Evaluation of organo [18F]fluorosilicon tetrazine as a prosthetic group for the synthesis of PET radiotracers

Article abstract of DOI:10.3390/molecules25051208

Fluorine-18 is the most widely used positron emission tomography (PET) radionuclide currently in clinical application, due to its optimal nuclear properties. The synthesis of ¹⁸F-labeled radiotracers often requires harsh reaction conditions, limiting the use of sensitive bio- and macromolecules as precursors for direct radiolabeling with fluorine-18. We aimed to develop a milder and efficient in vitro and in vivo labeling method for trans-cyclooctene (TCO) functionalized proteins, through the bioorthogonal inverse-electron demand Diels-Alder (IEDDA) reaction with fluorine-18 radiolabeled tetrazine ([¹⁸F]SiFA-Tz). Here, we used TCO-modified bovine serum albumin (BSA) as the model protein, and isotopic exchange (IE) (¹⁹F/¹⁸F) chemistry as the labeling strategy. The radiolabeling of albumin-TCO with [¹⁸F]SiFA-Tz ([¹⁸F]6), providing [¹⁸F]fluoroalbumin ([¹⁸F]10) in high radiochemical yield (99.1 ± 0.2%, n = 3) and a molar activity (MA) of 1.1 GBq/μmol, confirmed the applicability of [¹⁸F]6 as a quick in vitro fluorination reagent for the TCO functionalized proteins. While the biological evaluation of [¹⁸F]6 demonstrated defluorination in vivo, limiting the utility for pretargeted applications, the in vivo stability of the radiotracer was dramatically improved when [¹⁸F]6 was used for the radiolabeling of albumin-TCO ([¹⁸F]10) in vitro, prior to administration. Due to the detected defluorination in vivo, structural optimization of the prosthetic group for improved stability is needed before further biological studies and application of pretargeted PET imaging.

