A Fluorine-18 Radiolabeling Method Enabled by Rhenium(I) Complexation Circumvents the Requirement of Anhydrous Conditions

Article abstract of DOI:10.1002/chem.201700440

Azeotropic distillation is typically required to achieve fluorine-18 radiolabeling during the production of positron emission tomography (PET) imaging agents. However, this time-consuming process also limits fluorine-18 incorporation, due to radioactive decay of the isotope and its adsorption to the drying vessel. In addressing these limitations, the fluorine-18 radiolabeling of one model rhenium(I) complex is reported here, which is significantly improved under conditions that do not require azeotropic drying. This work could open a route towards the investigation of a simplified metal-mediated late-stage radiofluorination method, which would expand upon the accessibility of new PET and PET-optical probes.

Full text of DOI:10.1002/chem.201700440

A Journal of
Accepted Article
Title: A fluorine-18 radiolabelling method enabled by rhenium(I)
complexation circumvents the requirement of anhydrous
conditions
Authors: Mitchell Ashley Klenner, Giancarlo Pascali, Bo Zhang, Tiffany
Russel Sia, Lawson Kyle Spare, Anwen M. Krause-Heuer,
Janice Aldrich-Wright, Ivan Greguric, Adam Guastella,
Massimiliano Massi, and Benjamin Hugh Fraser
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To be cited as: Chem. Eur. J. 10.1002/chem.201700440
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A fluorine-18 radiolabelling method enabled by rhenium(I)
complexation circumvents the requirement of anhydrous
conditions
Mitchell A. Klenner[a,b], Giancarlo Pascali*[a,c], Bo Zhang[a,d], Tiffany R. Sia[a,c], Lawson K. Spare[e],
Anwen M. Krause-Heuer[a], Janice R. Aldrich-Wright[e], Ivan Greguric[a], Adam Guastella[c],
Massimiliano Massi[b] and Benjamin H. Fraser*[a]
Abstract: Azeotropic distillation is typically required to achieve
fluorine-18 radiolabelling during the production of positron emission
tomography (PET) imaging agents. However, this time consuming
process also limits fluorine-18 incorporation, due to radioactive
decay of the isotope and its adsorption to the drying vessel. In
addressing these limitations, we herein report the fluorine-18
radiolabelling of one model rhenium(I) complex, which is significantly
improved under conditions that do not require azeotropic drying. This
work could open towards the investigation of a simplified metal-
mediated late stage radiofluorination method, which would expand
upon the accessibility of new PET and PET-optical probes.
[18F]fluoride in an aqueous environment, that is typically
unreactive towards nucleophilic substitution; nonetheless, the
interest in employing this unfavorable aqueous environment has
inspired some innovative approaches.[3] Traditionally, in order to
provide an anhydrous environment, the [18F]fluoride in water is
trapped on an anion exchange resin (thus also removing
radioactive cationic impurities), eluted with a complexing agent
in aqueous/organic solvent mixture, and azeotropically dried in a
distillation vial. This azeotropic drying is an inefficient and time
consuming process (>15 mins[4]) during which period,
radioactivity (≥10%) is lost due to the radioactive decay of the
fluorine-18 isotope, as well as non-specific adsorption of
[18F]fluoride to the surface of the distillation vial, leading to lower
radiochemical yield (RCY) of the PET radiotracer.[3,5]
Main Text:
Positron emission tomography (PET) plays an important clinical
role towards unveiling the underlying biochemistry of diseases
via
the
administration
of
positron
(β+)
emitting
radiopharmaceuticals. Radiosynthesis of most PET tracers
needs to be performed quickly, in order to obtain a large enough
dose for a successful PET imaging experiment, due to the
radioactive decay of the β+ emitting isotope. Therefore the
production of PET radiotracers is performed using automated
synthesis modules, and many research groups have worked
towards integrating microfluidic technologies to reduce radiation
dose, miniaturise the workspace and improve production.[1]
Fluorine-18 is the most commonly employed PET radioisotope,
owing to its 97% β+ decay profile, ideal 109.7 min half-life and
low 0.64 MeV β+ energy.[2] However, one drawback is that
fluorine-18 is generally produced by the cyclotron mediated
18O(p,n)18F reaction on oxygen-18 enriched water, which affords
[a]
M. A. Klenner, Dr. G. Pascali, B. Zhang, T. R. Sia, Dr. A. M. Krause-
Heuer, Dr. I. Greguric, Dr. B. H. Fraser.
