Fully automated peptide radiolabeling from [18F]fluoride

Article abstract of DOI:10.1039/c8ra10541c

The biological properties of receptor-targeted peptides have made them popular diagnostic imaging and therapeutic agents. Typically, the synthesis of fluorine-18 radiolabeled receptor-targeted peptides for positron emission tomography (PET) imaging is a t

Full text of DOI:10.1039/c8ra10541c

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Fully automated peptide radiolabeling from [18F]
fluoride†
Cite this: RSC Adv., 2019, 9, 8638
ac
Ryan A. Davis,
Chris Drake,b Robin C. Ippisch,c Melissa Mooreb
acd
*
and Julie L. Sutcliffe
The biological properties of receptor-targeted peptides have made them popular diagnostic imaging and
therapeutic agents. Typically, the synthesis of fluorine-18 radiolabeled receptor-targeted peptides for
positron emission tomography (PET) imaging is a time consuming, complex, multi-step synthetic process
that is highly variable based on the peptide. The complexity associated with the radiolabeling route and
lack of robust automated protocols can hinder translation into the clinic. A fully automated batch
production to radiolabel three peptides (YGGFL, cRGDyK, and Pyr-QKLGNQWAVGHLM) from fluorine-18
using the ELIXYS FLEX/CHEM® radiosynthesizer in a two-step process is described. First, the prosthetic
group, 6-[18F]fluoronicotinyl-2,3,5,6-tetrafluorophenyl ester ([18F]FPy-TFP) was synthesized and
subsequently attached to the peptide. The [18F]FPy-peptides were synthesized in 13–26% decay
corrected yields from fluorine-18 with high molar activity 1–5 Ci mmolꢀ1 and radiochemical purity of
>99% in an overall synthesis time of 97 ꢁ 3 minutes.
Received 24th December 2018
Accepted 26th February 2019
DOI: 10.1039/c8ra10541c
rsc.li/rsc-advances
against a target epitope, facilitating identication and optimi-
zation of high affinity peptide ligands.5
Introduction
One of the rst examples of a peptide radiotracer translated
to the clinic was [123I]-Tyr3-octreotide, used to image somato-
statin receptor density in patients with neuroendocrine tumors
(NETs).6 Octreotide was later optimized and radiolabeled with
indium-111 for single photon computed tomography (SPECT),
and since then octreotide has been radiolabeled with PET-
radionuclides including gallium-68 and uorine-18, offering
greater sensitivity and higher resolution images that provide for
a more accurate diagnosis.6–11 Somatostatin imaging has
become the standard-of-care for patients with NETs, with
sensitivities and specicities of >90%, and the combination of
somatostatin imaging with computed tomography (CT) or
magnetic resonance imaging (MRI) has been reported to change
clinical treatment for 20–60% of patients.11 The FDA approval of
NETSPOT (68Ga-DOTATATE) in 2016 underscores the success of
peptide radiotracers currently used in the clinic.12
Several other disease associated cellular-receptors have been
targeted with peptide radiotracers for both imaging and radio-
therapy. For example the integrin-avb3, which is overexpressed
in a number of cancers, including melanoma, breast, and head
and neck as well as neo-angiogenic blood vessels.13–16 The
integrin-avb3 is involved in a number of cellular processes,
including cell proliferation, migration, and tumor metastasis,
and has been widely studied as a target for both imaging and
therapy using small peptides containing an arginine–glycine–
aspartic acid (RGD)-motif.13–19 Another member of the integrin
family of receptors that is currently being investigated is the
integrin-avb6, an epithelial-specic cell surface receptor that is
Peptide-based pharmaceuticals have applications in both
diagnostic imaging and targeted therapy with more than 60
approved by the US Food and Drug Administration (FDA), ꢂ140
in clinical trials (half of which are diagnostic imaging agents),
and approximately another 500 in pre-clinical development.1,2
Peptides have numerous characteristics that make them ideal
as radiotracers; in particular, their biological half-life ideally
matches the radioactive half-life of many radionuclides
currently being used for positron emission tomography (PET),
such as gallium-68, uorine-18, and copper-64.3 They are rela-
tively small in size (ꢂ0.5 to 10 kDa) and exhibit short in vivo
circulation times (min to h) that result in rapid clearance from
the blood pool and non-targeted tissues.4 Peptides have
demonstrated similar binding affinities as antibodies for their
respective targets but are simpler to prepare, typically non-
immunogenic, and can be easily chemically modied to
further improve affinity, selectivity, stability, and their phar-
macokinetic prole.3 Moreover, contemporary molecular
biology and peptide combinatorial library techniques allow
large peptide libraries to be generated and rapidly screened
aDepartment of Internal Medicine, Division of Hematology and Oncology, University of
California, Davis, CA, USA. E-mail: jlsutcliffe@ucdavis.edu; Fax: +1-916-734-7572;
Tel: +1-916-734-5536
bSoe Co., Culver City, CA, USA
cDepartment of Biomedical Engineering, University of California, Davis, CA, USA
dCenter for Molecular and Genomic Imaging, University of California, Davis, CA, USA
† Electronic supplementary information (ESI) available: HPLC-chromatograms
and mass spectra. See DOI: 10.1039/c8ra10541c
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undetectable in healthy adults, but is signicantly up-regulated both the intermediate 18F-prosthetic and the nal 18F-peptide
in a wide range of epithelial derived cancers including, radiotracer.4,31 For example, Thonon et al., used the
pancreas, colon, non-small cell lung cancer, ovarian, breast, FASTLab™ GE synthesis module for the production of [18F]SFB
prostate, and oral squamous cell carcinoma.20–26 Sutcliffe et al., via both a one- and two-pot method, followed by coupling to an
have focused signicant effort on the development of avb6-tar- RGD peptide (PRGD2).36 Ackermann et al. also reported the fully
geting peptides based on the 20 amino acid sequence derived automated radiolabeling of the protein glutathione with [18F]
from an envelope protein of the foot-and-mouth disease FBEM the thiol reactive prosthetic group on an iPHASE FLEX-
virus.23–26 Another example are the gastrin releasing peptide Lab module, however no extension of this methodology to
receptors (GRPRs) that are overexpressed in many cancers, radiolabeling a clinically-relevant peptide was included in this
including breast, pancreatic, and prostate, with GRPR-mediated paper.37 Both methods required some form of manual inter-
signalling being linked to several oncogenic processes such as vention, and to our knowledge a fully automated procedure for
invasiveness and proliferation.27,28 These receptors have been the radiosynthesis of a clinically relevant uorine-18 radio-
targeted by a series of bombesin peptides for both imaging and labeled peptide has yet to be reported.4 Therefore, the goal of
therapeutic purposes with their efficacy being examined in this work was to develop a fully automated 18F-peptide radio-
several clinical studies.27,28
labeling synthesis and apply it to three clinically relevant
Fluorine-18 is a favored radioisotope for radiolabeling peptides (YGGFL (1), cRGDyK (2), and Pyr-QKLGNQWAVGHLM
peptides due to its nuclear and physiochemical characteristics (3)) (Fig. 1) with no user intervention. To achieve this, the [18F]
for in vivo imaging, including a half-life (109.77 min) that FPy-TFP prosthetic group was synthesized on an ELIXYS FLEX/
closely matches the pharmacokinetics of peptides, low b+ CHEM® radiosynthesizer automated module and subsequently
energy (0.64 MeV), high 97% b+ emission, and is readily avail- conjugated to each of the target peptides.
