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Objectives: The routine 18F labeling of biomacromolecules like peptides and proteins mainly exploits the use of bifunctional labeling precursors, also referred to as prosthetic groups. 2-[18F]Fluoro-2-deoxy-D-glucose ([18F]FDG) is the most important clinical PET radiotracer, but only very few examples using readily available [18F]FDG as a building block for the radiosynthesis of 18F-labeled compounds. The present study describes the use of [18F]FDG as a 18F building block for the direct labeling of various aminooxy-functionalized peptides via chemoselective oxime formation. The potential of this novel peptide labeling reaction was expemplified by means of various neurotensin NT(8-13) derivatives. Methods: The labeling reaction was performed using a 0.9% NaCl solution of [18F]FDG and aminooxy-functionalized peptide at different concentrations in a mixture of MeOH/H2O at 80 °C for 30 min. The reaction mixture was analyzed by radio-HPLC to determine the radiochemical yield of the conjugation reaction (Fig. 1). Results: Direct labeling of aminooxy-functionalized peptides with [18F]FDG was strongly dependent on the amount of used peptide. Monomeric NT(8-13) derivative gave best radiochemical yields of up to 80% based upon [18F]FDG. More complex dimeric and tetrameric neurotensin derivatives gave lower radiochemical yields at comparable peptide concentrations. Increase of [18F]FDG activity also lowered radiochemical yield due to an increasing competitive reaction with glucose originating from the [18F]FDG solution. Depending on the size of the used peptide, separation of [18F]FDG-labeled peptide from glucose-labeled peptide is possible by semi-preparative HPLC. The formation of isomers during the aminooxy-aldehyde conjugation reaction between [18F]FDG and aminooxy-functionalized peptides in aqueous media leads to the formation of isomers according to literature reports. Conclusions: For the first time, readily available PET radiotracer [18F]FDG was shown to be a suitable prosthetic group for direct labeling of aminooxy-functionalized peptides with fluorine-18 under mild conditions. The reaction is especially suitable for small peptides. However, application of larger peptides seems to be limited by increasing separation difficulties of the corresponding glucose-peptide conjugate.

The study describes the use of [F-18]FDG as F-18 building block for the direct labelling of various aminooxy-functionalised peptides via chemoselective oxime formation.

Introduction: Neurotensin receptors are expressed with high incidence in several human tumour entities. Thus, radiolabeled neurotensin derivatives might be used for tumour targeting. However, its application is limited by insufficient metabolic stability. Metabolic stability might be improved by the synthesis of multimeric peptides. Experimental: Three methods for 18F-labeling of dimeric and tetrameric neurotensin(8-13) derivatives were evaluated with respect to the labeling yield and the required peptide amounts. Results and Discussion: Labeling using N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) gave low radiochemical yield for the dimeric peptides. Coupling of the tetramer with [18F]SFB was not successful. Furthermore, labeling of aminooxy-functionalized neurotensin(8-13) derivatives using 4-[18F]fluorobenzaldehyde ([18F]FBA) was investigated. High yields of up to 96% were obtained for the dimer whilst coupling of the tetramer only gave low yields of up to 10%. In contrast to these findings, labelling of sulfhydryl-functionalized neurotensin(8-13) derivatives using the maleinimide 4-[18F]fluorobenzaldehyde O-[6-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-hexyl]-oxime ([18F]FBAM) resulted in high radiochemical yields for both, the dimer and the tetramer, being 94% and 40%, respectively. Therefore, [18F]FBAM seems to be the most suitable 18F-labeling agent for multimeric neurotensin(8-13) derivatives. The synthesized 18F-labeled multimeric neurotensin derivatives are depicted in Fig. 1. Conclusion: In summary, 18F-radiolabeling of dimeric and tetrameric NT(8-13) derivates was investigated. Suitable results were obtained by labeling of the sulfhydryl-functionalized peptides using [18F]FBAM. Using [18F]SFB or [18F]FBA as the labeling agent gave only low radiochemical yields. Furthermore, labeling of the dimer gave better radiochemical yields than labeling of the tetramer – independent from the labeling method which was used. Therefore, the nature of the peptide exhibits a strong influence on the coupling reaction.

