Chemoselective 18F-incorporation into pyridyl acyltrifluoroborates for rapid radiolabelling of peptides and proteins at room temperature

Article abstract of DOI:10.1039/c9cc08645e

A new prosthetic group is reported for ¹⁸F-labelling of peptides and proteins based on the chemoselective ligation of potassium acyltrifluoroborates (KATs) and hydroxylamines without any detectable ¹⁸F/¹⁹F isotope exchange at the acyltrifluoroborate moiety. The new building block is appended via a common amide bond at room temperature with no need for protecting groups which enables an effective orthogonal ¹⁸F-radiolabelling.

Full text of DOI:10.1039/c9cc08645e

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Chemoselective F-incorporation into pyridyl
acyltrifluoroborates for rapid radiolabelling of
peptides and proteins at room temperature†
Cite this:DOI: 10.1039/c9cc08645e
Received 5th November 2019,
Accepted 4th December 2019
Aristeidis Chiotellis,‡a Hazem Ahmed, ‡a Thomas Betzel,a Matthias Tanriver,b
a
Christopher J. White,b Haewon Song,b Sara Da Ros,b Roger Schibli,
a
Jeffrey W. Bode *b and Simon M. Ametamey
*
DOI: 10.1039/c9cc08645e
rsc.li/chemcomm
A new prosthetic group is reported for 18F-labelling of peptides and
proteins based on the chemoselective ligation of potassium acyltri-
fluoroborates (KATs) and hydroxylamines without any detectable
18F/19F isotope exchange at the acyltrifluoroborate moiety. The new
building block is appended via a common amide bond at room
temperature with no need for protecting groups which enables an
effective orthogonal 18F-radiolabelling.
Positron emission tomography (PET) can visualise, characterise and
quantify biological processes at the cellular and molecular levels
in vivo non-invasively, with high sensitivity and spatial resolution. It
has evolved into a powerful imaging tool, contributing decisively in
both basic research and medical decision-making.1 Fluorine-18 is
the most frequently utilised radionuclide for diagnostic PET imaging
since its decay properties (18F; b+ 0.635 MeV, 97% abundance,
Scheme 1 Overview of work on boron-based 18F prosthetic groups.4,5
t1/2 = 109.8 min) provide significant advantages over other PET natural amino acids (most notably couplings between activated
radionuclides.2 The ever-increasing use of biomolecular vector- carboxyl groups and lysines and Michael additions between
based imaging agents has brought forth a high demand for maleimides and cysteines).6–9 However, the complete loss of
18F-radiosynthesis strategies that are fast, mild and selective. regiochemical control during the labelling of a biomolecule
However, the harsh conditions required for direct 18F-labelling when more than one (unprotected) lysine or cysteine are
(high temperatures, basic pH, dry organic solvents), the extremely present requiring time-consuming protection and/or purifica-
low concentration levels of 18F and its relatively fast radioactive tion steps prompted radiochemists to shift their attention
decay make the development of 18F radiopharmaceuticals of towards ligations of higher specificity. As such, a wide panel
complex molecules a challenging task.
of chemoselective ligation reactions have been used in PET
Prosthetic groups have expanded the field of radioactive radioligand development.10 Lately, the oxime-ligation metho-
bioconjugation in recent years (Scheme 1).3 These are small dology has gained increased attention.11
bifunctional molecules that can be radiolabelled with a radio-
The fairly recent discovery of (almost) ideal orthogonal ligations
nuclide and subsequently appended to biomolecules. The great gave a new impetus in the development of advantageous
majority of prosthetic groups have relied upon reactions with 18F-labelling prosthetic groups. Among these reactions, the
Cu-catalyzed azide–alkyne cycloaddition (CuAAC),12 the strain-
promoted azide–alkyne cycloaddition (SPAAC, azides-cyclooctenes)13
a Center for Radiopharmaceutical Sciences ETH-PSI-USZ, Institute of
and the inverse electron-demand Diels–Alder reaction (IEDDA)14 are
Pharmaceutical Sciences ETH, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland.
