18F-Trifluoromethanesulfinate Enables Direct C-H 18F-Trifluoromethylation of Native Aromatic Residues in Peptides

Article abstract of DOI:10.1021/jacs.9b11709

¹⁸F labeling strategies for unmodified peptides with [¹⁸F]fluoride require ¹⁸F-labeled prosthetics for bioconjugation more often with cysteine thiols or lysine amines. Here we explore selective radical chemistry to target aromatic residues applying C-H ¹⁸F-trifluoromethylation. We report a one-step route to [¹⁸F]CF₃SO₂NH₄ from [¹⁸F]fluoride and its application to direct [¹⁸F]CF₃ incorporation at tryptophan or tyrosine residues using unmodified peptides as complex as recombinant human insulin. The fully automated radiosynthesis of octreotide[Trp(2-CF₂¹⁸F)] enables in vivo positron emission tomography imaging.

Full text of DOI:10.1021/jacs.9b11709

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Communication
18F-Trifluoromethanesulfinate enables Direct C–H 18F-
Trifluoromethylation of Native Aromatic Residues in Peptides
Choon Wee Kee, Osman Tack, Florian Guibbal, Thomas Wilson, Patrick Isenegger, Mateusz
Imio#ek, Stefan Verhoog, Michael Tilby, Giulia Boscutti, Sharon Ashworth, Juliette Chupin,
Roxana Kashani, Adeline Poh, Jane K. Sosabowski, Sven Macholl, Christophe Plisson, Bart
Cornelissen, Michael C. Willis, Jan Passchier, Benjamin G. Davis, and Veronique Gouverneur
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 08 Jan 2020
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Page 1 of 6
Journal of the American Chemical Society
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18F-Trifluoromethanesulfinate enables Direct C–H 18F-
Trifluoromethylation of Native Aromatic Residues in Peptides
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⊥
Choon Wee Kee,†,§, Osman Tack,†,§ Florian Guibbal,#,† Thomas C. Wilson,† Patrick G. Isenegger,†
Mateusz Imiołek,† Stefan Verhoog,† Michael Tilby,† Giulia Boscutti,‡ Sharon Ashworth,‡ Juliette
Chupin,‡,^ Roxana Kashani,^ Adeline W. J. Poh,† Jane K. Sosabowski,^ Sven Macholl, ‡,^ Christophe
Plisson,‡ Bart Cornelissen,# Michael C. Willis,† Jan Passchier,‡ Benjamin G. Davis,† Véronique Gou-
verneur†*
†Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK
# Department of Oncology, University of Oxford, Radiobiology Research Institute, Headington, Oxford OX3 7LJ, UK
‡Invicro Ltd, Du Cane Road, London W12 0NN, UK
^ Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London EC1M 6BQ, UK
Supporting Information Placeholder
structural alteration imposed by the 18F-prosthetic group is typi-
cally tolerated, it could alter efficacy and/or function.1c As such,
innovative methods that employ [18F]fluoride, and target native
residues in unmodified peptides with 18F ,22 or a minimally-sized
18F-prosthetic (e.g. [18F]CF3) are of considerable value.
ABSTRACT: 18F-Labeling strategies for unmodified peptides
with [18F]fluoride require 18F-prosthetics for bioconjugation more
often with cysteine thiols or lysine amines. Here, we explore
selective radical chemistry to target aromatic residues applying
C–H 18F-trifluoromethylation. We report a one-step route to
[18F]CF3SO2NH4 from [18F]fluoride, and its application to direct
[18F]CF3-incorporation at tryptophan or tyrosine residues using
unmodified peptides as complex as recombinant human insulin.
The fully automated radiosynthesis of octreotide[Trp(2-CF218F)]
enables in vivo PET imaging.
A. 18F-Radiolabeling of unmodified peptides at cysteine (2018)21
SCF218F
SH
S
TfO
CF218F
+H3N
-
-
+H3N
CO2
CO2
base
B. 18F-Radiolabeling of unmodified peptides at tyrosine and tryptophan (this work)
OH
OH
Positron Emission Tomography (PET) is a powerful molecular
imaging modality for diagnosis, monitoring disease progression,
studying biological processes in vivo, and investigating the effica-
cy of drugs.1–3 Among the radioisotopes employed for the prepa-
ration of PET probes, 18F is a widely used and clinically relevant
radionuclide.2 Due to its short half-life (t1/2 = 109.7 min), 18F must
be incorporated into tracer molecules at a late stage of the synthet-
ic process.4,5 Additional challenges imposed by radiochemistry
include low reaction concentration, solvent compatibility, and
cyclotron-produced 18F sources being limited to 18F-fluoride and
[18F]F2. These constraints are stringent for biomolecules.