able in sufficient quantities (10–20 Ci) and high molar activity
(1–10 Ci mmolꢀ1, required for receptor-targeting imaging).29,30
Radiolabeling of peptides with uorine-18 is mainly carried out
using a prosthetic group, a small, bifunctional molecule that is
rst reacted with the radioisotope with subsequent attachment
to the peptide at the N-terminal amine, the N3-amine of a lysine
Experimental
General
residue, or a thiol of a cysteine residue.4 Examples of amine- Solvents and reagents were purchased from Acros Organics
reactive prosthetic groups include N-succinimidyl 4-[18F]uo- (Pittsburg, PA), Sigma Aldrich (St Louis, MO), and NovaBiochem
robenzoate ([18F]SFB), 4-[18F]uorobenzoic acid ([18F]FBA), and (Darmstadt, Germany) unless otherwise stated, and used
6-[18F]uoronicotinic acid 2,3,5,6-tetrauorophenyl ester ([18F] without further purication. All solvents used for automated
FPy-TFP).4 Thiol-reactive prosthetic groups include maleimide- synthesis were anhydrous extra dry over molecular sieves.
containing moieties such as N-[2-(4-[18F]uorobenzamido) Chromatography solvents were purchased from EMD Millipore
ethyl]maleimide
([18F]FBEM)
and
N-[6-(4-[18F]uoro- (Chicago, IL) as HPLC-grade and used directly. All automated
benzylidene)aminooxyhexyl]maleimide ([18F]FBAM).4 Non- syntheses were performed on an ELIXYS FLEX/CHEM® radio-
canonical functional groups can also be incorporated into synthesizer (Soe Co., Culver City, CA) using the exact same
peptides to facilitate radiolabeling using 18F-prosthetics such as command sequence (S1). The 19F-reference standards were
4-[18F]uorobenzaldehyde, 2-[18F]uorodeoxyglucose ([18F] synthesized using commercially available 6-uoronicotinic acid
FDG), and click-moieties such as
[
18F]uoro-trans-cyclo- (TCI America, Portland, OR) and/or 2,3,5,6-tetrauorophenol
octene.29,30 The use of a prosthetic group to radiolabel peptides (Sigma-Aldrich, St. Louis, MO). These synthon's C12-RP-HPLC
from uorine-18 is generally required as direct incorporation is provided for reference (S2 and S3) as they are the major by-
into the peptide is not feasible due to the harsh reaction products observed during hydrolysis of the [18F]FPy-TFP pros-
conditions required for C–F bond formation (e.g. high temper- thetic group. Rink-amide resin, N-terminal uorenylmethox-
atures or strongly basic conditions).4 Traditionally, synthesis of ycarbonyl (Fmoc) protected amino acids and 1-
the prosthetic group is performed manually.31
[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyr-
Automation of the radiosynthesis can provide a safe, repro- idinium 3-oxid hexauoro-phosphate (HATU) were purchased
ducible, straight-forward route to radiolabel any peptide from from GL Biochem (Shanghai, China) or NovaBiochem (Darm-
uorine-18. Unfortunately to date automated procedures have stadt, Germany). The peptides used for radiolabeling, cRGDyK
primarily focused on small molecules or the production of (2) and Pyr-QKLGNQWAVGHLM (3) (Fig. 1) were purchased
prosthetic groups.4 Several automated syntheses of 18F- from AnaSpec (Fremont, California) and used without further
prosthetics used for peptide radiolabeling have been reported, purication. The uorine-18 was produced by PETNET Solu-
including [18F]SFB,32 [18F]FBEM,32 [18F]FBA,32 azido-[18F]uoro- tions (Sacramento, California). The aqueous uoride-18 (6–10
sugars,33 [18F]hexauorobenzene,34 and [18F]uoroethylazide.35 mCi per synthesis) was trapped onto a chromax-PS-HCO3
¨
The radiochemical yields for these automated productions are anion exchange cartridge (Macherey-Nagel, Duren, Germany)
comparable, if not better than the analogous manual proto- (conditioned with 2 mL water). Purication cartridges C18-
cols.32–35 Automated protocols capable of generating 18F- SepPak-Plus and Oasis MCX-Plus-mixed cation exchange
peptides from uorine-18 without manual interventions are cartridge were purchased from Waters (Milford, MA, USA)
less prevalent, as multiple purications are typically needed for (conditioned with 10 mL ethanol followed by 10 mL water).
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Fig. 1 Structures of the target peptides.
the solid washed with diethyl ether. The resulting solid was
suspended in dichloromethane (200 mL) and stirred vigorously
while trimethylsilyl triuoromethanesulfonate (TMSOTf, 10 g,
44.99 mmol) was added dropwise via a syringe. The suspension
was stirred for 15 min and the unreacted material ltered off
with the reaction contents concentrated under reduced pres-
sure and the resulting precipitate triturated with diethyl ether
(50 mL). The product was collected by suction ltration and
rinsed with diethyl ether (50 mL ꢃ 3). An X-ray crystal structure
was obtained to conrm counter anion exchange39 and the
HPLC retention time of the precursor (4) was 8.93 min (S4).
