Ahtinen, Helena

Airaksinen, Anu J.

Autio, Anu

Helariutta, Kerttuli

Jalkanen, Sirpa

Li, Xiang-Guo

Liljenbäck, Heidi

Roivainen, Anne

Chemical Communications

English

VAP-1 binding peptide Siglec-9 was glycosylated with 5-deoxy-5-[18F]fluororibose and used for in vivo PET imaging of experimental inflammation.</p

Xiang-Guo Li

Anu Autio

Helena Ahtinen

Kerttuli Helariutta

Heidi Liljenbäck

Sirpa Jalkanen

Anne Roivainen

Anu J. Airaksinen

Crossref

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

Helsingin yliopiston digitaalinen arkisto

3682 Chem. Commun., 2013, 49, 3682--3684 This journal is c The Royal Society of Chemistry 2013Cite this: Chem. Commun.,2013,49, 3682Translating the concept of peptide labeling with5-deoxy-5-[18F]fluororibose into preclinical practice:18F-labeling of Siglec-9 peptide for PET imaging ofinflammation†Xiang-Guo Li,*ab Anu Autio,b Helena Ahtinen,b Kerttuli Helariutta,cHeidi Liljenbäck,bd Sirpa Jalkanen,e Anne Roivainen*bd and Anu J. Airaksinen*cPeptide glycosylation with 5-deoxy-5-[18F]fluororibose was trans-lated into preclinical settings. The novel 18F-labeled Siglec-9peptide was produced using an automated synthesis procedure.The 18F-labeled Siglec-9 peptide showed favorable binding in theanimal model of inflammation in vivo.Positron emission tomography (PET) is a sensitive and non-invasive in vivo molecular imaging modality. Fluorine-18 (18F)has favorable physical properties to generate high-resolutionPET images and consequently 18F-labeled compounds are themost used tracers in clinical PET. Among the short-lived radio-nuclides, the physical half-life of 18F (T1/2 = 109.7 minutes)allows multi-step synthesis of tracers for the same-day use inhospitals.1 Versatile fluorinating agents have been developedfor the synthesis of fluorine containing pharmaceuticals.1However, only a small proportion of the existing fluorinationmethods have been (or can be) translated into radiosynthesis of18F-labeled compounds for preclinical and clinical applications.1aThis is especially true in the case of 18F-labeled biomolecules,e.g. peptides, proteins and antibodies. Although many strate-gies for 18F-labeling of peptides and proteins have shown somepromise during the last two decades, only a few have provedamenable for routine large scale production for clinical use.Oxime formation between an aldehyde (or a ketone) and anaminooxy-functionalized compound is a classical and favorablereaction in bioconjugation due to the high chemoselectivity.Indeed, oxime formation has been successfully applied for theproduction of [18F]fluciclatide (GE Healthcare) starting from4-[18F]fluorobenzaldehyde ([18F]FBA). [18F]Fluciclatide is one ofthe few 18F-labeled peptide tracers currently in clinical use.1bAmong the 18F-labeled aldehydes, [18F]FBA is probably the mostfrequently used prosthetic group for peptide labeling. However,[18F]FBA is volatile requiring additional measures to ensureradiosafety and to reduce radioactive emission during theproduction. The attachment of [18F]FBA may cause increasedlipophilicity, resulting in altered pharmacokinetics in vivo.1bIn addition to [18F]FBA, aldehydes 1–5 have recently beendeveloped for peptide labeling (Fig. 1, [a]).1b,2It has been well documented that glycosylation may conferfavorable characteristics to peptide tracers for in vivo imaging.The incorporation of a galactose fragment into the RGD peptidehas generated a very valuable peptide tracer, [18F]galacto-RGD.So far, most of the published clinical PET imaging data con-cerning 18F-labeled peptides has actually been generated usingthis tracer. However, the synthesis procedure of [18F]galacto-RGD is rather complex and is a major challenge for its wide-spread use.3 As 2-deoxy-2-[18F]fluoroglucose ([18F]FDG, Fig. 1,[b]) is available in virtually every PET radiochemistry lab, it is anexcellent concept to use [18F]FDG for peptide and proteinlabeling.4 [18F]FDG is an aldose and has an open chain aldehydeFig. 