18F-Radiolabeled peptides can be used to measure the distribu-
tion and pharmacokinetics of peptide-based therapeutics, and
serve as imaging biomarkers for therapy.6,7 These benefits have
encouraged the development of methods for tagging peptides with
radioactive functional groups.8-10 Fluorine-18 is incorporated into
pre-functionalized peptides via direct C–18F, B–18F and Si–18F
bond formation, or chelation with Al–18F.11-14 Alternatively, an
18F-labeled prosthetic group is prepared prior to bioconjugation.
To preserve function, this latter conjugation ideally proceeds
under mild reaction conditions.15-19 Such strategies require han-
dles with unique reactivity either by e.g., prior installation of
unnatural amino acids or by taking advantage of the inherent
reactivity of natural amino acids. To date, the latter has almost
exclusively exploited the nucleophilicity of cysteine thiols20 or
lysine amines21 to attach the 18F-prosthetic group. Although the
CF218F
+H3N
+H3N
-
-
CO2
CO2
H
H
18FF2C
CF218F
N
N
This Work
+H3N
CF218F
+H3N
-
-
CO2
CO2
CF218FSO2-NH4
+(one step automated radiosynthesis)
from
new fit-for-purpose reagent for radical C–H 18F-trifluoromethylation
C–H 18F-trifluoromethylation of unmodified peptides
highly chemoselective for tryptophan and tyrosine
process amenable to automation and in vivo imaging
Figure 1. Direct 18F-trifluoromethylation of native residues in unmodified
peptides.
We reported the 18F-trifluoromethylation of native peptides
with
5-18F-(trifluoromethyl)dibenzothiophenium
trifluoro-
methanesulfonate, a method modifying cysteine thiols (Fig. 1A).23
We also applied tuned radical chemistry to program C–H 19F-
trifluoromethylation of aromatic residues in proteins.24a Sodium
trifluoromethanesulfinate (NaTFMS, Langlois’ reagent) displayed
selective reactivity for tryptophan under redox initiation. Recent-
ly, Krska et al. have demonstrated that Zn(TFMS)2 (Baran’s
reagent), when activated with a stoichiometric oxidant or via
visible photoredox catalysis, enabled trifluoromethylation of
tyrosine in peptides that do not contain tryptophan residues.25
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Journal of the American Chemical Society
Page 2 of 6
These precedents encouraged us to produce 18F-
trifluoromethanesulfinate for selective C–H
18F-
trifluoromethylation of these aromatic amino acid residues within
unmodified peptides. This approach would generate non-
canonical [18F]CF3-tryptophan and -tyrosine residues, a transfor-
mation unmatched by alternative 18F-labeling methods (Fig. 1B).
[18F]CF3SO2NH4 in >99% radiochemical purity. This protocol
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furnished up to 900 MBq of [18F]CF3SO2NH4 from 10 GBq of
[18F]fluoride. The overall nondecay corrected activity yield (AY)
of isolated [18F]CF3SO2NH4 calculated from [18F]fluoride was
11% ± 1% (n = 6, synthesis time = 70 mins). The identity of
[18F]CF3SO2NH4 was established by HPLC and ESI-MS analysis
–
(m/z) calc. for [19F]CF3SO2 : 133.0; found: 133.1).32
Routes towards trifluoromethanesulfinic acid salts include
metal or electro-reduction of a mixture of SO2 and CF3Br in
DMF,26 the treatment of CF3Cl with Na2S2O4,27 or multistep
syntheses from trifluoromethylsulfone precursors (Scheme 1A).28
For 18F-radiochemistry, these approaches would require a route
towards the [18F]CF3-precursor, and one or more reactions post-
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–
labeling. Our design plan was to construct [18F]CF3SO2 in one
step applying a multi-component approach that combines 18F-
fluoride, a difluorocarbene source, and SO2. The formation of
–
[18F]CF3 from difluorocarbene and [18F]F– is known,29-31 so the
challenge was to validate a protocol that couples in situ generated
[18F]CF3– with SO2 (or a surrogate of this gaseous and toxic rea-
gent) (Scheme 1B).
