Site-Specific Deoxyfluorination of Small Peptides with [18F]Fluoride

Article abstract of DOI:10.1002/anie.201807983

Radiolabeled receptor-binding peptides are an important class of positron emission tomography tracers owing to achievable high binding affinities and their rapid blood clearance. Herein, a method to introduce a 4-[¹⁸F]fluoro-phenylalanine residue into peptide sequences is reported, by chemoselective radio-deoxyfluorination of a tyrosine residue using a traceless activating group. The replacement of only one hydrogen atom with [¹⁸F]fluoride results in minimal structural perturbation of the peptide, which is desirable in the labeling of tracer candidates.

Full text of DOI:10.1002/anie.201807983

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Accepted Article
Title: Site-Specific Deoxyfluorination of Small Peptides with
[18F]Fluoride
Authors: Tobias Ritter and Jens Rickmeier
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201807983
Angew. Chem. 10.1002/ange.201807983
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COMMUNICATION
Site-Specific Deoxyfluorination of Small Peptides with
[18
F]Fluoride
Jens Rickmeier and Tobias Ritter*
Abstract: Radiolabeled receptor-binding peptides are an important
class of positron emission tomography tracers owing to achievable
high binding affinities and their rapid blood clearance. Here, we
18
report a method to introduce a 4-[ F]fluoro-phenylalanine residue
into peptide sequences, by chemoselective radio-deoxyfluorination
of
a tyrosine residue using a traceless-activating group. The
18
replacement of only one hydrogen atom with [ F]fluoride results in
minimal structural perturbation of the peptide, which is desirable in
the labeling of tracer candidates.
Radiolabeled peptides are advantageous positron emission
tomography (PET) tracers, and have been applied in preclinical
target evaluation, noninvasive diagnosis of diseases, and in the
study of biochemical processes.[1] The wide-spread use of small-
peptide tracers is due to the favorable prospects of achieving
high binding affinities, rapid blood clearance, and facile
accessed by automated solid phase peptide synthesis (SPPS).[2]
The labeling of native peptides without affecting their properties
is desirable and can be achieved using 11C, 13N, and 15O
radionuclide-labeled structures, however, the short half-lives of
2
0 minutes or less of these nuclei often prevents meaningful
imaging.[ The 110 min half-life of 18F is more appropriate for
PET imaging with peptides, but native peptides with fluorine
substitution are unknown.[4] One of the smallest structural
perturbations to a native peptide with 18F is the displacement of
a single hydrogen atom.[5] Despite the recent development of
several fluorination reactions, no method, conventional or
modern, has been successful in achieving this goal for
3]
Figure 1. Peptide radiolabeling approaches.
peptides, such as the somatostatin-targeting compounds
edotreotide and DOTA-TATE often contain ligands that are
useful to chelate radioactive metals for therapy and diagnosis
(Figure 1B).[8] Despite their utility, the large polar ligands
substantially alter the structure of the native peptide and can
1
8
polypeptide labeling with [ F]fluoride. The lack of a conceptual
breakthrough in this area is rooted in the dense functionality of
peptides that can result in deprotonation by basic [ F]fluoride
and side reactions of reagents or catalysts with the peptide. With
available methods, polypeptide labeling with [ F]fluoride is
restricted to the use of prosthetic groups, which can be very
useful, but alter the properties of the peptide as a consequence
of the approach itself.[6] Here we report a method to label small
peptides by formally replacing a single hydrogen, the para-
1
8
1
8
[6b]
have substantial effects on the pharmacokinetic profile. There
are several examples of the introduction of smaller labeled
prosthetic groups to peptides, which can be expected to alter the
properties of the peptides to a smaller extent.[9] For example,
Hooker and Buchwald reported a two-step protocol to label
1
8
11
hydrogen in phenylalanine, with [ F]fluoride (Figure 1A). The
method is robust, tolerates all 20 canonical amino acids, can
make use of conventional solid phase peptide synthesis, and
labels at a single, pre-defined site due to the use of a traceless
activating ruthenium group. While ruthenium-mediated
deoxyfluorination of simple phenols has been developed