Australian Nuclear Science and Technology Organisation (ANSTO)
New Illawarra Rd, Lucas Heights, New South Wales, Australia.
E-mail: Gianp@ansto.gov.au, bfr@ansto.gov.au.
M. A. Klenner, Dr. M. Massimiliano.
Department of Chemistry, Curtin University
Kent St, Bentley, Western Australia, Australia.
Dr. G. Pascali, T.R, Sia, Prof. A. Guastella.
Brain and Mind Centre – The University of Sydney
Mallett St, Camperdown, New South Wales, Australia.
B. Zhang.
[b]
[c]
[d]
[e]
Scheme 1. Synthesis of radiolabelling precursors 3 and 5, and non-radioactive
standards 4 and 6. Refer to SI sections 1.1 - 1.5 for detailed procedures.
Monash University
Wellington Road, Clayton, Victoria, Australia.
L.K. Spare, Prof. J. R. Aldrich-Wright.
Having been inspired by recent efforts to couple PET imaging
with an optical tracer to complement disease diagnosis[6], and
given our experience in the use of phosphorescent rhenium(I)
tricarbonyl diimine systems as optical markers for live cell
imaging and diagnosis of pathologies[7], we sought to investigate
School of Science and Health, Western Sydney University
Penrith, New South Wales, Australia.
Supporting information (SI) for this article is given via a link at the
end of the document.
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the fluorine-18 radiolabelling of this class of rhenium complexes
as potential precursors for the future development of PET-optical
multimodal markers. Scheme 1 shows the synthetic route used
to synthesise the ligand (3) and complex (5) precursors, as well
as the non-radioactive standards 4 and 6. N-Oxidation of 1,10-
phenanthroline allowed for selective chlorination in the 2-
position by the Vilsmeier reagent to afford 3. Nucleophilic
aromatic substitution with azeotropically dried fluoride, using 18-
crown-6 as a phase transfer catalyst, then afforded the novel 2-
fluoro-1,10-phenanthroline ligand (4). Ligands 3 and 4 were then
chelated to a Re(I) metal centre to attain complexes 5 and 6,
respectively (SI 1.1 - 1.5).
The radiosyntheses of [18F]4 and [18F]6, from 3 and 5 precursors
respectively, were automated via a flow chemistry set-up
adapted from our previous work[8] as shown in Figure 1. Within
this set-up, aqueous [18F]fluoride was first passed through an
MP1 cartridge and eluted with tetraethylammonium bicarbonate
–
(TEA+HCO3 ) in 90% CH3CN:H2O solution. The resulting
[18F]TEA+F– complex was then either azeotropically dried (dry
conditions) or not azeotropically dried (wet conditions), before
being loaded into the microreactor alongside the desired
precursor solution (3 or 5).
The RCYs obtained for the radiosynthesis of [18F]4 from 3 under
varying temperature conditions are illustrated in Figure 2. These
conditions employed the same residence times (47 s) and
precursor amounts (0.08 - 0.12 µmol) within the microreactor.
Under traditional dry conditions, the RCY of ligand [18F]4
followed the expected trend, whereupon the yield increases as a
function of temperature.[8] In this instance, [18F]4 only formed at
temperatures greater than 130°C, providing a maximum RCY of
61% at 190°C. The radiosynthesis of complex [18F]6 from 5 was
then attempted under analogous dry conditions, and the
dependence of RCY from temperature is illustrated in Figure 3.
Figure 1. Simplified schematic of the flow chemistry configuration assembled
for the automated syntheses of compounds [18F]4 and [18F]6.
Figure 3. Temperature dependence of nucleophilic aromatic substitution of
complex 5 with [18F]fluoride under azeotropically dried (red crosses, dry) and
not azeotropically dried (blue squares, wet) conditions.
Intriguingly, in this case we observed an opposite trend to the
ligand, whereupon the complex [18F]6 only formed at
temperatures below 150°C (in contrast to the ligand [18F]4 which
required higher temperatures to afford a radioproduct). The
RCYs for the complex [18F]6 were also noticeably lower than that
of the ligand [18F]4, with a maximum of 19% RCY obtained at
30°C; in addition, it was clearly noticeable the appearance of a
UV-absorbing degradation product (Figure 4). We suspected
that this by-product could be due to base-mediated degradation
of 5, and we indeed verified this by analyzing in HPLC a DMSO
solution of 5 spiked with 5 µL of 5N NaOH. Therefore, we sought
to suppress this undesired process by modifying the basicity of
the reaction media. Thus, the radiosynthesis of [18F]6 was
performed under wet conditions, whereupon azeotropic drying
was excluded while preparing the radiofluorinating solution,
Figure 2. Temperature dependence of nucleophilic aromatic substitution of
ligand 3 with [18F]fluoride under azeotropically dried (red crosses) and non-
azeotropically dried (blue squares) conditions.