E-mail: simon.ametamey@pharma.ethz.ch
the most prevalent. The selectivity, ease and modularity of these
ligations make them well-suited for the construction of complex
18F-labelled tracers. But despite their utility, these reactions
exhibit several flaws that limit their universal application (e.g., use
of toxic additives for CuAAC and formation of bulky hydrophobic
linkages for SPAAC and IEDDA).15 Therefore novel approaches to
b Laboratory of Organic Chemistry, Department of Chemistry and Applied
Biosciences, Vladimir-Prelog-Weg 3, 8093 Zurich, Switzerland.
E-mail: bode@org.chem.ethz.ch
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9cc08645e
‡ These authors made equal contributions to the paper.
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Table 1 Results of the 18F-incorporation yields with and without DABCO
facilitate 18F-labelling and diversify the pre-existing library of
18F-radiotracers are still needed.16
Labelled
prosthetic 8F-Incorporation with DABCOa
group
without DABCOa (140 mM)
18F-Incorporation
In 2012, Bode and Molander showed that the reaction between
potassium acyltrifluoroborates (KATs) and various hydroxylamines
leads to the formation of amide bonds under aqueous conditions at
Entry Precursor
1
room temperature with a second-order rate constant of 20 MÀ1 sÀ1 17
.
0%
n/a
The reaction shows excellent chemoselectivity and tolerates all
common unprotected functional groups typically found on proteins
and peptides. KAT ligation is faster than the majority of the ligations
in common use and it has the distinct advantage of using chemically
stable, easily handled functional groups.18 The Bode group
successfully used this method for the modification of a synthetic
GLP1 analogue19 and for the two-step modification of recombinant
proteins.20 In order to harness the advantages of the KAT ligation
for radiochemistry applications, we sought to develop a new
18F-labelling prosthetic group based on this chemistry.
2
3
3.5% Æ 2.5, n = 3 35.5% Æ 6.8, n = 5
2.3% Æ 1.1, n = 3 3.2% Æ 0.7, n = 3
23.6% Æ 1.1, n = 3 44.7% Æ 6.2, n = 4
4
Despite the advantages of KATs for rapid and chemoselective
ligation, installation of the 18F could be complicated by 18F/19F
isotopic exchange at the trifluoroborate group which would
inevitably lower the overall 18F-incorporation yield. There is
already an impressive body of work on 18F exchange with
organotrifluoroborates. Most notably, Perrin and co-workers have
employed 18F/19F isotopic exchange on trifluoroborates for PET
5
12.0% Æ 3.1, n = 3
n/a
a
10 mmol 1a–1e, [18F]CsF-K2.2.2, dry DMSO (300 mL), 150 1C, 10 min; n =
number of trials.
During the course of our experiments, Pike et al. reported
imaging.21,22 Their studies showed that alkyl acyltrifluoroborates the enhancing effects of 1,4-diazabicyclo[2.2.2]octane (DABCO) on
lose a fluoride with a long solvolysis half-life of 42000 min.23 We 18F-radiolabelling of substituted 2-halopyridines.28 As anticipated,
hypothesised that [18F]fluoride ions would favour nucleophilic the addition of DABCO (140 mM) to the reaction mixture of the
aromatic substitution over 18F/19F isotopic exchange under non- chloro-precursor 1c only marginally improved the 18F-incorporation
aqueous solvents at higher pH, as these isotopic exchange reactions (entry 3), as this method is effective only for 2-halopyridines. In
are usually carried out under aqueous acidic conditions or with contrast, a remarkable improvement was observed for precursor 1b
strong Lewis acids.6,24,25
and 1d (entries 2 and 4), with product 2d exhibiting the highest
For the purpose of this study, five precursors were evaluated 18F-incorporation of 44.7%. In light of these findings, 1d became
for 18F-incorporation (Table 1): KAT derivatives 1a–d and mono- the precursor for our new prosthetic group 2d, termed [18F]FPAT
fluoroacylboronate 1e. For the substrate scope, we selected targets (6-fluoropyridyl acyltrifluoroborate).