previously,[ our method represents a conceptual advance
because it enables access to an important class of 18F-labeled
molecules that has hitherto remained elusive to all known
fluorination methods. Currently, clinically used radiolabeled
unprotected peptides on cysteine with a 4-[ C]cyano-phenyl
substituent.[10] Unnatural peptides have been labeled through
1
8
biorthogonal 1,3-dipolar cycloaddition with [ F]-containing
alkynes (Figure 1C).[ The direct labeling with fluorine-18 can
be achieved successfully with prefunctionalized peptides that
11]
1
8
are able to capture [ F]fluoride under mild conditions by the
formation of C–F, Si–F, B–F or Al–F bonds.[6d, 12] Introduction of
fluorine-18 via carbon–sulfur bond formation was shown by the
Gouverneur group:[13] synthesis of the [ F]Umemoto reagent in
5% non-decay corrected radiochemical yield (RCY) enabled the
subsequent direct labeling of unmodified peptides with fluorine-
7]
18
18
1
3
8 at cysteine or homocysteine residues with [ F]CF to afford
radiolabeled thioethers (Figure 1D). The prosthetic group
methods have also been applied successfully for large
molecules such as proteins, however, structural modification has
[*]
J. Rickmeier, Prof. Dr. T. Ritter
Max-Planck-Institut für Kohlenforschung
Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr (Germany)
E-mail: ritter@mpi-muelheim.mpg.de
[6a]
a larger effect for smaller molecules, such as small peptides.
Supporting information for this article is given via a link at the end
of the document.
The Groves group disclosed a method to label small organic
molecules including one example of a dipeptide with [ F]fluoride
1
8
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COMMUNICATION
peptide synthesis. The presence of additional phenylalanine,
tyrosine, or tryptophan residues in the peptide does not pose a
challenge in reactivity or site selectivity in the ensuing labeling
reaction. Likewise, ruthenium migration from one side-chain to
another aromatic side-chain has not been observed in any case
of this study.
Radiolabeling procedures with 18F typically begin with an
1
8
aqueous elution of [ F]fluoride trapped on an anion exchange
cartridge, followed by azeotropic drying of the fluoride. In the
context of this project, we have found a simple, useful method to
avoid aqueous elution and azeotropic drying when elution was
executed with the peptide ruthenium complex in a solvent
mixture of ethanol and pivalonitrile directly. Key to successful
elution was the use of bis(trimethylneopentylammonium) oxalate
Scheme 1. Synthesis and application of novel amino acid building block 1.
(
3) (Table 1, entry 1). The elution efficiency dropped from 81%
through direct C–H bond fluorination, but the method was not
shown to be extendable to larger peptides.[14] Direct C–H
fluorination of peptides has been shown by the Britton group,
with oxalate 3 to 47% if 3 was not added to the eluent.
Alternatively a mixture of Kryptofix 222 and potassium oxalate
®
can be used (elution efficiency: 74%). No special precautions
towards air and moisture are required; addition of 10 µL
exogenous water to the 450 µL reaction volume resulted only in
a small decrease in RCY, from 84% to 78% (Table 1, entry 4).
For typical experiments, we employed 5 µmol of peptide
precursor, which corresponds to about 6–12 mg; lowering the
amount to 1.5 µmol (ca. 2 mg) resulted in 44% RCY, which may
be acceptable if the peptide is precious. The ideal reaction
temperature was determined to be 130 °C. Initially, we were
concerned about the high temperature, however, for all the
peptides evaluated, substantial decomposition was not an issue
during the 30 minutes time required for the reaction. Lowering
1
8
however, through the use of [ F]fluorine gas, which is more
18
[15]
difficult to make and handle than [ F]fluoride.
The first
method achieving peptide labeling without structural
modifications was reported by Långström, who labeled
1
1
11
homocysteine with [ C]MeI to afford a [ C]methionine residue
half-life of 11C: 20 min).[16] Despite recent progress, there is still
(
1
8
no approach that introduces a single [ F]fluoride into an
otherwise native polypeptide. Here we describe the first such
method; the only structural necessity for its successful
implementation is the presence of a phenylalanine residue in the
18
native peptide, which would be replaced by a [ F]-4-fluoro-
phenylalanine in the labeled peptide, obtained through radio-
deoxyfluorination of a tyrosine residue, introduced via SPPS.
