Site-specific addition of an18F-N-methylaminooxy-containing prosthetic group to a vinylsulfone modified peptide

Article abstract of DOI:10.1002/Jlcr.1686

Numerous strategies employing prosthetic groups for the radiosynthesis of ¹⁸F-fluorinated peptides for positron emission tomography have been investigated in recent years. We have previously reported a novel [ ¹⁸F]prosthetic group be

Full text of DOI:10.1002/Jlcr.1686

Research Article
Received 27 April 2009,
Revised 14 June 2009,
Accepted 24 August 2009
Published online 7 October 2009 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/Jlcr.1686
Site-specific addition of an
18F-N-methylaminooxy-containing prosthetic
group to a vinylsulfone modified peptide
Ã
Dag Erlend Olberg,a Ole Kristian Hjelstuen,a,b Magne Solbakken,b
Joseph M. Arukwe,b Knut Dyrstad,b and Alan Cuthbertsonb
Numerous strategies employing prosthetic groups for the radiosynthesis of 18F-fluorinated peptides for positron emission
tomography have been investigated in recent years. We have previously reported a novel [18F]prosthetic group bearing the
N-methylaminooxy functionality capable of reacting in a site-selective manner with peptides functionalized with Michael-
acceptors. In a further extension of this methodology we demonstrate that O-[2-(2-[18F]fluoroethoxy)ethyl]-N-methyl-N-
hydroxylamine, [18F]4, reacts chemoselectively with a vinylsulfone functionalized peptide. The conjugation yields were
studied with respect to reaction time, level of radioactivity, peptide concentration and purity of the [18F]prosthetic group
used in the conjugation reaction. Incubation at 701C gave conjugation yields of around 80% with high radiochemical purity
after 70 min at pH 5 in acetate buffer.
Keywords: [18F]prosthetic group; peptide; PET; conjugation; vinylsulfone
product. Examples include 4-[18F]fluorobenzaldehyde conjuga-
Introduction
tions to peptides bearing either aminooxy or hydrazine
Biomolecules such as peptides, labelled with [18F]fluoride, are
gaining increasing importance as tracers for positron emission
tomography (PET). For example, tracers targeting a variety of
receptor types associated with tumour growth are currently
under evaluation including radiolabelled RGD-peptides for
detection of integrin receptors, vasoactive intestinal peptide
(VIP) derivatives for VIP-receptor detection and octreotide
analogues for visualization of somatostatin-receptor expres-
sion.1
To date, [18F]fluoride is the most widely applied positron
emitting radionuclide for the labelling of peptides mainly due to
its favourable half-life of 110 min and well established produc-
tion method.2 Direct labelling of peptides with [18F]fluoride
typically results in poor incorporation yields and the harsh
conditions required often result in degradation of the peptide
precursor. Peptides are therefore typically labelled in two steps
by first incorporating fluoride-18 into a prosthetic group
followed by conjugation to the peptide precursor under mild
aqueous conditions.3
groups7,8 or copper catalyzed Huisgen 1,3-dipolar cycloaddition
reactions of o-[18F]-fluoroalkynes to peptides carrying an azido-
moiety.9
Recently, our group reported a new [18F]prosthetic group
based on the reactivity of the N-methylaminooxy functionality
with Michael acceptors.10 Here, we report a further extension of
the utility of this prosthetic group using the model peptide
(vinylsulfonyl)acetyl-Lys-Gly-Phe-Gly-Lys 6. It was anticipated
that the introduction of the sulfone moiety would increase the
hydrophilicity of the product compared to our previously
published maleimide and nitrostyrene systems. In this report
we demonstrate effective site-specific conjugation with O-[2-(2-
[18F]fluoroethoxy)ethyl]-N-methyl-N-hydroxylamine, [18F]4, yiel-
ding the [18F]fluoro-peptide conjugate [18F]7 in good yield. The
efficiency of the conjugation step was evaluated with respect to
peptide concentration, level of radioactivity, reaction time and
purity of the prosthetic group.