Full text of DOI:10.3390/molecules25051208

molecules
Article
Evaluation of Organo [18F]Fluorosilicon Tetrazine as a
Prosthetic Group for the Synthesis of
PET Radiotracers
Sofia Otaru 1, Surachet Imlimthan 1 , Mirkka Sarparanta 1 , Kerttuli Helariutta 1
,
Kristiina Wähälä 1,2 and Anu J. Airaksinen 1,3,
*
1
Department of Chemistry, Radiochemistry, University of Helsinki, 00100 Helsinki, Finland;
sofia.otaru@helsinki.fi (S.O.); surachet.imlimthan@helsinki.fi (S.I.); mirkka.sarparanta@helsinki.fi (M.S.);
Kerttuli.Helariutta@helsinki.fi (K.H.); kristiina.wahala@helsinki.fi (K.W.)
Department of Biochemistry and Developmental Biology, Faculty of Medicine, University of Helsinki,
00100 Helsinki, Finland
Turku PET Centre, Department of Chemistry, University of Turku, 20500 Turku, Finland
Correspondence: anu.airaksinen@helsinki.fi
2
3
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Academic Editors: Anne Roivainen and Xiang-Guo Li
Received: 19 February 2020; Accepted: 5 March 2020; Published: 7 March 2020
Abstract: Fluorine-18 is the most widely used positron emission tomography (PET) radionuclide
currently in clinical application, due to its optimal nuclear properties. The synthesis of 18F-labeled
radiotracers often requires harsh reaction conditions, limiting the use of sensitive bio- and
macromolecules as precursors for direct radiolabeling with fluorine-18. We aimed to develop
a milder and efficient in vitro and in vivo labeling method for trans-cyclooctene (TCO) functionalized
proteins, through the bioorthogonal inverse-electron demand Diels-Alder (IEDDA) reaction with
fluorine-18 radiolabeled tetrazine ([18F]SiFA-Tz). Here, we used TCO-modified bovine serum albumin
(BSA) as the model protein, and isotopic exchange (IE) (19F/18F) chemistry as the labeling strategy.
The radiolabeling of albumin-TCO with [18F]SiFA-Tz ([18F]
in high radiochemical yield (99.1 0.2%, n = 3) and a molar activity (MA) of 1.1 GBq/
the applicability of [18F]
as a quick in vitro fluorination reagent for the TCO functionalized proteins.
While the biological evaluation of [18F]
demonstrated defluorination in vivo, limiting the utility for
pretargeted applications, the in vivo stability of the radiotracer was dramatically improved when
[18F] was used for the radiolabeling of albumin-TCO ([18F]10
in vitro, prior to administration. Due
6
), providing [18F]fluoroalbumin ([18F]10
)
±
µmol, confirmed
6
6
6
)
to the detected defluorination in vivo, structural optimization of the prosthetic group for improved
stability is needed before further biological studies and application of pretargeted PET imaging.
Keywords: fluorine-18; positron emission tomography (PET), defluorination; isotopic exchange;
silicon-based fluoride acceptor; bioorthogonal chemistry; tetrazine; inverse electron-demand
Diels-Alder ligation
1. Introduction
Fluorine-18 is an ideal radionuclide for labeling of radiopharmaceuticals for positron emission
tomography (PET), due to its nuclear and physical characteristics, including the relatively long half-life
(109.7 min), the low energy levels of emitted positrons (Emax = 0.635 MeV), and high positron
decay probability (97%) [1
]. Fluorine-18 can be produced via the 18O(p,n)18F reaction, by irradiating
18O-enriched water with protons, yielding high molar activity [18F]fluoride, in aqueous solutions.
Several advances in 18F-fluorinations, such as metal-mediated (e.g., Pd, Cu, Ag) aromatic, aliphatic,
and aryl boronic ester radiofluorinations, as well as TiO2-catalyzed 18F-fluorinations in aqueous
media, have been recently presented [
2
–
4
]. The incorporation of nucleophilic [18F]fluoride into
Molecules 2020, 25, 1208; doi:10.3390/molecules25051208
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1208
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molecules generally requires alkaline conditions and elevated temperatures. Faster, milder, and more
selective radiolabeling methodologies are desired, especially for the radiolabeling of compounds
sensitive to temperature and higher pH. Fast and efficient catalyst-free click-reactions, such as the
inverse electron-demand Diels-Alder (IEDDA) reaction, have been applied as effective tools for the
selective incorporation of radiolabels, such as 18F, into bio- and macromolecules, via small radiolabeled
prosthetic groups. Other widely known click-reactions, such as the copper(I)-catalyzed azide-alkyne
cycloaddition (CuAAC) and the stainpromoted azide-alkyne cycloaddition (SPAAC) have been the
starting point for the modification of chemoselective biomolecules [5]. The CuAAC reaction was first
reported in 2002 by Sharpless et al. [ ] and Meldal et al. [ ]. The aim of these studies was to utilize the
6
7
CuAAC reaction for the formation of an enormous variety of five-membered heterocycles, triazoles,
and peptide derivatives. The use of CuAAC led to the investigation on the utility of these highly
efficient reactions for the labeling of biomolecules in living systems. However, the toxicity of copper
limited the feasibility of CuAAC in biological applications. SPAAC was developed in 2004 by Bertozzi
et al. who demonstrated the chemical modification of live Jurkat cells with an azide-modified sugar
for the subsequent conjugation of alkyne-biotin for fluorescent labeling with FITC-avidin, without any
apparent decline in cell viability [8]. Since then, SPAAC has served as a catalyst-free alternative to
overcome the cytotoxicity concerns of the CuAAC reaction. However, both CuAAC and SPAAC have
relatively slow reaction kinetics, which renders the reactions unsuitable for labeling of biomolecules
in a living system, for applications lile in vivo pretargeting. In 1959, Lindsey et al. reported the
ability of tetrazines to react chemoselectively with unsaturated compounds through a 1,4-cycloaddition
reaction [9]. These findings introduced a new and highly reactive bioorthogonal IEDDA click-reaction
as a pivotal tool for synthetic modification of biomolecules.
The bioorthogonal IEDDA-reaction has been successfully used for various pretargeted in vivo
radiolabeling applications [10–12]. Pretargeted imaging has found exceptional utility with imaging
agents with slow pharmacokinetics, such as antibodies and nanomaterials, which when directly
radiolabeled would require the use of long-lived radioisotopes (such as 89Zr or 111In, both with ~3-day
physical half-lives), to track their biodistribution in vivo. In the pretargeted approach, the targeting
vector (such as an IgG antibody) is first modified by one reactant of the IEDDA reaction, and allowed
to distribute in the body after administration. Next, it is tracked using the other reactant that is
radiolabeled with a short-lived radioisotope, with improved image contrast and lower radiation burden
to the subject [13
tetrazine (Tz) and trans-cyclooctene (TCO) (k
,
14]. The fastest IEDDA-reaction reported so far is from the conjugation between
106 M−1 −1) [
], rendering this reactive pair of utmost
≈
s
5
interest in the fields of chemical biology, nuclear imaging, and radiotracer development. However,
the sensitivity of tetrazines towards alkaline conditions and elevated temperature renders the direct
radiolabeling of tetrazines with fluorine-18 challenging. Therefore, the use of prosthetic groups for
radiolabeling, such as the glycoconjugate [18F]-5-fluoro-5-deoxyribose (FDR) or Al[18F]F [15
necessary to ensure that radiolabeling conditions preserve the reactivity of the Tz.
–17], is
The silicon-fluoride acceptor (SiFA) chemistry relies on 19F/18F-isotopic exchange for introducing
fluorine-18 into radiotracers, and has emerged as a fast and mild radiolabeling tool, especially for
sensitive molecules [18]. The small lipophilic SiFA compounds mainly utilized in the radiolabeling
of larger constructs such as peptides, proteins, and nanoparticles, in most cases have demonstrated
excellent stability against in vivo defluorination [19–23]. The lipophilic character of the SiFA-derivatives
can be used to tailor the pharmacokinetics of biomolecular tracers. To our knowledge, there is only
one compound containing SiFA bound to a tetrazine, SiFA-OTz, which has been reported to date,
but its enzymatic stability in vitro and in vivo has not yet been studied [24]. Nevertheless, the tracer
SiFA-OTz demonstrated good stability under the radiolabeling conditions. We have reported the
development of highly stable and highly hydrophilic 18F-tetrazines from sugar analogues, such as
[18F]fluorodeoxyribose ([18F]FDR-Tz) and 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG-Tz) [17
,25]. We
also previously studied the feasibility of utilizing the 18F-FDR-Tz for the in vivo IEDDA pretargeting
of antibodies and nanoparticles and were able to prove this approach to be highly successful [26,27].

Molecules 2020, 25, 1208
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Here, we investigated the use of a SiFA as a reaction strategy for the [18F]fluorination of tetrazines,
under mild reaction conditions, with the possibility to yield a more hydrophobic tetrazine variant for
the regulation of the pharmacokinetics of biomolecules labeled with [18F]SiFA-Tz. The aim of this
study was to investigate [18F]SiFA-Tz as a standalone tracer, to reveal its potential for pretargeted
imaging and its applicability for the rapid in vitro radiolabeling of TCO-containing biomolecules,
under physiological conditions.
2. Results
2.1. Chemistry
SiFA-Tz (
(Scheme 1) [14]. The first step comprised of an amide coupling reaction under argon, between
a carboxylic acid and an amine, forming the t-Boc protected aminooxy-tetrazine , in 65% yield after
silica gel column chromatography purification. The next reaction step was the deprotection of
with hydrochloric acid in methanol, to give as a pink solid in cold diethyl ether in 65% yield. This
hydrochloric salt intermediate was used such for the next reaction, the oxime bond formation between
compounds and generating an imine double bond such as E- and Z-isomers of SiFA-Tz ( ) [28].
in
6) was synthesized via a three-step route, providing good yields for each step
3
3
4
4
5
6
The reaction mixture was purified with semi-preparative HPLC, providing the desired product
6
1
90% yield. H-NMR, 13C-NMR, 19F-NMR spectra and ESI-TOF-MS were acquired for characterization
of the final product, SiFA-Tz (6).
Scheme 1. Synthesis of the precursor SiFA-Tz (
DIPEA, DMF, room temperature, 20 h, Argon (65%),
MeOH (65%), and (
) Aniliniumacetate-buffer pH 4.6, 25 ◦C, 15 min (90%).
6
). Reagents and conditions:
) 1 M HCl Et2O 25 ◦C, 24 h,
(HATU;
(a) HATU,
(b
c
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate,
DIPEA; N,N-Diisopropylethylamine, DMF; Dimethylformamide).
Bovine serum albumin (7) was functionalized with N-hydroxysuccinimide (NHS) ester of
transcyclooctene (TCO) (200 eq), from the lysine residues available in the protein structure (Scheme 2).
The analysis of the TCO:albumin ratio was carried out with MALDI-MS, which revealed a TCO:albumin
ratio 40:1. The MALDI-MS analysis indicates that 65% of the total 62 lysine residues were functionalized
with a TCO in one albumin molecule. The IEDDA cycloaddition product of SiFA-Tz (
albumin-TCO ( ), fluoroalbumin (10), was synthesized as a reference for the radiolabeling studies by
mixing the two reagents together at room temperature.
6) and
9