The non-natural, ruthenium-containing amino acid building
block 1 can be prepared from tyrosine and can be purified on
gram-scale without the need for chromatography (Scheme 1A).
the reaction temperature or shortening the reaction time resulted
in a different major product, likely the ruthenium-complexed 18F-
labeled peptide, which, if formed, can be readily separated by
18
HPLC. In a typical experiment, [ F]fluoride is trapped from an
aqueous solution on a quaternary methyl ammonium (QMA)
cartridge, followed by removal of residual water by pushing 1 mL
[
17]
Simple precipitation of the neutral zwitterion 1 provides
analytically clean material that can participate readily in
conventional SPPS using standard HBTU coupling. As a result,
various peptides can be accessed rapidly with 1 by modular
synthesis to incorporate the ruthenium directing group (Scheme
18
of acetonitrile through the QMA cartridge. The [ F]fluoride is
eluted from the cartridge with a solution of the peptide complex,
oxalate 3, and chloroimidazolium chloride 4. Then the cartridge
is eluted sequentially with a mixture of veratrole and pivalonitrile
1B). The ruthenium and phenol functionality in 1 act as
spectators in SPPS and did not engage in unwanted side
reactions, such as ester bond formation at the phenolic group or
undesired ruthenium leaching. The inert behavior may be a
result of the tight η6 binding mode of the ruthenium to the π-
system of tyrosine, and the resulting lower nucleophilicity of the
phenol.[18] After SPPS on a 2-chlorotrityl resin the ruthenium-
containing peptides were cleaved with a mixture of 20%
hexafluoroisopropanol (HFIP) in DCM to afford the fully
protected peptide. The peptide ruthenium complexes are stable
towards air and moisture and can be purified by preparative high
performance liquid chromatography (HPLC). When the peptide
change of reaction conditions
elution efficiency[a]
RCY[b]
84%
73%
76%
78%
44%
none
without oxalate 3
kryptofix® 222, K
81%
47%
74%
57%
67%
2 2 4
C O instead of 3
complexes were stored at −20 °C in
a sealed vial, no
degradation was observed for at least two months. Introduction
of the ruthenium complex as part of an individually labeled
amino acid building block during SPPS allows chemoselective,
site-specific labeling of peptides as opposed to labeling of a
native, assembled peptide. The ease and efficiency of SPPS
mitigates the additional synthetic effort required to incorporate 1,
and presents itself as an advantage in modularity and pace of
10 µL H
2
O added
2 mg of 5 instead of 7 mg
Table 1. Reaction optimization. Reaction conditions: 5 (5.0 µmol), 3 (14
µmol), 4 (15 µmol) solv. = pivalonitrile:veratrole:ethanol (450 µL, 4:4:1,
v:v:v),. [ determined by measuring the activity on the cartridge before and
after elution [ determined by multiplying radio-TLC conversion of fluorine-18
with radio-HPLC purity (n = 2).
a]
b]
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Table 2. Substrate table for ruthenium-mediated 18F-deoxyfluorination of peptides. Reaction conditions: All reactions were performed starting with 5.0 µmol
peptide precursor in a solvent mixture of pivalonitrile, veratrole, and ethanol (4:4:1, v:v:v). The decay-corrected radiochemical yields (RCYs) were determined by
measuring the product activity of HPLC purified material versus starting [18F]fluoride, and are averaged over n experiments.
Scheme 2. Fully automated peptide labeling.
into the same vial. The vial, containing 450 µL of reaction
mixture, was sealed and heated for 30 min at 130 °C to afford
carboxypeptidases.[21] The use of peptides containing C-terminal
primary amides provide generally high yields, such as for
octapeptide 9, an analog of the gastrin releasing peptide (GRP)-
receptor tracer MG11, which was isolated in 24% RCY after
HPLC purification.[22] The neuromedine B analog 10 containing
10 amino acids could be isolated after HPLC purification in 29%
RCY. Pentapeptide 11 contains a histidine residue that could
function as internal base to result in epimerization;[23] synthesis
of 11 was accomplished in 39% RCY after HPLC purification
and showed no epimerization through comparison with authentic
samples of both diastereomers within the limits of detection. The
generality of our protocol is highlighted by successful labeling of
all substrates under identical reaction conditions. All 20
canonical amino acids are compatible with the protocol, and the
ruthenium activation group can be positioned N-terminally, C-
terminally, or within the core part of the peptide. The use of
protected peptide precursors is crucial to achieve radio-
deoxyfluorination in high yield.
