Original synthesis of radiolabeling precursors for batch and on resin one-step/late-stage radiofluorination of peptides

Article abstract of DOI:10.1039/c9cc09434b

Radiolabeling of peptides with fluorine-18 is hurdled by their chemical sensitivity and complicated processes. Original triflyl-pyridine intermediates afforded ammonium precursors that were radiolabeled at low temperature. From that study, a generic tag h

Full text of DOI:10.1039/c9cc09434b

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DOI: 10.1039/C9CC09434B
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Original synthesis of radiolabeling precursors for batch and on
resin one-step/late stage radiofluorination of peptides
Mylène Richard, Simon Specklin, Mélanie Roche, Françoise Hinnen and Bertrand Kuhnast*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Radiolabeling of peptides with fluorine-18 is hurdled by their with the handling of short-lived positron emitters, a multistep
chemical sensitivity and complicated processes. Original triflyl- radiosynthesis is a major drawback for a fully automated
pyridine intermediates afforded ammonium precursors that were process, which is an important consideration in the
radiolabeled at low temperature. From that study, a generic tag has development of radiotracers. Such limitations question
been designed to allow a simple one-step/late-stage radiolabelling whether a direct introduction of fluorine-18 in a single step
of peptides. The strategy has been transposed to an automated could be considered for labeling peptides. Molecular fluorine
“on-resin” radiolabelling.
[18F]F2 or reagents derived were used for the direct
radiolabeling of peptides but with a limited extent due to the
limitations associated with the production of these electrophilic
species in terms of radioisotope supply or need of specific
equipment9. Nucleophilic fluorine-18 sources, e.g. [18F]F- in
H2[18O] or the classical K[18F]F/K222 complex, are preferred and
have been extensively exploited for direct radiolabeling. This
was illustrated either via silicon-10, boron-11 or aluminium-
fluoride12 bonds on a modified peptide or using adequate
peptide-precursors to undergo site-specific deoxyfluorination13
(Figure 1).
Radiolabeled peptides are increasingly demanded in molecular
imaging and nuclear medicine1. They are used in both diagnosis2
and therapy3 depending on the disintegration properties of the
selected radioisotope. Thanks to the synthesis options peptides
offer, their affinity and stability can be tuned thus providing
highly potent ligands with minimized toxicity for promising
applications in precision medicine4. Positron emission
tomography (PET) is one of the most sensitive functional
nuclear molecular imaging technique for diagnosis. Among the
available positron emitters, fluorine-18 meets numerous
advantages in terms of ease of production in biomedical
cyclotrons (at several GBq levels), half-life (109.8 min) and
disintegration properties (low positron energy of 635 keV; 97%
+ branching) for optimal image generation. Peptides appeared
in the molecular toolbox of nuclear imaging more than four
decades ago and they were initially radiolabeled by use of the
prosthetic approach5. The major strength of this sequential
strategy is the dissociation of the preparation of a small
radiofluorinated reagent, often necessitating harsh reaction
conditions, from its conjugation with the peptide in mild and
aqueous conditions. The reliability and robustness of this
approach was illustrated for more than 30 years by the diversity
of biologics that have been thus radiolabeled6 and the regular
upgrades, introducing click chemistry in the late 2000’s7 or the
regioselective labeling of “zero size” radiofluorinated motifs8
more recently. In return, regarding the constraints associated
Al-[18F]F
Si-[18F]F
-
CO2
COOH
18F
X
K[18F]F
K222
N
N
Si
Si
COO-
Al[18F]F2+
CO2H
18F
[Al ]F
N
N
N
N
-OOC
-
CO2
peptide
C-[18F]F
B-[18F]F
F
18F]F-
F
[
Cl-Im[18F]F
Ru
B
B
19F
F
18F
F
18F
-O
This work
EWG
N
EWG
K[18F]F·K222
18F
peptide
peptide
NR3
TfO-
DMSO
25 °C or 40 °C
N
Figure 1. Late-stage fluorination of peptides.
Nevertheless, apart from the boron-fluoride bond formation,
elevated temperatures are required and final molar activity
remains a concern when isotopic exchange was exploited.