Molecules 2020, 25, 1208
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Scheme 2. Functionalization of bovine serum albumin (7) with trans-cyclooctene (8) to form
albumin- trans-cyclooctene (TCO) (9), followed by the inverse-electron demand Diels-Alder (IEDDA)
cycloaddition with SiFA-Tz (6) to form fluoroalbumin (10) (molar ratio 1:1.5 SiFA-Tz:fluoroalbumin).
2.2. Radiochemistry
Two different synthetic sequences were investigated for the radiosynthesis of [18F]
direct radiolabeling of compound and a two–step method where we first radiolabeled the SiFA
moiety , before linking it to the tetrazine (Scheme 3). The one-step radiolabeling of , resulted in
21% radiochemical yield (n = 1), but the yield was detected to decrease rapidly as a function of time,
indicating decomposition of the precursor in the reaction mixture, under the alkaline conditions (pH
8.5–9). In the two-step method, the radiolabeling of the SiFA-moiety resulted in incorporation yields
ranging from 89 to 99.6 0.5% (n = 4), at the optimal time point (2 min) analyzed by radio-TLC. The
6
—a one–step
6
5
4
6
6
5
±
radiochemical impurities (maximum 11% of total radioactivity) formed in the first step were analyzed
by radio-HPLC and are shown in the Supplementary Data (Figure S6). The SiFA radiolabeling was
followed by an oxime bond formation between the [18F]SiFA ([18F]
5
) and tetrazine oxyamine
4
at 42.7
revealed
being predominant with the amount of
0.35% (n = 16). The final product [18F]
6 was isolated at
±
14.2% (n = 8) yield in the reaction mixture (Figure S7). Radio-TLC analysis of product [18F]
6
the formation of two isomers, with the (E)-isomer of [18F]
6
the radiolabeled
6
(Z)-isomer only 1.45
±
>98% radiochemical purity (Figure S9, radio-TLC) and was subsequently used as a prosthetic group to
chemoselectively radiolabel the TCO-functionalized albumin in vitro. The [18F]fluorinated albumin
[18F]10 was radiolabeled at 99.1
and good molar activity (1.1
of added [18F]
(0.26 nmol), minimum of 0.27 nmol of albumin-TCO was to be used. This resulted
in 2.5% of the TCOs in the albumin-TCO labeled with [18F]
. A radiochemical yield of >99% of
(0.27 nmol)
and [18F]10 are presented in
±
0.2% radiochemical yield (RCY) (n = 3) (Figure S12, radio-TLC)
±
0.2 GBq/ mol, n = 2). To achieve 99% RCY and the total consumption
µ
6
6
[18F]10 was achieved by incubating increasing amounts of albumin–TCO with [18F]
6
(Figure 1). The radio-HPLC chromatograms of the purified tracers [18F]
the Supplementary Data (Figures S8 and S12).
6
2.3. In Vitro Studies of [18F]6 and [18F]10.
The stability of [18F]
detachment of fluorine-18 (<1%) during the incubation (Figure S10). Plasma protein binding and
metabolic stability of [18F]
were also evaluated by incubating the radiotracer in plasma and analyzing
6 in 0.01M PBS pH 7.4 was shown to be excellent at 90 min, with minimal
6
the deproteinized samples through radio-TLC and radio-HPLC methods, at selected time-points after
incubation (Figure 2).

Molecules 2020, 25, 1208
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Scheme 3. Radiosynthesis of [18F]SiFA-Tz ([18F]
ACN, K[1 8F]F-[K2.2.2], (RCY 99%) and (b) anilinium acetate buffer pH 4.6, 25 ◦C, 15 min (RCY 43%).
6) by a two-step method. Reagents and conditions: (a)
Figure 1. Radiochemical yield (RCY) of [18F]fluoroalbumin ([18F]10) during titration of [18F]SiFA-Tz
([18F]
), with albumin-TCO revealed a maximal radiochemical yield of >99% with 0.26 nmol
6
of albumin-TCO.
Through radio-TLC analysis, [18F]
detachment of the radiolabel over 180 min (94.9
6
demonstrated good stability in plasma with up to 6%
1.6 % intact tracer, n = 2). The slight difference in the
±
stability profiles between radio-TLC and radio-HPLC analysis can be explained due to their different
ability to quantify free fluoride from the samples. The retention of free fluoride on the silica-based
C18 HPLC-column material makes the quantification of free fluoride with HPLC less accurate.
Representative radio-TLC and radio-HPLC chromatograms from the experiment are shown in the
Supplementary Data (Figures S15 and S16). A radio-HPLC analysis revealed no other radiometabolites
of [18F]
6 during the 180 min incubation (Figure 3), in addition to a highly polar component, most likely

Molecules 2020, 25, 1208
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the free fluoride. Despite the observed minor defluorination, the enzymatic stability was found to be
sufficient for proceeding into in vivo evaluation.
Figure 2. Free fraction of the radiotracer [18F]SiFA-Tz ([18F]
6) during 240 min incubation in human and
mouse plasma demonstrated comparable profiles with ~50% of the radioactivity in the free fraction at
the end of incubation.
Figure 3. In vitro stability of [18F]SiFA-Tz ([18F] ) after incubation in human plasma at 37 ◦C,
6
demonstrated excellent stability with ~95% intact tracer at 180 min after start of incubation. The amount
of parent [18F]6 was measured through radio-TLC and radio-HPLC analysis.

