Fluorine-18 labeled aldehydes as prosthetic groups for oxime coupling with a FVIIa protein

Article abstract of DOI:10.1002/jlcr.3900

¹⁸F-labeled aldehydes can be utilized for oxime coupling with chemically sensitive proteins, such as coagulation factor VII (FVII), if they are sufficiently reactive and can react in an aqueous solvent. New ¹⁸F-labeled nonvolatile aldehyde prosthetic groups derived from [¹⁸F]F-Py-TFP and spirocyclic iodonium (III)ylide precursors for late stage ¹⁸F-labeling were developed. These precursors were characterized, ¹⁸F-labeled, and compared in reactivity for oxime coupling. Oxime coupling was performed on an amino-oxy modified inhibited factor VII (FVIIai-ONH₂) in low concentration to prove the applicability of the proposed method.

Full text of DOI:10.1002/jlcr.3900

Jeppesen Troels (Orcid ID: 0000-0002-5009-4522)
Title
Fluorine-18 labelled aldehydes as prosthetic groups for oxime
coupling with a FVIIa protein
Authors
Troels E. Jeppesen1, Jesper B. Kristensen2, Carsten Behrens2, Jacob Madsen1, Andreas
Kjaer1,*.
1Dept. of Clinical Physiology, Nuclear Medicine & PET and Cluster for Molecular
Imaging, Dept. of Biomedical Sciences, Rigshospitalet and University of Copenhagen, DK-
2200, Copenhagen N and 2Novo Nordisk A/S, DK-2760, Måløv
*)Correspondence:
Prof. Andreas Kjaer, MD, PhD, DMSc, Dept. of Clinical Physiology, Nuclear Medicine &
PET, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. E-mail:
akjaer@sund.ku.dk
Abstract
18F-labelled aldehydes can be utilized for oxime coupling with chemically sensitive proteins,
such as coagulation factor VII (FVII), if they are sufficiently reactive and can react in an
aqueous solvent. New 18F-labelled non-volatile aldehyde prosthetic groups derived from
[18F]F-Py-TFP and spirocyclic iodonium(III)ylide precursors for late stage 18F-labelling were
developed. These precursors were characterized, 18F-labelled and compared in reactivity for
oxime coupling. Oxime coupling was performed on an amino-oxy modified inhibited factor
VII (FVIIai-ONH2) in low concentration to prove the applicability of the proposed method.
Introduction
Oxime coupling, catalyzed by aniline or derivatives thereof is a mild bioorthogonal coupling
reaction that forms a stable bond at physiological conditions.1–5 The resulting increased
reaction rate has made oxime coupling applicable to radiochemistry with short-lived isotopes
such as fluorine-18.6 Several 18F-labelled aldehydes have been proposed including 4-
[18F]fluorobenzaldehyde7, the reducing sugar [18F]FDR8 and other 18F-labelled aldehydes9. 4-
[18F]fluorobenzaldehyde is easy to produce, but the poor solvability in water is troublesome
and therefore 4-[18F]fluorobenzaldehyde is unattractive for labelling of chemically sensitive
biomolecules.10,11 [18F]FDR and the other 18F-labelled aldehydes requires high concentrations
(at least 200-300 µM) of the amino-oxy modified biomolecule8,9,12,13, heating (70 C)9,12,14
and preferably low pH (pH 4-5)8,9,12–14. We have recently developed a non-volatile fluorine-
18 labelled aldehyde that can react in aqueous buffer with an amino-oxy modified protein at
concentrations as low as 52 µM.11 The radio-synthesis consists of 5 steps, where [18F]SFB
(N-succinimidyl-4-[18F]Fluorobenzoate) alone accounts for 3 of 5 steps. Consequently, only
moderate radiochemical yields are obtained. As an alternative to [18F]SFB, the nicotinic
analogue, [18F]F-Py-TFP, has been suggested.15 For this precursor, it is possible to avoid
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azeotropic drying of [18F]fluoride, by eluting [18F]fluoride with the precursor, yielding the
purified [18F]F-Py-TFP in less than 35 minutes.16 In recent years, several late-stage 18F-
fluorination approaches have been suggested, for labelling of electron rich arenes.17–19 Of
these, spirocyclic iodonium(III)ylide precursors are stable, and easy to label without the need
for a mediator.20 In this work, Me3N-Py-TFP and spirocyclic iodonium(III)ylide precursors
were synthetized and fluorine-18 labelled to yield 18F-labelled aldehyde prosthetic groups in a
significantly shorter synthesis time than reported previously11. Radiolabelling efficiency and
oxime coupling capability were compared for the 18F-labelled aldehydes produced. Oxime
coupling was in the end conducted on amino-oxy modified inhibited factor VII (FVIIai-
ONH2).
Results and Discussion
As a starting point for a 18F-labelled aldehyde, the prosthetic group [18F]F-Py-TFP15 was used
as an alternative to [18F]SFB, because the synthesis time can be shortened considerably
compared to [18F]SFB by eluting the [18F]fluoride directly from the anion exchange cartridge
with the precursor, avoiding the normal azeotropic drying.21 This approach produces the
active ester [18F]F-Py-TFP in a single step in only 35 min16, compared to the typical synthesis
time for [18F]SFB of 55a-9022 minutes. Several literature approaches were tried to shorten
total synthesis time of the prosthetic group. The different methods were compared for the
synthesis of [18F]F-Py-TFP and are summarized in the supporting information, Table S1. In
general, all of the approaches trap [18F]fluoride in water on the anion exchange cartridge,
which is washed with an anhydrous solvent or solvent mixture. [18F]fluoride is then eluted
from the anion exchange cartridge by dissolving an onium-salt precursor in the specific
solvent. Either the elution solvent (for example MeOH) is evaporated followed by addition of
the desired reaction solvent or the elution solvent is used as the reaction solvent.