Easy-access to fluorine-18-radiolabeled peptides by late stage
fluorination is still a challenge today. Shifting from the standard
prosthetic concept of “radiolabel first and then conjugate” to
Université Paris Saclay, CEA, INSERM, CNRS, BioMaps, Service Hospitalier Frédéric
Joliot,
* Bertrand KUHNAST, 4 place du général Leclerc, 91401 ORSAY, France
+ 33 1 69 86 77 36
bertrand.kuhnast@cea.fr .
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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“conjugate first and then radiolabel”, we report herein observed between ammonium precursors 2 or 3 (ESI : II.2-Table
View Article Online
advances in the one-step/late stage toward “minimalist” S1). The kinetic profile shows a plateau rDeaOcI:h1e0d.10a3s9e/Ca9rClyCa0s94534toB
approaches to radiolabel peptides at low temperature. Our 10 min for the most reactive precursors.
approach is based on the higher reactivity of the pyridine ring14,
which is boosted in presence of electron-withdrawing groups15
(Figure 1).
EWG
EWG
EWG
EWG
NMe3 or
DABCO
K[18F]F·K222
18F
N
TfO
2
NMe3
N
TfO
3
N
N
OTf
N
THF
0 °C
DMSO
25 °C or 40 °C
We wanted first to examine the influence of the electron-
withdrawing groups (EWGs) on the kinetics of radiofluorination
at low temperature by SNAr using trialkylammonium as a
leaving group. However, we initially faced a synthetic obstacle
to access these ammonium precursors 2, as quaternarization of
such electron-deficient pyridines proved to be highly
challenging (ESI: I.5-Preliminary studies). Methylation of the
corresponding dimethylamines was surprisingly completely
ineffective, even with a single ethyl ester as EWG and using
highly electrophilic methyl sources. We next tried to reproduce
reported procedures involving the direct introduction of
trimethylamine on the pyridine ring by chlorine substitution14c,
15b, 16. In our case, this method led to the observation of the
desired ammonium chlorides as intermediates only, quickly
evolving to the dimethylamine derivatives by chloride
nucleophilic attack to one of the methyl group of the
ammonium.
N
1
4
O
O
NC
CN
18F
EtO
BnO
18F
18F
18F
N
4a
N
N
N
4b
4c
4d
65% ± 16%
94% ± 1%
93% ± 3%
96% ± 2%
73% ± 13%
85% ± 2%
70% ± 6%
85% ± 3%
2c
2d
2a
67%
2b
70%
81%
50%
68% ± 8%
84% ± 2%
70% ± 6%
82% ± 6%
76% ± 13%
90% ± 1%
82% ± 8%
93% ± 1%
3c
3d
3a
94%
3b
85%
84%
79%
F
F
O
F
O
O
O
O
CN
N
BnHN
EtO
O
F
O
O
18F
18F
18F
N
4f
18F
N
N
N
4g
4h
4e
48% ± 2%
85% ± 3%
90% ± 1%
94% ± 1%
57% ± 2%
66% ± 5%
4% ± 1%
4% ± 1%
2g
84%
2h
93%
2e
47%
2f
88%
46% ± 10%
69% ± 14%
82% ± 5%
93% ± 0%
50% ± 3%
57% ± 2%
15% ± 1%
19% ± 1%
3g
87%
3h
87%
3e
89%
3f
90%
This last observation prompted us to investigate a new route to
pyridine ammoniums 2 by the direct addition of trimethylamine
to 2-triflyl pyridines 1, the triflate group being a highly efficient
nucleofuge and a very poor nucleophile. If the nucleophilic
aromatic substitution of (hetero)aryl triflates by primary and
secondary amines have been reported17, addition of tertiary
amines leading to the corresponding ammonium was only
described for triazine triflates.18. By applying this strategy to our
electron-deficient pyridines, a surprisingly good conversion was
observed for the formation of 2 as a stable ammonium. With
this new method in hand, we next designed a library of 16
pyridine derivatives displaying different EWGs and divided in
two series of quaternary ammonium precursors:
trimethylammoniums 2 and ammoniums 3 derived from DABCO
amine, which are largely underexploited in radiochemistry
(Scheme 1). As EWGs, we selected i) nitrile and ester (ethyl and
benzyl) that are widely encountered in 18F-radiochemistry, and
a combination of both; ii) amide (benzyl) to mimic a peptide
Radiofluorination at 25 °C
Radiofluorination at 40 °C
Scheme 1. Synthesis of precursors 2 and 3 and their radiofluorination at
low temperatures. Conversions after 15 minutes at 25 or 40 °C (n = 3)
Nitriles and esters, or combination of both, gave the highest
conversions reaching more than 90% even at room temperature
after 15 min reaction for precursors 2d and 2f in particular. Due
to the softer electron-withdrawing character of amide
compared to ester or nitrile, a lower conversion was observed
starting from precursors 2e and 3e. Activated esters (NHS and
TFP) gave the lowest yields because of their relative instability
under the radiofluorination conditions (Scheme 1). Non-
radioactive by-products were characterized and correspond to
6-NHS and 6-TFP15a derivatives.