Several classes of prosthetic groups with different reactivities
have been assessed in recent years. The activated carboxylate
esters of 4-[18F]fluorobenzoate have been well documented in
the literature, and are widely used to label peptides bearing free
amino groups.4,5,6 However, the synthesis of the carboxylate
esters are quite laborious and a peptide carrying more than one
amino group will produce a complex mixture of 18F-fluorinated
products. More recently, generic methodologies have been
described where the prosthetic group is chosen to react site-
specifically with the peptide precursor yielding a single labelled
Supporting Information may be found in the online version of this article.
aDepartment of Pharmaceutics and Biopharmaceutics, University of Tromsø,
Institute of Pharmacy, N-9037 Tromsø, Norway
bGE Healthcare, Medical Diagnostics R&D, P.O. Box 4220, 0401 Oslo, Norway
*Correspondence to: Dag Erlend Olberg, Department of Pharmaceutics and
Biopharmaceutics, University of Tromsø, Institute of Pharmacy, N-9037 Tromsø,
Norway.
E-mail: dag-erlend.olberg@farmasi.uit.no, dag-erlend.olberg@ge.com
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D. E. Olberg et al.
and 2 M HCl in diethyl ether (0.5 mL) into a reaction vessel.
Heating the eluate at 701C under a gentle stream of nitrogen
simultaneously removed excess organic solvent and cleaved the
Boc-group liberating [18F]4. Although some carry over of the
alcohol was expected from this cartridge-based purification, we
had not previously determined accurate concentrations of 3 and
so the true impact on conjugation yield was unknown. In
addition, the efficiency of the solid-phase extraction was a
critical parameter affecting our ultimate aim of running the
radiochemistry on an automated synthesizer. In order to
understand the impact and to set a specification limit for by-
product 3 we decided to develop a preparative HPLC method
for the isolation of pure [18F]2. Using an isocratic reversed-
phase chromatography with 44% acetonitrile in water, all
radioactive and nonradioactive impurities were efficiently
removed before the conjugation step. This allowed the direct
comparison of the conjugation yield of [18F]7 obtained from
pure [18F]2 with the yields from the Oasis HLB SPE process as
shown in Scheme 2.
Results and discussion
Chemistry
The organic synthesis of precursor 1 and the solid-phase
synthesis of the model peptide KGFGK have been described in
detail earlier.10
Radiochemistry
The radiochemical synthesis of prosthetic group [18F]4 as shown
in Scheme 1 was carried out in good yields with high
reproducibility.10 In short, nucleophilic fluorination of the Boc-
protected tosylate precursor 1 (8 mmol) typically gave around
70% [18F]fluoride conversion into [18F]2 in a closed reaction
vessel at 901C for 10 min using acetonitrile as a solvent. A major
by-product produced during the labelling reaction was the
hydrolysis product 3 formed from tosylate 1. We had previously
demonstrated that the removal of this by-product was critical to
achieve acceptable yields in the conjugation step. We therefore
introduced a separation step utilizing solid-phase extraction on
an Oasis HLB Plus Sep-Pak cartridge (225 mg/60 mm), which
appeared to significantly reduce the amount of alcohol carried
over to the conjugation reaction.
After cooling and dilution with water (9 mL), the components
of the reaction mixture containing [18F]2 and 3 could be readily
trapped on the Oasis HLB cartridge. Subsequent washing of the
SPE cartridge with 50 mL of 25% methanol in water was
sufficient to remove unreacted [18F]fluoride as well as the main
bulk of hydrolysis product 3. Labelled compound [18F]2 was
then eluted with a mixture comprising dichloromethane (1 mL)
The pure fraction from the HPLC containing [18F]2 was first
diluted to 10% acetonitrile in water before trapping on a C18
Sep-Pak. After thorough drying of the column using N2-gas,
[
18F]2 was eluted in 1 mL acetonitrile. Addition of 2 M HCl in
diethyl ether (0.5 mL) and evaporation of the eluate at 701C
under a stream of nitrogen simultaneously removed the solvent
and Boc-protection group, liberating free [18F]4.
Conjugation of HPLC purified [18F]2 to peptide 6
Three solutions of the peptide precursor containing 0.5, 2, and
5 mg in 1 mL of 0.4 M acetate buffer pH 5 were added to the
Scheme 1. [18F]Fluorination of precursor 1, Oasis HLB purification and acidolysis.
