Synthesis and Application of [18F]FDG-Maleimidehexyloxime ([18F

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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† Frank Wuest,* Mathias Berndt, Ralf Bergmann, Joerg van den Hoff, and Jens Pietzsch Research Center Dresden-Rossendorf, Institute for Radiopharmacy, PF 510 119, D-01314 Dresden, Germany. Received January 9, 2008; Revised Manuscript Received February 25, 2008

2-[18F]Fluoro-2-deoxy-D-glucose ([18F]FDG) as the most important PET radiotracer is available in almost every PET center. However, there are only very few examples using [18F]FDG as a building block for the synthesis of 18 F-labeled compounds. The present study describes the use of [18F]FDG as a building block for the synthesis of 18 F-labeled peptides and proteins. [18F]FDG was converted into [18F]FDG-maleimidehexyloxime ([18F]FDGMHO), a novel [18F]FDG-based prosthetic group for the mild and thiol group-specific 18F labeling of peptides and proteins. The reaction was performed at 100 °C for 15 min in a sealed vial containing [18F]FDG and N-(6aminoxy-hexyl)maleimide in 80% ethanol. [18F]FDG-MHO was obtained in 45-69% radiochemical yield (based upon [18F]FDG) after HPLC purification in a total synthesis time of 45 min. Chemoselecetive conjugation of [18F]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 ([18F]FDG-MHO-anxA5) was obtained in 43-58% radiochemical yield (based upon [18F]FDG-MHO, n ) 6), and [18F]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.

INTRODUCTION Compounds labeled with short-lived positron-emitting radionuclides have extensively been utilized to target selective physiological and biochemical processes at the molecular and cellular level in vivo by means of positron emission tomography (PET) as the most advanced noninvasive imaging methodology. Besides numerous small molecular weight compounds, in recent years several radiolabeled molecules such as peptides, proteins, and aptamers have been proposed as highly target-specific PET imaging probes. In this context, various radiolabeled peptides and proteins, including antibodies and antibody fragments, have been used in nuclear medicine for imaging of tumors and inflammatory processes. Within the spectrum of available positron emitters, fluorine-18 (18F) is a particularly attractive radionuclide due to its favorable nuclear and chemical properties. 18F can be produced in high amounts via several nuclear reactions in particle accelerators (cyclotrons) at high specific radioactivity. The low positron energy of 0.64 MeV results in low radiation doses and short tissue range, and the relatively long 109.7 min half-life allows complex and multistep radiosyntheses and scanning protocols extending 4 to 6 h. However, radiochemistry with 18F focused on peptide and protein labeling also implies specific challenges. Thus, direct incorporation of 18F at high specific radioactivity as [18F]fluoride into peptides and proteins is hampered due to the harsh reaction conditions occurring during [18F]fluoride †

A part of this report has been presented at the 53rd annual meeting of the Society of Nuclear Medicine, San Diego 2006, June 3-7, and has been published in abstract form: M. Berndt, C. Hultsch, R. Bergmann, J. Pietzsch, and F. Wuest. A novel role for [18F]FDG: Synthesis and application of a [18F]FDG-based prosthetic group for peptide and protein labeling. J. Nucl. Med. 2006, 47, 83. * Address for correspondence: Frank Wuest, Institute of Radiopharmacy, Research Center Dresden-Rossendorf, P.O.-Box 51 01 19, 01314 Dresden, Germany. ([email protected].)