1 18F-Labeled prosthetic groups for oxime formation.18F-Fluorinatedsugars are presented in their cyclic forms.1b,2a Department of Pharmacology, Drug Development and Therapeutics,University of Turku, FI-20014 Turku, Finland. E-mail: xiali@utu.fib Turku PET Centre, University of Turku and Turku University Hospital,FI-20521 Turku, Finland. E-mail: anne.roivainen@utu.fic Laboratory of Radiochemistry, Department of Chemistry, University of Helsinki,FI-00014 Helsinki, Finland. E-mail: anu.airaksinen@helsinki.fid Turku Center for Disease Modeling, University of Turku, FI-20014 Turku, Finlande MediCity Research Laboratory and Department of Medical Microbiology andImmunology, 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/c3cc40738aReceived 29th January 2013,Accepted 15th March 2013DOI: 10.1039/c3cc40738awww.rsc.org/chemcommChemCommCOMMUNICATIONOpen Access Article. Published on 15 March 2013. Downloaded on 02/10/2017 11:46:32. View Article OnlineView Journal  | View IssueThis journal is c The Royal Society of Chemistry 2013 Chem. Commun., 2013, 49, 3682--3684 3683form in solution.1b However, the conjugation between [18F]FDGand aminooxy-functionalized peptides generally needs elevatedtemperature and low pH and forms a complex product profile.1b,4bVery recently, it has been discovered that 5-deoxy-5-[18F]fluoro-ribose ([18F]FDR, Fig. 1, [b]) conjugates to peptides much moreefficiently than [18F]FDG does.5 Although we were able to produce[18F]FDR in large scale in an automated procedure, we were notable to perform the conjugation step efficiently enough forpreclinical PET studies. Furthermore, the quality of radiolabeledpeptides was not acceptable (e.g. low chemical purity) for in vivoanimal studies. Thus, peptide glycosylation with [18F]FDRremained at a conceptual stage with respect to preclinical andclinical utilities. In this communication, we report the trans-lation of [18F]FDR-based bench chemistry into practical pre-clinical use, and exemplify this utility on PET imaging ofvascular adhesion protein 1 (VAP-1), which is a promisingnew target protein for PET imaging of inflammation.In the previous work, [18F]FDR was prepared in high radio-chemical purity (>98%) from precursor 6 (Scheme 1, [a]), andthe synthesis was scaled up to 4 GBq of radioactivity. However,chemical purity of the synthesized [18F]FDR was not optimaland the final product contained ribose in a mM-range, originat-ing from precursor 6. Subsequently, only a small proportion ofthe obtained [18F]FDR (8–20 MBq) was conjugated to modelpeptides. In the presence of an appropriate amount of peptides(e.g. at 23 mM of concentration), the conjugation reachedcompletion in 10 minutes in sodium acetate buffer (pH 4.6)at rt.5 Scaling up the conjugation reaction was hampered due tothe high ribose content of the produced [18F]FDR. Whenstarting with 100 MBq-4 GBq of [18F]FDR, only 5% conversionyields were achieved in an hour, even at high concentrations ofpeptide (e.g. 40 mM). Since only nano- or picomoles of non-carrier added and high specific radioactivity [18F]FDR was used,the ribose was present in the conjugation mixture in a largeexcess. Its presence as a competing aldehyde source resultedin the inefficient conjugation of [18F]FDR (Scheme 1, [b]).This prompted us to derive a strategy to produce [18F]FDR freeof ribose ([18F]FDR 8, Scheme 1, [a]). After the fluorination ofprecursor 6 with K18F-K222, intermediate 7 was isolated using apreparative HPLC column. The method gave an excellentseparation between the starting material 6 (retention time =10.6 min) and intermediate 7 (retention time = 4.9 min) in ashort HPLC protocol. Thus, the synthesis procedure was notsignificantly prolonged. The HPLC analysis of the isolated 7proved the success of the intermediate purification step (ESI†).After the acid-catalyzed hydrolysis of 7, [18F]FDR 8 was obtainedin high radiochemical purity (>98%). Subsequently, the pH of[18F]FDR 8 solution was adjusted to 4.6 at which the efficiencyof peptide conjugation was optimal. The total preparation timeof the high purity [18F]FDR 8 was typically 85 min starting fromend-of-bombardment (EOB), including a concentration step ofthe [18F]FDR solution before the conjugation reaction. In addi-tion to 6, 1,2,3-tri-O-acetyl-5-O-tosyl ribose (AcRib, ESI†) wastested as a precursor to produce [18F]FDR 8. Unfortunately, onlyresidual amount of [18F]FDR 8 was obtained from AcRib. In thisrespect, compound 6 was a very applicable precursor in radio-synthesis (n = 40). The next step was to prepare VAP-1 targeting[18F]FDR-Siglec-9 peptide for in vivo imaging of inflammationin rats (Scheme 1, [a] and [c]). VAP-1 is a 90 kDa glycoproteinand is a unique target in inflammation.6 Inflammation ishighly relevant in major diseases including cancer, diabetes,obesity and Alzheimer’s disease.6b In endothelial cells, VAP-1stays in the intracellular storage granules under normalphysiological conditions. Upon inflammation, VAP-1 relocatesrapidly to the endothelial cell surface. This makes VAP-1 anideal target for diagnostic imaging of inflammation. Recently,it has been discovered that sialic acid-binding Ig-like lectin 9(Siglec-9) is a leukocyte ligand of VAP-1 and 68Ga-labeledSiglec-9 motif peptide can be used as a PET tracer for in vivoimaging of inflammation and cancer.7 Accordingly, peptide 9(ESI†) was designed in order to enable conjugation to [18F]FDR8 in this work.In general, oxime formation is not the most efficientchemical reaction and the substrates need to be used atrelatively high concentrations in order to reduce the reactiontime. Specific radioactivity of the cyclotron produced 18F is highand the amount of the synthesized [18F]FDR 8 is in a nano- topicomolar level. Following the reported conjugation procedure,5the initial tests for the synthesis of [18F]FDR-Siglec-9 peptidewere carried out in sodium acetate buffer (pH 4.6, 90 mM) at rt.However, the concentration of peptide 9 used (2.1 kDa) had tobe at over 15 mM (32.2 mg ml1), which was absolutely notapplicable. In a previous study, aniline was used to catalyze theoxime formation between [18F]FBA and leptin.8 Accordingly, wetested aniline-catalysis in the conjugation reactions between5-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 catalyzeoxime formation with a rapid transimination mechanism.1bThe input concentration of peptide 9 could be as low as 0.3 mMScheme 1 Synthesis and preclinical utilization of [18F]FDR-Siglec-9.Communication ChemCommOpen Access Article. Published on 15 March 2013. Downloaded on 02/10/2017 11:46:32. View Article Online3684 Chem. Commun., 2013, 49, 3682--3684 This journal is c The Royal Society of Chemistry 2013(0.6 mg ml1) and the conversion was 95% in 10 min at rt. Theconcentration of the anilinium buffer also had an influence onthe conjugation efficiency (entries 2–4). Our results togetherwith the previous work demonstrated that aniline-catalyzedoxime formation is chemoselective in the presence of anyamino acid side chain from all the 20 standard amino acidsunder 18F-labeling conditions.8A general challenge in developing peptide PET tracers is toisolate the labeled products (e.g. [18F]FDR-Siglec-9) from theirunlabeled peptide precursors (e.g. 9). The possible residualpeptide precursors may compete with the tracer on bindingto the target protein or even saturate the target to be imaged.Bearing this in mind, we developed a reproducible HPLCmethod for efficient separation of [18F]FDR-Siglec-9 from 9 ina preparative scale (ESI†). Finally, [18F]FDR-Siglec-9 was pre-pared from the conjugation of [18F]FDR 8 to 9 (0.3 mM) inanilinium buffer (pH 4.6) at rt and the conversion was 50–60%in 10 min. The final product