Scheme 2. Conjugation of the prosthetic group [18F]4 to peptide 6.
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D. E. Olberg et al.
dried residue of [18F]4. The conjugation mixture was heated to reduce the yield from 84% to around 50% after 70 min
701C and aliquots from the reaction mixture were analyzed at incubation.
10, 30, 50 and 70 min. Formation of [18F]7 was monitored by
radio-HPLC and the identity of the product peak confirmed by Conjugation of Oasis HLB purified [18F]4 to peptide 6
co-elution with the nonradioactive standards. Figure 1 shows
In this final set of experiments, the effectiveness of the peptide
the results from a typical set of experiments.
conjugation with [18F]4 preparations from the Oasis HLB SPE-
based purification was studied by employing a mixed level
Conjugation of HPLC purified [18F]2 spiked with alcohol 5
to peptide 6
chemometric design with centre point (see supplemental
information). Variables were peptide precursor 6 concentrations,
levels of starting [18F]fluoride activity and reaction time (Table 1).
In a second set of experiments, the effect on conjugation yield
by spiking the pure HPLC purified [18F]4 preparation with
The conjugation reaction was again carried out at 701C in 0.4 M
known amounts of alcohol 5 was put under investigation. These
experiments were designed to understand the impact of
carryover from the SPE procedure of known concentrations of
5 on conjugation yield. We therefore repeated the previous
experiment using HPLC purified [18F]4 and a peptide concen-
tration of 7.5 mM, but spiked the conjugation mixture with 5 at
concentrations of 1, 4 and 8 mmol. The 8 mmol level corre-
sponded to 3 mg of precursor and represents a scenario where
almost complete hydrolysis of the tosylate precursor 1 has
occurred. The results of the spiked conjugation studies are
shown in Figure 2. In all cases, the conjugation yield was
significantly reduced and even 1 mmol carry over was enough to
acetate buffer of pH 5 and aliquots were removed from the
conjugation mixture at 10, 30, 50 and 70 min followed by the
immediate analysis by radio-HPLC.
The results from the experiment are shown in Figure 3.
Perhaps unsurprisingly the amount of radioactivity used in the
experiment had no significant impact on conjugation yield as
both the 0.75 mM incubations at 200 and 2000 MBq gave
identical kinetics with around 10% conversion at the 70 min time
point. Interestingly the 7.5 mM/2000 MBq sample gave similar
conjugation yield (76%) to the HPLC purified [18F]4 (84%). We
therefore concluded that the Oasis HLB purification was capable
of removing significant amounts of the alcohol 3. The lower
yield observed for the 7.5 mM/200 MBq (63%) may reflect
carryover variation in the manual process, something that
possibly could be eliminated on a fully automated protocol. As
anticipated the 3 mM/1000 MBq sample gave a yield of 38%
clearly demonstrating the dependency of peptide concen-
tration as can be directly read from the response surface plot
in Figure 4.
No degradation of [18F]7 was observed during the experi-
ments. A chromatogram of the conjugation following Oasis HLB
purification of [18F]2 at 7.5 mM peptide after 70min at 701C is
showed in Figure 5. The radiochromatogram (upper trace) shows
the clean conversion of [18F]4 (small peak) to [18F]7 (large peak).
Interestingly, a direct comparison of the HPLC retention time
of [18F]7 with the products from our earlier reported work using
nitrostyrene and maleimide Michael-acceptors revealed the
hydrophilic nature of the vinylsulfone system.10 The peptide
[
18F]7 had a retention time of 8.8 min compared to 10.8 and
Figure 1. Conjugation yield (% product formed) of the HPLC purified [18F]4 with
peptide precursor 6 at 701C in acetate buffer at pH 5. Incubation at 701C for 70 min
gave 84% conversion to product [18F]7 at a peptide concentration of 7.5 mM (ꢀ)
while 40 and 15% yields were obtained at 3 mM ( & ) and 0.75 mM (m),
respectively.
9.5 min for the nitrostyrene and maleimide groups, respectively.
