Ultrafast Photoclick Reaction for Selective18F-Positron Emission Tomography Tracer Synthesis in Flow

Article abstract of DOI:10.1021/jacs.1c02229

The development of very fast, clean, and selective methods for indirect labeling in PET tracer synthesis is an ongoing challenge. Here we present the development of an ultrafast photoclick method for the synthesis of short-lived¹⁸F-PET tracers

Full text of DOI:10.1021/jacs.1c02229

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18
Ultrafast Photoclick Reaction for Selective F‑Positron Emission
Tomography Tracer Synthesis in Flow
Youxin Fu,‡ Hugo Helbert,‡ Nadja A. Simeth, Stefano Crespi, Gerbren B. Spoelstra, Jan Maarten van Dijl,
Marleen van Oosten, Luiza Reali Nazario, Dion van der Born, Gert Luurtsema, Wiktor Szymanski,*
Philip H. Elsinga,* and Ben L. Feringa*
Cite This: J. Am. Chem. Soc. 2021, 143, 10041−10047
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ABSTRACT: The development of very fast, clean, and selective methods for indirect labeling in PET tracer synthesis is an ongoing
challenge. Here we present the development of an ultrafast photoclick method for the synthesis of short-lived 18F-PET tracers based
on the photocycloaddition reaction of 9,10-phenanthrenequinones with electron-rich alkenes. The respective precursors are
synthetically easily accessible and can be functionalized with various target groups. Using a flow photo-microreactor, the photoclick
reaction can be performed in 60 s, and clinically relevant tracers for prostate cancer and bacterial infection imaging were prepared to
demonstrate practicality of the method.
introduced in an attempt to address the issues related to slow
ositron emission tomography (PET) is a key molecular
Pimaging technique, characterized by unparalleled sensi- reaction rate and copper toxicity (Figure 1A).31,32 Considering
tivity.1 It targets the tissue of interest with tracers function-
the challenge to develop very fast, clean, and selective methods
for indirect labeling, photochemical click reactions would
provide a particularly appealing alternative (Figure 1B).
Highly beneficial is that photoclick reactions can combine
important requirements to provide a practical indirect labeling
protocol, such as high functional group tolerance, ambient
reaction conditions, and easy operation in a photoflow reactor,
and, importantly, one might achieve extremely high reaction
rates without the need for additional reagents or catalysts. The
outstanding possibilities offered by photochemical reactions33
have recently been recognized in several radiochemical
applications, i.e., a methylation protocol for 11C-PET ligand
synthesis,34 photo-redox catalysis for 18F−C bond formation,35
photoactivatable aryl azides, and photo-triggered reaction of
tetrazoles for radiosynthesis of 89Zr-labeled proteins.36−39
However, so far a very limited number of photochemical
transformations has been utilized as a key step in the indirect
labeling of 18F-PET tracers,40−42 and none of them provides
the modularity and selectivity typical of click reactions.
alized with radioactive, short half-life positron-emitting
nuclides for detection by gamma cameras. Therefore, the
development of radiopharmaceuticals for PET is highly
dependent on our ability to introduce radionuclides efficiently
and rapidly into the target chemical structures. The workhorse
radionuclide for PET is fluorine-18, which is characterized by a
half-life suitable for radiosynthesis and biodistribution (t1/2
=
109.8 min). Emission of low-energy positrons (Emean = 0.64
MeV) accompanies the decay of 18F, allowing for relatively
high image resolution.1 Consequently, 18F is the most widely
used radionuclide for the clinical labeling of PET radiophar-
maceuticals. Labeling methods to introduce 18F can be divided
into direct and indirect techniques. Most direct labeling
strategies rely on the use of 18F− to functionalize a wide range
of substrates, introducing 18F-aryl, 18F-alkyl, 18F-CF3,2,3 or, very
recently, 18F-SO3 groups.4 These new direct labeling strategies
greatly expanded the applicability of the method in the past
years;5,6 however, the need for elevated reaction temperatures
or the low functional group tolerance7−10 stimulated recent
efforts toward milder direct labeling methods involving
chelation of Al18F and 18F-19F-exchange reactions on
heteroatoms like Si and B.11−14 Hence, indirect labeling is
often the preferred option for particularly sensitive sub-
strates.15,16 This approach is based on the fluorination of a
prosthetic group that is subsequently attached to a tracer in a
bioorthogonal reaction.
