Efficient Method for Site-Specific (18)F-Labeling of Biomolecules Using the Rapid Condensation Reaction between 2-Cyanobenzothiazole and Cysteine

An efficient method based on a rapid condensation reaction between 2-cyanobenzothiazole (CBT) and cysteine has been developed for (18)F-labeling of N-terminal cysteine-bearing peptides and proteins. An (18)F-labeled dimeric cRGD ([(18)F]CBTRGD(2)) has been synthesized with an excellent radiochemical yield (92% based on radio-HPLC conversion, 80% decay-corrected, and isolated yield) and radiochemical purity (>99%) under mild conditions using (18)F-CBT, and shown good in vivo tumor targeting efficiency for PET imaging. The labeling strategy was also applied to the site-specific (18)F-labeling of a protein, Renilla lucifierase (RLuc8) with a cysteine residue at its N-terminus. The protein labeling was achieved with 12% of decay-corrected radiochemical yield and more than 99% radiochemical purity. This strategy should provide a general approach for efficient and site-specific (18)F-labeling of various peptides and proteins for in vivo molecular imaging applications.     

3,4,6-Tri-O-acetyl-2-deoxy-2-[18F]fluoroglucopyranosyl phenylthiosulfonate: a thiol-reactive agent for the chemoselective 18F-glycosylation of peptides

3,4,5-Tri-O-acetyl-2-[18F]fluoro-2-deoxy-d-glucopyranosyl 1-phenylthiosulfonate (Ac3-[18F]FGlc-PTS) was developed as a thiol-reactive labeling reagent for the site-specific 18F-glycosylation of peptides. Taking advantage of highly accessible 1,3,4,6-tetra-O-acetyl-2-deoxy-2-[18F]fluoroglucopyranose, a three-step radiochemical pathway was investigated and optimized, providing Ac3-[18F]FGlc-PTS in a radiochemical yield of about 33% in 90 min (decay-corrected and based on starting [18F]fluoride). Ac3-[18F]FGlc-PTS was reacted with the model pentapeptide CAKAY, confirming chemoselectivity and excellent conjugation yields of >90% under mild reaction conditions. The optimized method was adopted to the 18F-glycosylation of the alphavbeta3-affine peptide c(RGDfC), achieving high conjugation yields (95%, decay-corrected). The alphavbeta3 binding affinity of the glycosylated c(RGDfC) remained uninfluenced as determined by competition binding studies versus 125I-echistatin using both isolated alphavbeta3 and human umbilical vein endothelial cells (Ki = 68 +/- 10 nM (alphavbeta3) versus Ki = 77 +/- 4 nM (HUVEC)). The whole radiosynthetic procedure, including the preparation of the 18F-glycosylating reagent Ac3-[18F]FGlc-PTS, peptide ligation, and final HPLC purification, provided a decay-uncorrected radiochemical yield of 13% after a total synthesis time of 130 min. Ac3-[18F]FGlc-PTS represents a novel 18F-labeling reagent for the mild chemoselective 18F-glycosylation of peptides indicating its potential for the design and development of 18F-labeled bioactive S-glycopeptides suitable to study their pharmacokinetics in vivo by positron emission tomography (PET).     

Click chemistry for (18)F-labeling of RGD peptides and microPET imaging of tumor integrin alphavbeta3 expression