Molecules 2020, 25, 1208
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The LogDpH7.4 of [18F]
6
(1.6
±
0.2, n = 9) was determined by the shake-flask method, as previously
reported [16]. It has been shown that lipophilic compounds tend to exhibit a higher plasma-protein
binding than the more hydrophilic compounds [29], which support the findings of the measured
LogD-value and the value obtained for the plasma protein-bound [18F]6, in this study.
2.4. Biodistribution of [18F]6 and [18F]10
The biodistribution of [18F]
After intravenous administration into the lateral tail vein, [18F]
10% EtOH and 0.5% Solutol HS 15 in 0.01 M PBS pH 7.4) exhibited hepatobiliary excretion and a fast
clearance from the blood (Figure 4, Figure S13). The highest percentage of the injected dose per gram
6
was investigated in healthy 11 to 12 week-old female CD-1 mice.
(14.4 0.5 MBq/animal in ~200 L of
6
±
µ
of tissue (%ID/g) for urine (229.5
post-injection. In addition to the high radioactivity detected in the urine, gallbladder, liver, and the
feces, a high bone uptake of 18F− was observed 60 min post-injection (13.4
1.6% ID/g). No major
passage of [18F]
through the blood–brain barrier was observed (0.7 0.2% ID/g) at 5 min post-injection.
±
204.5) and gallbladder (143.9
±
103.1) was found to be at 60 min
±
6
±
In order to study the metabolism of [18F]6, the blood samples were collected at (t = 5, 30, and 60 min)
post-injection. The deproteinized plasma samples were analyzed through radio-HPLC and radio-TLC
methods, which revealed the formation of a highly polar metabolite (Rf 0.00 on TLC) that was retained
at the origin. On HPLC, a metabolite ([18F]M1) eluting at 7 min was also detected. Furthermore, there
was no indication of more lipophilic metabolites (Figure S17).
Figure 4. Biodistribution of radioactivity after the intravenous injection of [18F]SiFA-Tz ([18F]
6, n =
3), demonstrating a high bone uptake of radioactivity, 60 min post-injection. (S.M.—skeletal muscle;
bone—tibia, S.I.—small intestine; L.I.—large intestine).
In order to investigate how the conjugation of [18F]
defluorination rate, we used [18F] to synthesize [18F]fluoroalbumin ([18F]10) and evaluated its
biodistribution in CD-1 mice. Intravenously injected [18F]10 (0.6
0.1 MBq/animal in ~100
of 0.01 M PBS, pH 7.4) had a prolonged residence time in blood with 7.1 0.3% ID/g, at 60-min
6 onto a macromolecule influences its in vivo
6
±
µL
±

Molecules 2020, 25, 1208
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post-injection and a plasma half-life of 49 min (Figure 5). The highest % ID/g for urine (54.7
±
25.8)
and gallbladder (275.3
±
185.6), after intravenous injection of [18F]10 was shown to be at 60 min
post-injection, for both tissues.
Figure 5. Biodistribution of radioactivity after the intravenous administration of [18F]fluoroalbumin.
([18F]10, n = 5) demonstrating a prolonged residence time of radioactivity in whole blood and reduced
bone accumulation, indicating resistance to defluorination in vivo. (S.M.—skeletal muscle; bone—tibia;
S.I.—small intestine; L.I.—large intestine).
Stability of [18F]10 was further investigated by separating molecules with molecular weight of
30
≥
kDa from plasma samples collected after intravenous administration of [18F]10 through ultrafiltration.
The proportion of radiolabeled molecules with a MW of
30 kDa (presenting intact [18F]10) was
≥
over 90% until 180 min post-injection, after which it decreased to 40% over the next 60 min (240 min
incubation in total), as shown in Figure 6.
By comparing the bone uptake of 18F in these two biodistribution studies, it was seen that the
18F-Si bond in [18F]10 was noticeably more stable than in the [18F]SiFA-Tz ([18F]
6) alone (Figure 7).
The bone uptake for both tracers peaked around 120 min after administration and the radioactivity
persisted in the bone until the last time point of the biodistribution study, 240 min post-injection. This
was an indication of the uptake of free [18F]fluoride, which was a result of defluorination of the SiFA
moiety, in vivo [30].

Molecules 2020, 25, 1208
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Figure 6. The ex vivo distribution (%) of
30 kDa 18F-radiolabeled molecules in mouse plasma after
≥
intravenous injection of [18F]fluoroalbumin ([18F]10), separated with a molecular weight cut-off filter
revealed a highly stable albumin tracer until 2 h post-injection. At 240 min post-injection around 40%
of radioactivity was of
lower molecular weight.
≥
30 kDa size, with almost 60% of the radioactivity being comprised of species of
✱✱
✱✱✱
Figure 7. Comparison of the radioactivity accumulated in bone (tibia) in the biodistribution studies of
[
18F]SiFA-Tz ([18F] ) and [18F]fluoroalbumin ([18F]10). A significant enhancement in stability was seen
6
for the albumin-bound [18F]SiFA group in [18F]10 (** p ≤ 0.01, *** p ≤ 0.001), compared to [18F]6.
Furthermore, the rate of defluorination was substantially diminished when [18F]
to chemoselectively radiolabel albumin in vitro through the IEDDA ligation. The plasma protein
radiotracer [18F]10, which acted as a model protein in this study, demonstrated a significantly (p
6 was used
≤