The resulting [18F]F-Py-TFP (n = 5) was obtained in 90  5% radiochemical yield as
measured by radioTLC. The combined yield, taking both elution efficiency and RCY (by
TLC) into account, was 73  8% and the isolated RCY was 58  7% in a total synthesis time
of 14 minutes. From [18F]F-Py-TFP the synthesis of [18F]F-nicotinamidealdehyde [18F]3 was
performed (as from [18F]SFB),11 by addition of 4,4-diethoxybutan-1-amine and deprotection
of the corresponding acetal, [18F]2, Figure 1.
Figure 1. Synthesis of [18F]F-nicotinamidealdehyde ([18F]3). (i) [18F]Fluoride on an anion
exchange cartridge, eluted with N,N,N-trimethyl-5-((2,3,5,6-
aSynthesized on Tracerlab MX using casette and reagents from ABX GmbH
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tetrafluorophenoxy)carbonyl)pyridin-2-aminium trifluoromethanesulfonate (1) in t-
BuOH/MeCN. (ii) 4,4-diethoxybutan-1-amine, MeCN, 5 min. (iii) 10% TFA/H2O, 5 min.
[18F]F-nicotinamidealdehyde ([18F]3) was purified by preparative HPLC. The solvent was
exchanged to MeCN using a Chromafix HR-P cartridge before evaporation of the solvent.
Co-elution with the corresponding non-radioactive compounds by HPLC confirmed the
identity of [18F]F-Py-TFP and [18F]F-nicotinamidealdehyde ([18F]3), supporting information.
[18F]F-nicotinamidealdehyde ([18F]3) was produced in 78 minutes starting from 3.1 – 7.5
GBq of 18F water, with an isolated RCY of 20 ± 10% (up to 604 MBq, n = 3), which is
significantly faster and in a comparable yield to [18F]F-butyracetal ([18F]13), vide infra,
produced from [18F]SFB (150 min, 20% RCY, up to 2109 MBq).11
Spirocyclic iodonium(III)ylide precursors offer the advantage that fluorine-18 can be
introduced late in the synthesis. Cyclopentyl substituted meldrums acid (SPI5) and adamantyl
substituted meldrums acid (SPIAd) has been proposed as auxillary groups.20,23,24 SPIAd
precursors are more stable in reaction mixtures than SPI5 and can resist higher temperatures
needed in labelling of more electron rich arenes. The auxiliary groups were synthesized as
described in the literature.23,25 From 4-iodobenzoic acid the desired acetals (4-7) were formed
by amide coupling via EDC in CH2Cl2. Oxidation of I(I) to I(III) was achieved by mCPBA,
and reaction with the auxiliary groups were performed in a one pot reaction in aqueous basic
media.20 It was however not possible to directly produce the aldehyde spirocyclic
iodonium(III)ylide precursors in our hands, probably due to oxidation of the aldehyde moiety
by the conditions needed for oxidation of I(I) to I(III). Instead 18F-fluorination was therefore
performed on the acetal protected aldehydes. Synthesis of 5 acetal spirocyclic iodonium(III)
ylide precursors (8-12) was performed in 7-18% yield, Figure 2. Labelling was performed
with dried 18F-[K222K]+ dissolved in DMF, where an aliquot of this mixture was added to
spirocyclic iodonium(III)ylide precursor and TEMPO dissolved in DMF.25 TEMPO has
previously been shown to increase labelling yields of iodonium salts, perhaps by preventing
decomposition.26
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Figure 2. Synthesis and labelling with fluorine-18 of spirocyclic iodonium(III)ylide
precursors. (i) EDC (1-1.3 eq.), 4,4-diethoxybutan-1-amine, 1-amino-3,3-diethoxypropane,
aminoacetaldehyde diethyl acetal or aminoacetaldehyde dimethyl acetal (1 eq.), CH2Cl2, r.t.,
over night. (ii) mCPBA (2 eq.), CH2Cl2, r.t. 4h. then SPI5 or SPIAd in 10% Na2CO3, r.t. 4h.
(iii) 18F-[K222K]+, TEMPO, DMF, 5 min, 120 C. (iv) 10-90 % TFA/H2O, r.t., 5 min.
[18F]F-butyracetal ([18F]13), labelled from the precursors (8) and (9) was examined by radio-
TLC at reaction time points from 2 to 20 minutes, Figure 3A (n = 2-3 where error bars are
present). The two spirocyclic iodonium(III)ylide precursors gave similar RCY’s, and a
reaction time of 5 minutes at 120 C was sufficient. In contrast to SPI5, SPIAd is not easily
dissolvable in DMF, and the SPI5 auxiliary group is, therefore, more suitable in this case.
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Figure 3. RCY determined by TLC of 18F-fluorination with the spirocyclic
iodonium(III)ylide precursors (8-12). (i) 18F-[K222K]+, TEMPO, DMF, 120 C. A, 18F-
labelling of SPI5-butyracetal (8) and SPIAd-butyracetal (9) at timepoints from 2 to 20
minutes. B, RCY of 18F-labelled acetals from spirocyclic iodonium(III)ylide precursors (8-
12).
The RCY’s of 18F-labelled acetals from the corresponding spirocyclic iodonium(III)ylide
precursors (8-12) are summarized in Figure 3B. Precursors (8) and (9) gave [18F]13 in 76.2 
10.2 % (n = 3) and 85.2  5.1 % (n = 3), respectively, while precursor (10) gave [18F]14 in
8.4  4.5 % (n = 2), precursor (11) gave [18F]15 in 83.0  4.2 % (n = 2) and precursor (12)
gave [18F]16 in 87.1  4.6 % (n = 3). The SPI5-propylacetal (10) resulted in a lower RCY
than the other precursors, which show comparable RCY. The reason for the lower yield was
not investigated further. Formation of the desired 18F-aldehydes was accomplished by
treatment of the corresponding 18F-labelled acetals with TFA/H2O. The identity of the formed
18F-labelled aldehydes was confirmed by HPLC co-elution with the corresponding non-
radioactive aldehyde compounds, supporting information.