−
Scheme 2. A. Initial studies towards one step synthesis of [18F]CF3SO2 .
B. Radiosynthesis, purification and isolation of [18F]CF3SO2NH4.
Next, we studied the C–H 18F-trifluoromethylation of model
peptides containing L-tryptophan and/or L-tyrosine residues using
[18F]CF3SO2NH4 and t-butyl hydroperoxide (TBHP) as the oxi-
dant. In 19F-mode, CF3SO2Na is added in large excess (up to ~
200 equiv) to enable C–H trifluoromethylation of peptides and
proteins.24,35 These conditions are not compatible with 18F-
radiochemisty due to inherent constraints on concentrations for
both large peptides and [18F]CF3SO2NH4, the latter being by far
the limiting reagent. An additional complication was competitive
Scheme 1. A. Multi-step syntheses towards trifluoromethanesulfinic acid
salts, M = metal. B. Proposed one-step radiosynthesis towards 18F-
trifluoromethanesulfinate.
–
–
oxidation of [18F]CF3SO2 into [18F]CF3SO3 with the initiation
oxidant. For 19F-trifluoromethylation, this issue is solved using an
excess of CF3SO2Na with respect to TBHP, or via slow addition
of TBHP to the reaction mixture.36 These solutions are not suita-
ble for 18F-labeling because [18F]CF3SO2NH4 is the limiting rea-
gent, and operational simplicity is paramount for 18F-
radiochemistry.
Exploratory studies performed with 19F-fluoride provided
useful information.32 The difluorocarbene and SO2 sources were
–
found to be critical in enabling the construction of CF3SO2 . The
reaction of 2,2,-difluoro-2-(triphenylphosphonio)acetate (PDFA)
with either 1,4-diazabicyclo[2.2.2]-octane bis(SO2) adduct
(DABSO)33 or N-methyl-morpholine SO2 (NMM SO2) in the
•
•
The treatment of L-Tyr with [18F]CF3SO2NH4 and TBHP in
AcOH/aq. ammonium formate did not lead to C–H 18F-
trifluoromethylation after 20 mins, even at 60 °C.32 Extensive
optimization overcame 18F-labeling constraints, and led to L-
Tyr(3-CF218F) in 14% RCC when the reaction was performed in
presence of KF/K222 in DMF at 100 °C afforded the ammonium
−
salt of CF3SO2 in 31% and 44% yields after isolation by semi-
preparative HPLC, respectively. A saturated solution of SO2 in
DMF did not lead to product formation, whilst ClF2CCO2Me in
combination with PPh3 was the only alternative difluorocarbene
source found suitable for this process. For 18F-labeling, PDFA
was elected as the optimal reagent. In contrast to experiments
carried out with fluoride, DABSO and PDFA afforded
[18F]CF3SO2K in trace amounts (Scheme 2A). However, the
the presence of TBHP and Fe(NO3)3 9H2O36 in DMSO/aq. ammo-
•
nium formate at 40 °C for 20 mins (Scheme 3).32 18F-
Trifluoromethylation on the C2-position was detected in 2% RCC.
These two regioisomers are separable by HPLC. The RCC of L-
Tyr(3-CF218F) increased to 53% when the reaction was performed
at 60 °C. When FeCl3 was used at 40 °C instead of
combination of PDFA, NMM SO2 and [18F]KF/K222 gave
•
[18F]CF3SO2K in 22% radiochemical conversion (RCC). These
results encouraged the development of a manual protocol to
prepare, purify and isolate this novel 18F-reagent for subsequent
use (Scheme 2B). PDFA is thermally unstable and poorly soluble
Fe(NO3)3 9H2O, L-Tyr(3-CF218F) was formed in 7% RCC. The
•
C–H 18F-trifluoromethylation of L-Trp was also successful with
[18F]CF3SO2NH4 when activated by either Fe(NO3)3•9H2O or
FeCl3 in the presence of TBHP. Applying these conditions, L-
Trp(2-CF218F) was obtained in 22% and 18% RCC, respectively.