1
8
the F-labeled, fully protected peptide. After concentration of the
reaction mixture to dryness, the acid-labile protecting groups
were removed with a mixture of trifluoroacetic acid (TFA, 0.44
mL), DL-dithiothreitol (DTT, 25 mg), water (25 µL), and
triisopropylsilane (TIPS, 13 µL). The resulting deprotected
peptides were purified by HPLC.
The procedure is amenable to labeling complex small
peptides that could potentially be used as PET tracers (Table 2).
Cyclic peptide 7 containing the Arg-Gly-Asp (RGD) motif,
commonly used as a binding site in angiogenesis monitoring
PET tracers, could be labeled in 25% decay corrected RCY after
HPLC purification.[19] The presence of
a C-terminal free
carboxylic acid during radiolabeling is tolerated, but tends to
lower the yield.[20] For example, octapeptide 8 was labeled in
11% RCY after HPLC purification. The protection of the free
carboxylic acid as a p-methoxybenyzl (PMB) ester increased the
RCY to 39%. Most peptide radiotracers contain C-terminal
primary amides, which often mimics more closely the native
peptide, and often have higher metabolic stability towards
Initial attempts to automate the process on the cassette-based
radiochemical synthesizer ELIXYS FLEX/CHEM connected to a
PURE/FORM purification and formulation unit (Sofie
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COMMUNICATION
Biosciences) were low yielding. To obtain the same results as in
the manual reaction, the set temperature was set to 150 °C,
possibly an artefact of a temperature difference between set and
actual temperature. Starting from 11.4 GBq (308 mCi) of
aqueous [ F]fluoride, we were able to isolate 1.28 GBq (34.6
mCi) of HPLC-purified peptide 12 within 99 min in 21% decay
corrected radiochemical yield, formulated in ethanolic saline
[7]
8]
M. H. Beyzavi, D. Mandal, M. G. Strebl, C. N. Neumann, E. M. D'Amato,
J. Chen, J. M. Hooker, T. Ritter, ACS Cent. Sci. 2017, 3, 944.
a) E. P. Krenning, D. J. Kwekkeboom, W. H. Bakker, W. A. P. Breeman,
P. P. M. Kooij, H. Y. Oei, M. van Hagen, P. T. E. Postema, M. de Jong,
J. C. Reubi, T. J. Visser, A. E. M. Reijs, L. J. Hofland, J. W. Koper, S.
W. J. Lamberts, Eur. J. Nucl. Med. Mol. Imaging 1993, 20, 716; b) S. R.
Banerjee, M. G. Pomper, Appl. Radiat. Isot. 2013, 76, 2;
[
1
8
[9]
C. L. Charron, J. L. Hickey, T. K. Nsiama, D. R. Cruickshank, W. L.
Turnbull, L. G. Luyt, Nat. Prod. Rep. 2016, 33, 761.
(
Scheme 2). Determination of the molar activity in this project is
1
8
important because, while no-carrier-added [ F]fluoride is used,
conventional SPPS employs reagents with PF −
counter-anions
[10] W. Zhao, H. G. Lee, S. L. Buchwald, J. M. Hooker, J. Am. Chem. Soc.
2017, 139, 7152.
6
[11] a) J. Marik, J. L. Sutcliffe, Tetrahedron Lett. 2006, 47, 6681; b) M.
Glaser, E. Årstad, Bioconjugate Chem. 2007, 18, 989.
in large excess that could, in principle, substantially dilute the
molar activity. Gratifyingly, the molar activity of peptide 12 was
[
12] a) J. Becaud, L. Mu, M. Karramkam, P. A. Schubiger, S. M. Ametamey,
K. Graham, T. Stellfeld, L. Lehmann, S. Borkowski, D. Berndorff, L.
Dinkelborg, A. Srinivasan, R. Smits, B. Koksch, Bioconjugate Chem.