The cell adhesion molecule integrin alpha vbeta 3 plays a key role in tumor angiogenesis and metastasis. A series of (18)F-labeled RGD peptides have been developed for PET of integrin expression based on primary amine reactive prosthetic groups. In this study, we report the use of the Cu(I)-catalyzed Huisgen cycloaddition, also known as a click reaction, to label RGD peptides with (18)F by forming 1,2,3-triazoles. Nucleophilic fluorination of a toluenesulfonic alkyne provided (18)F-alkyne in high yield (nondecay-corrected yield: 65.0 +/- 1.9%, starting from the azeotropically dried (18)F-fluoride), which was then reacted with an RGD azide (nondecay-corrected yield: 52.0 +/- 8.3% within 45 min including HPLC purification). The (18)F-labeled peptide was subjected to microPET studies in murine xenograft models. Murine microPET experiments showed good tumor uptake (2.1 +/- 0.4%ID/g at 1 h postinjection (p.i.)) with rapid renal and hepatic clearance of (18)F-fluoro-PEG-triazoles-RGD 2 ( (18)F-FPTA-RGD2) in a subcutaneous U87MG glioblastoma xenograft model (kidney 2.7 +/- 0.8%ID/g; liver 1.9 +/- 0.4%ID/g at 1 h p.i.). Metabolic stability of the newly synthesized tracer was also analyzed (intact tracer ranging from 75% to 99% at 1 h p.i.). In brief, the new tracer (18)F-FPTA-RGD2 was synthesized with high radiochemical yield and high specific activity. This tracer exhibited good tumor-targeting efficacy and relatively good metabolic stability, as well as favorable in vivo pharmacokinetics. This new (18)F labeling method based on click reaction may also be useful for radiolabeling of other biomolecules with azide groups in high yield.     

Efficient (18)F-Labeling of Large 37-Amino-Acid pHLIP Peptide Analogues and Their Biological Evaluation

Solid tumors often develop an acidic microenvironment, which plays a critical role in tumor progression and is associated with increased level of invasion and metastasis. The 37-residue pH (low) insertion peptide (pHLIP) is under study as an imaging platform because of its unique ability to insert into cell membranes at a low extracellular pH (pH(e) < 7). Labeling of peptides with [(18)F]-fluorine is usually performed via prosthetic groups using chemoselective coupling reactions. One of the most successful procedures involves the alkyne-azide copper(I) catalyzed cycloaddition (CuAAC). However, none of the known "click" methods have been applied to peptides as large as pHLIP. We designed a novel prosthetic group and extended the use of the CuAAC "click chemistry" for the simple and efficient (18)F-labeling of large peptides. For the evaluation of this labeling approach, a d-amino acid analogue of WT-pHLIP and an l-amino acid control peptide K-pHLIP, both functionalized at the N-terminus with 6-azidohexanoic acid, were used. The novel 6-[(18)F]fluoro-2-ethynylpyridine prosthetic group, was obtained via nucleophilic substitution on the corresponding bromo-precursor after 10 min at 130 °C with a radiochemical yield of 27.5 ± 6.6% (decay corrected) with high radiochemical purity ≥98%. The subsequent Cu(I)-catalyzed "click" reaction with the azido functionalized pHLIP peptides was quantitative within 5 min at 70 °C in a mixture of water and ethanol using Cu-acetate and sodium l-ascorbate. [(18)F]-d-WT-pHLIP and [(18)F]-l-K-pHLIP were obtained with total radiochemical yields of 5-20% after HPLC purification. The total reaction time was 85 min including formulation. In vitro stability tests revealed high stability of the [(18)F]-d-WT-pHLIP in human and mouse plasma after 120 min, with the parent tracer remaining intact at 65% and 85%, respectively. PET imaging and biodistribution studies in LNCaP and PC-3 xenografted mice with the [(18)F]-d-WT-pHLIP and the negative control [(18)F]-l-K-pHLIP revealed pH-dependent tumor retention. This reliable and efficient protocol promises to be useful for the (18)F-labeling of large peptides such as pHLIP and will accelerate the evaluation of numerous [(18)F]-pHLIP analogues as potential PET tracers.     

Synthesis, 18F-labeling, and in vitro and in vivo studies of bombesin peptides modified with silicon-based building blocks