We have previously shown that conjugate hydrophilicity/
hydrophobicity has
a significant impact on the in vivo
biodistribution profile of peptide tracers significantly reducing
liver and increasing kidney excretion rates.7 The vinylsulfone/N-
methylaminooxy prosthetic group combination may therefore
offer a simple approach to enhancing the in vivo characteristics
of peptide tracers.
Experimental
General
All the reagents and solvents were purchased from Acros, Fluka,
or Sigma-Aldrich and were used without further purification.
Mobile phase solvents in all instances were A: water/0.1%
trifluoroacetic acid and B: acetonitrile/0.1% trifluoroacetic acid.
Mass spectra were recorded on a LCQ DECA XP MAX
instrument using electrospray ionization (ESI) operated in a
positive mode coupled to Finnigan Surveyor PDA chromato-
graphy system. Accurate mass spectra were recorded on a QToF-
micro orthogonal acceleration time-of-flight mass spectrometer
Figure 2. Percent incorporation of HPLC purified [18F]4 into peptide precursor 6 at
701C in acetate buffer pH 5 spiked with 1 mmol (m), 4 mmol ( & ) and 8 mmol (~) of 5.
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D. E. Olberg et al.
Table 1. Experimental design setup
Radioactivity (MBq)
1000 (medium)
Peptide conc. (mM)
200 (low)
2000 (high)
0.75 (low)
3 (medium)
7.5 (high)
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Asterisk indicates combination tested. Samples were analysed at 10, 30, 50 and 70 min.
Figure 3. Percent [18F]7 formed from [18F]4 with peptide precursor 6 at 701C at
pH 5 in acetate buffer over time from the combinations from Table 1.
Figure 5. Chromatograms of the crude reaction mixture after 70 min at 701C and a
concentration of 7.5 mM peptide precursor (a): small peak, unreacted [18F]4; large
peak, conjugate [18F]7; (b): corresponding UV trace of the sample at 214 nm.
(44% acetonitrile in water) was performed using the TRACERLab
integrated HPLC system equipped with a Phenomenex Luna
C(18)2 column (150 Â 10 mm2, 5 mm; flow rate 5 mL/min).
Analytical radio-HPLC was performed on an Agilent system
(1100 series) with UV detection (214 and 254 nm) equipped in
series with a g-detector (Bioscan flow-count) on a Phenomenex
Luna C18(2) column (150 Â 4.6 mm2, 5 mm) with a flow rate of
1.0 mL/min using gradient 0–40% B over 15 min. [18F]Fluoride
was produced by a cyclotron (GE PETtrace 6) using 18O(p,n)18F
nuclear reaction with a 16.5 MeV proton irradiation of an
enriched [18O]H2O target.
Figure 4. Response surface plot for time and peptide concentration as variables
for experiments based on Sep-Pak purification presented in Figure 3. Radioactive
level was excluded from the model, since it was not found significant for the levels
studied.
(Micromass UK Ltd.) coupled to an Agilent 1100 chromatogra-
phy system (Agilent Technology) using Leucine Enkephalin as a
reference compound. Preparative HPLC purification was per-
formed on a Phenomenex Luna C18(2) column (250 Â 21.2 mm2,
5 mm) with a flow rate of 10 mL/min using gradient 0–40% B
over 60 min. LC-MS analyses were performed on a Phenomenex
Luna C18(2) column (20 Â 2 mm2, 3 mm) with a flow rate of
0.6 mL/min and gradient 0–30% B over 5 min. UV detection were
at 214 and 254 nm. NMR-spectra were recorded at 251C in 5 mm
tubes on a Bruker Avance III 400 NMR spectrometer in CDCl3
with tetramethylsilane (TMS) as a reference standard. Data
interpretation and regression analysis were done by using the
The Unscrambler software (Ver. 9.2).
Preparation of the K[18F]F-K222 complex
Aqueous [18F]fluoride (1 mL, 200–2000 MBq) was passed
through an anion-exchange resin (Sep-Pak light Waters Accell
Pluss QMA Cartridge, preconditioned with K2CO3). The [18F]fluor-
ide was then eluted from the resin into the TRACERLab reaction
vessel using a solution of Kryptofix-222 (56 mg) and K2CO3
(10 mg) in a mixture of water (215 mL) and acetonitrile (785 mL).
