Translating the concept of peptide labeling with 5-deoxy-5-[ 18F]fluororibose into preclinical practice: 18F-labeling of Siglec-9 peptide for PET imaging of inflammation

Article abstract of DOI:10.1039/c3cc40738a

Peptide glycosylation with 5-deoxy-5-[¹⁸F]fluororibose was translated into preclinical settings. The novel ¹⁸F-labeled Siglec-9 peptide was produced using an automated synthesis procedure. The ¹⁸F-labeled Siglec-9 peptide

Full text of DOI:10.1039/c3cc40738a

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Translating the concept of peptide labeling with
5-deoxy-5-[18F]fluororibose into preclinical practice:
18F-labeling of Siglec-9 peptide for PET imaging of
inflammation†
Cite this: Chem. Commun., 2013,
49, 3682
Received 29th January 2013,
Accepted 15th March 2013
Xiang-Guo Li,*ab Anu Autio,b Helena Ahtinen,b Kerttuli Helariutta,c
DOI: 10.1039/c3cc40738a
Heidi Liljenback,bd Sirpa Jalkanen,e Anne Roivainen*bd and Anu J. Airaksinen*c
¨
www.rsc.org/chemcomm
Peptide glycosylation with 5-deoxy-5-[18F]fluororibose was trans- production of [18F]fluciclatide (GE Healthcare) starting from
lated into preclinical settings. The novel 18F-labeled Siglec-9 4-[18F]fluorobenzaldehyde ([18F]FBA). [18F]Fluciclatide is one of
peptide was produced using an automated synthesis procedure. the few 18F-labeled peptide tracers currently in clinical use.1b
The 18F-labeled Siglec-9 peptide showed favorable binding in the Among the 18F-labeled aldehydes, [18F]FBA is probably the most
animal model of inflammation in vivo.
frequently used prosthetic group for peptide labeling. However,
[18F]FBA is volatile requiring additional measures to ensure
Positron emission tomography (PET) is a sensitive and non- radiosafety and to reduce radioactive emission during the
invasive in vivo molecular imaging modality. Fluorine-18 (18F) production. The attachment of [18F]FBA may cause increased
has favorable physical properties to generate high-resolution lipophilicity, resulting in altered pharmacokinetics in vivo.1b
PET images and consequently 18F-labeled compounds are the In addition to [18F]FBA, aldehydes 1–5 have recently been
most used tracers in clinical PET. Among the short-lived radio- developed for peptide labeling (Fig. 1, [a]).1b,2
nuclides, the physical half-life of 18F (T1/2 = 109.7 minutes)
It has been well documented that glycosylation may confer
allows multi-step synthesis of tracers for the same-day use in favorable characteristics to peptide tracers for in vivo imaging.
hospitals.1 Versatile fluorinating agents have been developed The incorporation of a galactose fragment into the RGD peptide
for the synthesis of fluorine containing pharmaceuticals.1 has generated a very valuable peptide tracer, [18F]galacto-RGD.
However, only a small proportion of the existing fluorination So far, most of the published clinical PET imaging data con-
methods have been (or can be) translated into radiosynthesis of cerning 18F-labeled peptides has actually been generated using
18F-labeled compounds for preclinical and clinical applications.1a this tracer. However, the synthesis procedure of [18F]galacto-
This is especially true in the case of 18F-labeled biomolecules, RGD is rather complex and is a major challenge for its wide-
e.g. peptides, proteins and antibodies. Although many strate- spread use.3 As 2-deoxy-2-[18F]fluoroglucose ([18F]FDG, Fig. 1,
gies for 18F-labeling of peptides and proteins have shown some [b]) is available in virtually every PET radiochemistry lab, it is an
promise during the last two decades, only a few have proved excellent concept to use [18F]FDG for peptide and protein
amenable for routine large scale production for clinical use. labeling.4 [18F]FDG is an aldose and has an open chain aldehyde
Oxime formation between an aldehyde (or a ketone) and an
aminooxy-functionalized compound is a classical and favorable
reaction in bioconjugation due to the high chemoselectivity.