The gastrin-releasing peptide receptor (GRPr) is overexpressed on various human tumors. The goal of our study was the synthesis of new 18F-labeled bombesin analogues for the PET imaging of GRPr expression in prostate tumor using a silicon-based one-step n. c. a. radiolabeling method. The silicon-containing building blocks were efficiently coupled to the N-terminus of the peptides via solid-phase synthesis. Radiolabeling of the obtained peptide precursors proceeded smoothly under acidic conditions (34-85% conversion). Using the di-tert-butyl silyl building block as labeling moiety, products containing a hydrolytically stable 18F-label were obtained. In in vitro receptor binding experiments 2-(4-(di-tert-butylfluorosilyl)phenyl)acetyl-Arg-Ava-Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH 2 ( 4b, IC50 = 22.9 nM) displayed a 12-fold higher binding affinity than 2-(4-(di-tert-butylfluorosilyl)phenyl)acetyl-Arg-Ava-Gln-Trp-Ala-Val-Gly-His(3Me)-Sta-Leu-NH2 ( 3b, IC50 = 276.6 nM), and 4b was therefore chosen for further evaluation. In vitro and ex vivo metabolite studies of [18F]4b showed no significant degradation. In biodistribution experiments, tumor uptake of [18F]4b was low and unspecific, whereas the GRPr-rich pancreas revealed a high and specific accumulation of the radiotracer. This study demonstrates the applicability of our silicon-based one-step n. c. a. radiolabeling method for the synthesis of new 18F-labeled bombesin derivatives. This innovative approach represents a general, straightforward access to radiolabeled peptides as PET imaging probes.     

"Click labeling" with 2-[18f]fluoroethylazide for positron emission tomography

As an effort in the development of more flexible (18)F-labeling chemistry, we report herein on the use of the Cu(I)-catalyzed Huisgen cycloaddition, also known as the "click reaction", to form (18)F-labeled 1,2,3-triazoles. Nucleophilic fluorination of 2-azidoethyl-4-toluenesulfonate followed by distillation provided 2-[(18)F]fluoroethylazide in 55% radiochemical yield (decay-corrected). 2-[(18)F]fluoroethylazide was reacted with a small library of terminal alkynes in the presence of excess Cu(2+)/ascorbate or copper powder. The most reactive alkyne, N-benzylpropynamide provided nearly quantitative incorporation of 2-[(18)F]fluoroethylazide after 15 min at ambient temperature, whereas the majority of the alkyne substrates provided excellent yields of the corresponding (18)F-labeled 1,2,3-triazoles following heating to 80 degrees C. Using the method described, a model peptide was obtained in 92.3 +/- 0.3% (n = 3) radiochemical yield (decay-corrected) after purification by semipreparative HPLC.     

Preparation of 18F-labeled peptides using the copper(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition

An optimized procedure for preparing fluorine-18 ((18)F)-labeled peptides by the copper-catalyzed azide-alkyne 1,3-dipolar cyloaddition (CuAAC) is presented here. The two-step radiosynthesis begins with the microwave-assisted nucleophilic (18)F-fluorination of a precursor containing a terminal p-toluenesulfonyl, terminal azide and polyethylene glycol backbone. The resulting (18)F-fluorinated azide-containing building block is coupled to an alkyne-decorated peptide by the CuAAC. The reaction is accelerated by the copper(I)-stabilizing ligand bathophenanthroline disulfonate and can be performed in either reducing or nonreducing conditions (e.g., to preserve disulfide bonds). After an HPLC purification, (18)F-labeled peptide can be obtained with a 31 ± 6% radiochemical yield (n = 4, decay-corrected from (18)F-fluoride elution) and a specific activity of 39.0 ± 12.4 Ci μmol(-1) within 77 ± 4 min.     

Synthesis and application of [18F]FDG-maleimidehexyloxime ([18F]FDG-MHO): a [18F]FDG-based prosthetic group for the chemoselective 18F-labeling of peptides and proteins

2-[(18)F]Fluoro-2-deoxy-D-glucose ([(18)F]FDG) as the most important PET radiotracer is available in almost every PET center. However, there are only very few examples using [(18)F]FDG as a building block for the synthesis of (18)F-labeled compounds. The present study describes the use of [(18)F]FDG as a building block for the synthesis of (18)F-labeled peptides and proteins. [(18)F]FDG was converted into [(18)F]FDG-maleimidehexyloxime ([(18)F]FDG-MHO), a novel [(18)F]FDG-based prosthetic group for the mild and thiol group-specific (18)F labeling of peptides and proteins. The reaction was performed at 100 degrees C for 15 min in a sealed vial containing [(18)F]FDG and N-(6-aminoxy-hexyl)maleimide in 80% ethanol. [(18)F]FDG-MHO was obtained in 45-69% radiochemical yield (based upon [(18)F]FDG) after HPLC purification in a total synthesis time of 45 min. Chemoselecetive conjugation of [(18)F]FDG-MHO to thiol groups was investigated by the reaction with the tripeptide glutathione (GSH) and the single cysteine containing protein annexin A5 (anxA5). Radiolabeled annexin A5 ([(18)F]FDG-MHO-anxA5) was obtained in 43-58% radiochemical yield (based upon [(18)F]FDG-MHO, n = 6), and [(18)F]FDG-MHO-anxA5 was used for a pilot small animal PET study to assess in vivo biodistribution and kinetics in a HT-29 murine xenograft model.     