The 19FPy-TFP reference standard was prepared as previously
described by Olberg et al.,38 6-uoronicotinic acid (200 mg, 1.4
mmol) was reacted with DCC (439 mg, 2.1 mmol) in THF (4 mL)
for 5 minutes followed by dropwise-addition of 2,3,5,6-tetra-
uorophenol (285 mg, 1.7 mmol) dissolved in THF (2 mL). Aer
stirring overnight, the dicyclohexylurea was ltered off, the
solvent concentrated, and the resulting 19FPy-TFP ester puried
by silica-gel column chromatography in 90%-n-hexanes and
10%-ethyl acetate. The 19FPy-TFP ester reference standard was
characterized per previous literature38 and had an HPLC-
retention time of 13.73 min (S5).
HPLC conditions
All peptides were puried and characterized using C12-RP-
HPLC using a Beckmann Coulter Gold HPLC system equipped
with a 2 mL loop and a C12-Phenomenex-Jupiter 5 mm Proteo 90
˚
A column (250 ꢃ 4.6 mm, 4 mm) with an ultraviolet (UV)
absorbance detector (220 nm) and radioactivity detector (pho-
tomultiplier tube; Bioscan) connected in series. Mobile phase
solvents were 0.05% triuoroacetic acid in water (solvent A) and
100% acetonitrile (solvent B) with a gradient starting from 100%
solvent A to 10% solvent B over 2 min, then to 90% solvent B
over 10 min, held at 90% B for 5 min, then returned to 100%
solvent A over 5 min, held at 100% solvent A for 3 min with
a ow rate of 1.5 mL minꢀ1
.
PyTFP-precursor (4) and 19FPy-TFP reference standard
synthesis
The precursor N,N,N-trimethyl-5-((2,3,5,6-tetrauorophenoxy)
carbonyl)pyridine-2-amminiumuoro-methanesulfonate
(4,
Scheme 1) was synthesized using the slightly modied proce-
dure as described by Olberg et al.38 6-Chloronicotinic acid (5.2 g,
33.0 mmol) was activated with N,N-dicyclohexylcarbodiimide
(DCC, 10.22 g, 49.51 mmol) in THF (100 mL) for 5 minutes
followed by dropwise addition of 2,3,5,6-tetrauorophenol
(6.58 g, 39.61 mmol) in THF (35 mL) and reacted overnight. The
Peptide synthesis of YGGFL (1)
precipitate of N,N-dicyclohexylurea was removed by ltration The NH2-YGGFL peptide (1, Fig. 1) was assembled on rink
and the reaction contents concentrated, washed with a satu- amide resin using traditional solid-phase
rated solution of sodium bicarbonate in dichloromethane, uorenylmethyloxycarbonyl (Fmoc)-chemistry with reiterative
concentrated under reduced pressure and puried by silica-gel couplings (1.5 h) with Fmoc-amino acids (3 equiv.), HATU (2.8
chromatography with a gradient solvent system starting from equiv.) and DIPEA (6 equiv.) and Fmoc removal (2 ꢃ 15 min)
10%-ethyl acetate in n-hexanes increasing to 50%-ethyl acetate with 20%-piperidine in N,N-dimethylformamide (DMF,
1
in n-hexanes. The resulting ester (7.8 g, 25.57 mmol) was dis- mL).40,41 Coupling and deprotection was monitored with a picryl
solved in THF (100 mL), stirred vigorously while trimethylamine sulfonic acid colorimetric test.41 Aer assembly, the peptide was
gas was bubbled through the reaction for 3 hours and stirred globally deprotected and cleaved from the resin using tri-
overnight. The reaction contents were then concentrated and uoroacetic acid/triisopropylsilane/water (TFA/TIPS/H2O, 95/
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Scheme 1 (A) Radiolabeling of peptides with [18F]FPy-TFP (5). (B) Structures of radiolabeled [18F]FPy-peptides (6–8). Reagents and conditions: (a)
[
18F]F, K222, K2CO3, 4 : 1 tBuOH : MeCN, 40 ꢄC, 10 min or (b) [18F]F, TBA-HCO3, 4 : 1 tBuOH : MeCN, 40 ꢄC, 10 min. (c) Peptide (1 mg), DMSO,
DIPEA, 40 ꢄC, 15 min.
2.5/2.5, v/v/v, 1 mL, 3 h) and puried by HPLC. The puried was 9.52 min (S11) and the HR-ESI-MS m/z: [M + H]+ calc'd for
peptide (NH2-YGGFL (1)) was characterized by HPLC and high
resolution electrospray ionization mass spectrometry (HR-ESI-
C
34H41FN7O7 678.3046; found 678.3048 (S12).
cRGDyK(19FPy). Following previously reported protocol,42
MS) in positive mode on an Orbitrap (Thermo-Fisher Scien- cRGDyK was synthesized and cyclized on resin preloaded with
tic, San Diego, CA, USA). The HPLC retention time was the aspartic acid (D), connected via its side-chain and pro-
7.48 min (S9) and the HR-ESI-MS m/z: [M + H]+ calc'd for tected as the allyl ester for orthogonal deprotection and
C
28H39N6O6 555.2926; found 555.2906 (S10).
cyclization. The sidechain N3-amine of the lysine residue was
protected with 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-
methylbutyl (ivDde) protecting group, that was removed with
hydrazine aer cyclization. The cRGDyK peptidyl resin was
coupled with 6-uoronicotinic acid (10 mg) by reaction with
HATU (10 mg) and DIPEA (50 mL) in DMF (1 mL) for 1.5 h and
cleaved from the resin with TFA/TIPS/H2O (95/2.5/2.5, v/v/v, 1
mL, 3 h). The reference standard cRGDyK(19FPy) was HPLC
puried and characterized by HPLC and high resolution
electrospray ionization mass spectrometry (HR-ESI-MS) in
positive mode on an Orbitrap (Thermo-Fisher Scientic, San
Diego, CA, USA). The retention time of cRGDyK(19FPy) was
7.30 min (S16) and the HR-ESI-MS m/z: [M + H]+ calc'd for
19FPy-peptide reference standards
19FPy-NH-YGGFL-CONH2. The 19FPy-YGGFL reference stan-
dard was prepared using peptidyl resin (25 mg). The N-terminal
Fmoc was removed by 20% piperidine in DMF (2 ꢃ 15 min) and
6-uoronicotinic acid (10 mg) added via activation with HATU
(10 mg) and DIPEA (25 mL) in DMF (1 mL). The peptidyl resin
was allowed to react for 1.5 h before being rinsed with DMF (3ꢃ)
followed by methanol (3ꢃ) and dried on the lyophilizer (1 h).