Indeed, oxime formation has been successfully applied for the
a Department of Pharmacology, Drug Development and Therapeutics,
University of Turku, FI-20014 Turku, Finland. E-mail: xiali@utu.fi
b Turku PET Centre, University of Turku and Turku University Hospital,
FI-20521 Turku, Finland. E-mail: anne.roivainen@utu.fi
c Laboratory of Radiochemistry, Department of Chemistry, University of Helsinki,
FI-00014 Helsinki, Finland. E-mail: anu.airaksinen@helsinki.fi
d Turku Center for Disease Modeling, University of Turku, FI-20014 Turku, Finland
e MediCity Research Laboratory and Department of Medical Microbiology and
Immunology, University of Turku, FI-20014 Turku, Finland
† Electronic supplementary information (ESI) available: Radiosynthesis of ribose-
free [18F]FDR 8 and [18F]FDR-Siglec-9 and PET imaging in rats. See DOI: 10.1039/
Fig. 1 18F-Labeled prosthetic groups for oxime formation.18F-Fluorinated
sugars are presented in their cyclic forms.1b,2
c3cc40738a
c
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form in solution.1b However, the conjugation between [18F]FDG This prompted us to derive a strategy to produce [18F]FDR free
and aminooxy-functionalized peptides generally needs elevated of ribose ([18F]FDR 8, Scheme 1, [a]). After the fluorination of
temperature and low pH and forms a complex product profile.1b,4b precursor 6 with K18F-K222, intermediate 7 was isolated using a
Very recently, it has been discovered that 5-deoxy-5-[18F]fluoro- preparative HPLC column. The method gave an excellent
ribose ([18F]FDR, Fig. 1, [b]) conjugates to peptides much more separation between the starting material 6 (retention time =
efficiently than [18F]FDG does.5 Although we were able to produce 10.6 min) and intermediate 7 (retention time = 4.9 min) in a
[18F]FDR in large scale in an automated procedure, we were not short HPLC protocol. Thus, the synthesis procedure was not
able to perform the conjugation step efficiently enough for significantly prolonged. The HPLC analysis of the isolated 7
preclinical PET studies. Furthermore, the quality of radiolabeled proved the success of the intermediate purification step (ESI†).
peptides was not acceptable (e.g. low chemical purity) for in vivo After the acid-catalyzed hydrolysis of 7, [18F]FDR 8 was obtained
animal studies. Thus, peptide glycosylation with [18F]FDR in high radiochemical purity (>98%). Subsequently, the pH of
remained at a conceptual stage with respect to preclinical and [18F]FDR 8 solution was adjusted to 4.6 at which the efficiency
clinical utilities. In this communication, we report the trans- of peptide conjugation was optimal. The total preparation time
lation of [18F]FDR-based bench chemistry into practical pre- of the high purity [18F]FDR 8 was typically 85 min starting from
clinical use, and exemplify this utility on PET imaging of end-of-bombardment (EOB), including a concentration step of
vascular adhesion protein 1 (VAP-1), which is a promising the [18F]FDR solution before the conjugation reaction. In addi-
new target protein for PET imaging of inflammation.
tion to 6, 1,2,3-tri-O-acetyl-5-O-tosyl ribose (AcRib, ESI†) was
In the previous work, [18F]FDR was prepared in high radio- tested as a precursor to produce [18F]FDR 8. Unfortunately, only
chemical purity (>98%) from precursor 6 (Scheme 1, [a]), and residual amount of [18F]FDR 8 was obtained from AcRib. In this
the synthesis was scaled up to 4 GBq of radioactivity. However, respect, compound 6 was a very applicable precursor in radio-
chemical purity of the synthesized [18F]FDR was not optimal synthesis (n = 40). The next step was to prepare VAP-1 targeting
and the final product contained ribose in a mM-range, originat- [18F]FDR-Siglec-9 peptide for in vivo imaging of inflammation
ing from precursor 6. Subsequently, only a small proportion of in rats (Scheme 1, [a] and [c]). VAP-1 is a 90 kDa glycoprotein
the obtained [18F]FDR (8–20 MBq) was conjugated to model and is a unique target in inflammation.6 Inflammation is
peptides. In the presence of an appropriate amount of peptides highly relevant in major diseases including cancer, diabetes,
(e.g. at 23 mM of concentration), the conjugation reached obesity and Alzheimer’s disease.6b In endothelial cells, VAP-1
completion in 10 minutes in sodium acetate buffer (pH 4.6) stays in the intracellular storage granules under normal
at rt.5 Scaling up the conjugation reaction was hampered due to physiological conditions. Upon inflammation, VAP-1 relocates
the high ribose content of the produced [18F]FDR. When rapidly to the endothelial cell surface. This makes VAP-1 an
starting with 100 MBq-4 GBq of [18F]FDR, only 5% conversion ideal target for diagnostic imaging of inflammation. Recently,
yields were achieved in an hour, even at high concentrations of it has been discovered that sialic acid-binding Ig-like lectin 9
peptide (e.g. 40 mM). Since only nano- or picomoles of non- (Siglec-9) is a leukocyte ligand of VAP-1 and 68Ga-labeled
carrier added and high specific radioactivity [18F]FDR was used, Siglec-9 motif peptide can be used as a PET tracer for in vivo
the ribose was present in the conjugation mixture in a large imaging of inflammation and cancer.7 Accordingly, peptide 9
excess. Its presence as a competing aldehyde source resulted (ESI†) was designed in order to enable conjugation to [18F]FDR
in the inefficient conjugation of [18F]FDR (Scheme 1, [b]). 8 in this work.
In general, oxime formation is not the most efficient
chemical reaction and the substrates need to be used at
relatively high concentrations in order to reduce the reaction
time. Specific radioactivity of the cyclotron produced 18F is high
and the amount of the synthesized [18F]FDR 8 is in a nano- to
picomolar level. Following the reported conjugation procedure,5
the initial tests for the synthesis of [18F]FDR-Siglec-9 peptide
were carried out in sodium acetate buffer (pH 4.6, 90 mM) at rt.