was isolated by HPLC with highradiochemical 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 thedecay-corrected radiochemical yield was 27% starting from18F-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 mmol1at the end-of-synthesis (EOS) (ESI†). [18F]FDR-Siglec-9 wasformulated in phosphate-buffered saline (PBS) for intravenousinjection. In PBS, [18F]FDR-Siglec-9 was stable for >4 hours andthus suitable for in vivo imaging studies. The [18F]FDR-Siglec-9(18.3  5.1 MBq) was intravenously injected into rats havingsterile, turpentine oil induced inflammation.7 Dynamic PETimaging lasting for 1 hour was performed by using a HighResolution Research Tomograph (Siemens). The inflammationfocus on the right shoulder area of the rats (n = 8) was clearlyvisualized (Fig. 2). The target-to-muscle ratio was 9 : 1. Theobserved liver uptake and rapid excretion through kidneysto 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-basedoxime formation into the production of [18F]FDR-Siglec-9 peptidefor preclinical use. [18F]FDR-Siglec-9 has been produced in highradiochemical 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 andthe whole synthesis procedure can be automated. Thus,[18F]FDR-based glycosylation should be generally applicable inthe development of peptide PET tracers.We thank the Academy of Finland (no. 133127, 136805,119048 and 258814) for financial support. The study wasconducted within the Finnish Centre of Excellence in MolecularImaging in Cardiovascular and Metabolic Research supportedby the Academy of Finland, the University of Turku, the TurkuUniversity Hospital and the Åbo Akademi University.Notes and references1 (a) M. Tredwell and V. Gouverneur, Angew. Chem., Int. Ed., 2012,51, 11426; (b) X.-G. Li, M. Haaparanta and O. Solin, J. Fluorine Chem.,2012, 143, 49, and references therein; (c) X.-G. Li, J. Domarkas andD. O’Hagan, Chem. Commun., 2010, 46, 7819.2 J. A. H. Inkster, K. Liu, S. Ait-Mohand, P. Schaffer, B. Gurin, T. J. Ruthand T. Storr, Chem.–Eur. J., 2012, 18, 11079.3 A. J. Beer and M. Schwaiger, J. Nucl. Med., 2011, 52, 335.4 (a) O. Boutureira, G. J. L. Bernardes, F. D’Hooge and B. G. Davis,Chem. Commun., 2011, 47, 10010; (b) C. Hultsch, M. Schottelius,J. Auernheimer, A. Alke and H.-J. Wester, Eur. J. Nucl. Med.Mol. Imaging, 2009, 36, 1469.5 X.-G. Li, S. Dall’Angelo, L. F. Schweiger, M. Zanda and D. O’Hagan,Chem. Commun., 2012, 48, 5247.6 (a) M. Salmi and S. Jalkanen, Science, 1992, 257, 1407;(b) A. Roivainen, S. Jalkanen and C. Nanni, Eur. J. Nucl. Med. Mol.Imaging, 2012, 39(suppl. 1), S68; (c) Http://www.biotie.com.7 K. Aalto, A. Autio, E. A. Kiss, K. Elima, Y. Nymalm, T. Z. Veres,F. Marttila-Ichihara, H. Elovaara, T. Saanijoki, P. R. Crocker,M. Maksimow, E. Bligt, T. A. Salminen, M. Salmi, A. Roivainen andS. Jalkanen, Blood, 2011, 118, 3725.8 R. R. Flavell, P. Kothari, M. Bar-Dagan, M. Synan, S. Vallabhajosula,J. M. Friedman, T. W. Muir and G. Ceccarini, J. Am. Chem. Soc., 2008,130, 9106.Table 1 The influence of aniline on conjugation efficiency of [19F]FDR to peptide9 in 10 min of reaction (pH 4.6, rt)EntryConcentration of[19F]FDR and 9a (mM) Buffer (M)Conversionb(%)1 0.3 Sodium acetate (0.3) 52 0.3 Anilinium acetate (0.3) 953 0.3 Anilinium acetate (0.2) 894 0.3 Anilinium acetate (0.05) 635 0.1 Anilinium acetate (0.3) 48a The two reagents were used in equal amounts. b Conversions weredetermined using HPLC.Fig. 2 Representative sagittal (left), transaxial (middle) and coronal (right)multiplane PET images of [18F]FDR-Siglec-9 biodistribution in a rat. The imagesare summation from 10–60 min post-injection.ChemComm CommunicationOpen Access Article. Published on 15 March 2013. Downloaded on 02/10/2017 11:46:32. View Article Online

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

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

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