The peptide was globally deprotected and cleaved from the resin
with TFA/TIPS/H2O (95/2.5/2.5, v/v/v, 1 mL, 3 h) and puried by
HPLC and characterized by both HPLC and high resolution
electrospray ionization mass spectrometry (HR-ESI-MS) in
positive mode on an Orbitrap (Thermo-Fisher Scientic, San
Diego, CA, USA). The retention time of [19F]FPy-YGGFL-CONH2
C
33H44FN10O9 743.3271; found 743.3269 (S17).
Pyr-QK(19FPy)LGNQWAVGHLM. The bombesin peptide (Pyr-
QKLGNQWAVGHLM (3, Fig. 1), 0.25 mg) was reacted in solution
DMF (200 mL) with HATU (5 mg), DIPEA (25 mL), and 6-uo-
ronicotinic acid (5 mg) for 2 h. The peptide was then puried by
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HPLC and characterized by HPLC and matrix assisted laser pressure etc.) can be dened, was created for the synthesis of the
desorption ionization time of ight mass spectrometry (MALDI- [18F]FPy-peptides (6–8) from [18F]uoride via conjugation with
TOF-MS) with a matrix of sinapinic acid (Sigma Aldrich, St. [18F]FPy-TFP (5) (S1†). The ELIXYS radiosynthesizer was
Louis, MO, USA). The HPLC retention time of Pyr-QK(19FPy) congured with two identical cassettes for each 18F-peptide
LGNQWAVGHLM was 8.40 min (S21) and the MALDI-TOF-MS radiosynthesis that were connected via an external gross dilu-
m/z: [M + H]+ calc'd for C77H113FN23O19S 1714.8282; found tion vessel (25 mL-pear shaped round bottom sealed with rubber
1715.1168 (S22).
septum) to enable the high volume dilution (15 mL) required for
trapping of [18F]FPy-TFP (5) on the SPE-cartridge. This dilution
vessel was connected inline between reactors 1 and 2 and posi-
tioned before the purication MCX-cartridge. A small stir-plate
was placed under the dilution vessel to enable stirring of the
reaction mixture during the dilution step. The rst cassette was
joined from the “T” position on cassette 1 into the gross dilution
vessel containing an outlet line composed of a long stainless steel
syringe needle (150 mm) positioned at the bottom of the dilution
ask leading out of the ask with the SPE-cartridge inline that
feeds back into cassette 1 at the “C2-O” position, allowing for the
dilution volume and SPE-washes to be directed to the waste.
Cassette 1 was attached to cassette 2 from the “O” position of
cassette 1 into the “I-1” position on cassette 2.
Description of ELIXYS FLEX/CHEM® radiosynthesizer set up
All radiosyntheses were carried out on an ELIXYS FLEX/CHEM®
radiosynthesizer (Fig. 2). The ELIXYS FLEX/CHEM® radio-
synthesizer has three identical, independent reactors that
support heating and cooling (room temperature to 200 ꢄC) with
stirring capabilities. The reactors move in the Y, Z-direction to
engage with a universal disposable reagent cassette, which can
hold up to 12 different reagents (36 for full system) of 3 mL
volume in disposable septum sealed vials. The disposable
cassette houses all reagents paths, and tubing assemblies for
routing liquid, including two external addition ports, and an
inlet for delivery of aqueous uoride-18. Additionally, the
underside of the cassette mates with the reactor v-vial at various
positions for robot arm-assisted reagent addition (ADD
REAGENT, 2 positions), gas and vacuum assisted evaporation
(EVAPORATE, 1 position), fully-sealed reactions (REACT, 2
ELIXYS FLEX/CHEM® radiosynthesizer synthesis of [18F]FPy-
TFP (5) and radiolabeling of peptides
Synthesis of [18F]FPy-TFP (5). A solution of [18F]uoride in O-
positions, supporting up to 150 psi),43 and transfer of contents 18 water (ꢂ1 to 2 mL, 6–10 mCi) was delivered into the system
out of the reactor v-vial to various other positions in the using 5 psi positive nitrogen gas pressure and trapped onto
synthesizer (TRANSFER, 1 position).
a PS-HCO3 anion exchange cartridge. The radioactivity was
The reactor v-vial is monitored at all times by a live camera eluted from the cartridge into reactor 1 with either (i) kryptox
feed displayed on the graphical user interface of the soware. (K222, 20 mg) and potassium carbonate (K2CO3, 4 mg) in 94 : 6
Likewise, radioactivity within the system is monitored by radi- acetonitrile : water (MeCN : H2O, 2 mL), or (ii) 0.075 M tetra-
ation sensors positioned throughout the system. The reagents, butylammonium bicarbonate (TBA-HCO3, 300 mL, ABX, GmBH,
their location, and the cassettes used are listed in Table 1 for the Radeburg, Germany) solution in 94 : 6 MeCN : H2O (2 mL)
automated peptide radiolabeling with [18F]FPy-TFP (5). The (reagent cassette 1, position 1). Aer azeotropic drying with
synthesis program (“sequence”), consisting of individual “unit acetonitrile (1 mL ꢃ 2, reagent cassette 1, positions 2 and 3)
operations” wherein specic conditions (temp., inert gas under vacuum and constant stirring, the precursor 4 (10 mg,
Fig. 2 (A) ELIXYS FLEX/CHEM® radiosynthesizer (B) zoom-in of a reagent cassette above one of the reactors containing the v-vial.
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Table 1 List of reagents in each reagent cassette of the ELIXYS FLEX/CHEM® radiosynthesizer
Cassette #
Reagent position
Reagent
222/K2CO3(20 mg/4mg) in 94 : 6 MeCN : H2O (2 mL)a
1
1
1
1
1
1
1
2
1
2
3
4
5
6
7
1
K
1.0 mL MeCN
1.0 mL MeCN
[
18F]Py-TFP precursor (4, 10 mg) in 4 : 1 tBuOH : MeCN (1 mL)
2.5 mL water
2.5 mL water
3.0 mL MeCN
Peptide (1 mg) in 0.5 mL DMSO and 10 mL DIPEA
a
TBA-HCO3 (0.075 M, 300 mL) in 94 : 6 MeCN : H2O (2 mL) was also used to elute uoride-18 as another alternative with both methods providing
identical results.