The photoreactions showcased for PET tracer synthesis
often lack in functional group tolerance41,42 or are performed
with short irradiation wavelength that can be absorbed by, or
damage, common biomolecules.43 With these challenges in
mind, we were aiming to identify and evaluate a fast photoclick
reaction that can be conveniently used for the versatile
preparation of 18F-PET tracers under visible light irradiation.
Due to the limited half-life of 18F, this final coupling step has
to be very efficient and fast.16,17 Hence, the copper-catalyzed
azide−alkyne cycloaddition (CuAAC) reaction became an
attractive labeling method.18−20 Its metal-free labeling variants,
such as strain-promoted click chemistry (SPAAC)21−25 and
tetrazine trans-cyclooctene cycloadditions (IEDDA),26−30 were
Received: March 5, 2021
Published: June 28, 2021
© 2021 The Authors. Published by
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https://doi.org/10.1021/jacs.1c02229
J. Am. Chem. Soc. 2021, 143, 10041−10047
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Figure 1. Overview of different “click” reactions used in PET tracer synthesis: (A) the established CuAAC, SPAAC, and IEDDA techniques and
(B) the novel ultrafast photoclick reaction presented here.
An interesting candidate for such a process is provided by
photocycloaddition between 9,10-phenanthrenequinones
(PQs) and suitably substituted alkenes (see Figure 1B).44
This “photoclick” [4+2] cycloaddition44−47 was already
discovered in the 1940s. However, the need for long reaction
times hampered its application in synthesis.47 Recently, using a
suitable high-power LED light source able to excite PQ (λmax at
395 nm), the transformation was performed in the minute
range in a biological environment.48 We envisioned that this
fast and clean photoclick reaction holds tremendous potential
for indirect labeling of tracers with short-lived radioisotopes to
produce 18F-PET radiopharmaceuticals (Figure 1B). Here, we
present the development of the PQ photoclick reaction into
highly efficient batch and flow methodology for ultrafast
radiosynthesis and its application for the preparation of 18F-
labeled compounds, including a prostate cancer biomarker and
a bacterial infection imaging tracer.
reactivity with PQ of a series of hitherto unexplored cyclic
VEs: 3,4-dihydro-2H-pyran-2-methanol (VE2), 2,3-dihydrofur-
an (DF), and 2,3-dihydropyran (DP, Figure 2A) were reacted
under 395 nm light irradiation with the diketone (for detailed
information, see SI, section 4). All substrates showed high
reactivity toward PQ and formed adducts exhibiting strong
blue fluorescence, even visible by naked eye. The photoclick
reactions were monitored by fluorescence spectroscopy
(Figure 2A) and reached completion in less than 5 min.
Formation of the cycloaddition products was confirmed by
NMR and high-resolution mass spectroscopy (see SI, sections
2−4). Gratifyingly, the cyclic vinyl ethers VE2, DF, and DP
exhibited significantly higher reaction rates compared to the
linear vinyl ether (VE1). The full conversion could be achieved
in around 90 s for the cyclic vinyl ethers and 180 s for VE1,
respectively. Indeed, the electron properties of the vinyl ether
greatly influence the reaction rate (see Figure 2A). The
computed energies (SMD(MeCN)-ωB97X-D/def2-SVP level)
of the HOMO of the various traps match the observed rates
found in the experiments (see Figure 2B and SI, section 5).
The cyclic vinyl ethers are more nucleophilic than the linear
analogs and, consequently, more prone to react with the lowest
unoccupied β-spin orbital of the triplet PQ.
By irradiation of PQ with 395 nm light in the presence of
electron-rich vinyl ethers (VEs, see Figure 2), the photo-
cycloaddition, proceeding via the triplet state of PQ, furnishes
PQ-VE adducts. Establishing the reaction conditions between
PQ and VE1 (Figure 2A) allowed the synthesis of the
corresponding photocycloadduct PQ-VE1 (see SI, Figure S59)
after only 180 s of irradiation (see SI, sections 2 and 4, for
1
We then proceeded to extend the scope of this fast
photoclick reaction to the fluorinated vinyl ethers F-VE1
(linear) and F-VE2 (cyclic; see Figure 2A). The conversion of
both substrates with PQ was monitored by fluorescence
spectroscopy (Figure 2A), showing a similar trend as observed
for the VE compounds, with the cyclic compound reacting
faster (full conversion in 1 min for F-VE2 and 3 min for F-
VE1). Formation of the cycloaddition products was confirmed
photoproduct PQ-VE1 synthesis and characterization by H
and 13C NMR and HRMS). We also discovered ultra-fast
reactivity of cyclic vinyl ethers (Figure 2A). Moreover, control
experiments using light−dark cycles showed that changes in
the absorption spectrum and product formation follow
exclusively a photochemical pathway (see SI, Figure S43).