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which realized a 10% v/v content of H2O. Such wet conditions
have been shown previously to be influential in the formation of
different reaction products[9] or improved RCYs; to our delight,
the radiosynthesis of [18F]6 under wet conditions was successful
and we achieved as large as 78% RCY at a temperature as low
as 50°C. Residence times and precursor amounts were also
optimized in order to maximize the RCY (refer to SI 3.1), yielding
[18F]6 in as high as 87% RCY when using 0.38 µmol of precursor
within a residence time of 47 s. Significantly less of the
degradation product from 5 was observed under these wet and
low temperature conditions, confirming the desired switch from
degradation to improved nucleophilic displacement by
[18F]fluoride in these aqueous conditions. To verify that the Re(I)
centre was influential in facilitating nucleophilic substitution in
the 2- position in such extremely mild conditions, we also
performed the radiosynthesis of the ligand [18F]4 under identical
wet conditions which, as expected, afforded no radioproduct at
all as shown in Figure 2. It is suspected that the electron
withdrawing nature of the Re(I) metal centre, due to the π-
acceptance of the CO ligands, may activate the 2-position of
phenanthroline for improved nucleophilic substitution at low
temperatures. Mechanistic studies are currently underway in our
labs to explain why the presence of water favors the
radiofluorination reaction so greatly.
equivalents of Re(CO)5Cl compared to 3, as shown in Figure 6,
despite the residual basic conditions from the reaction in the first
microreactor.
Therefore,
this
unprecedented
two-step
microfluidic approach indeed led to the desired final product by
marginally increasing total process time (4 vs. 10 min), but
provided
a reduced RCY compared to the direct milder
radiofluorination of 5. A process involving the purification of
[18F]4 and performance of a separate 2nd step reaction was not
attempted, as it would have involved a much longer and more
complex system resulting in further radioactive decay.
Figure 5. Simplified schematic of the flow chemistry configuration assembled
for the automated two-step radiosynthesis of [18F]6.
Before performing the radiolabelling using microfluidic systems,
we were unable to synthesize 6 from 5 using the usual heated
and anhydrous conditions which afforded 4 from 3. Given the
resulting formation of [18F]6 from 5 under wet, low temperature
conditions, we revisited the bulk non-radioactive synthesis and
attempted the reaction of 5 to 6 under analogous conditions. UV
reaction monitoring revealed that complex 6 did indeed form
under these wet and low temperature conditions (32% HPLC
conversion) thus demonstrating how fast and efficient
microfluidic optimization can be also used to identify the best
reaction conditions for bulk syntheses.
Figure 4. UV absorbance (254nm, solid blue) and superimposed radioactive
(dashed red) profiles, for dry and wet analogous conditions (top and bottom,
respectively). UV active degradation by-product (Rt: 3 min) is reduced under
wet conditions. Rt: [18F]F− (RAD, 1 min), [18F]6 (RAD, 3.7 min) 5 (UV, 6.2 min).
The direct radiolabelling of the Re(I) tricarbonyl diimine complex
was also compared to a two-step automated radiosynthetic
process.[10] Using microfluidic technology, we assembled a
second microreactor in series, alongside a fourth pump to supply
the Re(CO)5Cl precursor, as shown in Figure 5. RCYs as great
as 35% were able to be achieved using ≥ 5× stoichiometric
Figure 6. Temperature dependence (2nd step) of the two-step radiofluorination
to form complex [18F]6 under azeotropically dried conditions using one (blue
squares), five (green circle) and eight (red crosses) stoichiometric equivalents
of the Re(CO)5Cl precursor.
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Photophysical properties of 6 were also assessed, in order to
investigate the effect of replacing a chloro for a fluoro substituent
in the 2- position of phenanthroline, given our initial research
incentives to develop PET-optical probes. We therefore verified
that these Re(I) complexes afforded similar quantum yields (Φ =
0.3%), lifetimes (τ = 27 - 41 ns), emission maxima (λex = 410
nm, λem = 653 - 654 nm) and monoexponential decay profiles in
DMSO (SI 4.1 - 4.3).