Scheme 1), dissolved in 4 : 1 tBuOH : MeCN (1 mL) (reagent C18SepPak-MCX), eluted in acetonitrile, analysed by analytical
cassette 1, position 4), was added to reactor 1 and heated to radioHPLC (S7) and identity conrmed by co-injection with
40 C for 10 min with constant stirring. Aer reaction comple- 19FPy-TFP reference standard (S8). In each case, the SPE-
ꢄ
tion, an aliquot (100–200 mL) was taken out of the reaction, cartridges were positioned per Fig. 3.
diluted to 1 mL with solvent A and the crude reaction mixture
analyzed by analytical radio-HPLC (S6).
Synthesis of [18F]FPy-peptides (6–8). Peptides (NH2-YGGFL
(1), cRGDyK (2), or Pyr-QKLGNQWAVGHLM (3), 1.0 mg, 1.8
Purication of [18F]FPy-TFP (5). Aer formation of [18F]FPy- mmol, 1.6 mmol, and 0.6 mmol respectively, Fig. 1) were dissolved
TFP (5), the crude reaction mixture was transferred from in 0.5 mL dimethylsulfoxide (DMSO) and DIPEA (10 mL, reagent
reactor 1 into the dilution vessel, pre-loaded with 15 mL of cassette 2, position 1) and added to the dried down MCX-
water; the diluted mixture was then passed through an MCX puried [18F]FPy-TFP (5) in reactor 2 (Scheme 1). The reaction
SPE cartridge, trapping [18F]FPy-TFP (5). The cartridge was then was heated at 40 ꢄC for 15 min with constant stirring. The crude
rinsed with water (5 mL, reagent cassette 1, positions 5 and 6) by reaction mixture was diluted to ꢂ1.2 mL with solvent A and
rst adding water in 2 portions to reactor 1, and then trans- injected remotely onto an analytical radio-HPLC (S13, S18, and
ferring it into the external dilution vessel and through the S23). The puried radiolabeled peptide was collected, radioac-
cartridge. Similarly, the [18F]FPy-TFP (5) was eluted off the MCX- tivity measured, and then re-injected onto the analytical radi-
cartridge with acetonitrile (MeCN, 3 mL, reagent cassette 1, oHPLC to assess overall yield and purity of nal product (S14,
position 7) (Fig. 3) into reactor 2, where the solvent was removed S19 and S24). The molar activity was determined by injection of
in preparation for peptide coupling. During method develop- known amounts of each of the nonradioactive 19FPy-peptide
ment, various preconditioned SPE-cartridges (C18 SepPak-Plus reference standard and comparing the HPLC integration of
cartridge (Waters) and Oasis MCX-Plus cartridge (Waters) and the UV-peak (220 nm) to the associated UV-peak (220 nm) of the
their respective combinations) were explored for the purica- radioactive peak of the corresponding [18F]FPy-peptide (6–8).
tion of [18F]FPy-TFP (5). The diluted crude [18F]FPy-TFP (5) The identity of all radiolabeled [18F]FPy-peptides (6–8, Scheme
reaction mixture was trapped on four different SPE-cartridge 1) was conrmed by co-elution with non-radioactive 19FPy-
combinations (MCX, C18SepPak, MCX-C18SepPak, and peptide reference standards (S15, S20 and S25).
Results
Synthesis of [18F]FPy-TFP (5) on ELIXYS FLEX/CHEM®
radiosynthesizer
[18F]FPy-TFP (5) was generated in decay-corrected yields of 76.1
ꢁ 3.5% (n ¼ 6) aer MCX-cartridge purication with a synthesis
time of 42 ꢁ 6 minutes (Scheme 1). Radiouorination utilized
10 mg of the PyTFP-precursor (4), heated with [18F]uoride for
ꢄ
10 minutes at 40 C in tBuOH : MeCN (4 : 1, v/v). Purication
was achieved using a single MCX-SPE-cartridge; an external
gross dilution vessel was required to sufficiently dilute the
crude reaction mixture prior to trapping on the SPE, however no
manual intervention was required. Identical yields of [18F]FPy-
Fig. 3 Diagram of gross dilution set-up. Set-up allows [18F]FPy-TFP (5)
TFP (5) were obtained using either K2.2.2 (10 mg mLꢀ1) and
purification via a 15 mL water dilution in an external flask, trapping on
an Oasis MCX cartridge followed by a 5 mL water wash and 3 mL
acetonitrile elution. All operations are fully automated via ELIXYS FLEX/
K2CO3 (2 mg mLꢀ1) in a mixture of MeCN and H2O (94 : 6, v/v; 2
mL), or TBAHCO3 (300 mL of a 0.075 M solution) in the same
CHEM® radiosynthesizer and no manual interventions required.
solvent system.
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corresponding to the radiolabeled [18F]FPy-peptide (Fig. 6A,
peak A) and hydrolyzed
Purication of [18F]FPy-TFP (5)
[
18F]FPy-TFP (5) to 6-[18F]uo-
Purication of [18F]FPy-TFP (5) was required to remove excess
unreacted PyTFP-precursor (4) and to limit formation of by-
product 2,3,5,6-tetrauorophenyl-6-(2,3,5,6-tetrauorophenoxy)
nicotinate (11) (Fig. 4).38 Both PyTFP-precursor (4) and by-
product (11) can compete with [18F]FPy-TFP (5) for peptide
conjugation, hindering the radiochemical yield of [18F]FPy-
peptides. The purication of [18F]FPy-TFP (5) was explored
using various SPE cartridge(s) (C18 SepPak-Plus cartridge, Oasis
MCX-Plus cartridge, and combinations of the two) by moni-
toring UV impurities by HPLC. The crude HPLC-chromatogram
of the [18F]FPy-TFP (5, Fig. 5A, peak :) showed large amounts of
unreacted PyTFP-precursor (4, Fig. 5A, peak *), along with
2,3,5,6-tetrauorophenol (9, Fig. 5B, peak -), no by-product
(11) was observed as previously by Olberg et al.38 Purication
via MCX-cartridge removed excess PyTFP-precursor (4) (Fig. 5B),
with the only remaining impurity being the UV-impurity of
2,3,5,6-tetrauorophenol (9, Fig. 5B, peak -). Further attempts
to improve the purication using combinations of C18 SepPak-
Plus and Oasis MCX-Plus cartridges in series, did not afford
higher purities than the MCX-cartridge alone. In fact, all dual-
cartridge purication systems examined, led to a higher
percentage of hydrolysis of [18F]FPy-TFP (5, Fig. 5C, peak :), as
demonstrated by increased amounts of 6-[18F]uoronicotinic
acid (Fig. 5C, peak C). In summary, a single Oasis MCX-Plus
cartridge provided the optimal purication and had the best
elution efficiencies (>80%) with minimal hydrolysis of [18F]FPy-
TFP (5) to 6-[18F]uoronicotinic acid.
ronicotinic acid (Fig. 6A, peak C). Fig. 6B is a representative
HPLC chromatogram of puried radiolabeled [18F]FPy-YGGFL
(6) peptide's co-injected with its 19FPy-YGGFL peptide refer-
ence standard (S15, S20, and S25), showing that all radiolabeled
peptides could be puried to >99% radiochemical purity.