Molecules 2020, 25, 1208
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0.0001) longer blood circulation time in healthy CD-1 mice than the 18F-labeled tetrazine [18F]
6 alone
(Figure 8), with a biological half-life similar to what has been reported for other [18F]SiFA-radiolabeled
serum albumins.
✱✱✱✱
✱✱✱✱
Figure 8. Radioactivity (%ID/g) in ex vivo blood samples at selected time–points, post-injection
for [18F]SiFA-Tz ([18F] ) and [18F]fluoroalbumin ([18F]10), demonstrated a significantly prolonged
6
circulation time for the plasma protein tracer ([18F]10) (**** p ≤ 0.0001).
3. Discussion
Two PET-tracer candidates, [18F]SiFA-Tz ([18F]
and evaluated in vivo. Compound [18F]
was synthesized with two different synthesis methods,
6
) and [18F]fluoroalbumin ([18F]10) were synthesized
6
from which the two-step approach was selected, due its higher RCY and good reproducibility. In
the one-step radiolabeling approach, the RCY of the product was observed to decrease rapidly as a
function of time, indicating decomposition of the precursor in the alkaline reaction mixture. In the
two-step method, aminooxy tetrazine
4 was introduced into the reaction mixture at pH 4.6, which was
found to be an advantage to avoid any unnecessary decomposition of the tetrazine group. In addition
to the two-step method presented in this study, alterative elution protocols with milder reagents,
such as copper salts or weak base solutions, as described by Scott et al., should be investigated for
the radiolabeling of base sensitive precursors [31]. The in vivo metabolic profile of [18F]
6
displayed
hepatobiliary elimination, which is characteristic for compounds with low hydrophilicity. Nevertheless,
no observable passage of [18F]
through the blood–brain barrier was detected (0.7 0.2% ID/g) at 5
min post-injection, despite the favorable lipophilicity of the tracer (LogD = 1.56 0.20). Based on
the radio-HPLC metabolite analysis (Supplement. Figure S17) from ex vivo blood samples and the
detection of radioactivity in the bone, we concluded that [18F]
underwent rapid biotransformation,
6
±
±
6
generating highly polar metabolites, one of which was most likely free [18F]fluoride detached from the
radiotracer. Furthermore, the accumulation of radioactivity in bone is a characteristic indication of fast
defluorination in vivo. After defluorination, the free fluoride is sequestered rapidly from circulation
and either binds into the surface of the bone or accumulates irreversibly into the hydroxyapatite
Ca10(PO4)6(OH)2, forming fluorapatite (Ca10(PO4)6F2) [30]. Thus, it was evident that unexpected

Molecules 2020, 25, 1208
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and relatively fast defluorination was observed in vivo. Free fluoride was also excreted into the
urine, in vivo. Defluorination of 18F-radiolabeled tracer [18F]
could be detected in the bone as early
as 10–20 min after injection [30]. The observed rapid defluorination in vivo limited the utility of
[18F]
for pretargeted PET imaging, and further structural optimization was warranted to stabilize
the structure towards the defluorination. However, since the stability of biomacromolecular SiFA
conjugates has been reported to be good, the possibility of using [18F]
as a prosthetic group for the
in vitro bioorthogonal radiolabeling of proteins was investigated through administration of [18F]10
to healthy mice. Stability of the [18F]SiFA-Tz group against in vivo defluorination was dramatically
6
6
6
,
improved when the group was bound to albumin (13.4
±
1.6% ID/g in bone for [18F]
6
vs. 3.4
±
1.5%
ID/g in bone for [18F]10, at 60 min post-injection). Furthermore, the blood circulation half-life was 48
min, which is in the order of the reported plasma half-life of 60 min, for the bovine serum albumin in
mice [32,33].
There are some examples of small molecular SiFA derivatives and a SiFA-conjugated peptide that
have exhibited detectable in vivo defluorination, but not at the level observed in our study [34,35].
Rat serum albumin (RSA) radiolabeled with [18F]SiFA, through isothiocyanate modification of
lysine residues has been shown to be relatively stable with only a low rate of defluorination,
until 90 min after administration [36]. It has also been shown that the conjugation position of
the [18F]SiFA-moiety on the albumin could have an influence on the rate of defluorination in vivo. A
more stable maleimido-[18F]SiFA conjugated to RSA via thiol groups is an example of the enhanced
stability of the radiolabel in a [18F]SiFA-radiolabeled serum albumin [37]. Thus, this radiolabeling
system could be further improved by using a more selective conjugation chemistry (maleimide over
N-hydroxysuccinimide) for the addition of the TCO to albumin, while simultaneously optimizing the
TCO:albumin ratio and availability of the TCO moiety to the IEDDA reaction, with [18F]
6. Nevertheless,
our results demonstrated the feasibility of using the highly selective and rapid bioorthogonal reaction
strategy for the radiolabeling of biomacromolecules with fluorine-18, under mild reaction conditions.
4. Materials and Methods
All reagents and solvents were purchased from commercial providers and used as received
without further purification. Hyox-18 18O–enriched water (98%) was purchased from Rotem Industries
Limited (Arava, Israel). Ultrapure water (18.0 MΩ) was produced with a Milli-Q Integral Water
Purification System (Merck Millipore, Burlington, MA, USA). HATU, DMF, DIPEA, DMSO, LiCl,
methanol, aniline, Kryptofix 2.2.2, 1 M HCl in diethylether, formic acid, ethylacetate and boc-aminooxy
acetic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). MgSO4 was purchased from
Merck Millipore (Darmstadt, Germany). TCO-PEG4-NHS ester was purchased from either Jena
Biosciences (Jena, Germany) or Conju-Probe (San Diego, CA, USA). Tetrazine amine was purchased
from either BroadPharm (San Diego, CA, USA) or Conju-Probe (San Diego, CA, USA). DNA synthesis
quality anhydrous acetonitrile (max. 10 ppm water) was purchased from Merck (Kenilworth, NJ,
USA). SiFA-aldehyde was purchased from Enamine (Monmouth, NJ, USA). Bovine serum albumin
was purchased from Merck (Kenilworth, NJ, USA). Moisture or air sensitive reactions were carried out
under an argon atmosphere in oven-dried glassware. Reactions were monitored by TLC Silica gel 60
F254 Merck Millipore (Darmstadt, Germany). Silica gel TLC-plates were run in EtOAc:heptane (7:3) as
eluent. [18F]SiFA-Tz ([18F]6) Rf = 0.59, [18F]fluoroalbumin ([18F]10) Rf = 0.00.
1H-, 13C-, and 19F-NMR spectra were acquired with a Varian Mercury spectrometer (300 MHz,
500 MHz, 600 MHz) (Palo Alto, CA, USA). Chemical shifts (δ) are reported in ppm units, using the
solvent residual signal as a reference. Coupling constants (J) are expressed in hertz (Hz). The purities
of radiolabeled compounds were determined through RP-HPLC with photodiode array (PDA)-, and
radiodetector and through silica TLCs analyzed with a Fujifilm FLA 5100 scanner (Fujifilm Life sciences,
Cambridge, MA, USA). The excised tissue samples were measured with 1480 Wallac Wizard® 3”
(PerkinElmerTM Life Sciences, Waltham, MA, USA) gamma counter for 60 s per sample.