In summary, the fluorine-18 radiolabelling of a ligand complexed
to Re(I) was found to afford optimal RCYs under remarkably
mild conditions, without the need for azeotropic distillation and
with a reduction of >100°C for the optimal temperature. Further
investigations will clarify the involvement of the Re(I) centre in
the radiofluorination mechanism, and will seek to apply this
radiolabelling strategy toward improving the RCY of existing
PET tracers, as well as toward accessing new PET and PET-
optical probes.
Keywords: fluorine-18 radiolabelling • microfluidics • PET
chemistry • rhenium complexes • late stage fluorination
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Experimental Section
Aqueous
[
18F]fluoride was produced on an IBA Cyclone 18 Twin
cyclotron (ANSTO, Camperdown, Australia) using the 18O(p,n)18F nuclear
reaction. Microfluidic radiosyntheses were performed in Discovery Mode
using a NanoTek LF Microfluidic Synthesis System (Advion, Ithaca, NY)
connected to a standard laptop using the NanoTek software, V1.4.0 GMP
Lite. Microreactors were made of fused silica tubing (100 µm × 2 m),
coiled tightly into a brass ring containing a thermoresistent polymer to
hold the tubing in place. RadioHPLC analyses were carried out using a
Shimadzu system comprised of a CBM-20 controller, LC-20AD pump,
SIL-20AHT autoinjector, SPD-M20A PDA detector (for UV analyses) and
a Lablogic Posi-RAM gamma detector. Analyses were performed using
isocratic conditions consisting of CH3CN/H2O/TFA mobile phases
(7.95:91.95:0.10% v/v for [18F]4 and 64.95:34.95:0.10% v/v for [18F]6), on
a Chromolith RP column (monolith system, Merck, 50 × 4.6 mm). RCYs
were calculated using the radio-HPLC profiles, integrating the
radioproduct peak against all the other radioactive peaks (inclusive of the
unreacted [18F]fluoride), using Laura V4.1.70 SP2 HPLC data analysis
software.
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The microfluidic experiments were carried out on the scale of 29±10 MBq,
and afforded [18F]6 with a molar activity of 71±12 MBq.nmol-1.
[7]
Experimental details concerning the general syntheses, structure
elucidation, radiochemistry and photophysical characterisations of the
reported compounds can be found within the SI sections 5.1 – 5.3.
Acknowledgements
[8]
[9]
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P. A. Salvadori and I. Greguric, Nat. Protoc., 2014, 9, 2017–29.
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Greguric and P. A. Salvadori, J. Flow Chem. 2014, 4, 86–91; b) T. R.
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This work has been partially funded by the Australian Institute
for Nuclear Science and Engineering (AINSE) via the provision
of a Postgraduate Research Award (PGRA), and the Australian
Research Council (ARC) – Linkage grant LP1590101307. We
also acknowledge Nikolas Paneras for the cyclotron production,
as well as Dr. Lidia Matesic and Iveta Kurlapski for laboratory
support.
[10] G. Pascali, G. Nannavecchia, S. Pitzianti and P. A. Salvadori, Nucl.
Med. Biol. 2011, 38, 637 – 644.
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Entry for the Table of Contents (Please choose one layout)
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A novel fluorine-18 radiolabelling
method has been found to circumvent
the need for preliminary azeotropic
distillation. The method involves
complexation to a rhenium(I) centre,
and is of particular importance in the
Mitchell A. Klenner, Giancarlo
Pascali*, Bo Zhang, Tiffany R. Sia,
Lawson K. Spare, Anwen M. Krause-
Heuer, Janice Aldrich-Wright, Ivan
Greguric, Adam Guastella,
Massimiliano Massi and Benjamin H.
Fraser*
((Insert TOC Graphic here))
expanded scope of positron emission
tomography (PET) imaging agent
design. Results were verified through
the monitoring of multiple reaction
conditions by employing microfluidic
technologies in flow.
Page No. – Page No.
A fluorine-18 radiolabelling method
enabled by rhenium(I) complexation
circumvents the requirement of
anhydrous conditions
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Author(s), Corresponding Author(s)*
((Insert TOC Graphic here))
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Title
Text for Table of Contents
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