This kinetic study showed that EWG-substituted pyridines could
be radiolabeled at low temperatures and demonstrated the
relevance of designing a pyridine-based tag that could be
conjugated to a peptide for a direct radiofluorination. In that
end, the first step was to assess the chemical stability of
radiolabeled pyridines 4 regarding their potential inclination to
defluorination (ESI: II.6-Stability studies). The hydrolytic stability
was checked by incubation of all labeled pyridines 4 in PBS for
30 min, but no trace of free fluoride-18 was observed in any
case. A second control was performed by incubation of 4c in
foetal bovine serum up to 60 min, again without observable
defluorination, indicating a presumptive satisfying chemical
stability for future in vivo application. We then undertook the
synthesis of a generic tag displaying a nitrile group as EWG for
the functionalization of peptides. Compound 5 was prepared in
6 steps from commercial 3-cyano-2-pyridone. The “conjugate
first and then radiolabel” strategy was then evaluated with
three model compounds: two peptides (glutathione, c(RDGfK))
bond;
and
iii)
N-hydroxysuccinimide
(NHS)
and
tetrafluorophenyl (TFP) activated esters. Compound 4g, acting
as acylating prosthetic reagent, would thus be prepared in a
simpler way than its ubiquitous benzene homologue 4-
[18F]fluorobenzoate ([18F]SFB), prepared in a multistep time-
consuming procedure.19 Such a simplified approach was already
proposed for the TFP activated ester.15a The triflate
intermediates 1 were synthesized from the fairly accessible
pyridones in good to excellent yields varying from 53 to 91 %.
Triflates 1 were next treated with either trimethylamine or
DABCO affording the final quaternary ammonium triflate
precursors (2 and 3) in good to excellent yields.
The performance of each precursor toward radiofluorination
was then evaluated at 40 °C and even at room temperature (ESI:
II.2-Kinetic studies). As expected, the conversions were higher
at 40 °C, whatever the EWG and no drastic differences could be
2 | J. Name., 2012, 00, 1-3
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and a PSMA ligand, the latter being extensively used in nuclear observed during this step. Precursors were thenVsieuwbAjreticclteeOdnlitnoe
medicine for theranostic applications. Each compound was radiofluorination with K[18F]F/K222 for 30 min at 40 °C (Scheme
DOI: 10.1039/C9CC09434B
functionalized with the cyanopyridine tag
5 providing 2). Peptides 4i, 4k and 4j were obtained with isolated yields
selectively the precursors 3i-k, resulting from the coupling of ranging from 42 to 55 % and their identity was checked by
the peptides or PSMA with the NHS-ester. No aromatic radioHPLC (ESI: II.4 Fig S2, Fig S3 and Fig S4).
substitution at the pyridine ring with the free amine was
Scheme 2. Late-stage fluorination of peptides at low temperature via a pyridine tag.