Molecules 2020, 25, 1208
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High performance liquid chromatography was carried out with a Shimadzu HPLC system
consisting of a DGU-20A degasser, an LC-20AD UPLC LC unit, a SIL-20A HT autosampler, a CTO20
AC column oven, a CBM-20A communications bus module, a Scionix Holland scintillation detector
with a 51 BP 51/2 NaI(Tl) crystal and an SPD-M20A diode array detector. For the [18F]SiFATz, a Waters
Symmetry semi-preparative C18 column (300
×
7.8 mm, 7 µm) was used, with 0.01 M H3PO4:ACN
(20:80, 3 mL/min) as the eluent. Phenomenex BioSep SEC s3000 size exclusion column was used, with
0.1 M phosphate buffer pH 7 (0.8 mL/min) as the eluent, to analyze the conjugated albumin-TCO and
radiolabeled protein tracer [18F]fluoroalbumin ([18F]10).
Preparative high performance liquid chromatography was carried out using a system consisting
of a Phenomenex DegassexTM DG-4400 degasser, Merck LaChrom L-7100 pump, in-house prepared
remote-controlled injection system, Amersham pharmacia biotech REC 112 dual channel chart recorder,
Carroll & Ramsey Associates 101-H-DC3 multi-channel radiation detector, and a Knauer Azura UVD
2.1S detector. A waters Symmetry semi-preparative C18 column (300
H3PO4:ACN (20:80, 3 mL/min flowrate) as the eluent was used for the preparative purification of the
[18F]SiFA-Tz radiotracer ([18F]6).
×
7.8 mm, 7 µm), with 0.01 M
4.1. Synthesis
Tert-butyl(2-{[4-(1,2,4,5-tetrazin-3-yl)ben-zyl]amino}-2-oxoethoxy)carbamate (
mol) in DMF (0.5 mL) was added to a solution of (32 mg, 168 mol) in dry DMF (0.7 mL), under
argon. The mixture was stirred at room temperature for 10 min, then (25 mg, 112 mol) in DMF (2.5
mL) was added, followed by DIPEA (49 L, 281 mol). After 20 h mixing at room temperature, ethyl
acetate (15 mL) was added and the organic phase was extracted with ultrapure water (10 mL). The
organic phase was extracted with 5% LiCl solution (3 10 mL, 1 20 mL), dried over MgSO4, and
3). HATU (77 mg, 201
µ
1
µ
2
µ
µ
µ
×
×
concentrated in vacuo. The crude product was purified through silica gel column chromatography
(EtOAc:heptane) to afford 3 as a pink solid (26 mg, 72 µmol, 65% yield).
1H-NMR (300 MHz, CDCl3, Supplement Figure S1)
δ 10.20 (d, J = 0.6 Hz, 1H), 8.79 (s, 1H), 8.61–8.49
(m, 2H), 7.72 (s, 1H), 7.60–7.51 (m, 2H), 4.63 (d, J = 6.1 Hz, 2H), 4.40 (s, 2H), 1.42 (d, J = 0.6 Hz, 9H).
13C-NMR (75 MHz, CDCl3, Supplement Figure S2) 169.29, 166.53, 158.19, 157.98, 144.09, 130.73, 128.00,
83.63, 68.00, 42.83, 28.27 ppm.
N-[4-(1,2,4,5-tetrazin-3-yl)benzyl]-2-(aminooxy)acetamide hydrochloride (
4
). 1 M HCl in diethyl ether (50
mol) in MeOH (4 mL). After 24 h, the solution was
4 as a pink solid. The solid was dissolved in 2 mL of methanol and
mL) was added to a solution of
3 (22 mg, 61 µ
concentrated in vacuo, to afford
20 mL of cold diethyl ether was added to the solution. The closed flask was kept in +4 ◦C overnight,
to facilitate the crystallization of the purified product. The recrystallized solid (10 mg, 39
yield) was suction filtered and used as such in the next reaction step.
µmol, 65%
(E,Z)-N-[4-(1,2,4,5-tetrazin-3-yl)benzyl]-2-({[4-(di-tert-butylfluorosilyl)benzylidene]-amino}oxy)acetamide (
6).
Compound (8.5 mg, 32 mol) in ACN (0.5 mL) was added into a solution of (4 mg, 15 mol) in 0.3
5
µ
4
µ
M anilinium acetate buffer pH 4.6 (2 mL). After 15 min, ultrapure water (10 mL) was added to the
reaction mixture, concentrated with two Sep-pak C18 Light cartridges (Milford, MA, USA) and eluted
with ACN (5 mL). The crude product was purified through RP-HPLC (0.01 M H3PO4:CAN, 20:80, 3
mL/min) to afford 6 as a pink solid (7 mg, 14 µmol, 90% yield).
1H-NMR (300 MHz, CDCl3, Supplement Figure S3)
7.69–7.49 (m, 6H), 4.76 (s, 2H), 4.68 (d, J = 6.1 Hz, 2H), 1.05 (d, J = 1.1 Hz, 18H). 19F-NMR (282 MHz,
CDCl3 Supplement Figure S4) 170.10,
-189.13. 13C-NMR (126 MHz, CDCl3, Supplement Figure S5)
δ 10.21 (s, 1H), 8.57 (d, J = 8.5 Hz, 2H), 8.21 (s, 1H),
δ
δ
166.58, 158.15, 151.49, 143.92, 134.80, 134.77, 132.32, 131.18, 129.02, 128.65, 126.67, 77.61, 77.56, 77.36,
77.11, 73.68, 42.93, 27.61, 20.64, 20.54. ESI-TOF MS: Calculated for C26H34FN6O2Si [M + H]+ 509.24911
m/z, found 509.2147 m/z. Calculated [M + Na]+ 531.22378 m/z, found 531.1967 m/z. Calculated [M +
K]+ 547.19771 m/z, found 547.1706 m/z.