labeling reactions. In order to circumvent this obstacle, peptide and protein labeling with 18F is usually carried out by means of prosthetic groups, also referred to as bifunctional labeling agents (1–6). This approach involves 18F incorporation into a small organic molecule capable of being linked to peptides, proteins, antibodies, and oligonucleotides under mild conditions. Such linkage of 18F-labeled prosthetic groups can be accomplished via acylation (6–18), amidation (19), imidation (20), alkylation (19–32), photochemical conjugation (6, 33–35), and oxime (36, 37) or hydrazone formation (38, 39). Every linking method has its own advantage and limitations. Depending on the conjugation mechanism and the multitude of functional groups abundant in proteins (e.g., lysine residues), the chemoselective and regioselective introduction of the radiolabel into a defined position represents a special challenge. Moreover, the laborious, time-consuming, and multistep synthesis of most 18Flabeled prosthetic groups represents another drawback, which has limited the availability and applicability of prosthetic groups in routine 18F-labeling reactions. As a consequence, the routine 18F labeling of molecules like peptides and proteins should preferentially involve simple and efficient radiolabeling methods based on readily available prosthetic groups while allowing the chemo- and regioselective introduction of the radiolabel. 2-[18F]Fluoro-2-deoxy-D-glucose ([18F]FDG) is the most important radiotracer for PET imaging in nuclear medicine. Its highly efficient radiosynthesis makes this radiotracer available in almost every PET center worldwide. However, despite the convenient automated synthesis and almost unlimited availability of [18F]FDG there are only very few examples using [18F]FDG and peracetylated [18F]FDG (1,3,4,6-tetra-O-acetyl-2-[18F]fluoro2-deoxyglucose) as building blocks for the radiosynthesis of 18 F-labeled compounds (Figure 1). One of the first reports dealing with [18F]FDG as 18F building block was written by Borman et al. on the synthesis of β-O18 D-galactopyranosyl-(1,4′)-2′-[ F]fluoro-2′-deoxy-D-glucopyra-

10.1021/bc8000112 CCC: $40.75  2008 American Chemical Society Published on Web 05/16/2008

Synthesis and Application of [18F]FDG-MHO)

Figure 1. Structures of 18F-labeled radiotracers synthesized starting from [18F]FDG or peracetylated [18F]FDG.

nose (2′-[18F]fluoro-deoxylactose, [18F]FDL) using an enzymatic reaction starting from [18F]FDG (40). Although the obtained radiochemical yield was quite low (3.4% decay-corrected, starting from [18F]FDG), the authors could perform preliminary biological evaluation of [18F]FDL involving biodistribution studies in mice. Starting from peracetylated [18F]FDG as 18F building block, Patt and co-workers developed a FDG-based radiotracer for imaging hypoxia (41). HPLC-purified peracetylated [18F]FDG was treated with 2-nitroimidazole in the presence of Hg(CN)2 as a source for cyanide anion and the Lewis acid SnCl4 to give a glucose-coupled 2-nitroimidazole intermediate. After removal of the acetyl protecting groups of the intermediate applying reaction conditions similar to the [18F]FDG production method, the desired [18F]FDG-coupled 2-nitroimidazole could be isolated and used in subsequent biological studies. The use of peracetylated [18F]FDG as building block in the radiosynthesis of 18F-glycosylated amino acids serine and threonine has been reported by Prante et al. (42, 43). The authors describe two different reaction pathways involving either the reaction of an intermediate glucopyranosyl bromide with FmocSer-OH or Z-Ser-OBn in the presence of AgOTf or direct conversion of peracetylated [18FDG] with Fmoc-protected serine or threonine in the presence of Lewis acid BF3 into glycosylated amino acids. The glycosylation reaction involving a glucopyranosyl bromide intermediate according to a Koenigs-Knorr reaction afforded radiochemical yields of 67%, whereas the BF3mediated method provided significantly lower radiochemical yields of 27% suggesting the suitability of Koenig-Knorr reaction conditions for 18F-glycosylation of protected bioactive peptides. Another reaction with peracetylated [18F]FDG was described by the same author aiming at the enzymatic synthesis of uridine5′-diphospho-2-deoxy-2-[18F]fluoro-R-D-glucopyranose (UDP[18F]FDG) (44). Starting from peracetylated [18F]FDG, a MacDonald synthesis was used to prepare [18F]FDG-1phosphate which was successfully subjected to an enzymatic reaction to prepare UDP-[18F]FDG. This method is also discussed as a highly selective and mild 18F-labeling method for glycosylated biomolecules. Very recently, 3,4,5-tri-O-acetyl-2-[18F]fluoro-2-deoxy-Dglucopyranosyl 1-phenylthiosulfonate (Ac3-[18F]FGLc-PTS) was