Two additional regioisomers resulting from competitive 18F-
labeling on the C4- and C7-positions were also formed giving a
•
in DMF, so a mixture of this reagent and NMM SO2 was added as
a suspension in a suitable solvent to azeotropically-dried 18F-
fluoride. Amongst all solvents tested, propylene carbonate (PC)
was best when used with DMF.34 Additional optimization tuning
reagents, ratios of various components, and concentrations proved
beneficial. The optimal process consisted of reacting PDFA (0.16
•
combined RCC of 10% or 9% when Fe(NO3)3 9H2O or FeCl3 was
employed, respectively.37
18
•
A series of dipeptides was evaluated focusing on feasibility
and selectivity (Scheme 3).32 For reactions leading to more than
one 18F-labeled product, identification was made by comparison
of HPLC traces with fully characterized references prepared
independently. Dipeptides Tyr-Trp and Trp-Tyr underwent
[18F]CF3 incorporation exclusively at Trp, a result corroborating
mmol) and NMM SO2 (0.06 mmol) with [ F]KF/K222 (up to 10
GBq) in 350 L PC/DMF mixture at 110 °C. Initial purification
of [18F]CF3SO2− using a weak anion exchange cartridge (WAX)
removed most of the unreacted [18F]fluoride and organic by-
products. Elution with a solution of ~0.4 M ammonium hydroxide
in EtOH followed by reverse phase HPLC purification afforded
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Journal of the American Chemical Society
our previous studies.24 For Tyr-Trp, 18F-labeling experiments
ry effect on endocrine secretion, was 18F-labeled in 20% RCY.41
The 18F-trifluoromethylation of the 26-residue venom peptide
melittin42 and of octreotide,43 an octapeptide that mimics natural
somatostatin, was equally successful (14% RCC and 29% RCC,
respectively). Tyrosine-containing peptides were examined next.
Angiotensin fragment 1-7, a peptide with anti-inflammatory
properties,44,45 and c(RGDyK), a peptide ligand of integrin v3
receptors,46 both underwent 18F-labeling at Tyr in 16% and 33%
RCC, respectively. The C–H 18F-trifluoromethylation of a much
larger peptide was considered with recombinant human insulin
(MW: 5808 Da).47 This experiment was carried out with insulin
(5.2 μmol), Fe(NO3)3∙9H2O (5.8 equiv) and TBHP (11.5 equiv) in
DMSO/aq. ammonium formate affording [18F]CF3-insulin as a
mixture of four products, resulting from [18F]CF3 incorporation at
all tyrosine residues, in 34% overall RCC. The main site of 18F-
trifluoromethylation was at chain A residue Y19, a result con-
sistent with the report of Krska et al.25
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3
4
5
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7
8
performed with [18F]CF3SO2NH4 and TBHP with either
Fe(NO3)3∙9H2O or FeCl3 gave 40% and 55% RCC, respectively.
For dipeptide Phe-Trp, 18F-trifluoromethylation occurred at Trp,
affording Phe-Trp(2-CF218F) in 43% RCC. [18F]CF3-incorporation
on Trp occurred at the C2-, C4- and C7-positions (C2 major) while
18F-labeling on Phe was not observed. 18F-Labeling at His was not
detected for either Tyr-His or His-Trp. Met oxidation was mini-
mized for the 18F-trifluoromethylation of Met-Trp or Met-Tyr by
decreasing TBHP:Fe(III) ratio (1:1). Oxidative dimerization of
cysteine by disulfide formation is unavoidable.24,25 Next, we
studied biologically relevant peptides of increasing complexity.