Molecules 2020, 25, 1208
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Albumin trans-cyclooctene (albumin-TCO (
9)). TCO-PEG4-NHS ester (5 mg, 9.7
µmol) in DMSO
: 0.5 M borate buffer (1:1, 1 mL) pH 9 was added to a solution of bovine serum albumin
7 (3.5 mg,
53 nmol) in borate buffer pH 9.0 (1 mL). After 1 h, the reaction mixture was purified with a PD-10
size-exclusion column (GE Healthcare, Chicago, IL, USA), using ultrapure water as the eluent. The
collected fractions were analyzed by a SEC-column HPLC, using 0.1 M PBS as an eluent, with a flow
rate of 0.8 mL/min, for identifying the fractions containing albumin-TCO. The fractions containing
albumin-TCO were lyophilized to afford
9 as white solid (n = 3). Albumin-TCO (1 mg) was dissolved
in 1 mL of ultrapure water and analyzed with MALDI-TOF-MS (calculated for bovine serum albumin
66,338 Da, measured for albumin-TCO 82039-82265 Da).
4.2. Radiochemistry
No-carrier-added 18F-Fluoride was produced in-house with Cyclone 10/5 cyclotron (IBA,
Louvain-la-Neuve, Belgium) through a 18O(p,n)18F nuclear reaction, by bombarding H218O with 10
MeV protons. The radiosynthesis was carried out in a semiautomatic synthesis unit (DM Automation),
with an integrated preparative HPLC system for the purification of the radiotracer. The nucleophilic
18F– was trapped on a Waters QMA Light ion-exchange cartridge, followed by elution with a basic
K[18F]FK2.2.2-complex solution. Water residue was evaporated azeotropically by adding anhydrous
ACN, followed by heating, under a 40-mL/min argon flow.
4.2.1. Radiosynthesis of [18F]6
In the one-step method, precursor 6, dissolved in 500 µL of anhydrous acetonitrile, was added
into the dried K[18F]F/K2.2.2 and incubated for 2 min (25 ◦C). The reaction mixture was diluted with
an additional 500 µL of anhydrous acetonitrile for the radio-TLC and radio-HPLC analysis.
In the two-step method, after evaporation of the solvent, SiFA (
added into the reaction vial containing K[18F]F/K2.2.2 and incubated at room temperature for 2 min.
Tetrazine oxyamine ( ) in 0.3 M anilinium acetate buffer pH 4.6 (200 L) and ACN (50 L) was added
into the reaction mixture, and the reaction was further incubated at room temperature, for 15 min (RCY
74%, radio-TLC), and purified by preparative HPLC, providing 385 MBq of [18F]
at 115 min from
end-of-bombardment (EOB, 14.8 1.6% DCY) and 5 GBq/ mol at end-of-bombardment (EOS). The
purified (RCP 98.8 was formulated (10% ethanol, 0.5% Solutol HS 15 in
1.3%, n = 16) product [18F]
5) in anhydrous ACN (0.5 mL) was
3
µ
µ
6
±
µ
±
6
0.01 M PBS), sterile filtered (0.22 µm), and used as such for injections.
4.2.2. Radiosynthesis of [18F]10
The formulated [18F]
6 was added onto the lyophilized albumin-TCO (350 µg) and incubated at
room temperature for 15 min. The produced [18F]fluoroalbumin ([18F]10) was purified by centrifugation
(10,000 g, Eppendorf Centrifuge 5430, Ag, Hamburg, Germany) with molecular weight cut-off (MWCO)
filters (30K, VWR®, Radnor, PA, USA), with 0.01 M PBS as the eluent, sterile-filtered and used as such
for injections. The apparent molar activity was 1.1 GBq/µmol of protein at EOS. The radio-HPLC
chromatogram of the purified [18F]10 is presented in the Supplementary Data (Figure S11).
4.3. In Vitro Experiments
4.3.1. LogDpH7.4 Determination for [18F]6
A total of 25
microtube. The mixture was shaken mechanically (500 rpm) for 10 min and centrifuged (1000 g, 5 min),
and the layers were separated. A sample of 500 L of each layer were pipetted into pre-weighed
µ 6 was added to a mixture of 1-octanol and 0.02 M PBS (pH 7.4) in a 1.5 mL
L of [18F]
µ