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reported as a novel thiol-reactive reagent for the chemoselective 18 F-glycosylation of peptides (45). Ac3-[18F]FGLc-PTS was prepared in three steps in radiochemical yield of 33%. The feasibility of using Ac3-[18F]FGLc-PTS as a thiol-reactive agent was demonstrated through chemoselective bioconjugation to a cycteine-containing model pentapeptide and to the integrin RVβ3affine peptide c(RGDfC). Both peptides could be labeled in conjugation yields greater than 90%. The present study aims at the use of [18F]FDG as a 18F building block for the convenient synthesis of a thiol-reactive prosthetic group for the chemoselective conjugation to cysteinecontaining peptides and proteins. In recent years, several maleimide-functionalized prosthetic groups have been shown to be especially suitable as chemoselective agents for the sitespecific 18F labeling of cysteine-containing peptides under mild reaction conditions (19–22, 32, 46). Herein, we report on the use of [18F]FDG as a suitable building block for the synthesis of 18F-labeled peptides and proteins. The presented approach involves the design and synthesis of [18F]FDG-maleimidehexyloxime ([18F]FDG-MHO) as a novel [18F]FDG-based prosthetic group for the mild and thiol group-specific 18F labeling of peptides and proteins. The feasibility of [18F]FDG-MHO as thiol group-reactive prosthetic group was assessed through the reaction with cycteine-containing tripeptide glutathione and with single thiol group-containing protein annexin A5 (anxA5). The present work is concluded with a pilot small animal PET study to assess biodistribution and kinetics of chemoselectively radiolabeled anxA5 in a human colorectal adenocarcinoma (HT-29) murine xenograft model in vivo.

EXPERIMENTAL METHODS General. All commercial reagents and solvents were used without further purification unless otherwise specified. Nuclear magnetic resonance spectra were recorded on a Varian Unity 300 MHz spectrometer. 1H and 19F chemical shifts were given in ppm and were referenced with the residual solvent resonances relative to tetramethylsilane (TMS). Mass spectra were obtained on a Quattro/LC mass spectrometer (MICROMASS) by electrospray ionization. Thin-layer chromatography (TLC) was performed on Merck silica gel F-254 aluminum plates, with visualization under UV (254 nm). [18F]FDG was prepared according to refs (47, 48). HPLC analyses were carried out with a SUPELCOSIL LC-18S column (4.6 × 250 mm, 5 mm) using isocratic elution with CH3CN/ 0.1 M ammonium formate (30/70) gradient pump L2500 (Merck, Hitachi) with a flow rate of 1 mL/min. The products were monitored by an UV detector L4500 (Merck, Hitachi) at 254 nm and by γ-detection with a scintillation detector GABI (X-RAYTEST). Semipreparative HPLC was performed with a Merck LiChroCart 250-10 column at a flow rate 4 mL/min. Solvent A: CH3CN; solvent B: 0.1 M ammonium formate. The following gradient was used: 0 min 15% A, 20 min 70% A, 30 min 70% A. Radio-TLC analyses of glutathione coupling with [18F]FDG-MHO was performed on Merck RP18 F-254 aluminum plates. Radio-sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to published literature procedure (32). Aminooxy-maleimide hydrochloride 1 (32) and [18F]FDG (47, 48) were prepared according to literature. 2-Fluoro-3,4,5,6-tetrahydroxyhexanal-O-[3-(2,5-dioxo-2,5dihydropyrrol-1-yl)hexyl]oxime (FDG-MHO). 2-Fluorodeoxyglucose (50 mg, 275 µmol) and aminooxymaleimide × HCl 1 (69 mg, 277 µmol) were dissolved in 5 mL of EtOH/H2O (4:1) and the resulting solution was refluxed for 1 h. The mixture was concentrated under reduced pressure. After the addition of water (50 mL), the mixture was passed through a C18 SepPak