However, the concentration of peptide 9 used (2.1 kDa) had to
be at over 15 mM (32.2 mg mlÀ1), which was absolutely not
applicable. In a previous study, aniline was used to catalyze the
oxime formation between [18F]FBA and leptin.8 Accordingly, we
tested aniline-catalysis in the conjugation reactions between
5-deoxy-5-fluororibose ([19F]FDR) and 9 (Table 1). Being the
‘‘cold’’ counterpart of [18F]FDR 8, [19F]FDR was used to prepare
[19F]FDR-Siglec-9, which was needed as a reference in radio-
synthesis of [18F]FDR-Siglec-9. In anilinium buffer (0.3 M,
pH 4.6), the conjugation efficiency was dramatically enhanced
(Table 1, entries 1 and 2). Aniline has been proposed to catalyze
oxime formation with a rapid transimination mechanism.1b
The input concentration of peptide 9 could be as low as 0.3 mM
Scheme 1 Synthesis and preclinical utilization of [18F]FDR-Siglec-9.
c
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Table 1 The influence of aniline on conjugation efficiency of [19F]FDR to peptide
9 in 10 min of reaction (pH 4.6, rt)
decay-corrected radiochemical yield was 27% starting from
18F-fluoride (EOB). The two HPLC-purifications were well inte-
grated in the protocol and were performed with remote-control.
Both radiation exposure and radioactive emission were mini-
mal. The synthesis was scaled up to 1.1 GBq of the final product
[18F]FDR-Siglec-9. The specific radioactivity was 36–43 GBq mmolÀ1
at the end-of-synthesis (EOS) (ESI†). [18F]FDR-Siglec-9 was
formulated in phosphate-buffered saline (PBS) for intravenous
injection. In PBS, [18F]FDR-Siglec-9 was stable for >4 hours and
thus suitable for in vivo imaging studies. The [18F]FDR-Siglec-9
(18.3 Æ 5.1 MBq) was intravenously injected into rats having
sterile, turpentine oil induced inflammation.7 Dynamic PET
imaging lasting for 1 hour was performed by using a High
Resolution Research Tomograph (Siemens). The inflammation
focus on the right shoulder area of the rats (n = 8) was clearly
visualized (Fig. 2). The target-to-muscle ratio was 9 : 1. The
observed liver uptake and rapid excretion through kidneys
to the urine were in line with our previous studies with
[68Ga]-DOTA-Siglec-9.7 Very importantly, bones were not visua-
lized, which was an indication that there was no in vivo defluori-
nation of the 18F-label on the prosthetic group [18F]FDR 8.
In conclusion, we have successfully translated [18F]FDR-based
oxime formation into the production of [18F]FDR-Siglec-9 peptide
for preclinical use. [18F]FDR-Siglec-9 has been produced in high
radiochemical quality and sufficient specific radioactivity facili-
tating in vivo PET imaging of experimental inflammation.
Further development of the tracer for clinical use is warranted.
[18F]FDR-conjugation is independent of peptide sequence and
the whole synthesis procedure can be automated. Thus,
[18F]FDR-based glycosylation should be generally applicable in
the development of peptide PET tracers.
Concentration of
[
19F]FDR and 9a (mM) Buffer (M)
Conversionb
(%)
Entry
1
2
3
4
5
0.3
0.3
0.3
0.3
0.1
Sodium acetate (0.3)
Anilinium acetate (0.3)
Anilinium acetate (0.2)
Anilinium acetate (0.05) 63
Anilinium acetate (0.3)
5
95
89
48
a
b
The two reagents were used in equal amounts. Conversions were
determined using HPLC.
We thank the Academy of Finland (no. 133127, 136805,
119048 and 258814) for financial support. The study was
conducted within the Finnish Centre of Excellence in Molecular
Imaging in Cardiovascular and Metabolic Research supported
by the Academy of Finland, the University of Turku, the Turku
Fig. 2 Representative sagittal (left), transaxial (middle) and coronal (right)
multiplane PET images of [18F]FDR-Siglec-9 biodistribution in a rat. The images
are summation from 10–60 min post-injection.
(0.6 mg mlÀ1) and the conversion was 95% in 10 min at rt. The University Hospital and the Åbo Akademi University.
concentration of the anilinium buffer also had an influence on
the conjugation efficiency (entries 2–4). Our results together
with the previous work demonstrated that aniline-catalyzed
oxime formation is chemoselective in the presence of any
Notes and references
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A general challenge in developing peptide PET tracers is to
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peptide precursors may compete with the tracer on binding
to the target protein or even saturate the target to be imaged.
Bearing this in mind, we developed a reproducible HPLC
method for efficient separation of [18F]FDR-Siglec-9 from 9 in
a preparative scale (ESI†). Finally, [18F]FDR-Siglec-9 was pre-
pared from the conjugation of [18F]FDR 8 to 9 (0.3 mM) in
anilinium buffer (pH 4.6) at rt and the conversion was 50–60%
in 10 min. The final product was isolated by HPLC with high
radiochemical purity (>98%). The amount of the peptide pre-
cursor 9 was under the detection limit of the UV-detection.
The total synthesis time was typically 120 minutes and the
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c
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