Discussion
The unique properties of receptor-targeting peptides, such as
their high target affinity, rapid clearance from the circulation,
and relative ease of synthesis, has made them attractive agents
for both receptor imaging and targeted radiotherapy.3,4 With
favorable nuclear and physiochemical characteristics, 18F-
radiolabeled receptor-targeting peptides have shown great
promise as imaging agents in oncology. However, there remains
an unmet need to develop a reliable, reproducible, fully auto-
mated approach that is amenable to cGMP guidelines and can
be applied to a wide range of peptides.4 Therefore, the goal of
this work was to develop a fully automated radiolabeling
protocol to generate 18F-peptides from [18F]uoride requiring
no manual user interventions, using [18F]FPy-TFP and the
ELIXYS FLEX/CHEM® radiosynthesizer.
The [18F]FPy-TFP prosthetic group is an activated ester
prepared in a single step from [18F]uoride with a single
cartridge based purication, and can be coupled to any peptide
bearing a free amine, either at the N-terminus or at the N3 on
a lysine residue in the peptide sequence.38 To date, the batch
production of [18F]FPy-TFP and subsequent peptide radio-
labeling has been carried out using manual synthesis tech-
niques.38,44–46 Although Olberg, Malik, and others have
Synthesis of [18F]FPy-peptides (6–8) via ELIXYS FLEX/CHEM®
radiosynthesizer
Coupling of peptides (1–3, Fig. 1) to puried [18F]FPy-TFP (5) demonstrated the effectiveness of the [18F]FPy-TFP prosthetic
was achieved via reaction with 1 mg of peptide in 0.5 mL DMSO group for radiolabelling various peptides, intermediate puri-
and 10 mL of DIPEA at 40 ꢄC for 15 min. Puried [18F]FPy- cation of the [18F]FPy-TFP was required prior to conjugation to
peptides (6–8, Scheme 1) were obtained from [18F]uoride in a peptide in order to remove unreacted PyTFP-precursor that
a total synthesis time of 97 ꢁ 3 minutes, and with decay cor- interferes with peptide radiolabeling.38,44–46 In addition, Olberg
rected yields (including 25 min HPLC purication) ranging et al. showed that hydrolysis of the PyTFP-precursor resulted in
between 13–26% (n ¼ 4). Radiochemical purities were high formation
of
2,3,5,6-tetrauorophenyl-6-(2,3,5,6-
(>99%, Table 2), and molar activities ranged from 1–5 Ci tetrauorophenoxy)nicotinate (by-product (11)), which can
mmolꢀ1. The crude radioHPLC chromatogram for each peptide hinder efficient radiolabeling of the peptide.38 To facilitate
(S13, S18 and S23) showed only two radiochemical peaks, purication, the ELIXYS FLEX/CHEM® radiosynthesizer can
Fig. 4 Mechanism for by-product (11) formation during [18F]FPy-TFP (5) synthesis.
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Fig. 5
[
18F]FPy-TFP (5) HPLC-chromatograms (PMT (gamma)-red, UV (220 nm)-green). (A) Crude HPLC chromatogram (peak C-6-[18F]fluo-
ronicotinic acid, peak *-PyTFP-precursor (4), peak --2,3,5,6-tetrafluorophenol (9), peak :-[18F]FPy-TFP (5)). (B) HPLC chromatogram after
MCX-cartridge purification (peak C-6-[18F]fluoronicotinic acid, peak --2,3,5,6-tetrafluorophenol (9), peak :-[18F]FPy-TFP (5)). (C) HPLC
chromatogram after MCX-C18SepPak double cartridge purification (peak C-6-[18F]fluoronicotinic acid, peak --2,3,5,6-tetrafluorophenol (9),
peak :-[18F]FPy-TFP (5)). (D) Purified HPLC-chromatogram co-injected with [19F]FPy-TFP (peak :-[18F]FPyTFP (5)).
Table 2 Summary of [18F]Py-TFP (5) radiolabeling yields of peptides
corrected yields of 35 ꢁ 8% (98 min), 69 ꢁ 8% (78 min), and 44
(1–3)
ꢁ 9% (71 min) respectively, using the ELIXYS FLEX/CHEM®
radiosynthesizer.32
Radiochemical
purity
Radiolabeling of peptides with [18F]FPy-TFP were previously
Peptide
Amount
% Yielda
performed manually using a variety of peptide coupling condi-
tions.38,44–46 The original manual peptide radiolabeling with
6
7
8
1.0 mg, 1.8 mmol
1.0 mg, 1.6 mmol
1.0 mg, 0.6 mmol
25.6 ꢁ 2.7
16.0 ꢁ 5.6
13.0 ꢁ 5.2
>99%
>99%
>99%
[18F]FPy-TFP by Olberg et al. utilized
a
1 : 1 : 1
DMSO : MeCN : phosphate buffer (pH-9.0), 3–5 mg of peptide,
and a reaction temperature of 40 ꢄC for 15 minutes to generate
the [18F]FPy-peptide in a 22.5 ꢁ 6.0% yield (total synthesis time
¼ 85 minutes).38 Malik et al. manually radiolabeled a DUPA-
peptide (2 mg) with [18F]FPy-TFP in 5 : 3 MeCN : H2O, con-
taining 5.5 mg of sodium bicarbonate as a base, at 60 ꢄC for 10
minutes, resulting in a 48 ꢁ 0.9% non-decay corrected yield
(total synthesis time ¼ 50 minutes).44 Basuli et al. manually
reacted 3–5 mg of peptide at 50 ꢄC for 10 minutes with [18F]FPy-
TFP in a 2.75 : 2.5 : 1 mixture of MeCN : H2O : tBuOH contain-
ing either 10–15 mg of sodium bicarbonate or 5 mL of triethyl-
amine, generating the 18F-peptide in non-decay corrected yields
of 25–43% (total synthesis time ¼ 30 minutes).45,46 To avoid
solubility issues and the potential to clog lines and needles of
the automated system, DIPEA was used over sodium bicar-
bonate as the base. DMSO was selected as a solvent, as previous
literature suggested that peptide conjugation in buffers seemed
to result in lower yields.36 Using these conditions, acceptable
radiochemical yields (13–26%) were achieved using minimal
amounts of peptide (1 mg).