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cartridge. The cartridge was washed with water (10 mL) and the product was eluted with EtOH (2 mL). The solvent was evaporated to give 77 mg (75%) of the desired compound FDGMHO as a colorless glass. 1H NMR (400 MHz, CD3OD) δ 7.48 (dd, 1H, JH-F ) 7.5 Hz, JH-H ) 6.1 Hz; dNHOR), 6.79 (s, 2H, CHdCH), 5.03 (dt, 1H, JH-F ) 48.4 Hz, JH-H ) 7.5 Hz; CH-F), 4.08 (dd, 1H, J ) 7.1 Hz, J ) 1.6 Hz), 4.05 (t, 2H, J ) 6.6 Hz; dNOsCH2), 3.77 (dd, 1H, J ) 11.2 Hz, J ) 3.4 Hz), 3.69 (m, 1H), 3.58-3.66 (m, 2H), 3.48 (t, J ) 7.1 Hz, 2H; CH2-maleimide), 1.53-1.67 (m, 4H, CH2), 1.26-1.42 (m, 4H, CH2). 13C NMR (100 MHz, CD3OD) δ 26.1, 27.3, 28.6, 29.3, 38.5, 62.5, 71.2 (d, JC-F ) 7.9 Hz), 71.7, 72.5, 91.4 (d, JC-F ) 169.9 Hz), 134.2, 145.8 (d, JC-F ) 23.7 Hz), 172.7. 19 F-NMR (376 MHz, CD3OD) δ -61.67 (ddd, J ) 45.8 Hz, J ) 12.2 Hz, J ) 6.1 Hz), LR-MS (ESI, positive): m/z 377.4 [M+H]+, 399.3 [M+Na]+. 2-[18F]Fluoro-3,4,5,6-tetrahydroxyhexanal-O-[3-(2,5-dioxo2,5-dihydropyrrol-1-yl)hexyl]oxime ([18F]FDG-MHO). [18F]FDG solution in saline (1 mL, 400-700 MBq) was added to vial containing aminooxymaleimide hydrochloride 1 (5 mg, 13.3 µmol) in EtOH (4 mL). The vial was sealed and heated at 100 °C for 15 min. After evaporation of the solvent and addition of water (20 mL), the mixture was passed through a SepPak cartridge. The crude prudct was eluted with acetonitrile (1 mL) and subjected to semipreparative HPLC for purification. The peak eluting at 18-19 min was collected, diluted with water (20 mL), and passed through a SepPak cartridge. Elution with EtOH (1 mL) gave the desired product in high radiochemical purity (>95%). In a typical experiment, starting from 420 MBq of [18F]FDG, 135 MBq of [18F]FDG-MHO (42% radiochemical yield based upon [18F]FDG) could be obtained within 45 min. RadiolabelingofGluthathionewith[18F]FDG-MHO. [18F]FDGMHO in ethanol (25 µL, 8 MBq) was added to 900 µL of glutathione solutions (1.0 mg/mL up to 10 ng/mL) in phosphatebuffer (pH ) 7.2) and incubated at room temperature. The progress of the reaction was monitored by radio-TLC after 10 min. Radio-TLC: Rf ) 0.8, CH3CN/water (80/20). Radiolabeling of anxA5 with [18F]FDG-MHO. [18F]FDGMHO in ethanol (25 µL, 18-20 MBq) was added to 100 µL of anxA5 solution (1.0 mg/mL recombinant AnxA5 (kind gift from Dr. Nediljko Budisa), isolated from Escherichia coli B834 (DE3), transformed with plasmid pASET-pp4, as described by Burger et al. (49)) in 0.05 mol/L Tris-HCl (pH 7.4) and incubated for 30 min at room temperature. The reaction mixture ¨ kta Prime plus, was applied to size exclusion chromatography (A HiTrap Desalting column 5 mL) and eluted with phosphatebuffered saline (pH 7.2; 0.5 mL/min). The collected high molecular weight fractions were combined to give 6-8 MBq (44-58%, decay-corrected, based upon [18F]FDG-MHO) of radiolabeled anxA5. Radiochemical purity radiolabeled anxA5 was greater 95% as determined by radio-sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The isolated product was directly used for small animal PET imaging studies. Small Animal PET Imaging of 18F-labeled anxA5. Dynamic small animal PET studies were performed with a dedicated PET scanner for small animals (microPET P4, Siemens Preclinical Solutions, Knoxville, TN, USA). For imaging studies, mice were anesthetized with a desflurane 10% oxygen/40% air mixture and catheters with hypodermic needle were placed into a tail vein. The animals were then positioned and immobilized prone with their medial axis parallel to the axial axis of the scanner on a heated bed. In a typical experiment, 1 MBq of [18F]FDG-MHO-anxA5 was administered in a total volume of 0.5 mL over 1 min using a programmable syringe pump 30 s after start of the PET data acquisition. Dynamic PET scanning was carried out for 14 400 s (4 h), using the following time intervals (frames) for sinogram generation: 15 × 10 s, 5

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Figure 2. Proposed mutarotation of glucose analogue [18F]FDG via an acyclic form.