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Kersemans, S. Smart, M. Tredwell, B. G. Davis, V. Gouverneur, J. Am.
Chem. Soc. 2018, 140, 1572.
-1
9
9 GBq · µmol-1 (2.7 Ci · µmol ). The ruthenium content in the
reformulated sample was 1.8 µg, which is more than fivefold
below the specified limit of 10 µg per day considered safe for
injection into humans.[24] If desired, the residual ruthenium can
be further reduced by additional C-18 purification before
preparative HPLC purification, or the use of commercial metal
scavengers such as Quadrasil™ AP to 0.09 µg and 0.11 µg
respectively, roughly two orders of magnitude below accepted
limits, compared to 0.03 µg of a blank sample.
[
[
14] W. Liu, X. Huang, M. S. Placzek, S. W. Krska, P. McQuade, J. M.
Hooker, J. T. Groves, Chem. Sci. 2018, 9, 1168.
In summary, we report the first protocol to directly label
[
15] Z. Yuan, M. B. Nodwell, H. Yang, N. Malik, H. Merkens, F. Bénard, R. E.
Martin, P. Schaffer R. Britton, Angew. Chem. Int. Ed.,
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1
8
polypeptides with [ F]fluoride by formally replacing a single
hydrogen atom with fluorine-18, which results in only minimal
perturbation of the structure of the corresponding native peptide.
We anticipate that our protocol will accelerate the development
of novel peptide tracers due to its efficiency, predictability,
robustness, and the imminent commercial availability of
compound 1, Aldrich catalog number 902314.
[16] B. Långström, S. Sjöberg, U. Ragnarsson, J. Label. Compd.
Radiopharm. 1981, 18, 479.
[
17] D. S. Perekalin, E. E. Karslyan, P. V. Petrovskii, Y. V. Nelyubina, K. A.
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[
[
[
[
19] H. Cai, P. S. Conti, J. Labelled Compd. Radiopharm. 2013, 56, 264.
20] C. N. Neumann, J. M. Hooker, T. Ritter, Nature 2016, 534, 369.
21] L. Di, AAPS J. 2015, 17, 134.
Acknowledgements
We thank the Max-Planck-Institut für Kohlenforschung for fund-
ing. We thank Dr. Matthew Tredwell, Prof. Robert H. Grubbs and
Prof. Jennifer G. Murphy for helpful discussions. We thank Nele
Kronau and Sonja Irena Klein for synthesis of reagents and sub-
strates. We thank A. Deege, M. S. Sterling and H. Hinrichs
22] S. Good, M. A. Walter, B. Waser, X. Wang, J. Müller-Brand, M. P. Béhé,
J. C. Reubi, H. R. Maecke, Eur. J. Nucl. Med. Mol. Imaging 2008, 35,
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[23] J. H. Jones, W. I. Ramage, M. J. Witty, Int. J. Pept. Protein Res. 2009,
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1
[
24] a) https://www.gmp-compliance.org/gmp-news/ich-q3d-implemented-in-
the-european-pharmacopoeia-revision-of-two-general-monographs-
with-regard-to-elemental-impurities
(
Max-Planck-Institut für Kohlenforschung) for LC analysis.
6
Keywords: deoxyfluorination • η activation • fluorine • peptide
labeling • radiochemistry
b) International Conference on Harmonization. 2009. Final Concept
Paper Q3D: Impurities: Guideline for elemental impurities. Accessed
August 08, 2018. www.ich.org
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Entry for the Table of Contents
Layout 2:
COMMUNICATION
Jens Rickmeier and Tobias Ritter*
Page No. – Page No.
Site-Specific Deoxyfluorination of
1
8
Small Peptides with [ F]Fluoride
Peptide labeling with minimal perturbation in structure: Peptide labeling with
1
8
[
F]fluoride is achieved via chemoselective radio-deoxyfluorination of a tyrosine
residue bearing a traceless ruthenium activating group. The activated tyrosine was
introduced during solid phase peptide synthesis by application of a novel amino
acid building block. The method is robust, tolerates all 20 canonical amino acids
and results in a minimal perturbation of the peptide structure.
For internal use, please do not delete. Submitted_Manuscript
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