N-Succinimidyl 4-[(18)F]-fluoromethylbenzoate-labeled dimeric RGD peptide for imaging tumor integrin expression

RGD peptides, radiolabeled with (18)F, have been used in the clinic for PET imaging of tumor angiogenesis in cancer patients. RGD peptides are typically labeled using a prosthetic group such as N-succinimidyl 4-[(18)F]-fluorobenzoate ([(18)F]SFB) or 4-nitrophenyl 2-[(18)F]-fluoropropionate ([(18)F]NPFP). However, the complex radiosynthetic procedures have impeded their broad application in clinical studies. We previously radiolabeled proteins and peptides with the prosthetic group, N-succinimidyl 4-[(18)F]-fluoromethylbenzoate ([(18)F]SFMB), which was prepared in a simple one-step procedure. In this study, we labeled a PEGylated cyclic RGD peptide dimer, PEG(3)-E[c(RGDyK)](2) (PRGD2), using [(18)F]SFMB and evaluated for imaging tumor αvβ3 integrin expression with positron emission tomography (PET). [(18)F]SFMB was prepared in one step using [(18)F]fluoride displacement of a nitrobenzenesulfonate leaving group under mild reaction conditions followed by HPLC purification. The (18)F-labeled peptide, [(18)F]FMBPRGD2 was prepared by coupling PRGD2 with [(18)F]SFMB in pH 8.6 borate buffer and purified with HPLC. The direct labeling on BMBPRGD2 was also attempted. A Siemens Inveon PET was used to image the uptake of the [(18)F]FMBPRGD2 into a U87MG xenograft mouse model. [(18)F]FMBPRGD2, was prepared with a 15% overall radiochemical yield (uncorrected) in a total synthesis time of 90?min, which was considerably shorter than the preparation of [(18)F]SFB- and [(18)F]NPFP-labeled RGD peptides. The direct labeling, however, was not successful. High quality microPET images using [(18)F]FMBPRGD2 clearly visualized tumors by 15?min with good target to background ratio. Early tracer accumulation in the bladder suggests fast renal clearance. No obvious bone uptake can be detected even at 4-h time point indicating that fluorine attachment is stable in mice. In conclusion, N-succinimidyl 4-[(18)F]-fluoromethylbenzoate ([(18)F]SFMB) prosthetic group can be a good alternative for labeling RGD peptides to image αvβ3 integrin expression and for labeling other peptides.     

Solid-phase synthesis of 2-[18F]fluoropropionyl peptides

The 2-[(18)F]fluoropropionic (2-[(18)F]FPA) acid is used as a prosthetic group for radiolabeling proteins and peptides for targeted imaging using positron emission tomography (PET). Radiolabeling of compounds with more than one acylable functional group can lead to complex mixtures of products; however, peptides can be labeled regioselectively on the solid phase. We investigated the use of a solid-phase approach for the preparation of 2-[(18)F]fluoropropionyl peptides. [(18)F]FPA was prepared and conjugated to the peptides attached to the solid phase support. The (18)F-labeled peptides were obtained in 175 min with decay corrected yields of 10% (related to [(18)F]fluoride) and with a purity of 76-99% prior HPLC purification. The suitability of various coupling reagents and solid supports were tested for radiolabeling of several peptides of various lengths.     

