 16346−7.
Notes
(28) Spicer, C. D.; Davis, B. G. Chem. Commun. 2011, 47, 1698−1700.
(29) Spicer, C. D.; Triemer, T.; Davis, B. G. J. Am. Chem. Soc. 2012,
134, 800−803.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(30) Dumas, A.; Spicer, C. D.; Gao, Z.; Takehana, T.; Lin, Y. A.;
Yasukohchi, T.; Davis, B. G. Angew. Chem., Int. Ed. 2013, 52, 3916−
3921.
We thank EPSRC, CRUK for funding; Advion BioSystems for
supplying the NanoTek Chemistry System; Drs. M. Tredwell
and A. Dumas for discussion and technical assistance; C. Spicer,
L. Lercher, Dr. S. van Berkel, and Dr. K. Yamamoto for
discussions; and Drs. T. L. Collier, and Y. Weng for technical
assistance.
(31) Spicer, C. D.; Davis, B. G. Chem. Commun. 2013, 49, 2747−2749.
(32) Knust, E. J.; Kupfernagel, C.; Muller-Platz, C.; Stocklin, G. J.
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