a
Radiochemical decay corrected yields from [18F]uoride of puried
radiolabeled peptide reported as the average yield ꢁ SD (n ¼ 4).
accommodate SPE-cartridges in multiple congurations,
enabling intermediate cartridge based purications within the
reaction sequence. However, in order to purify [18F]FPy-TFP, the
initial organic reaction mixture required dilution with 15 mL
water prior to trapping on the SPE-cartridge. To achieve this
a pre-lled external vessel was incorporated upstream of the
Oasis MCX-SPE-cartridge, with minimal modications to the
ELIXYS FLEX/CHEM® radiosynthesizer system. As a result, [18F]
FPy-TFP was produced with average yields of 76.1 ꢁ 3.5% and
sufficiently high radiochemical and chemical purities. These
yields are comparable to other automated 18F-prosthetics group
productions used for peptide radiolabeling. For example, the
automated synthesis of [18F]FBEM on an Eckert & Ziegler
module took 95 minutes to afford a 17.3 ꢁ 7.1% non-decay
corrected yield.47 The automated synthesis of [18F]SFB on
a TRACERlab FXFN synthesizer (GE Healthcare) produced non-
decay corrected yields ranging from 25–35% in 40 minutes.48
Thonon, et al. also automated the synthesis of [18F]SFB to yield
42% decay corrected yield in 57 minutes on a FASTLab™ (GE
Healthcare).36 Other 18F-prosthetics such as [18F]FBEM, [18F]
SFB, and [18F]F-TCO were previously produced with decay
The radiolabeling yields for YGGFL using the ELIXYS FLEX/
CHEM® radiosynthesizer were 25.6 ꢁ 2.7% and were compa-
rable to the previously reported radiolabeling yield of YGGFL
with [18F]FPy-TFP using the Advion NanoTek microuidic
device, which produced a 26% incorporation yield in 12
minutes.49 However, the microuidics device could not readily
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Fig. 6 HPLC chromatogram of radiolabeled [18F]FPy-YGGFL (6) (PMT(gamma)-red, UV(220 nm)-green). (A) Crude radioHPLC chromatogram
(peak C-6-[18F]fluoronicotinic acid, peak A-[18F]FPy-YGGFL (6)). (B) Purified HPLC chromatogram co-injected with [19F]FPy-YGGFL (peak
A-[18F]FPy-YGGFL (6)).
be used to produce “batch” amounts of [18F]FPy-peptides due to produce radiochemical yields of 31.4 ꢁ 4.6% with a total
practical limitations to scaling up, such as volume and radia- synthesis time of 150 ꢁ 20 minutes from uorine-18.57 Most
tion shielding restrictions. The radiolabeling of cRGDyK using recently, a kit-radiolabeling approach of a bombesin derivative
the
ELIXYS
FLEX/CHEM®
radiosynthesizer
yielded with [18F]-NOTA-AlF afforded 50–60% radiochemical yields, but
cRGDyK([18F]FPy) in 16.0 ꢁ 5.6% using 1.6 mmol of peptide. required a high temperature of 95 ꢄC.58 The previously reported
Manual radiolabelings of cRGDfK with [18F]FPy-TFP by Basuli, 18F-radiolabelings of bombesin have produced higher yields
et al. afforded an non-decay corrected yields of 32–43% in a total than the presented automated [18F]FPy-TFP radiolabeling of
of 30 minutes, but required 3–5 times the amount of peptide.46 bombesin using various radiochemical approaches. The most
Other manual approaches for radiolabeling various cRGD- similar method used was the manual two-step indirect radio-
peptides with uorine-18 have provided yields between 10– labeling approach utilizing a different activated ester [18F]SFB.57
35% with total reaction times ranging between 90–218 This manual [18F]SFB radiolabeling of bombesin57 employed
minutes.50–55 The yield obtained for cRGDyK([18F]FPy) was also a different reaction solvent, concentration, and a reaction time
similar to the previously reported manual solid phase radio- of 30 minutes, double that of [18F]FPy-TFP.
labeling of cRGDyK with 6-[18F]uorobenzoic acid (14 ꢁ 2%
Several groups have attempted to develop automated proto-
yield in 90 minutes).42 Overall, using the ELIXYS FLEX/CHEM® cols for the production of 18F-labeled peptides. One of the most
radiosynthesizer was faster, with a total synthesis time under complete examples reported to date by Thonon et al., used the
a 100 minutes including HPLC purication, and provided FASTLab (GE Healthcare) to produce [18F]SFB via both a one-
similar radiochemical yields.
The radiolabeling yields for [18F]FPy-bombesin using the (PRGD2).36 Higher yields and fewer by-products were observed
ELIXYS FLEX/CHEM® were 13.0
5.2%. Other reports with the 2-pot approach, which generated [18F]PRGD2 in decay-
and two-pot method, followed by coupling to an RGD peptide
ꢁ
describing the synthesis of radiolabeled bombesin analogues corrected yields of 13 ꢁ 3% aer 130 minutes with high
have predominantly used radiometals (64Cu, 68Ga, and 111In) radiochemical purity (>98%) and high molar activity (3873 ꢁ
and chelation chemistry, with only a handful describing the 1081 Ci mmolꢀ1). This semi-automated method included
manual synthesis of 18F-bombesin analogues.56–58 For example, a single manual intervention; the addition of the peptide to the
Lys3-bombesin,
was
radiolabeled
using
4-[18F]uo- FASTLab module.36 Tang et al. also carried out a semi-
18F]SFB was produced on
roazidobutane in a strain promoted copper free 1,3-dipolar automated synthesis wherein
[
cyclization reaction which proceeded at room temperature in 15 a TRACERlab FXFN synthesizer (GE Healthcare) and then
minutes to give a 37% radiochemical yield.56 Zhang et al. re- manually coupled to a cell penetrating peptide in a 30 minutes
ported the radiolabelling of Lys3-bombesin using [18F]SFB in reaction to afford a 10–20% non-decay corrected yields of [18F]
sodium borate buffer (pH ¼ 8.5) at 40 ꢄC for 30 minutes, to SFB-peptide.48 Valdivia et al. generated the [18F]F-pyridyl alkyne
8646 | RSC Adv., 2019, 9, 8638–8649
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prosthetic
[
18F]FPyKYNE and subsequently coupled it to
2 K. Fosgerau and T. Hoffmann, Peptide therapeutics: current
status and future directions, Drug Discovery Today, 2015,
20(1), 122–128.