× 30 s, 5 × 60 s, 4 × 300 s, and 21 × 600 s. The PET data were reconstructed using a 3D OSEM MAP iteration algorithm provided by the PET scanner producer. The image data were evaluated with the program ROVER (ABX GmbH, Radeberg, Germany) to get 3D regions of interest (ROI) of tissues of interest. The time activity concentration curves and the activity amounts were further processed using software written in the R language (R Development Core Team: R: A Language and Environment for Statistical Computing, http://www.R-project.org, R Foundation for Statistical Computing, Vienna, Austria (2007). ISBN 3-900051-07-0.).

RESULTS AND DISCUSSION [18]FDG as the most important PET radiotracer for in vivo imaging of glycolysis in nuclear medicine has found multiple applications in the fields of oncology, neurology, and cardiology. In aqueous solutions, [18F]FDG consists of a mixture of R/β anomers. The process behind the equilibration between the R anomer and the β anomer is known as mutarotation. Mechanistically, the isomerization must proceed with ring opening via a small concentration of the acyclic form (Figure 2). To the best of our knowledge, however, the exact amount of each isomeric form of [18F]FDG in aqueous solution is still not known yet. In the case of D-glucose, in aqueous solution at room temperature the amounts of the R and β anomers were determined to be 38% and 62%, respectively. The amount of the acyclic form is reported to be as little as 0.003% (50). A comparable situation should be expected for the glucose analogue [18F]FDG. Although presumably available in only very small amounts, in principle the open chain aldehyde form of [18F]FDG should allow chemoselective aldehyde group-based reactions. A very prominent and chemoselective reaction represents the aminooxy-aldehyde coupling reaction to yield the corresponding oxime. Recently, the chemoselective oxime formation was shown to be a promising and versatile approach for both the convenient synthesis of 18F-labeled prosthetic groups (20, 32) and the direct 18F-labeling of aminooxy groupfunctionalized peptides (36) with readily available 4-[18F]fluorobenzaldehyde. Likewise, chemoselective reaction between the acyclic aldehyde form of [18F]FDG with aminooxy group-containing compound 1 should lead to the corresponding oxime derivative [18F]FDG-MHO. However, in the case of hexoses as aldehyde components in the aminooxy-aldehyde coupling reaction the formation of isomeric products must be considered. Hence, Ramsay et al. observed the formation of three isomeric products with identical molecular mass when they investigated the reaction of O-(4-(nitrobenzyl)-hydroxylamine with glucose at pH 4 (51). The observed isomers were proposed to be a mixture of E- and Z- acyclic oximes and R- and β-cyclic products (pyranoses and furanoses, respectively.). Accordingly, chemoselective condensation between the hexose FDG and aminooxyfunctionalized maleimide 1 should also lead to a mixture of isomers as shown in Figure 3. Synthesis of the Reference Compound FDG-MHO. Reference compound FDG-MHO was prepared by heating equimolar amounts of [19]FDG and aminooxy compound 1 in EtOH/H2O (4:1) for two hours. After solid-phase extraction using a C18



Figure 3. Proposed isomer formation through aminooxy-aldehyde coupling reaction involving FDG.

Figure 4. HPLC analysis of isolated product of the aminooxy-aldehyde coupling reaction with FDG.

SepPak cartridge and evaporation of the solvent, the desired reference compound FDG-MHO was obtained as a glass in 79% yield. HPLC analysis of the product indicated, however, the formation of several compounds consisting of a major product (>90%, tR ) 5.6 min) accompanied by at least two smaller peaks at retention times of 5.1 and 6.0 min, respectively (Figure 4). The three peaks were collected and subjected to mass spectrometric analysis (ESI, positive). The peaks at 5.1 and 5.6 min showed identical mass peaks of m/z 377.4 ([M+H]+) and m/z 399.35 ([M+Na]+), respectively, suggesting the formation of at least two isomers. The formation of isomers during the aminooxy-aldehyde conjugation reaction between the hexose FDG and aminooxy compound 1 in aqueous media is in accordance with the report by Ramsay et al. (51). However, NMR analysis of the isolated reaction product in CD3OD shows only one distinct compound. In the 1H NMR spectra, the observed characteristic doublet-doublet coupling pattern (JH-F ) 7.5 Hz, JH-H ) 6.1 Hz) at 7.48 ppm correlates with one proton as expected for an oxime proton (dNHsOR). No signals were found which would refer to the formation of related pyranose or furanose isomers as depicted in Figure 3. It is known from the literature that oxime protons of E- and Z-isomers show characteristic chemical shift differences, being, e.g., 7.56 ppm