To go one step further, the reactivity of ammonium precursors [18F]fluoride and pyridines 4 as radioactive species. This enabled
2 and 3 was harnessed for the development of an advanced the isolation of pyridines 4 with a high radiochemical purity
late-stage radiofluorination process for peptide labeling. By after only a simple formulation on C18 or Al2O3 SPE cartridge.
immobilization of the trialkylamine on a solid support, we We however noted the presence of the corresponding 2-
interrogated whether our method allowed the loading of hydroxypyridines as the major non-radioactive byproduct,
triflates 1 on a resin by SNAr and if the corresponding supported resulting from the competitive SNAr with water. With this
ammoniums would retain their reactivity toward process, pyridines 4b, 4c and 4j were obtained with low to
radiofluorination. Such ammonium triflates could also act as an moderate radiochemical yield, demonstrating the proof of
anion exchanger to directly trap the [18F]fluorides from the concept of the on resin radiolabeling. A longer air-drying, the
aqueous solution produced by the cyclotron. This strategy use of other polar organic solvents or other types of resins are
would facilitate the whole radiolabeling process with an easy options for improvement. To further simplify this method, resin
translation to automation by loading the solid support in a 7b was also prepared via slow elution of a cartridge filled with
cartridge, as well as short synthesis time and selective release resin 6 with a solution of 1-triflyl pyridine 1b and radiolabeling
of the labeled pyridine from the resin. Although anion was performed without any alteration of the yield of 4b. This
metathesis of [18F]fluorides with a labeling precursor on a solid method radically differs from previous reports describing the
support is known,20 immobilization of the precursor and its use of resins either in a solid phase peptide synthesis
labeling on a resin with [18F]F-/H2O have never been reported approach21 or in a solid phase organic radiosynthesis approach
yet. To evaluate this new concept, we prepared a supported that always involved standard K[18F]F-K222 complex, organic
quinuclidine amine 6 in a few steps from a commercial solvents, elevated temperatures 22
(aminomethyl)polystyrene resin (Figure 2).
.
The grafting of this resin by SNAr was studied with three
selected 1-triflyl pyridines 1b, 1c and the PSMA derivative 1j.
Incubation in DMF at room temperature of these pyridines with
resin 6 was quite efficient and provided resins 7b, 7c and 7j in
satisfying grafting yields with a final ammonium loading
comprised between 0.46 and 0.90 mmol/g. The potential of
these supported precursors for radiofluorination was explored
by loading the resins in cartridges that were installed on a PET
synthesizer (ESI:II.5 Fig S6 and S7). Trapping efficiency of resins
7 was first determined, after elution of the [18F]fluoride
aqueous solution produced by the cyclotron, 23-35% of the
starting activity remained on the cartridge, presumably by anion
metathesis between [18F]fluoride and triflate anion. After a
short air-drying of the cartridge, a moderately slow elution with
DMSO was performed. Analysis of the resulting eluate indicated
a conversion of 75% in the desired labeled pyridine, with only
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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In summary, we have developed an original synthesis exploiting
View Article Online
DOI: 10.1039/C9CC09434B
a triflyl intermediate to prepare trimethylammonium and
DABCO-derived precursors for the radiolabeling with fluorine-
18. We have demonstrated that such precursors, displaying an
appropriated electron-withdrawing group, could be
radiolabeled at room temperature. Taking advantage of this, we
have designed a generic tag that allowed for a one-step/late
stage radiofluorination of model peptides and a PSMA ligand at
moderate temperature with satisfactory yields. Inspired by the
DABCO-derived ammonium precursor and the high reactivity of
the triflyl intermediate, we established the proof of concept of
an “on-resin” radiofluorination of peptides in an innovative,
simple and automatable way. This approach may contribute to
popularize radiolabeled peptides in molecular imaging and
opens new opportunities to radiolabel compounds accepting an
auxiliary group to incorporate fluorine-18.
3
Figure 2. Automated on-resin radiolabeling at room temperature using aquTehoiussw[1o8Frk]fliusoinridpeasrotlsuutipopnoprrtoeddubceydAbNyRthfeuncydcilnogtrMon ODALITy (ANR-
17-CE18-0018-01) and the CEA DRF-Impulsion program.
8
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Conflicts of interest
There are no conflicts to declare.
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