likely to be metabolically stable and not prone to defluorination,
Subsequently, further optimisation experiments were per-
in contrast to alkyl fluorides.26,27 Defluorination is particularly formed, the results of which are summarized in the ESI.† We
undesired in the context of in vivo PET imaging, due to bone first established that the same high radiochemical conversions
uptake of free [18F]fluoride.
could be obtained at 120 1C with a lower amount of the precursor
All precursors for the radiolabelling (Table 1, 1a–e) and their (1 mg, 3.4 mmol). Furthermore, aqueous DMSO (0.1–2% H2O v/v) had
respective 19F-reference compounds were synthesised according to a positive impact on 18F-incorporation. Most importantly, the yield
the protocols developed by the Bode group (see ESI†). The precursors could be enhanced by avoiding the conventional azeotropic drying
were subjected to typical conditions for nucleophilic aromatic step. We were delighted to see that these optimisations significantly
substitution – [18F]fluoride ions were eluted with a solution of improved the conversion to 71.1% Æ 12.5 (n = 11). This increase in
Kryptofixs2.2.2/Cs2CO3 from an anion exchange cartridge then yield can be attributed to less adsorption and higher availability of
azeotropically dried with CH3CN. Precursors 1a–e in DMSO were [18F]fluoride in the reaction medium. Moreover, the tolerance
added and the reaction mixture was heated at 150 1C for 10 min. towards H2O could be rationalised by the strong charge interactions
18F-Incorporation yield and product identity were determined by between the free [18F]fluoride ion and the positively charged qua-
UPLC (see ESI†) and HPLC respectively, equipped with a radio- ternary nitrogen of DABCO at the expense of weaker interactions
detector. KAT 1a with the electron-withdrawing nitro leaving with water. Employing these conditions with NBu4HCO3 instead of
group failed to afford the desired labelled compound since the Cs2CO3 further improved the radiochemical conversion to 90.5% Æ
electron-withdrawing effect of the KAT was not sufficient to 5.2 (n = 6). However, this caused broadening of the peaks and poor
activate the aromatic ring. Both precursors 1b and 1c bearing separation of [18F]FPAT during the HPLC purification step.
the KAT group in meta-position to the halogen showed less than
Exchanging the base to K2CO3 afforded [18F]FPAT with
5% 18F-incorporation (entries 2 and 3). Precursor 1d afforded 84.2% Æ 1.2 (n = 5) 18F-incorporation while maintaining a clean
product 2d with 23.6% 18F-incorporation (entry 4), confirming a HPLC purification profile. This procedure was deemed optimal for
synergistic effect of the two activating groups. Acylmonofluoro- [18F]FPAT, affording the prosthetic group in good molar activities of
boronate 1e gave product 2e with 12% yield (entry 5).
81 Æ 26 (n = 7) GBq mmolÀ1 and isolated RCYs of 58% Æ 2.5 (decay
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Scheme 3 Radiolabelling of hydroxylamine peptide 3b with [18F]FPAT.
Yields determined by g-HPLC.
Fig. 1 18F-Labelling of bromo-precursor 1d. (a) UV-HPLC chromatogram;
(b) g-HPLC chromatogram (crude).
To further extend this method towards larger biomolecules,
we modified sfGFP(S147C)20 with a maleimide functionality
bearing the hydroxylamine group at the exposed cysteine
residue (Scheme 4). Due to the low concentrations of the
protein used, small amounts of [18F]FPAT (50–100 MBq) were
used respectively to avoid interference of the cold [19F]FPAT. At
70 mM, the reaction successfully afforded the labelled protein
4c in quantitative yields, while a three-fold lower concentration
led to 80% conversion to the conjugation product.
corrected, n = 3). Unlike similarly structured 18F-nicotinamide based
prosthetic groups (e.g., [18F]FPy-TFP)29 and other amine reactive
building blocks (e.g., 18F-SFB),30 which all non-selectively react with
Lys residues, [18F]FPAT reacts chemoselectively with hydroxylamines
without the need for protecting groups.