for the E-isomer and 6.95 ppm for the Z-isomer (52). The observed more downfield shifted signal at 7.48 ppm would agree with an E-isomer oxime proton. Consequently, we conclude that the product possesses an open-chain E-oxime structure. Radiosynthesis of [18F]FDG-MHO. The radiosynthesis of [18F]FDG-MHO is given in Figure 5. The reaction is based on treatment of a [18F]FDG solution, which was prepared according to the well-established [18F]FDG synthesis (47, 48), with aminooxy-functionalized compound 1. Notably, the used [18F]FDG solution contains substantial amounts of D-glucose (50-70 µg per milliliter [18F]FDG solution) as typically formed during the [18F]FDG production process starting from mannose triflate and subsequent basic hydrolysis. Therefore, formation of the corresponding glucose product must be considered. The formation of the glucose product was confirmed through LC-MS (ESI, positive) analysis of the collected mass peak (m/z 375.2 [M+H]+, m/z 397.4 [M+Na]+) in the semipreparative HPLC (tR ) 19-21 min). To avoid complete consumption of the amino-oxy labeling precursor by the reaction with glucose, the amount of labeling precursor has to be sufficiently high to give the desired [18F]FDG-MHO compound in satisfactory radiochemical yields. Thus, best results were obtained when at least 10 mg (40 µmol) of compound 1 per milliliter [18F]FDG solution was used. The


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Figure 5. Radiosynthesis of [18F]FDG-MHO.

Figure 6. Radiolabeling of GSH with [18F]FDG-MHO. Table 1. Radiolabeling of GSH with [18F]FDG-MHO GSH concentration

1 mg/mL

100 µg/mL

10 µg/mL

1 µg/mL

100 ng/mL

10 ng/mL

1 ng/mL

% Product

>95

>95

>95

>95

92

47

38

reaction proceeded best in 80% ethanol as the solvent at 100 °C for 15 min. After semipreparative HPLC purification desired [18F]FDG-MHO could be isolated in 45-69% radiochemical yield based upon [18F]FDG (decay-corrected) within 45 min in high radiochemical purity (greater than 95%). The obtained radiochemical yield of 45-69% for [18F]FDGMHO is comparable with related maleimide-containing 18F prosthetic groups such as N-{4-[(4-[18F]fluorobenzylidene)aminooxy]butyl}maleimide (20), [18F]FBAM (32), [18F]FPyME (21), and [18F]FBEM (46). However, the facile availability of [18F]FDG as the labeling precursor in the coupling reaction circumvents the laborious preparation of 18F-labeled precursors such as 4-[18F]fluorobenzaldehyde, [18F]fluoropyridine derivatives, or [18F]SFB as intermediates required for the synthesis of N-{4-[(4-[18F]Fluorobenzylidene)aminooxy]butyl}maleimide, [18F]FBAM, [18F]FPyME, and [18F]FBEM, respectively. Thus, the simple single step reaction sequence starting from easily available [18F]FDG make the synthesis of [18F]FDGMHO a very attractive alternative to previously published maleimide-containing 18F prosthetic groups. Radiolabeling of Glutathione (GSH). Various thiol-reactive agents containing a maleimide group have successfully been used for the chemoselective modification of peptides and proteins. Thiol-specific Michael reaction of maleimide-containing prosthetic groups provides high chemoselectivity when compared with amine and carboxylate-reactive labeling agents. The labeling properties of the bifunctional labeling agent [18F]FDG-MHO for chemoselective conjugation toward thiol groups was exemplified by the reaction with the cysteinecontaining tripeptide glutathione (GSH) and the single thiol group-containing protein anxA5 (Figure 6). The 18F-labeling reaction was performed at room temperature for 10 min in a phosphate buffer solution (pH 7.2) containing various GSH concentrations ranging from 1 ng/mL to 1.0 mg/ mL. Conversion of [18F]FDG-MHO into conjugate [18F]FDGMHO-GSH was monitored by radio-TLC analysis as previously reported (32). The conversion refers to as the percentage of radioactivity area of coupling product [18F]FDG-MHO-GSH