As the RCY of [18F]F-ethylacetal ([18F]15 and [18F]16) from either precursors (11) or (12) is
comparable, precursor (12) was used as a higher yield was achieved in the precursor
synthesis.
[18F]F-butyraldehyde ([18F]17) was produced from SPI5-butyracetal (8) in 67 minutes
starting from 2.4 - 11 GBq of 18F water, with an isolated RCY of 32 ± 3% (up to 2 GBq, n =
3), and [18F]F-ethylaldehyde ([18F]19) was produced from SPI5-ethyl methoxyacetal (12) in
89 minutes starting from 0.6 – 5.6 GBq of 18F water, with an isolated RCY of 9 ± 4 % (up to
129 MBq, n = 3).
The oxime coupling ability of the formed [18F]F-nicotinamidealdehyde ([18F]3), [18F]F-
butyraldehyde ([18F]17) and [18F]F-ethylaldehyde ([18F]19) was tested using O-
benzylhydroxylamine (O-Bn-ONH2) as a model compound. O-Bn-ONH2 was mixed at
different concentrations with [18F]17, [18F]19 or [18F]3 and 1% aniline was added as
mediator. The mixtures were left for 30 minutes at room temperature to evaluate the
reactivity of the 18F-labelled aldehydes in oxime coupling, Figure 4.
Figure 4. A, Oxime reaction with [18F]F-nicotinamidealdehyde ([18F]3), [18F]F-
butyraldehyde ([18F]17) or [18F]F-ethylaldehyde ([18F]19) and O-Bn-ONH2 (20, 50, 100 and
500 µM), with 1% aniline for 30 minutes. B, zoom of A in the 0-100 µM region. Reactions
were analysed by HPLC after 30 min.
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[18F]F-butyraldehyde ([18F]17) reacts with 20, 50 and 100 µM O-Bn-ONH2 with a higher
radiochemical yield than [18F]F-ethylaldehyde ([18F]19). Only at 500 µM of O-Bn-ONH2,
does [18F]F-ethylaldehyde ([18F]19) give a comparable yield. In general, [18F]F-
nicotinamidealdehyde ([18F]3) gives comparable yield to [18F]F-butyraldehyde ([18F]17).
Acetals [18F]2 and [18F]13 and aldehyde [18F]3 and [18F]17 has the potential to cyclize, as
previously described.11 However, [18F]3 and [18F]17 displays a higher reaction rate towards
O-Bn-ONH2 than [18F]19, indicating that even if cyclization occurs, it does not impact
reactivity.
The reactivity on oxime coupling and isolated RCY of [18F]3 and [18F]17 is similar, but in
this case a more direct labelling method in less radioactive steps is more attractive. However,
[18F]F-Py-TFP is easy to produce in high yield, and is therefore well suited for a library-like
approach to 18F-label aldehyde prosthetic groups.
[18F]F-butyraldehyde ([18F]17) prepared from SPI5-butyracetal (8) was reacted with FVIIai-
ONH2 (52 µM) as a proof-of-principle reaction for chemically sensitive proteins, Figure 5.
MeCN was evaporated in the presence of DMSO (5 µL), FVIIai-ONH2 was added, and the
reaction was left at room temperature for 30 minutes, prior to purification with a PD-10
desalting column. The radiochemical purity of labelled 18F-FVIIai was >98%, had a pseudo
molar activity of 27 GBq/µmol, a protein bound fraction of 96.9% and an isolated RCY of
11% (80 MBq) from [18F]F-butyraldehyde ([18F]17). The isolated RCY from 18F-water was
3.5 %. The radiochemical purity, pseudo molar activity, protein bound fraction and isolated
RCY were comparable to [18F]F-butyraldehyde ([18F]17) prepared from 18F-SFB, but the
aldehyde was prepared in less than half the time, 67 minutes compared to 150 minutes.11 The
identity of the formed 18F-FVIIai was confirmed by HPLC comparison with native FVIIai,
supporting information, Figure S19.
Figure 5. A. Reaction scheme with [18F]F-butyraldehyde ([18F]17) and FVIIai-ONH2.
FVIIai- ONH2 (500 μg, 192.5 μL, 52 μM, 2.6 mg/mL, 50 mM Hepes, 200 mM NaCl, 10 mM
CaCl2, pH 7.0), aniline (1% v/v), 30 minutes, room temperature. B. Radio-chromatogram of
the crude reaction mixture. C. Radio-chromatogram of purified 18F-FVIIai, by PD-10.
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Conclusion
Two approaches for producing reactive 18F-aldehydes suitable for oxime coupling on
chemically sensitive proteins were developed. One approach, starting from the known
synthesis of [18F]F-Py-TFP without azeotropic drying16, can be used as a “library-like”
approach, yielding [18F]F-nicotinamidealdehyde ([18F]3). The other approach, starting from a
spirocyclic iodonium(III)ylide precursor, has fewer steps and would therefore be preferred for
single-substance labelling, yielding three different 18F-aldehydes, including [18F]F-
butyraldehyde ([18F]17). [18F]17 showed superior reactivity towards O-Bn-ONH2 and the
synthesis time was decreased to around 60 minutes, compared to 150 minutes for
synthesizing [18F]17 from [18F]SFB11. [18F]17 was coupled to FVIIai-ONH2 in a moderate
yield (11%), high purity (>98%) and acceptable pseudo molar activity (27 GBq/µmol). This
approach is thus suitable for oxime coupling with sensitive amino-oxy modified proteins.