Neurotensin receptors are expressed with high incidence in several human tumour entities. Thus, radiolabelled neurotensin derivatives might be used for tumour targeting. However, its application is limited by insufficient metabolic stability. Metabolic stability might be improved by the synthesis of multivalent peptides. Three methods for 18F-labelling of dimeric and tetrameric neurotensin(8-13) derivatives were evaluated with respect to the labelling yield and the required peptide amounts. Labelling using N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) gave low radiochemical yield for the dimeric peptides. Coupling of the tetramer with [18F]SFB was not successful. Furthermore, labelling of aminooxy-functionalized neurotensin(8-13) derivatives using 4-[18F]fluorobenzaldehyde ([18F]FBA) was investigated. High yields were obtained for the dimer whilst coupling of the tetramer only gave low yields. In contrast to these findings, labelling of sulfhydryl-functionalized neurotensin(8-13) derivatives using the maleinimide 4-[18F]fluorobenzaldehyde O-[6-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-hexyl]-oxime ([18F]FBAM) resulted in high radiochemical yields for both, the dimer and the tetramer. Therefore, [18F]FBAM seems to be the most suitable 18F-labelling agent for multivalent neurotensin(8-13) derivatives.

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Amadori products are formed in the early stage of the so-called Maillard reaction between reducing sugars and amino acids or proteins. Such nonenzymatic glycosylation may occur during the heating or storage of foods, but also under physiological conditions. N-epslion-fructoselysine is formed via this reaction between the epsilon-amino group of peptide-bound lysine and glucose. Despite the fact that, in certain heated foods, up to 50% of lysyl moieties may be modified to such lysine derivatives, up to now, very little is known about the metabolic fate of alimentary administered Amadori compounds. In the present study, N-succinimidyl-4-[18F]fluorobenzoate was used to modify N-epsilon-fructoselysine at the a-amino group of the lysyl moiety. The in vitro stability of the resulting 4-[18F]fluorobenzoylated derivative was tested in different tissue homogenates. Furthermore, the 4-[18F]fluorobenzoylated N-epsilon-fructoselysine was used in positron emission tomography studies, as well as in studies concerning biodistribution and catabolism. The results show that the 4-[18F]fluorobenzoylated N-epsilon-fructoselysine is phosphorylated in vitro, as well as in vivo. This phosphorylation is caused by fructosamine 3-kinases and occurs in vivo, particularly in the kidneys. Despite the action of these enzymes, it was shown that a large part of the intravenously applied radiolabeled N-epsilon-fructoselysine was excreted nearly unchanged in the urine. Therefore, it was concluded that the predominant part of peptide-bound lysine that was fructosylated during food processing is not available for nutrition.

Isopeptide bonds between the varepsilon-amino group of lysine and the gamma-carboxamide group of glutamine are formed during strong heating of pure proteins or, more important, by enzymatic reaction mediated by transglutaminases. Despite the wide use of a microbial transglutaminase in food biotechnology, up to now little is known about the metabolic fate of the isopeptide N(varepsilon)-(gamma-glutamyl)-L-lysine. In the present study, N-succinimidyl-4-[18F]fluorobenzoate was used to modify N(varepsilon)-(gamma-glutamyl)-L-lysine at each of its two alpha-amino groups, resulting in the 4-[18F]fluorobenzoylated derivatives, for which biodistribution, catabolism, and elimination were investigated in male Wistar rats. A significant different biochemical behavior of the two labelled isopeptides was observed in terms of in vitro stability, in vivo metabolism as well as biodistribution. The results suggest that the metabolic fate of isopeptides is likely to be dependent on how they are reabsorbed - free or peptide bound.