The solution was then concentrated to dryness by heating at
1001C under reduced pressure and a flow of nitrogen for 2 min.
Acetonitrile (0.8 mL) was added and evaporated off as above.
This step was repeated twice.
Labelling and synthesis of the 18F-precursor and peptide
conjugate were performed as described in the results section. Tosyl
precursor 1 and [19F]2 were synthesised as reported previously.10
Radiochemistry was performed using the TRACERLabTM
FxFN (GE Medical Systems) with manual interventions when
required. Semipreparative radio-HPLC using an isocratic system
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D. E. Olberg et al.
peak identified as the target compound. After 60 min at 701C
the reaction was complete. The crude product was purified by
reversed-phase preparative HPLC and isolated by lyophilization
giving reference compound [19F]7 as a white fluffy solid
3 mg (90%). HRMS: found m/z = 805.3951 (MH)1, calcd. for
C34H57FN8O11S m/z = 805.3930.
Synthesis of peptide precursor 6
¨
Vinylsulfonyl acetic acid was prepared according to Schoberl
et al.11 The compound was isolated by normal phase flash
chromatography instead of the original reported distillation
procedure. 1H NMR (400 MHz, CDCl3):d6.90 (dd, Jc = 9.9 Hz,
Jt = 16.6 Hz, 1H), 6.55 (dd, Jg = 0.6 Hz, Jt = 16.6 Hz, 1H), 6.28 (dd,
Jg = 0.6 Hz, Jc = 9.9 Hz, 1H), 4.07 (s, 2H).
Conclusion
Assembly of the resin-bound peptide sequence Lys(Boc)-Gly-
Phe-Gly-Lys(Boc)-OH was done using fully automated synthesis
(ABI 433A synthesis machine) on Fmoc-Lys(Boc)-Sasrin resin
(0.25 mmol) with 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyla-
minium hexafluorophosphate (HBTU) activation. Further deriva-
tization of the peptide to yield peptide precursor 6 was
performed on solid phase using a manual nitrogen bubbler
apparatus (0.1 mmol peptide loading) by activating the (vinyl-
sulfonyl)acetic acid (0.5 mmol) with (7-azabenzotriazol-1-yloxy)-
The vinylsulfonyl modified peptide, (vinylsulfonyl)acetyl-Lys-Gly-
Phe-Gly-Lys-OH, 6 in combination with the prosthetic group
O-[2-(2-[18F]fluoroethoxy)ethyl]-N-methyl-N-hydroxylamine [18F]4
is a useful extension to the toolbox of prosthetic groups in PET
chemistry. Conjugation reactions were found to progress with
high purity, and yields of around 80% were achievable under mild
acidic aqueous conditions at 701C.
tripyrrolidinophosphonium
hexafluorophosphate
(PyAOP)
(0.5 mmol) in presence of diisopropylethylamine (DIEA) 1 mmol
in DMF (5 mL).
Full conversion of the peptide was determined by the Kaiser
test after 2 h. Simultaneous removal of the peptide from the
resin and deprotection of side chain protecting groups was
carried out in trifluoroacetic acid containing triisopropylsilane
and water (95:2.5:2.5 v/v/v). After filtration, the solution was
concentrated under reduced pressure and the residue was
washed with diethyl ether. The crude product was purified by
reversed-phase preparative HPLC and isolated by lyophilization
yielding peptide precursor 6 as a white fluffy solid. Yield 51 mg
(76%). ESI-MS: found m/z 668.4 (M1H)1, calcd m/z 668.3.
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To a conical vial charged with [19F]2 (2 mg, 8 mmol), 0.3 mL of
clean trifluoroacetic acid was added, and the reaction mixture
was left at room temperature for 5 min removing the Boc-group
quantitatively to give [19F]4. Trifluoroacetic acid was removed by
reduced pressure and a solution of 6 (4 mg, 3 mmol) in 0.4 M
sodium acetate buffer of pH 5 (0.8 mL) was added. Progress of
¨
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J. Label Compd. Radiopharm 2009, 52 571–575
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