cRGDyK using a TRACERLab FXFN pro module.59 Following
HPLC-purication, the radiolabeled peptide was obtained in
12–18% decay-corrected yields in 125 minutes with high
radiochemical purities (>99%) and specic activities (980–2000
Ci mmolꢀ1).59 This report, however, uses copper-mediated
prosthetic coupling, which may not be suitable for all applica-
tions and due to the reagent's high toxicity additional puri-
cation maybe required to ensure copper levels are below the
FDA ICH Q3D guidelines. Recently, Inkster et al. introduced
a thiol-targeted [18F]uoro-2-cyanobenzothiazole prosthetic,
which was subsequently coupled to a cysteine-functionalized
cRGDfK peptide using a TRACERLab FXFN pro module.60
Moderate decay-corrected yields of 7 ꢁ 1% were reported aer
124–132 minutes, along with high radiochemical purities and
reasonable specic activities (ꢂ100 to 300 Ci mmolꢀ1).60 Over-
all, ELIXYS FLEX/CHEM® radiosynthesizer radiolabelings
produced similar yields within the same timeframe.
3 M. Fani, H. R. Maecke and S. M. Okarvi, Radiolabeled
peptides: Valuable tools for the detection and treatment of
cancer, Theranostics, 2012, 2(5), 481–501.
4 S. Richter and F. Wuest, 18F-Labeled peptides: The future is
bright, Molecules, 2014, 19(12), 20536–20556.
5 L. Y. Hu, K. A. Kelly and J. L. Sutcliffe, High-throughput
approaches to the development of molecular imaging
agents, Mol. Imaging Biol., 2017, 19(2), 163–182.
6 C. B. Johnbeck, U. Knigge and A. Kjaer, PET tracers for
somatostatin receptor imaging of neuroendocrine tumors:
current status and review of the literature, Future Oncol.,
2014, 10(14), 2259–2277.
7 M. W. Hanson, Scintigraphic evaluation of neuroendocrine
tumors for applied radiology, Appl. Radiol., 2001, 30(6), 11–
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8 W. A. Weber, Positron emission tomography as an imaging
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9 P. Laverman, et al. A novel facile method of labeling
octreotide with 18F-uorine, J. Nucl. Med., 2010, 51(3), 454–
461.
Conclusions
The ELIXYS FLEX/CHEM® radiosynthesizer was specically
designed to support good manufacturing practices (i.e. dispos-
able cassettes, soware with detailed batch record keeping, etc.) 10 M. Gabriel, et al. 68Ga-DOTA-Tyr3-octreotide PET in
for 18F-radiolabeled compounds, ultimately enabling their
rapid translation into the clinic. Until now the fully automated
synthesis of 18F-peptides from uorine-18 has been challenging
neuroendocrine tumors: Comparison with somatostatin
receptor scintigraphy and CT, J. Nucl. Med., 2007, 48(4),
508–518.
in part due to the complexity of synthesis and purication of 11 I. Velikyan, Prospective of 68Ga-radiopharmaceutical
both the intermediate prosthetic and the nal 18F-peptide. This
development, Theranostics, 2013, 4(1), 47–80.
work represents the fully automated batch production of three 12 M. Barrio, et al. The Impact of somatostatin receptor–
[18F]FPy-peptides in a two-step approach from uorine-18 using
the ELIXYS FLEX/CHEM® radiosynthesizer. Minimal amounts
of peptide were required to achieve good radiochemical yields
directed PET/CT on the management of patients with
neuroendocrine tumor: A systematic review and meta-
analysis, J. Nucl. Med., 2017, 58(5), 756–761.
13–26% with high molar activities 1–5 Ci mmolꢀ1 and radio- 13 R. Haubner, D. Finsinger and H. Kessler, Stereoisomeric
chemical purity >99%. In the future addition of the ELIXYS
PURE/FORM®, a HPLC purication module that interfaces with
the ELIXYS FLEX/CHEM® radiosynthesizer will enable the
whole process to be fully automated.
peptide libraries and peptidomimetics for designing
selective inhibitors of the avb3 integrin for a new cancer
therapy, Angew. Chem., Int. Ed., 1997, 36(13–14), 1374–1389.
´
14 F. Danhier, A. Le Breton and V. Preat, RGD-Based strategies
to target avb3 integrin in cancer therapy and diagnosis, Mol.
Pharmaceutics, 2012, 9(11), 2961–2973.
15 J. S. Desgrosellier and D. A. Cheresh, Integrins in cancer:
biological implications and therapeutic opportunities, Nat.
Rev. Cancer, 2010, 10(1), 9–22.
Conflicts of interest
Drs Drake and Moore are employees and owners of SOFIE Co.
16 E. Ruoslahti and M. D. Pierschbacher, New perspectives in
cell adhesion: RGD and integrins, Science, 1987, 238(4826),
491–497.
Acknowledgements
This work was supported by the Office of Science, United States
Department of Energy: (DE-SC0008385). Additional support for 17 R. Haubner and C. Decristoforo, Radiolabelled RGD
C. R. D. and M. M. was received from the National Cancer
Institute (HHSN261201400041C), National Institute of Mental
peptides and peptidomimetics for tumour targeting, Front.
Biosci., Landmark Ed., 2009, 14, 872–886.
Health (2R44MH097271), and National Institute of Biomedical 18 R. Haubner, S. Maschauer and O. Prante, PET
Imaging and Bioengineering (1R43EB023782-01).
radiopharmaceuticals for imaging integrin expression:
Tracers in clinical studies and recent developments,
BioMed Res. Int., 2014, 2014, 1–17.
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