Synthesis of a new heterobifunctional linker, N-[4-(aminooxy)butyl]maleimide, for facile access to a thiol-reactive 18F-labeling agent

A new heterobifunctional linker containing an aldehyde-reactive aminooxy group and a thiol-reactive maleimide group, namely N-[4-(aminooxy)butyl]maleimide, was synthesized as a stable HCl salt by O-alkylation of either N-hydroxyphthalimide or N-(4-monomethoxytrityl)hydroxylamine, followed by N-alkylation of maleimide, in an overall yield of 18% (seven steps) or 29% (five steps), respectively. This heterobifunctional linker allowed a simple and efficient synthesis of a maleimide-containing thiol-reactive (18)F-labeling agent. Thus, N-[4-[(4-[(18)F]fluorobenzylidene)aminooxy]butyl]maleimide (specific activity: approximately 3000 Ci/mmol at end of synthesis) was synthesized in two steps involving the preparation of 4-[(18)F]fluorobenzaldehyde, followed by its aminooxy-aldehyde coupling reaction to the heterobifunctional linker, with an overall radiochemical yield of approximately 35% (decay corrected) within approximately 60 min from end of bombardment. Initial (18)F-labeling experiments were carried out using a thiol-containing tripeptide glutathione (GSH) and a 5'-thiol-functionalized oligodeoxynucleotide (5'-S-ODN) in phosphate-buffered saline (PBS, pH 7.5). After standing at room temperature for 10 min, the (18)F-labeled GSH and 5'-S-ODN were obtained in (18)F-labeling yields of approximately 70% and approximately 5% (decay-corrected), respectively. The heterobifunctional linker is easy to synthesize and provides a facile access to the maleimide-containing thiol-reactive (18)F-labeling agent, which could be advantageously employed in the development of (18)F-labeled biomomolecules for use with positron emission tomography.     

Distribution and binding of 18F-labeled and 125I-labeled analogues of ACI-80, a prospective molecular imaging biomarker of disease: a whole hemisphere post mortem autoradiography study in human brains obtained from Alzheimer's disease patients

One of the major pathological landmarks of Alzheimer's disease and other neurodegenerative diseases is the presence of amyloid deposits in the brain. The early non-invasive visualization of amyloid is a major objective of recent diagnostic neuroimaging approaches, including positron emission tomography (PET), with an eye on follow-up of disease progression and/or therapy efficacy. The development of molecular imaging biomarkers with binding affinity to amyloid in the brain is therefore in the forefront of imaging biomarker and radiochemistry research. Recently, a dodecamer peptide (amino acid sequence=QSHYRHISPAQV; denominated D1 or ACI-80) was identified as a prospective ligand candidate, binding with high ex vivo affinity to L-Aβ-amyloid (K(d): 0.4 μM). In order to assess the ligand's capacity to visualize amyloid in Alzheimer's disease (AD), two (125)I labeled and three (18)F labeled analogues of the peptide were synthesized and tested in post mortem human autoradiography experiments using whole hemisphere brain slices obtained from deceased AD patients and age matched control subjects. The (18)F-labeled radioligands showed more promising visualization capacity of amyloid that the (125)I-labeled radioligands. In the case of each (18)F radioligands the grey matter uptake in the AD brains was significantly higher than that in control brains. Furthermore, the grey matter: white matter uptake ratio was over ~2, the difference being significant for each (18)F-radioligands. The regional distribution of the uptake of the various radioligands systematically shows a congruent pattern between the high uptake regions and spots in the autoradiographic images and the disease specific signals obtained in adjacent or identical brain slices labeled with histological, immunohistochemical or autoradiographic stains for amyloid deposits or activated astrocytes. The present data, using post mortem human brain autoradiography in whole hemisphere human brains obtained from deceased AD patients and age matched control subjects, support the visualization capacity of the radiolabeled ACI-80 analogues of amyloid deposits in the human brain. Further studies are warranted to explore the usefulness of the (18)F-labeled analogues as in vivo molecular imaging biomarkers in diagnostic PET studies.     