Experimental procedures
Chemicals were purchased from Sigma-Aldrich unless stated otherwise. Fluorine-18 was
produced by an 18O(p,n)18F reaction on a MC32 Scanditronix or on a RDS Eclipse,
CTI/Siemens apparatus. Analytical HPLC was performed on a Thermo Fischer Ultimate 3000
system and a gamma detector (Scansys Laboratorieteknik) connected in series. Data
collection and liquid chromatography control used the program Chromeleon 6.8. Analytical
HPLC was performed using either method 1: Gradient from 0-100% MeCN/H2O in 10 min.,
0.1% TFA, on a Phenomenex Luna C18(2) column (5 µm, 150 x 4.6 mm), or method 2:
Gradient from 10% to 100% MeCN/H2O 0.1% TFA in 16 min, on a Grace Vydac column
(C4 Protein column, 300 Å, 5 μm, 4.6 × 250 mm). Preparative HPLC was performed on a
Phenomenex Luna C18(2) column (5 µm, 250 x 10.00 mm) with a flow of 3 mL/min in 50%
MeCN/H2O with 0.1% TFA. NMR was performed on a 600 MHz Bruker Avance III HD, or a
400 MHz Bruker Avance III. MS (MALDI-TOF) was performed on Bruker microflex LT.
All data are presented as  SEM where applicable.
General procedure for synthesis of iodine-acetals
4-iodobenzoic acid and EDC was dissolved in CH2Cl2. The appropriate acetal-amine was
added and the reaction was stirred over night at room temperature. The reaction was followed
by TLC (EtOAc). The reaction was diluted with CH2Cl2 (50-80 mL) and washed with sat.
NaHCO3 (50-80 mL), 10% citric acid (50-80 mL) and sat. NaCl (50-80 mL). The organic
phase was dried over MgSO4, and the solvent was removed in vacuo. The substance was
recrystallized from hot heptane or from CH2Cl2/Heptane, yielding a white powder in all
cases.
N-(4,4-diethoxybutyl)-4-iodobenzamide (4)
4-iodobenzoic acid (2.0g, 8.1 mmol), EDC (1.55g, 8.1 mmol), CH2Cl2 (20 mL). 4,4-
diethoxybutan-1-amine (1.39 mL, 8.1 mmol). Yield: 58% (1.76 g). 1H NMR (400 MHz,
CDCl3) δ 7.80 – 7.74 (m, 2H), 7.51 – 7.46 (m, 2H), 6.46 (s, 1H), 4.56 – 4.48 (m, 1H), 3.66
(dq, J = 9.4, 7.1 Hz, 2H), 3.55 – 3.42 (m, 4H), 1.72 (td, J = 4.2, 2.0 Hz, 4H), 1.20 (t, J = 7.1
Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 166.77, 137.85, 134.37, 128.65, 102.84, 98.29,
61.79, 39.95, 31.23, 24.46, 15.48. Hires MALDI-TOF calc. [M+Na]+ 414.0536 found
414.0536.
N-(3,3-diethoxypropyl)-4-iodobenzamide (5)
4-iodobenzoic acid (1.0 g, 4 mmol), EDC (1.0 g, 5 mmol), CH2Cl2 (10 mL). 1-amino-3,3-
diethoxypropane (0.7 mL, 4 mmol). Yield: 25% (0.375 g). 1H NMR (400 MHz, CDCl3) δ
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7.83 – 7.72 (m, 2H), 7.53 – 7.42 (m, 2H), 7.06 (s, 1H), 4.64 (t, J = 4.9 Hz, 1H), 3.72 (dq, J =
9.4, 7.1 Hz, 2H), 3.62 – 3.46 (m, 4H), 1.98 – 1.88 (m, 2H), 1.23 (t, J = 7.0 Hz, 6H). 13C NMR
(101 MHz, CDCl3) δ 166.30, 137.80, 134.37, 128.59, 103.31, 98.24, 62.52, 36.11, 32.73,
15.55. Hires MALDI-TOF calc. [M+Na]+ 400.0380 found 400.0375.
N-(2,2-diethoxyethyl)-4-iodobenzamide (6)27
4-iodobenzoic acid (1.0 g, 4 mmol), EDC (1.0 g, 5 mmol), CH2Cl2 (10 mL).
Aminoacetaldehyde diethyl acetal (0.6 mL, 4 mmol). Yield: 30% (0.437 g). 1H NMR (600
MHz, CDCl3) δ 7.82 – 7.76 (m, 2H), 7.52 – 7.47 (m, 2H), 6.33 (s, 1H), 4.61 (t, J = 5.2 Hz,
1H), 3.75 (dq, J = 9.5, 7.1 Hz, 2H), 3.58 (td, J = 7.0, 2.8 Hz, 4H), 1.23 (t, J = 7.0 Hz, 6H).
13C NMR (151 MHz, CDCl3) δ 166.83, 137.96, 133.97, 128.68, 100.83, 98.59, 63.26, 42.64,
15.51.
N-(2,2-dimethoxyethyl)-4-iodobenzamide (7)28
4-iodobenzoic acid (0.66 g, 2.7 mmol), EDC (0.66 g, 3.5 mmol), CH2Cl2 (10 mL).
Aminoacetaldehyde dimethyl acetal (0.29 mL, 3.5 mmol). Yield: 38% (0.340 g). 1H NMR
(600 MHz, CDCl3) δ 7.82 – 7.75 (m, 2H), 7.53 – 7.47 (m, 2H), 6.29 (s, 1H), 4.48 (t, J = 5.2
Hz, 1H), 3.60 (t, J = 5.5 Hz, 2H), 3.43 (s, 6H). 13C NMR (151 MHz, CDCl3) δ 166.88,
137.95, 133.87, 128.72, 102.79, 98.65, 54.81, 41.67.
6,10-dioxaspiro[4.5]decane-7,9-dione (SPI5)25
Synthesized as described previously. Yield: 29% (4.7 g).1H NMR (400 MHz, CDCl3) δ 3.61
(s, 2H), 2.25 – 2.16 (m, 4H), 1.94 – 1.85 (m, 4H).