Apoptosis imaging with PET plays an increasing role in various medical fields like oncology, cardiology, transplant rejection, and inflammation, but the radiotracer distribution in the tis-sues is influenced by various mechanisms. In this study a recombinant annexin-V (courtesy N. Budisa, Max Planck Institute of Biochemistry) derivative and human serum albumin (HSA) were radiolabelled using N-succinimidyl-4-F-18-fluorobenzoate (SFB), and characterized. Mechanism and specificity of both F-18-annexin-V and F-18-HSA biodistribution and accu-mulation were examined in rodents. Recombinant annexin-V derivative and HSA were radiolabelled using SFB and the products were confirmed by size exclusion chromatography and SDS-PAGE. The radiotracer distribu-tions in animals were studied in rats and mice ex vivo by organ extraction, autoradiography, and in vivo with animal PET. For apoptosis imaging, F-18-annexin-V or F-18-HSA were in-travenously applied in 4 groups of mice that received either intraperitoneal 100 µg Anti-Fas antibody in 200 µL isotonic NaCl or 200 µL isotonic NaCl 2 hours before the radiotracer. The degree of liver apoptosis was characterized by plasma ALAT and ASAT activity measure-ments. The radiochemical yield was in the range of 10 to 30% (corrected for decay) with a specific activity of more than 20 GBq/µmol. The accumulation of F-18-annexin-V and F-18-HSA, respectively in the apoptotic livers (4 – 16%ID/g) were correlated to the ASAT and increased up to 4 times in comparison to control. The biodistribution of the tracers were comparable except for the renal elimination of F-18-annexin-V, which was up to 3 times higher than of F-18-HSA.

Oxidative modification of low density lipoprotein (LDL) is regarded as a crucial event in athergenesis. Data concerning the role of circulating oxidized LDL (oxLDL) in the development of atherosclerosis are scarce. One reason for this is the shortage of methaods for direct assessment of metabolism of oxLDL in vivo. We reprot an improved methodology for labelling of both native LDL (nLDL) and oxLDL with fluorine-18 (18F) by N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) and the use of LDL-[18]FB-conjugates in dynamic PET studies in Wistar rats. For labelling experiments, pools of chemically well characterized human nLDL and oxLDL, respectively, were used. Preparation of [18F]SFB was achieved within 40 min with radiochemical yields of 50±5% and purity of >95% using O-(N-succinimidyl)-N-N,N´,N´-tetramethyluronium tetrafluoroborate (TSTU) as activating reagent. LDL labelling with [18F]SFB resulted in radiochemical yields of 30±10%. The method was evaluated with respect to uptake of FB-conjugated nLDL in HepG2 cells and of FB-conjugated oxLDL in primary human macrophages, respectively. Biodistribution studies revealed high in vivo stability for the LDL-[18F]FB conjugates. The metabolic fate of LDL-[18F]FB conjugates in vivo was delineated by PET using a dedicated small animal tomograph (microPET; spatial resolution of 2 mm). In conclusion, [18F]SFB-labelling of LDL and the use of PET provide a valuable tool for assessment of metabolism of nLDL and oxLDL in vivo.

The isopeptide NEpsilon-(Gamma-glutamyl)-L-lysine 4 was labelled with 18F via N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB). A modified approach for the convenient synthesis of [18F]SFB was used, and [18F]SFB could be obtained in decay-corrected radiochemical yields of 44-53% (n=20) and radiochemical purity >95% within 40 min after EOB. For labelling NEpsilon-(Gamma-glutamyl)-L-lysine with [18F]SFB the effects of isopeptide concentration, temperature, and pH were studied to determine the optimum reaction conditions. The coupling reaction was shown to be temperature and pH independent while being strongly affected by the isopeptide concentration. Using the optimized labelling conditions, in a typical experiment 1.3 GBq of [18F]SFB could be converted into 447 MBq (46%, decay-corrected) of [18F]fluorobenzoylated isopeptide within 45 min, including HPLC purification.