Molecules 2020, 25, 1208
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polypropylene tubes and the activity in the samples was measured with a Wizard gamma counter. The
distribution of [18F]6 between octanol and PBS was calculated, according to
AcOCT
LogD7.4 = Log
AcPBS
where Acoct = activity concentration of octanol and AcPBS = activity concentration of PBS. LogDpH7.4
= 1.56 ± 0.20 (n = 5).
4.3.2. In Vitro Stability and Plasma Protein Binding for [18F]6
A total of 40
90 min, with mixing (400 rpm). At selected time-points (t = 5, 30, 60 and 90 min, n = 1), the samples
were injected into an HPLC with PDA- and radiodetector, for analysis. Radiotracer [18F]
(40 L) was
µ 6 was incubated in 0.01 M PBS at room temperature, in a microtube for
L of [18F]
6
µ
incubated in 50% human plasma (anonymous donor FFP-24 plasma provided by the Finnish Red Cross
Blood Service, Helsinki, Finland) in PBS at 37 ◦C, and in mouse plasma (separated from CD-1 mouse
blood). At selected time-points (5, 60, 120, and 240 min, n = 2 for each), 100
µL of the samples were
taken, diluted with 200 L of cold acetonitrile, and centrifuged at 10,000 g for 5 min. The radioactivity
µ
in the precipitated pellet (protein-bound fraction) and supernatant (free fraction) were measured with
a gamma counter and 100 µL of the samples were injected into HPLC, for radio-HPLC analysis.
4.4. Biological Studies
All animal experiments were conducted under a project license approved by the National Board
of Animal Experimentation in Finland (license number ESAVI/12132/04.10.07/2017). The animals were
group-housed in standard polycarbonate cages, on aspen bedding, in a HEPA-filtered housing unit
(UniProtect, Ehret, Emmendingen, Germany), with food (Envigo Teklad Global Diet 2016) and tap
water, available ad libitum. Conditions were maintained at 21
with a 12:12 lighting cycle. The biodistribution studies were conducted in healthy, female CD-1 mice
(weight 25–33 g, 11 to 12 weeks, Charles River). The radiotracers [18F] and [18F]10 were injected
±
1 ◦C and 55
±
15% relative humidity,
6
via the tail vein to CD-1 mice, in the following formulations—10% EtOH and 0.5% Solutol HS 15 in
0.01 M PBS pH 7.4 for [18F]6 and 0.01 M PBS pH 7.4 for [18F]10. At selected time-points (5, 60, 120,
and 240 min), the mice were euthanized with CO2 asphyxiation, followed by cervical dislocation,
and selected tissues were collected, weighed, and the radioactivity was measured on an automated
gamma counter.
4.4.1. Biodistribution of [18F]6
14.3
±
0.5 MBq (n = 12) (25.4
±
1.4
µ
g, 44.2
±
2.4 nmol) of 96.2% pure [18F]6 was injected into
the tail vein of healthy female CD-1 mice (n = 3 per time-point), to evaluate the biodistribution and
stability of the tracer in vivo. The mice were euthanized at selected time-points (t = 5, 60, 120, and 240
min) and the tissues were collected and measured with a gamma counter, as described above. For
metabolite studies, blood from a cardiac puncture was collected into an Eppendorf tube containing
2
µL of 1% heparin solution in 0.9% NaCl (aq.) and centrifuged at 1000 g, for 10 min, to separate the
plasma from the blood cells. Cold acetonitrile (twice the volume of separated plasma) was added into
the plasma and centrifuged (at 10 000 g for 5 min) to precipitate the proteins. A small sample (4
was applied onto a silica TLC and analyzed with digital autoradiography.
µL)
4.4.2. Biodistribution of [18F]10
A total of 0.6
±
0.1 MBq (n = 15) (43.2
±
1.4 µ
g of protein) of [18F]fluoroalbumin was injected into the
tail vein of female CD-1 mice (n = 3–4 per time point). The mice were euthanized at selected time-points
and the tissues were collected and measured with a gamma counter, as described above. Blood from a
cardiac puncture was collected into an Eppendorf tube containing 2 µL of 1% heparin solution in 0.9%

Molecules 2020, 25, 1208
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NaCl (aq) and was pretreated before analysis, as described above. After centrifugation, a small sample
(4 L) was applied onto a silica TLC plate and 100 L of the supernatant was injected into HPLC for
µ
µ
radiometabolite analysis. For the radiometabolite studies of [18F]10, the blood was collected into an
1.5-mL microtube containing 1% heparin solution in 0.9% NaCl (aq), and was centrifuged (at 1000 g
for 10 min). Plasma was separated from the cell pellet and added onto a 30-kDa molecular weight
cut-off (MWCO) centrifugal filter (VWR® Radnor, PA, USA) and centrifuged (at 6500 rpm for 10 min).
The filter with over 30 kDa 18F-labeled molecules and the microtube containing the below-30-kDa
18F-labeled molecules in the eluate, were measured with a gamma counter, to determine the percentage
of small molecular weight metabolites from over 30 kDa molecules representing the intact BSA in
the plasma.
4.4.3. Statistical Analysis
Statistical significance of 18F bone accumulation after intravenous administration and the
tracer blood circulation time were analyzed using an unpaired t-test (GraphPad Prism 8.0.1). The
values presented in the synthesis, biodistribution studies, and LogD-measurements are mean
standard deviation.
±
5. Conclusions
Despite of promising hydrolytic stability in vitro, [18F]SiFA-Tz ([18F]
6) demonstrated fast
defluorination in vivo, after intravenous administration in CD-1 mice limiting its utility as a standalone
radiotracer, for pretargeted PET imaging. However, the fluorine-18 label in the biomacromolecular
radiotracer [18F]fluoroalbumin ([18F]10), which was radiolabeled as a proof-of-concept model
6
compound with [18F] , was found to be metabolically more stable, suggesting the utility of [18F]SiFA-Tz
as a prosthetic group for in vitro radiolabeling of biomolecules of higher molecular weight. Based on
these findings, the structure of [18F]SiFA-Tz warrants further optimization, before it can be considered
for use as a radiolabeling tool for low molecular weight biomolecules or as a tracer for pretargeted
PET imaging.
Supplementary Materials: The following are available online, NMR spectra, HPLC chromatograms,
autoradiography profiles, and biodistributions of synthesized and radiolabeled compounds. Supplementary data
are provided as a separate .pdf document.
Author Contributions: Conceptualization, S.O., M.S., K.H., K.W., and A.J.A.; methodology, S.O., S.I., M.S., K.H.,
K.W., and A.J.A.; software, S.O.; validation, S.O., S.I.; formal analysis, S.O.; investigation, S.O., S.I.; resources, M.S.
and A.J.A.; data curation, S.O.; writing—original draft preparation, S.O.; writing—review and editing, S.O., S.I.,
M.S., K.H., K.W., and A.J.A.; visualization, S.O.; supervision, M.S., K.H., K.W., and A.J.A.; project administration,
M.S. and A.J.A.; funding acquisition M.S. and A.J.A. All authors have read and agreed to the published version of
the manuscript.
Funding: This work was supported by the Academy of Finland (decision numbers 306239, 298481, 278056) and a
University of Helsinki three-year research grant. Open access funding provided by University of Helsinki.
Acknowledgments: The authors would like to thank Osku Alanen and Outi Keinänen for assistance in the
experiments. The authors thank Eliza Lambidis and Petri Heinonen for the use of ESI–TOF MS and Dr. Sami
Heikkinen for the use of NMR (600 MHz).
Conflicts of Interest: The authors declare no conflict of interest.
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