Spiro[adamantane-2,2'-[1,3]dioxane]-4',6'-dione (SPIAd)23
Synthesized as described for 6,10-dioxaspiro[4.5]decane-7,9-dione. Yield: 55% (2.48 g). 1H
NMR (600 MHz, CDCl3) δ 3.61 (s, 2H), 2.19 (s, 2H), 2.14 (d, J = 12.5 Hz, 4H), 1.92 (s, 2H),
1.79 (d, J = 12.5 Hz, 4H), 1.75 (s, 2H).13C NMR (151 MHz, CDCl3) δ 163.15, 109.75, 37.79,
36.65, 36.44, 33.62, 26.20.
General procedure for synthesis of spirocyclic ylides
The synthesis was adapted and modified from references.20,25 mCPBA (2 eq.) was added to
the iodine (1 eq.) in CH2Cl2. The reaction was stirred at room temperature until TLC
(EtOAc/Heptane 1:1) showed conversion, generally 4 hours. Auxiliary dione (1 eq.) in 10%
Na2CO3 (0.33 M) was added and the reaction mixture was stirred vigorously until TLC (10%
EtOH/EtOAc) showed conversion, generally 4 hours. The mixture was diluted with water (25
mL) and extracted with 3 x CH2Cl2 (25 mL). The combined organic phases were washed with
3 x H2O (25 mL) and 3 x brine (25 mL). The combined organic phases were dried over
MgSO4 and concentrated in vacuo. The substance was recrystallized from EtOAc and
Heptane, yielding a pure off-white to white powder in all cases.
N-(4,4-diethoxybutyl)-4-((7,9-dioxo-6,10-dioxaspiro[4.5]decan-8-ylidene)-λ3-
iodaneyl)benzamide (8)
mCPBA (441 mg, 2.56 mmol), N-(4,4-diethoxybutyl)-4-iodobenzamide (500 mg, 1.28
mmol), CH2Cl2 (10 mL). 6,10-dioxaspiro[4.5]decane-7,9-dione (218 mg, 1.28 mmol), 10%
Na2CO3 (4.5 mL). Yield: 18% (126 mg). 1H NMR (600 MHz, CDCl3) δ 7.91 – 7.86 (m, 2H),
7.79 (d, J = 8.4 Hz, 2H), 6.81 (s, 1H), 4.53 (q, J = 4.7, 3.7 Hz, 1H), 3.70 – 3.62 (m, 2H), 3.56
– 3.43 (m, 4H), 2.19 – 2.13 (m, 4H), 1.83 – 1.78 (m, 4H), 1.73 (q, J = 3.2 Hz, 4H), 1.20 (t, J
= 7.0 Hz, 6H).. 13C NMR (151 MHz, CDCl3) δ 164.35, 163.22, 136.70, 132.22, 129.31,
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127.49, 115.22, 113.32, 101.65, 60.76, 39.02, 36.40, 30.09, 23.10, 22.38, 14.34. Hires
MALDI-TOF calc. [M+Na]+ 582.0959 found 582.0964.
4-((4',6'-dioxospiro[adamantane-2,2'-[1,3]dioxan]-5'-ylidene)-λ3-iodaneyl)-N-(4-ethoxy-
5-methoxypentyl)benzamide (9)
mCPBA (99 mg, 511 µmol), N-(4,4-diethoxybutyl)-4-iodobenzamide (100 mg, 256 µmol),
CH2Cl2 (2 mL). spiro[adamantane-2,2'-[1,3]dioxane]-4',6'-dione (61 mg, 256 µmol), 10%
Na2CO3 (0.8 mL). Yield: 13% (21 mg). 1H NMR (400 MHz, DMSO-d6) δ 8.58 (t, J = 5.6 Hz,
1H), 7.89 – 7.78 (m, 4H), 4.47 (s, 1H), 3.55 (dq, J = 9.5, 7.0 Hz, 2H), 3.41 (dq, J = 9.5, 7.0
Hz, 2H), 3.25 (p, J = 6.4, 5.4 Hz, 2H), 2.34 (s, 2H), 1.94 (d, J = 12.3 Hz, 4H), 1.80 (s, 4H),
1.66 (d, J = 11.5 Hz, 4H), 1.54 (q, J = 3.1 Hz, 4H), 1.09 (t, J = 7.0 Hz, 6H). 13C NMR (101
MHz, DMSO) δ 165.09, 162.50, 132.15, 129.43, 105.10, 101.92, 60.50, 36.45, 34.80, 33.19,
30.82, 25.95, 24.37, 15.29. Hires MALDI-TOF calc. [M+Na]+ 648.1428 found 648.1436.
N-(3,3-diethoxypropyl)-4-((7,9-dioxo-6,10-dioxaspiro[4.5]decan-8-ylidene)-λ3-
iodaneyl)benzamide (10)
mCPBA (92 mg, 530 µmol), N-(3,3-diethoxypropyl)-4-iodobenzamide (100 mg, 265 µmol),
CH2Cl2 (2 mL). 6,10-dioxaspiro[4.5]decane-7,9-dione (45 mg, 265 µmol), 10% Na2CO3 (0.9
mL). Yield: 6.7% (9.7 mg). 1H NMR (400 MHz, CDCl3) δ 7.86 (d, J = 8.4 Hz, 2H), 7.72 (t, J
= 7.5 Hz, 2H), 7.12 (s, 1H), 4.58 (t, J = 4.8 Hz, 1H), 3.66 (t, J = 6.6 Hz, 2H), 3.57 – 3.42 (m,
4H), 2.11 (d, J = 7.5 Hz, 4H), 1.87 (d, J = 6.2 Hz, 2H), 1.74 (s, 4H), 1.22 – 1.11 (m, 6H).