Rapid and simple one-step F-18 labeling of peptides

Labeling biomolecules with 1?F is usually done through coupling with prosthetic groups, which requires several time-consuming radiosynthesis steps and therefore in low labeling yield. In this study, we designed a simple one-step 1?F-labeling strategy to replace the conventional complex and the long process of multiple-step radiolabeling procedure. Both monomeric and dimeric cyclic RGD peptides were modified to contain 4-NO?-3-CF? arene as precursors for direct 1?F labeling. Binding of the two functionalized peptides to integrin α(v)β? was tested in vitro using the MDA-MB-435 human breast cell line. The most promising functionalized peptide, the dimeric cyclic RGD, was further evaluated in vivo in an orthotopic MDA-MB-435 tumor xenograft model. The use of relatively low amount of precursor (~0.5 μmol) gave reasonable yield, ranging from 7 to 23% (decay corrected, calculated from the start of synthesis) after HPLC purification. Overall reaction time was 40 min, and the specific activity of the labeled peptide was high. Modification of RGD peptides did not significantly change the biological binding affinities of the modified peptides. Small animal PET and biodistribution studies revealed integrin specific tumor uptake and favorable biokinetics. We have developed a novel one-step 1?F radiolabeling strategy for peptides that contain a specific arene group, which shortens reaction time and labor significantly, requires low amount of precursor, and results in specific activity of 79 ± 13 GBq/μmol. Successful introduction of 4-fluoro-3-trifluoromethylbenzamide into RGD peptides may be a general strategy applicable to other biologically active peptides and proteins.     

Peptide ligands can bind to distinct sites in integrin alphaIIbbeta3 and elicit different functional responses.

The spatial relationship between the binding sites for two cyclic peptides, cyclo(S,S)KYGCRGDWPC (cRGD) and cyclo(S,S)KYGCHarGDWPC (cHarGD), high affinity analogs for the RGD and HLGGAKQAGDV peptide ligands, in integrin alphaIIbbeta3 (GPIIb-IIIa) has been characterized. For this purpose, cRGD and cHarGD were labeled with fluorescein isothiocyanate and tetramethylrhodamine 5-isothiocyanate, respectively. Both cyclic peptides were potent inhibitors of fibrinogen binding to alphaIIbbeta3, particularly in the presence of Mn2+; IC50 values for cRGD and cHarGD were 1 and <0.1 nM in the presence of Mn2+. Direct binding experiments and fluorescence resonance energy transfer analysis using the purified receptor showed that both peptides interacted simultaneously with distinct sites in alphaIIbbeta3. The distance between these sites was estimated to be 6.1 +/- 0.5 nm. Although cRGD bound preferentially to one site and cHarGD to the other, the sites were not fully specific, and each cyclic peptide or its linear counterpart could displace the other to some extent. The binding affinity of the cHarGD site was dramatically affected by Mn2+. cRGD, but not cHarGD, bound to recombinant beta3-(95-373) in a cation-dependent manner, indicating that the cRGD site is located entirely within this fragment. With intact platelets, binding of c-RGD and cHarGD to alphaIIbbeta3 resulted in distinct conformational alterations in the receptor as indicated by the differential exposure of ligand-induced binding site epitopes and also induced the opposite on membrane fluidity as shown by electron paramagnetic resonance analyses using 5-doxylstearic acid as a spin probe. These data support the concept the two peptide ligands bind to distinct sites in alphaIIbbeta3 and initiate different functional consequences within the receptor itself and within platelets.     

A novel prosthetic group for site-selective labeling of peptides for positron emission tomography

Efficient methodologies for the radiolabeling of peptides with [(18)F]fluoride are a prerequisite to enabling commercialization of peptide-containing radiotracers for positron emission tomography (PET) imaging. It was the purpose of this study to investigate a novel chemoselective ligation reaction comprising conjugation of an [(18)F]-N-methylaminooxy-containing prosthetic group to a functionalized peptide. Twelve derivatives of general formula R1-CO-NH-Lys-Gly-Phe-Gly-Lys-OH were synthesized where R1 was selected from a short list of moieties anticipated to be reactive toward the N-methylaminooxy group. Conjugation reactions were initially carried out with nonradioactive precursors to assess, in a qualitative manner, their general suitability for PET chemistry with only the most promising pairings progressing to full radiochemical assessment. Best results were obtained for the ligation of O-[2-(2-[(18)F]fluoroethoxy)ethyl]-N-methyl-N-hydroxylamine 18 to the maleimidopropionyl-Lys-Gly-Phe-Gly-Lys-OH precursor 10 in acetate buffer (pH 5) after 1 h at 70 degrees C. The non-decay-corrected isolated yield was calculated to be 8.5%. The most encouraging result was observed with the combination 18 and 4-(2-nitrovinyl)benzoyl-Lys-Gly-Phe-Gly-Lys-OH, 9, where the conjugation reaction proceeded rapidly to completion at 30 degrees C after only 5 min. The corresponding non-decay-corrected radiochemical yield for the isolated (18)F-labeled product 27 was 12%. The preliminary results from this study demonstrate the considerable potential of this novel strategy for the radiolabeling of peptides.     

Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB)

Biomolecules, including peptides,^1-9 proteins,^10,11 and antibodies and their engineered fragments,^12-14 are gaining importance as both potential therapeutics and molecular imaging agents. Notably, when labeled with positron-emitting radioisotopes (e.g., Cu-64, Ga-68, or F-18), they can be used as probes for targeted imaging of many physiological and pathological processes.^15-18 Therefore, significant effort has devoted to the synthesis and exploration of ¹⁸F-labeled biomolecules. Although there are elegant examples of the direct ¹⁸F-labeling of peptides,^19-22 the harsh reaction conditions (i.e., organic solvent, extreme pH, high temperature) associated with direct radiofluorination are usually incompatible with fragile protein samples. To date, therefore, the incorporation of radiolabeled prosthetic groups into biomolecules remains the method of choice.^23,24N-Succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB),^25-37 a Bolton-Hunter type reagent that reacts with the primary amino groups of biomolecules, is a very versatile prosthetic group for the ¹⁸F-labeling of a wide spectrum of biological entities, in terms of its evident in vivo stability and high radiolabeling yield. After labeling with [¹⁸F]SFB, the resulting [¹⁸F]fluorobenzoylated biomolecules could be explored as potential PET tracers for in vivo imaging studies.¹ Most [¹⁸F]SFB radiosyntheses described in the current literatures require two or even three reactors and multiple purifications by using either solid phase extraction (SPE) or high-performance liquid chromatography (HPLC). Such lengthy processes hamper its routine production and widespread applications in the radiolabeling of biomolecules. Although several module-assisted [¹⁸F]SFB syntheses have been reported,^{29-32, 41-42} they are mainly based on complicated and lengthy procedures using costly commercially-available radiochemistry boxes (Table 1). Therefore, further simplification of the radiosynthesis of [¹⁸F]SFB using a low-cost setup would be very beneficial for its adaption to an automated process.Herein, we report a concise preparation of [¹⁸F]SFB, based on a simplified one-pot microwave-assisted synthesis (Figure 1). Our approach does not require purification between steps or any aqueous reagents. In addition, microwave irradiation, which has been used in the syntheses of several PET tracers,^38-41 can gives higher RCYs and better selectivity than the corresponding thermal reactions or they provide similar yields in shorter reaction times.³⁸ Most importantly, when labeling biomolecules, the time saved could be diverted to subsequent bioconjugation or PET imaging step.^28,43 The novelty of our improved [¹⁸F]SFB synthesis is two-fold: (1) the anhydrous deprotection strategy requires no purification of intermediate(s) between each step and (2) the microwave-assisted radiochemical transformations enable the rapid, reliable production of [¹⁸F]SFB.Download video file.^{(51M, mov)}     

Site-specific labeling of annexin V with F-18 for apoptosis imaging

Annexin V is useful in detecting apoptotic cells by binding to phosphatidylserine (PS) that is exposed on the outer surface of the cell membrane during apoptosis. In this study, we examined the labeling of annexin V-128, a mutated form of annexin V that has a single cysteine residue at the NH 2 terminus, with the thiol-selective reagent (18)F-labeling agent N-[4-[(4-[(18)F]fluorobenzylidene)aminooxy]butyl]maleimide ([(18)F]FBABM). We also examined the cell binding affinity of the (18)F-labeled annexin V-128 ([(18)F]FAN-128). [(18)F]FBABM was synthesized in two-step, one-pot method modified from literature procedure. (Toyokuni et al., Bioconjugate Chem. 2003, 14, 1253-1259). The average yield of [(18)F]FBABM was 23 +/- 4% (n = 4, decay-corrected) and the specific activity was approximately 6000 Ci/mmol. The total synthesis time was approximately 92 min. The critical improvement of this study was identifying and then developing a purification method to remove an impurity N-[4-[(4-dimethylaminobenzylidene)aminooxy]butyl]maleimide 4, whose presence dramatically decreased the yield of protein labeling. Conjugation of [(18)F]FBABM with the thiol-containing annexin V-128 gave [(18)F]FAN-128 in 37 +/- 9% yield (n = 4, decay corrected). Erythrocyte binding assay of [(18)F]FAN-128 showed that this modification of annexin V-128 did not compromise its membrane binding affinity. Thus, an in vivo investigation of [ (18)F]FAN-128 as an apoptosis imaging agent is warranted.     