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The dipeptide immunomodulator thymogen (OglufanideTM 38
) was
18F-trifluoromethylated at Trp with an isolated radiochemical
yield (RCY) calculated from [18F]CF3SO2NH4 of 39%. Endomor-
phin-1, a tetrapeptide associated with Alzheimer’s disease,39,40
underwent Trp-selective 18F-labeling in 23% RCY, and somato-
statin-14, a cyclic tetradecapeptidic hormone with broad inhibito-
OH
OH
CF218F
CO2H
NH
NH
or
or
H2N
[
18F]CF3SO2NH4, Fe(III) salt, TBHP
CF218F
HCO2NH4 (aq., 25 mM) / DMSO, 20 min, 40 °C
H2N
CO2H
H2N
CO2H
H2N
OH
CO2H
H
N
H
18FF2C
H
N
N
O
CF218F
H
O
O
CF218F
N
N
D R V
I H P
CO2H
O
H2N
18FF2C
HO
O
H2N
S
OH
H2N
OH
OH
18FF2C
N
N
H2N
S
OH
H
NH2
H
OH
O
N
H
18FF2C
O
O
H2N
O
HO
HO
L-Tyr(3-CF218F)
RCC 14% ± 5% (n = 2)[a]
RCC 7% ± 3% (n = 2)[b]
L-Trp(2-CF218F)
RCC: 22% ± 1% (n = 2)[a]
RCC: 18% ± 6% (n = 2)[b]
Tyr(3-CF218F)-His
Met-Tyr(3-CF218F)
RCC: 29% ± 7% (n = 4)[a]
Angiotensin I/II (1-7)[Tyr(3-CF218F)]
Met-Trp(2-CF218F)
RCC: 39% ± 6% (n = 3)[a]
RCC: 26% ± 8% (n = 4)[a]
RCC: 16% ± 4% (n = 3)[a]
H
OH
H
NH2
OH
N
18FF2C
N
18FF2C
H
18FF2C
18FF2C
N
HN
O
NH2
O
O
H2N
OH
O
NH
O
N
H2N
N
OH
H2N
OH
HN
H
N
H2N
N
OH
O
H
HN
O
H
N
H
O
O
H
HN
O
O
HO2C
O
HN
CF218F
Trp(2-CF218F)-Tyr
NH
N
HO
N
Thymogen[Trp(2-CF218F)]
RCY: 39% ± 3% (n = 3)[b]
Tyr-Trp(2-CF218F)
RCC: 55% ± 4% (n = 3)[b]
His-Trp(2-CF218F)
RCC: 56% ± 4% (n = 3)[a]
O
O
H
RCC: 24% ± 3% (n = 2)[a]
OH
c(RGDyK)[Tyr(3-CF218F)]
H
N
O
CF218F
RCY: 33% ± 1% (n = 3)[b]
H
H
N
N
18FF2C
F
NH2
HO2C
Y P
OH
18FF2C
O
H
O
NH2
N
F
O
O
H2N
N
OH
K
T
F
F
N
H
18FF2C
NH
O
H
N
H
A
N
O
HO2C
G
K
NH
OH
Endomorphin-1[Trp(2-CF218F)]
RCC: 11% ± 3% (n = 4)[b]
RCY: 23% (n = 1)[b]
T
HN
S
S
O
S
HN
HO
OH
NH
S
18FF2C
Phe-Trp(2-CF218F)
RCC: 43% ± 10% (n = 3)[a]
S
OH
NH
O
O
O
O
HN
Somatostatin-14[Trp(2-CF218F)]
HN
RCY: 20% ± 5% (n = 2)[b]
O
O
H
HN
H
N
G G G S
I A V L K V L T T L P A L I
I
K R K R
Q Q NH2
HO2C
Octreotide[Trp(2-CF218F)]
RCC: 29% ± 7% (n = 3)[a]
Recombinant Insulin[Tyr(CF218F)]
Melittin[Trp(2-CF218F)]
RCC: 14% ± 3% (n = 4)[b]
H2N
CF218F
RCC: 34% ± 9% (n = 7)[a]
N
H
Scheme 3. Substrate scope for C–H 18F-trifluoromethylation of native aromatic residues of peptides. Peptide (0.03 mmol). TBHP (2 or 4 equiv). [a]
Fe(NO3)3•9H2O (2 equiv). [b] FeCl3 (2 equiv). Synthesis time for the 18F-labeled peptide from [18F]CF3SO2NH4 = 90 mins.32
•
To date, automated radiosyntheses have focused on small mole-
cules, rarely on peptides.48 To demonstrate translational applica-
bility, we developed a fully automated radiosynthesis of oc-
treotide[Trp(2-CF218F)] on the Advion NanoTek® microfluidic
synthesis system (Figure 2).32 The automated radiosynthesis of
[18F]CF3SO2NH4 required optimization of selected steps. The
was required for NMM SO2. With these modifications, starting
from up to 45 GBq of [18F]fluoride, [18F]CF3SO2NH4 was pro-
duced in up to 6% ± 1% activity yield (non-decay corrected, n =
2), after semi-preparative HPLC (Am = 1.13 GBq/µmol, synthesis
time = 40 mins). Removal of HPLC solvents was necessary to
afford dry [18F]CF3SO2NH4 required for peptide 18F-labeling. This
critical drying step also required extensive modification. For
automation, [18F]CF3SO2NH4 was trapped on a WAX cartridge
and subsequently eluted with NH4OH in MeCN (1.4%) followed
by evaporation.