MALDI-TOF calc. [M+H]+ 546.09 found 546.07.
N-(2,2-diethoxyethyl)-4-((7,9-dioxo-6,10-dioxaspiro[4.5]decan-8-ylidene)-λ3-
iodaneyl)benzamide (11)
mCPBA (95 mg, 550 µmol), N-(2,2-diethoxyethyl)-4-iodobenzamide (100 mg, 275 µmol),
CH2Cl2 (2 mL). 6,10-dioxaspiro[4.5]decane-7,9-dione (47 mg, 275 µmol), 10% Na2CO3 (0.9
mL). Yield: 7.3% (10.7 mg). 1H NMR (400 MHz, CDCl3) δ 7.87 (d, J = 8.6 Hz, 2H), 7.73 (d,
J = 8.2 Hz, 2H), 6.34 (s, 1H), 4.54 (s, 1H), 3.69 (s, 2H), 3.52 (q, J = 9.8, 9.2 Hz, 4H), 2.09 (d,
J = 9.5 Hz, 4H), 1.78 – 1.69 (m, 4H), 1.19 (s, 6H). Hires MALDI-TOF calc. [M+Na]+
554.0646 found 554.0645.
N-(2,2-dimethoxyethyl)-4-((7,9-dioxo-6,10-dioxaspiro[4.5]decan-8-ylidene)-λ3-
iodaneyl)benzamide (12)
mCPBA (103 mg, 597 µmol), N-(2,2-dimethoxyethyl)-4-iodobenzamide (100 mg, 298 µmol),
CH2Cl2 (2 mL). 6,10-dioxaspiro[4.5]decane-7,9-dione (51 mg, 298 µmol), 10% Na2CO3 (0.9
mL). Yield: 13.1% (19.7 mg). 1H NMR (400 MHz, CDCl3) δ 7.95 – 7.86 (m, 2H), 7.84 – 7.75
(m, 2H), 6.48 (d, J = 6.0 Hz, 1H), 4.49 (t, J = 5.0 Hz, 1H), 3.61 (t, J = 5.4 Hz, 2H), 3.43 (s,
6H), 2.16 (td, J = 7.4, 6.0, 3.8 Hz, 4H), 1.85 – 1.76 (m, 4H). 13C NMR (151 MHz, CDCl3) δ
165.48, 164.17, 138.26, 133.38, 130.41, 128.59, 116.38, 114.38, 102.43, 54.71, 41.68, 37.43,
23.40. Hires MALDI-TOF calc. [M+H]+ 504.0513 found 504.0521.
2,3,5,6-tetrafluorophenyl 6-chloronicotinate15
The synthesis was modified slightly from reference. Briefly, 6-chloronicotinic acid (5.0 g,
31.7 mmol) was dissolved in dioxane (185 mL). 2,3,5,6-tetrafluorophenol (5.3 g, 31.7 mmol)
and EDC (9.7g, 31.7 mmol) was added, and the reaction was stirred overnight. The reaction
mixture was filtered and the solvent was evaporated in vacuo. The resulting oily substance
was recrystallized from hot heptane, to yield a white powder (83%, 8.0 g).
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N,N,N-trimethyl-5-((2,3,5,6-tetrafluorophenoxy)carbonyl)pyridin-2-aminium
trifluoromethanesulfonate (1)29
The synthesis was performed as described previously with minor modifications. 2,3,5,6-
tetrafluorophenyl 6-chloronicotinate (3.0 g, 9.8 mmol) was mixed with trimethylamine (1.0
M in THF, 147 mmol, 147 mL) and stirred under N2 overnight. The formed crystals were
filtered off and washed with Et2O. A halide test with silver nitrate was performed. A sample
of the crystals were dissolved in water, acidified with dilute nitric acid, and silver nitrate was
added. A precipitate formed. The formed precipitate dissolved upon addition of diluted
ammonia solution, confirming the presence of a free halide (no precipitate formed upon
halide test using 2,3,5,6-tetrafluorophenyl 6-chloronicotinate). The isolated crystals were
suspended in CH2Cl2 (150 mL) and TMSOTf was added slowly under N2. The precipitate
disappears after 10-15 minutes and the solvent was evaporated in vacuo. The resulting
crystals were washed with Et2O and dried under vacuum overnight. The product was
recrystallized in EtOAc, affording a white powder (83%, 3.9 g). A halide test was performed,
showing no precipitate and thereby no remaining Cl--counterion.
General synthesis of non-radioactive 4-fluorine acetal and aldehyde reference
compounds
4-Fluorine acetals were synthesized by mixing either 4-fluorobenzoic acid or 6-
fluoronicotinic acid and EDC in CH2Cl2. The appropriate amine-acetal was added and the
reaction was stirred over night or until TLC (EtOAc) showed complete conversion. The
reaction mixture was diluted with CH2Cl2 (20-50 mL) and washed with sat. NaHCO3 (20-50
mL), 10% citric acid (20-50 mL) and sat. NaCl (20-50 mL). The organic phase was
concentrated in vacuo and recrystallized from either heptane or CH2Cl2 and heptane, yielding
a white powder or needle shaped crystals in all cases. Deprotection to the corresponding
aldehyde was performed with 10-90% TFA/H2O in situ, Supporting Information.
N-(4,4-diethoxybutyl)-4-fluorobenzamide (13)11
4-fluorobenzoic acid (1.0 g, 7.14 mmol), EDC (1.37g, 7.14 mmol), 4,4-diethoxybutan-1-
amine (1.23 mL, 7.14 mmol). Yield: 41% (834 mg). 1H NMR (400 MHz, CDCl3) δ 7.75 –
7.66 (m, 2H), 7.09 – 6.98 (m, 2H), 6.33 (s, 1H), 4.49 – 4.42 (m, 1H), 3.60 (dq, J = 9.4, 7.0
Hz, 2H), 3.50 – 3.37 (m, 4H), 1.72 – 1.58 (m, 4H), 1.14 (t, J = 7.0 Hz, 6H). 13C NMR (101
MHz, CDCl3) δ 166.40, 163.38, 131.04, 129.19, 129.10, 115.63, 115.41, 102.73, 61.64,
39.81, 31.10, 24.41, 15.34. 19F NMR (376 MHz, CDCl3) δ -108.67 (tt, J = 10.3, 5.0 Hz).