To explore the scope of the photocycloaddition and enable
even higher reaction rates for PET labeling, we investigated the
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Communication
Figure 2. (A) Comparison of the conversion of the photo-
cycloaddition of PQ with different VEs over time followed by
fluorescence spectroscopy (1 cm cuvette, 2 mL sample volume, 25 °C,
sample interval 10 s. Concentration: 2.5 μM (PQ), 25 μM (VE), λex
=
365 nm, λobs = 403 nm). (B) Frontier orbitals of the species involved
in the reaction (HOMO of VEs and lowest unoccupied β-spin orbital
of the triplet PQ) at the SMD(MeCN)-ωB97X-D/def2-SVP level.
by NMR and high-resolution mass spectroscopy (see SI,
section 2).
These results indicate that F-VEs show excellent reactivity
toward PQ, quickly and selectively generating the desired
fluorogenic photocycloadducts. With the “cold” reaction
conditions in hand, we synthesized the 18F-radiolabeled
analogs of the two F-VEs. The fluorination of the
corresponding tosylates was performed by rapid (3 min)
nucleophilic substitution with azeotropically dried 18F−/K222
,
and the products were purified by distillation, affording 18F-
VE1 and 18F-VE2 in moderate to good radiochemical yield
(58% and 37%, respectively; for experimental details see SI,
section 6). Both compounds could be directly used for the
subsequent photoclick reaction. Irradiation of PQ in the
presence of both 18F-VE linkers (cf. Figure 3A) showed full
conversion of the radioactive starting material; however, the
expected 18F-VE-PQ was not formed. (For experimental
details of optimization, see also SI, section 4.)
1H NMR analysis of the reaction between VE2 and PQ
revealed that, without an excess of the trap, namely the VEs,
photooxidation degraded the product. Consequently, the
photochemical reactions at equimolar ratios or with excess of
PQ (such as in the radiolabeling experiments) were performed
with deoxygenated solvents. To our delight, degradation of the
product was prevented, and F-VE2-PQ remained unaffected
Figure 3. (A) Batch reaction setup. (B) Flow reaction setup. (C)
Scope of “hot” F-VE-PQs including the potential prostate cancer
tracer 18F-VE1-PQ-PSMA and the bacterial infection imaging agent
18F-VE1-PQ-Vanco (only one isomer displayed; see also SI). The
RCC values over three repetitions are reported with the
corresponding error associated with the measurements.
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even after 10 min of irradiation (see SI, Figure S51, F-VE2-
PQ). Applying oxygen-free conditions to the radiolabeling
procedure with 18F-VE1 resulted in 18F-labeled product 18F-
VE1-PQ in high radiochemical conversion (RCC, 69%) in 5
min. The use of cyclic 18F-VE2 resulted in lower conversion to
the product (RCC 20% to 18F-VE2-PQ) compared to the
linear VE.
To improve the efficiency of the photoclick reaction and to
further reduce the reaction time, the reaction was optimized in
a microfluidic flow photoreactor (for details regarding the
optimization condition, see SI, section 6). This allowed us to
have highly efficient irradiation (effective light penetration)
and to automate the protocol. Toward this goal, a
commercially available FlowSafe synthesis module20 was
equipped with two 395 nm LEDs (see Figure 3B). Indeed,
performing the reaction this way enhanced the irradiation
efficiency significantly and allowed us to achieve high
conversions even at residence times as short as 60 s. The
high conversion observed in batch for the synthesis of 18F-
VE1-PQ could be improved even further in flow, affording the
desired product in 77% RCC (Figure 3C).
to plasma protein after 3 h incubation. An in vitro assay was
performed using the Gram-positive bacterium Staphylococcus
aureus and the Gram-negative bacterium Escherichia coli. We
observed significant binding of the 18F-VE1-PQ-Vanco by S.
aureus, but not by E. coli, which is consistent with the mode of
action of the parent compound vancomycin. Specifically, after
30 min incubation, a 17-fold higher accumulated activity was
detected for S. aureus compared to E. coli (Figure 4).