•
addition of the suspension of PDFA and NMM SO2 in PC/DMF to
a vial containing [18F]KF was not compatible with automation.
This issue was solved by changing the difluorocarbene source to
ClF2CCO2Me,
a reagent activated with (2-biphenyl)di-tert-
butylphosphine (JohnPhos), and the solvent to DMA; no change
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Figure 2. Automated radiosynthesis of octreotide[Trp(2-CF218F)] from [18F]fluoride on the Advion NanoTek® microfluidic synthesis system.
Successful C-H 18F-trifluoromethylation in the presence of
Present Address
⊥C.W.K: Institute of Chemical and Engineering Sciences,
Fe(NO3)3∙9H2O (4 equiv) and TBHP (8 equiv) afforded up to 69
MBq octreotide[Trp(2-CF218F)] (n = 3, Am = 0.28 ± 0.08
A*STAR, 1 Pesek Road, Jurong Island, 627833 Singapore.
GBq/µmol) after purification by HPLC. The total synthesis time
from [18F]fluoride to octreotide[Trp(2-CF218F)] was 133 mins.
Authors Contributions
§C.W.K. and O.T. contributed equally to this work.
This automated protocol enabled an in vivo PET imaging experi-
ment with this [18F]CF3-peptide on naïve Sprague Dawley rats, a
Notes
preliminary study suggesting excretion via the gastro-intestinal
pathway and the kidneys.32,49,50,51
The authors declare no competing financial interests.
In conclusion, we report the first protocol enabling direct 18F-
labeling of unmodified peptides at tryptophan and tyrosine resi-
ACKNOWLEDGMENTS
dues (high selectivity for tryptophan) applying direct C–H 18F-
trifluoromethylation. This method is a new tool to accelerate the
Financial support was provided by the Agency for Science, Tech-
discovery of 18F-peptides as imaging agents as well as the devel-
nology and Research (A*STAR, Singapore, C.W.K.), BBSRC
opment of peptide-based drugs. The strategy required the novel
(BB/M016757/1, BB/P026311/1), European Union Horizon 2020
18F-reagent [18F]CF3SO2NH4 prepared in one step from
Marie Skłodowska-Curie grant agreement No. 675071 (M.I.),
[18F]fluoride, a difluorocarbene reagent, and an SO2 source. The
Swiss National Science Foundation P2BSP2_178609 (P.G.I.),
iron salt was essential to overcome the difficulties arising from
CRUK
(C5255/A16466),
Medical
Research
Council
[18F]CF3SO2NH4 being the limiting reagent, thereby enabling C–
18F-trifluoromethylation of peptides as complex as insulin. The
(MR/R01695X/1), and EPSRC (EP/L025604/1, NS/A000024/1,
EP/L015838/1). The authors thank Dr M. Tredwell for helpful
discussions.
H
automated radiosynthesis of octreotide[Trp(2-CF218F)] from
[18F]fluoride enabled in vivo PET imaging. This major milestone,
unrivalled by known methods making use of minimally-sized
labeled prosthetics,23,52,53 sets the stage for an in-depth investiga-
tion on clinically relevant peptides. Considering the number of
reactions relying on Langlois-type reagents, [18F]CF3SO2NH4
could expand considerably the radiochemical space for PET
applications beyond the peptides described herein.
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ASSOCIATED CONTENT
SUPPORTING INFORMATION
The Supporting Information is available free of charge on the
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Corresponding Author
*veronique.gouverneur@chem.ox.ac.uk
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Graphical Abstract TOC
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