Hires MALDI-TOF calc. [M+Na]+ 306.1476 found 306.1473.
N-(3,3-diethoxypropyl)-4-fluorobenzamide (14)30
4-fluorobenzoic acid (1.0 g, 7.14 mmol), EDC (1.37g, 7.14 mmol), 1-amino-3,3-
1
diethoxypropane (1.2 mL, 7.14 mmol). Yield: 29% (556 mg). H NMR (400 MHz, CDCl3) δ
7.76 – 7.64 (m, 2H), 7.08 – 6.97 (m, 2H), 6.95 (s, 1H), 4.58 (t, J = 4.9 Hz, 1H), 3.65 (dq, J =
9.4, 7.1 Hz, 2H), 3.56 – 3.39 (m, 4H), 1.92 – 1.82 (m, 2H), 1.16 (t, J = 7.0 Hz, 6H). 13C NMR
(101 MHz, CDCl3) δ 165.95, 163.35, 131.01 (d, J = 3.1 Hz), 129.10, 129.02, 115.56, 115.35,
103.16, 62.33, 35.98, 32.66, 15.39. 19F NMR (376 MHz, CDCl3) δ -107.35 – -112.73 (m).
N-(2,2-diethoxyethyl)-4-fluorobenzamide (15)
4-fluorobenzoic acid (1.0 g, 7.14 mmol), EDC (1.37g, 7.14 mmol), Aminoacetaldehyde
diethyl acetal (1.04 mL, 7.14 mmol). Yield: 55% (995 mg). 1H NMR (400 MHz, CDCl3) δ
77.83 – 7.73 (m, 2H), 7.15 – 7.04 (m, 2H), 6.45 (s, 1H), 4.57 – 4.49 (m, 1H), 3.67 (dq, J =
9.4, 7.0 Hz, 2H), 3.57 – 3.42 (m, 4H), 1.20 (t, J = 7.0 Hz, 6H). 13C NMR (101 MHz, CDCl3)
δ 166.43, 163.39, 131.04, 129.21, 129.13, 115.63, 115.41, 102.75, 61.65, 39.82, 15.35. 19F
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NMR (376 MHz, CDCl3) δ -105.48 – -112.95 (m). Hires MALDI-TOF calc. [M+Na]+
278.1161 found 278.1161.
N-(2,2-dimethoxyethyl)-4-fluorobenzamide (16)
4-fluorobenzoic acid (1.0 g, 7.14 mmol), EDC (1.37g, 7.14 mmol), Aminoacetaldehyde
dimethyl acetal (778 µL, 7.14 mmol). Yield: 31% (499 mg). 1H NMR (400 MHz, CDCl3) δ
7.84 – 7.74 (m, 2H), 7.17 – 7.06 (m, 2H), 6.27 (s, 1H), 4.49 (t, J = 5.2 Hz, 1H), 3.64 – 3.57
(m, 2H), 3.44 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 166.49, 163.55, 130.53, 129.36,
129.27, 115.74, 115.52, 102.74, 54.65, 41.55. 19F NMR (376 MHz, CDCl3) δ -108.14 (tt, J =
9.1, 5.1 Hz). Hires MALDI-TOF calc. [M+Na]+ 250.0850 found 250.0852.
N-(4,4-diethoxybutyl)-6-fluoronicotinamide (2)
6-fluoronicotinic acid (100 mg, 709 µmol), EDC (136 mg, 709 µmol), 4,4-diethoxybutan-1-
amine (122 µL, 709 µmol). Yield: 37% (74 mg). 1H NMR (400 MHz, CDCl3) δ 8.64 – 8.57
(m, 1H), 8.25 (ddd, J = 8.4, 7.5, 2.5 Hz, 1H), 7.00 (ddd, J = 8.6, 2.9, 0.7 Hz, 1H), 6.73 (s,
1H), 4.58 – 4.49 (m, 1H), 3.68 (dq, J = 9.4, 7.1 Hz, 2H), 3.59 – 3.44 (m, 4H), 1.74 (dd, J =
6.1, 3.4 Hz, 4H), 1.20 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 166.15, 164.40,
146.57, 140.83, 128.89, 109.84, 102.68, 61.82, 39.93, 31.13, 24.07, 15.34. Hires MALDI-
TOF calc. [M+Na]+ 307.1428 found 307.1429.
6-fluoronicotinic acid 2,3,5,6-tetrafluorophenyl ester (F-Py-TFP)15,29
6-fluoronicotinic acid (116 mg, 822 µmol) was dissolved in dioxane (10 mL). EDC (150 mg,
904 µmol) and 2,3,5,6-tetrafluorophenol (122 mg, 781 µmol) was added and the reaction
mixture was stirred overnight. The solvent was evaporated to give off-white crystals.
Recrystallized from hot heptane. Yield: 14 mg (6%). 1H NMR (400 MHz, CDCl3) δ 9.02 (d, J
= 2.4 Hz, 1H), 8.56 – 8.43 (m, 1H), 7.11 – 6.90 (m, 2H). 13C NMR (101 MHz, CDCl3) δ
167.88, 165.41, 160.31, 151.62 (d, J = 17.0 Hz), 147.69 – 147.00 (m), 145.52 – 144.36 (m),
143.46 (d, J = 9.7 Hz), 141.93 (d, J = 10.7 Hz), 139.43 (d, J = 20.6 Hz), 121.86 (d, J = 4.5
Hz), 110.25 (d, J = 37.6 Hz), 103.83 (t, J = 22.7 Hz). 19F NMR (376 MHz, CDCl3) δ -57.97, -
138.23 – -138.46 (m), -152.42 – -152.65 (m).