Synthesis and evaluation of radiolabeled AGI-5198 analogues as candidate radiotracers for imaging mutant IDH1 expression in tumors

Mutations in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) are commonly found in gliomas. AGI-5198, a potent and selective inhibitor of the mutant IDH1 enzyme, was radiolabeled with radioiodine and fluorine-18. These radiotracers were evaluated as potential probes for imaging mutant IDH1 expression in tumors with positron emission tomography (PET). Radioiodination of AGI-5198 was achieved using a tin precursor in 79?±?6% yield (n?=?9), and ¹⁸F-labeling was accomplished by the Ugi reaction in a decay-corrected radiochemical yield of 2.6?±?1.6% (n?=?5). The inhibitory potency of the analogous nonradioactive compounds against mutant IDH1 (IDH1-R132H) was determined in enzymatic assays. Cell uptake studies using radiolabeled AGI-5198 analogues revealed somewhat higher uptake in IDH1-mutated cells than that in wild-type IDH1 cells. The radiolabeled compounds displayed favorable tissue distribution characteristics in vivo, and good initial uptake in IDH1-mutated tumor xenografts; however, tumor uptake decreased with time. Radioiodinated AGI-5198 exhibited higher tumor-to-background ratios compared with ¹⁸F-labeled AGI-5198; unfortunately, similar results were observed in wild-type IDH1 tumor xenografts as well, indicating lack of selectivity for mutant IDH1 for this tracer. These results suggest that AGI-5198 analogues are not a promising platform for radiotracer development. Nonetheless, insights gained from this study may help in design and optimization of novel chemical scaffolds for developing radiotracers for imaging the mutant IDH1 enzyme.     

High-yielding aqueous 18F-labeling of peptides via Al18F chelation

The coordination chemistry of a new pentadentate bifunctional chelator (BFC), NODA-MPAA 1, containing the 1,4,7-triazacyclononane-1,4-diacetate (NODA) motif with a methylphenylacetic acid (MPAA) backbone, and its ability to form stable Al(18)F chelates were investigated. The organofluoroaluminates were easily accessible from the reaction of 1 and AlF(3). X-ray diffraction studies revealed aluminum at the center of a slightly distorted octahedron, with fluorine occupying one of the axial positions. The tert-butyl protected prochelator 7, which can be synthesized in one step, is useful for coupling to biomolecules on solid phase or in solution. High yield (55-89%) aqueous (18)F-labeling was achieved in 10-15 min with a tumor-targeting peptide 4 covalently linked to 1. Defluorination was not observed for at least 4 h in human serum at 37 °C. These results demonstrate the facile application of Al(18)F chelation using BFC 1 as a versatile labeling method for radiofluorinating other heat-stable peptides for positron emission imaging.     

Synthesis and biological evaluation of a series of novel N- or O-fluoroalkyl derivatives of tropane: potential positron emission tomography (PET) imaging agents for the dopamine transporter.

A series of novel fluoroalkyl-containing tropane derivatives was synthesized, and their binding affinities for the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET) were determined via competitive binding assays. Among these derivatives, the fluoropropyl ester of beta-CIT (19), the fluoroethyl ester of beta-CIT (20), the N-fluoropropyl derivative of beta-CBT (12), and the fluoropropyl ester of beta-CMT (18) displayed higher affinity and greater selectivity for the DAT versus SERT and NET than FP-CIT, which indicates that they are attractive candidates for the development of (18)F-labeled PET imaging agents for the DAT.