We were also curious to see whether the acyltrifluoroborate
engages in 18F/19F isotopic exchange. There was no observable
radiosignal corresponding to the bromo-precursor 1d (10.3 min) on
the HPLC after 18F-incorporation (Fig. 1). The high 18F-incorporation
yields indicated that overall there was a strong preference for
nucleophilic substitution over isotopic exchange under the radio-
labelling conditions. The origin of the remarkably slow exchange of
the fluorides on the KAT moiety is under further investigation.
We next evaluated the ligation efficiency of [18F]FPAT at ambient
temperature with various hydroxylamine substrates 3a–c (results
summarised in the ESI†). For all succeeding experiments, [18F]FPAT
was purified to avoid competing reactions with excess remaining
precursor. These ligations were performed in aqueous medium with
organic solvents (tBuOH or DMSO) as additives for solubilising the
substrates. Aqueous oxalic acid was also added since KAT ligations
are faster under acidic conditions.19 Complete conversion of
[18F]FPAT to amide 4a was observed within 3 min at 10 mM and
1 mM concentrations of the precursor. Decreasing the concentration
to 0.1 mM led to 87% conversion in 15 min and full conversion after
30 min (Scheme 2).
In order to establish that this method could be used to introduce
18F into biologically relevant molecules, we selected a short
eight-residue peptide. The peptide VSPTYRYL was synthesised
by standard Fmoc-SPPS on a Rink Amide resin, and the
N,N-diethylcarbamoylhydroxylamine functional group introduced
at the N-terminus. 18F-Labelling of 3b was carried out with
[18F]FPAT (1–4 GBq) using peptide concentrations typically reported
for similar substrates31 (Scheme 3). At 800 mM and 400 mM, the
ligation afforded the labelled peptide 4b quantitatively after only
15 min. Reducing the concentration to 200 mM resulted in 20%
incorporation after 15 min.
In order to confirm that [18F]FPAT is resistant towards
defluorination in vivo, which is particularly important for
applications in pretargeting, [18F]FPAT was injected into a C57BL/6
mouse for a pilot PET/CT study (Fig. 2). No bone uptake was
observed over the 90 minute time period, establishing that no
defluorination of the prosthetic group was evident, indicating the
high stability of the [18F]fluoride incorporated into this scaffold.
In summary, we developed a new radioconjugation strategy
for the 18F-radiolabelling of peptides and proteins based on KAT
ligation. The novel prosthetic group [18F]FPAT was prepared in high
radiochemical yield and good molar activity without any detectable
18F/19F isotopic exchange. [18F]FPAT coupled selectively within
minutes in aqueous medium with O-diethylcarbamoylhydroxyl-
amines at low concentrations with excellent conversions. Compared
to modern orthogonal 18F-ligation approaches (e.g., SPAAC, IEDDA)
where building blocks are appended to the biomolecules with
bulky hydrophobic linkages, [18F]FPAT is tethered via a robust
and innocuous amide bond which is expected to constitute a
minimal perturbation of the native biomolecule. Moreover,
compared to the increasingly used oxime ligation methodology,
Scheme 4 Radiolabelling of protein sfGFP(S147C) 3c with [18F]FPAT; (a)
g-HPLC chromatogram of [18F]FPAT; (b) g-HPLC chromatogram of ligation
(crude).
Scheme 2 Radiolabelling of hydroxylamine 3a with [18F]FPAT. Yields
determined by g-UPLC.
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[18F]FPAT appears advantageous since its conjugation with
hydroxylamine-functionalized biomolecules is unconditionally
fast, and the amide adducts are expected to possess increased
stability over a wide pH range in contrast to oxime conjugation
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effective orthogonal radiolabelling strategy and has the potential
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under investigation, as well as for applications in pretargeting.
This work was supported by ETH Research Grant ETH-44 17-2.
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Conflicts of interest
There are no conflicts to declare.
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