We subsequently explored if the substituted PQ derivatives
perform equally well in this developed ultrafast click method-
ology, envisioning the embedding of PQ derivatives into
targeting moieties of future tracers for reaction with the vinyl
prosthetic groups. A carboxylic acid on the PQ moiety as a
handle for further synthetic functionalization and linear 18F-
VE1 was selected as the reaction partner in the photo-induced
cyclization reaction. First, the effect of the amide substitution
on PQ was assessed by performing the labeling of compound
18F-VE1-PQ-Amide. The expected product was formed in
satisfactory radiochemical conversion (RCC 49%, Figure 3C)
after only 60 s of irradiation in flow. In order to test the
labeling of potential probes via a direct attachment using an
amide linkage, a well-established prostate-specific membrane
antigen (PSMA) binding motif was connected to the PQ core
structure as a model substrate. PSMA is a biological marker of
prostate cancer that is often targeted for diagnosis in PET
imaging20,49−51 (Figure 3C). Most of the PET probes
developed to target PSMA share the same lysine-urea-
glutamate binding motif.20
Finally, driven by a clinical need to detect bacterial
infections, non-invasively and with high sensitivity,52−54 the
labeling of the antibacterial agent vancomycin was undertaken.
A suitable PQ derivative, PQ-Vanco, was synthesized (for
details see SI) and isolated as a mixture of functional isomeric
products, as reported before for the vancomycin system (see SI
for the fragmentation pattern assignment).55,56 Gratifyingly,
application of our labeling strategy in flow led to successful
radiolabeling of this highly functionalized drug in 60 s, under
irradiation with 395 nm light (RCC 11 1%), without the
need of protecting groups. This is the first report of the
synthesis of a 18F-labeled vancomycin derivative, which
highlights the versatility and utility of the synthetic approach
for a large and complex multifunctional molecule of high
biomedical relevance.
Figure 4. Accumulation of radioactivity over time (0, 5, 15, 30, 60,
and 120 min) in S. aureus (orange) and E. coli (blue) grown in liquid
culture, corrected for colony forming units (CFUs). The fitting line
represents the mean
standard error of the mean (in black);
Student’s t test: *P < 0.05, ***P < 0.001.
Altogether, these results suggest that 18F-VE1-PQ-Vanco can
be of great value for the selective detection of infections caused
by S. aureus and other Gram-positive bacteria through PET
imaging.
In summary, we successfully developed a novel, extremely
fast photoclick reaction for the synthesis of short-lived 18F-PET
tracers. The respective 18F-VE and PQ precursors are
synthetically easily accessible. By functionalizing the PQ
moiety with a carboxylic acid handle, various target-specific
agents, such as the clinically relevant PSMA ligand and the
antibacterial agent vancomycin, can be readily attached via an
amide bond. By using a commercially available, automated
module equipped with a flow photo-microreactor, the reaction
time could be optimized to only 1 min under visible (violet)
light irradiation, representing a considerable improvement
compared to most current methods. Our strategy enables the
complete process, from the stage of dried 18F−/K222 to the
crude final product, to be performed in less than 10 min. From
a practical point of view, the method holds tremendous
potential as a novel radiolabeling procedure for 18F-tracers.
Moreover, exploiting the fluorescence properties of the
photocycloadduct offers prospects toward the development
of other (multimodal) imaging protocols.
To assess the potential of this novel vancomycin derivative,
the in vitro binding properties of 18F-VE1-PQ-Vanco were
determined. Lipophilicity (LogP) of 18F-VE1-PQ-Vanco was
found to be −0.68 0.10 (mean, standard deviation). Little
tracer degradation was observed in human plasma, with >90%
of tracer remaining intact after 2 h incubation at 37 °C. A
fraction of 22% of 18F-VE1-PQ-Vanco was found to be bound
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.1c02229.