Radiochemistry
RadioTLC was performed using silica gel in aluminium foil with fluorescent indicator,
developed in EtOAc and imaged with a Packard instant imager. All samples were quenched
by MeCN/H2O prior to analysis. Chromafix PS-30 cartridges were used without pre-
conditioning. All other cartridges were pre-conditioned with EtOH (5 mL) and H2O (10 mL)
prior to use. RCY’s are decay corrected. Isolated activity yields are non-decay corrected.
Pseudo molar activities are calculated from the isolated activity and 5 consecutive HPLC
18
analyses of FVIIai with a known concentration and compared to the purified F-FVIIai.
[18F]F-Py-TFP
Performed as described in the literature.16 Briefly, [18F]Fluoride was captured on a chromafix
-
PS-30 cartridge (HCO3 anion), washed with MeCN (3 mL) and eluted in the same direction
or reversed with N,N,N-trimethyl-5-((2,3,5,6-tetrafluorophenoxy)carbonyl)pyridin-2-aminium
trifluoromethanesulfonate in t-BuOH/MeCN (4:1), MeCN or MeOH (5-10 mg in 2 mL),
according to Table 1, vide supra. The mixture was diluted with H2O (15 mL), and passed
through an Oasis MCX cartridge. From here the resulting [18F]F-Py-TFP was eluted in MeCN
(1.5 mL).
[18F]6-fluoro-N-(4-oxobutyl)nicotinamide ([18F]3)
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To [18F]F-Py-TFP in MeCN, 4,4-diethoxybutan-1-amine (4.15 µL, 24 µmol) was added and
reacted for 5 min. 10% TFA/H2O (v/v, 1 mL) was added and left for 5 min. Preparative
HPLC was performed, using a Phenomenex Luna C18(2), 5 µm, 10 x 250 mm column, with a
solvent system of 25% MeCN/H2O, 0.1% TFA. The collected fractions were diluted with
H2O (20 mL) and passed through an Chromafix HR-P cartridge. [18F]6-fluoro-N-(4-
oxobutyl)nicotinamide was eluted with 2 mL MeCN.
Spirocyclic iodonium ylides
Radiolabelling was performed as described in the literature.25 Briefly, the [18F]fluoride was
-
trapped on a waters QMA cartridge (HCO3 anion), and eluted with a kryptofix mix (0.6 mL,
40 mM K222, 55 mM K2CO3 in H2O/MeOH 1:50), and dried azeotropically with MeCN (2 x 1
mL). The dried 18F-[K222K]+ complex was dissolved in DMF and used for several reactions.
Ylide (3-10 mg), and TEMPO (1 mg) was dissolved in DMF (0.1 mL) and 18F-solution (0.1-
0.2 mL) was added. The reaction was heated at 120C for 2-20 min. The mixture was cooled
to room temperature, and 10-90% TFA/H2O was added and left for 5 min. Preparative HPLC
was performed, using a Phenomenex Luna C18(2), 5 µm, 10 x 250 mm column, with a
solvent system of 20% MeCN/H2O, 0.1% TFA for 18F[19], 30% MeCN/H2O, 0.1% TFA for
18F[17] and 50% MeCN/H2O, 0.1% TFA for 18F[16]. The collected fractions were diluted
with H2O (20 mL) and passed through an Oasis HLB cartridge. The 18F-aldehyde was eluted
with MeOH or MeCN.
Reaction with O-Benxylhydrozylamine
[18F]F-aldehyde in MeOH or MeCN (33-150 µL) was diluted with MeCN to 150 µL and
added to 50 µL of 80, 200, 400 or 2000 µM O-Bn-ONH2 (giving a final concentration of 20,
50, 100 and 500 µM) in HEPES buffer, pH 7.5. 1 % aniline (2 µL) was added, the reaction
was shaken and left for 30 min. Analytical HPLC method 1 was used for evaluation.
Reaction with FVIIai-ONH2
[18F]F-butyraldehyde ([18F]13) was reacted with FVIIai-ONH2 as described earlier11, with
minor modifications. In brief, 5 µL DMSO was added to [18F]F-butyraldehyde ([18F]13) in
MeCN, and MeCN was evaporated at 80°C under N2 flow. FVIIai- ONH2 (500 μg, 192.5 μL,
52 μM, 2.6 mg/mL, 50 mM Hepes, 200 mM NaCl, 10 mM CaCl2, pH 7.0) and aniline (1%
v/v) was added and the reaction was left for 30 minutes at room temperature, prior to
purification by a PD-10 desalting column. 18F-FVIIai was eluted in gly-gy buffer (10 mM gly
gly, 150 mM NaCl, 10 mM CaCl2, pH 7.5). Analytical HPLC method 2 was used for
evaluation. The protein bound fraction was determined as described earlier.11
Acknowledgements
This project received funding from the European Union’s Horizon 2020 research and
innovation programme under grant agreements no. 670261 (ERC Advanced Grant) and
668532 (Click-It), the Lundbeck Foundation, the Novo Nordisk Foundation, the Innovation
Fund Denmark, the Danish Cancer Society, Arvid Nilsson Foundation, Svend Andersen
Foundation, the Neye Foundation, the Research Foundation of Rigshospitalet, the Danish
National Research Foundation (grant 126), the Research Council of the Capital Region of
Denmark, the Danish Health Authority, the John and Birthe Meyer Foundation and Research
Council for Independent Research.
Supporting Information
Supporting information is available in electronic form from the Wiley Online Library.
This article is protected by copyright. All rights reserved.

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