■
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Experimental procedures and characterization data for
all new compounds, photophysical and chemical studies,
10044
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Gert Luurtsema − Department of Nuclear Medicine and
Molecular Imaging, University of Groningen, University
Medical Centre Groningen, 9713 GZ Groningen, The
Netherlands; orcid.org/0000-0001-8599-8129
details regarding the computational calculation, and
detailed protocols of radiochemistry (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c02229
Wiktor Szymanski − Centre for Systems Chemistry, Stratingh
Institute for Chemistry, Faculty for Science and Engineering,
University of Groningen, 9747 AG Groningen, The
Netherlands; Department of Radiology, Medical Imaging
Center, University of Groningen, University Medical Centre
Groningen, 9713 GZ Groningen, The Netherlands;
orcid.org/0000-0002-9754-9248; Email: w.szymanski@
umcg.nl
Philip H. Elsinga − Department of Nuclear Medicine and
Molecular Imaging, University of Groningen, University
Medical Centre Groningen, 9713 GZ Groningen, The
Netherlands; orcid.org/0000-0002-3365-4305;
Email: p.h.elsinga@umcg.nl
Author Contributions
‡Y.F. and H.H. contributed equally.
Notes
The authors declare the following competing financial
interest(s): Dion van der Born is an employee of Future
Chemistry which produces the FlowSafe equipment used in
this work.
ACKNOWLEDGMENTS
■
Bram Maas is kindly acknowledged for operating the cyclotron
and providing the 18F. We thank Renze Sneep (University of
Groningen) for his help with the HRMS measurements. We
gratefully acknowledge the generous support from the Horizon
2020 Framework Program (ERC Advanced Investigator Grant
No. 694345 to B.L.F.), the Marie Skłodowska-Curie Actions
(Individual Fellowship 838280 to S.C.), the Alexander-von-
Humboldt Foundation (Feodor Lynen Fellowship to N.A.S.),
the Ministry of Education, Culture and Science of The
Netherlands (Gravitation Program No. 024.001.035 to B.L.F.),
and The Netherlands Organization for Scientific Research
(NWO, VIDI grant no. 723.014.001 for W.S.). This research
was supported by NWO domain TTW and Stryker European
Operations Ltd.
Ben L. Feringa − Centre for Systems Chemistry, Stratingh
Institute for Chemistry, Faculty for Science and Engineering,
University of Groningen, 9747 AG Groningen, The
Netherlands; orcid.org/0000-0003-0588-8435;
Email: b.l.feringa@rug.nl
Authors
Youxin Fu − Centre for Systems Chemistry, Stratingh Institute
for Chemistry, Faculty for Science and Engineering, University
of Groningen, 9747 AG Groningen, The Netherlands;
orcid.org/0000-0001-6942-6534
Hugo Helbert − Centre for Systems Chemistry, Stratingh
Institute for Chemistry, Faculty for Science and Engineering,
University of Groningen, 9747 AG Groningen, The
Netherlands; Department of Nuclear Medicine and Molecular
Imaging, University of Groningen, University Medical Centre
Groningen, 9713 GZ Groningen, The Netherlands;
orcid.org/0000-0003-0308-2841
Nadja A. Simeth − Centre for Systems Chemistry, Stratingh
Institute for Chemistry, Faculty for Science and Engineering,
University of Groningen, 9747 AG Groningen, The
Netherlands; orcid.org/0000-0001-8130-883X
Stefano Crespi − Centre for Systems Chemistry, Stratingh
Institute for Chemistry, Faculty for Science and Engineering,
University of Groningen, 9747 AG Groningen, The
Netherlands; orcid.org/0000-0002-0279-4903
Gerbren B. Spoelstra − Department of Nuclear Medicine and
Molecular Imaging, University of Groningen, University
Medical Centre Groningen, 9713 GZ Groningen, The
Netherlands; orcid.org/0000-0002-7063-2834
Jan Maarten van Dijl − Department of Medical Microbiology
and Infection Prevention, University of Groningen, University
Medical Center Groningen, 9713 GZ Groningen, The
Netherlands; orcid.org/0000-0002-5688-8438
Marleen van Oosten − Department of Medical Microbiology
and Infection Prevention, University of Groningen, University
Medical Center Groningen, 9713 GZ Groningen, The
Netherlands; orcid.org/0000-0002-7061-6324
Luiza Reali Nazario − Department of Nuclear Medicine and
Molecular Imaging, University of Groningen, University
Medical Centre Groningen, 9713 GZ Groningen, The
Netherlands; orcid.org/0000-0002-4983-5047
Dion van der Born − FutureChemistry, 6708 PW
Wageningen, The Netherlands; orcid.org/0000-0001-
7381-1160
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