relative to the total radioactivity area. The results are summarized in Table 1. More than 95% of the labeling reagent [18F]FDG-MHO was converted into the corresponding GSH conjugate at GSH concentrations ranging from 1.0 mg/mL to 1 µg/mL after 10 min at room temperature. At lower GSH concentrations of 100, 10, and 1 ng/mL, the conversions were still good to moderate, being 92%, 47%, and 38%, respectively. The dependency of the radiochemical yield upon the peptide concentration has also been reported in previous publications describing the 18Flabeling of peptides via prosthetic group labeling strategies (13). The labeling efficiency of [18F]FDG-MHO toward GSH in this work is superior to recently reported results in the literature involving maleimide-containing prosthetic groups for 18Flabelings, especially at very low GSH concentrations (20, 32). The determined radiochemical yields of 92% and 47% at 100 ng/mL and 10 ng/mL for [18F]FDG-MHO are significantly higher compared to the 20% and 5% radiochemical yield observed at similar GSH concentrations of 100 and 10 ng/mL in the case of the prosthetic group [18F]FBAM (32). Thus, the obtained excellent radiochemical yields even at very low GSH concentrations after 10 min at room temperature make the bifunctional labeling agent [18F]FDG-MHO an excellent 18F prosthetic group for the efficient labeling of thiol groups under mild reaction conditions compatible with peptides and proteins. Radiolabeling of Annexin A5 (anxA5). The feasibility of [18F]FDG-MHO as a thiol-reactive bifunctional labeling agent was further assessed by using the single thiol group-containing protein annexin A5 (anxA5) as a model. The mature human anxA5 is a 36 kDa, 319 amino acid protein which belongs to a family of calcium-dependent phospholipid-binding interfacial membrane proteins. In the past decade, anxA5 binding has become one of the standard procedures to measure apoptosis in histopathology (55). Moreover, an increasing number of reports deal with radiolabeled anxA5 as molecular probes for imaging apoptosis in vivo (56). In this line, 99mTc-, 68Ga-, 18F-, 123I-, and 124I-labeled annexin derivatives have been prepared and studied. In the case


Figure 7. Representative radio-SDS-PAGE pattern of the reaction mixture (A) and the purified 18F-labeled anxA5 (B). The figure shows radioactivity distribution as determined by radioluminography using a BAS 5000 scanner (Fujix, Japan). The radioluminograms were analyzed with the AIDA software package (Raytest GmbH, Germany).

of 18F-labeled anxA5 derivatives, all described radiosyntheses were based on the reaction of anxA5 with the amine-group reactive prosthetic group [18F]SFB (57–61). Consequently, incorporation of the 18F label was achieved via nonchemoselective, random [18F]fluorobenzoylation of variations of the total 22 lysine residues or the chemically accessible N-terminus of anxA5. On the other hand, mature anxA5 contains only one chemically accessible cysteine residue at position 315 (position 316, often cited in the literature, refers to the unprocessed precursor). Therefore, anxA5 should prove a suitable model for the chemoselective 18F radiolabeling of proteins possessing one single cysteine residue. Radiolabeling of anxA5 with the thiolgroup reactive agent [18F]FDG-MHO is expected not to lead to adverse modification of anxA5. Moreover, modification of the cysteine residue in anxA5 should not alter the biological activity and functionality of the protein. This assumption is supported by recent data from the patent literature reporting the same binding affinity of anxA5 or annexin conjugates for phosphatidylserine-containing membranes when cysteine 315 is deleted


or replaced with serine or other non-sulfur-containing amino acids (62, 63). The radiolabeling of anxA5 was performed through chemoselective alkylation of the cysteine residue in anxA5 with maleimide [18F]FDG-MHO. Treatment of anxA5 (100 µg, 2.8 nmol) in Tris buffer (pH ) 7.4) with [18F]FDG-MHO and subsequent size-exclusion chromatography afforded 43-58% (decay-corrected, based upon [18F]FDG-MHO, n ) 6) of [18F]FDG-MHO-anxA5 within 60 min. Considering the low anxA5 concentration used (2.25 µM) and the presence of a single cysteine residue as the sole potential reaction site, the radiochemical yield is remarkably good. The high efficiency of the chemoselective introduction of the 18F label via maleimidecontaining prosthetic group [18F]FDG-MHO into anxA5 is further confirmed when comparing the obtained 43-58% radiochemical yields with the reported yields of [18F]SFB-based anxA5 derivatives. Thus, conjugation reaction with [18F]SFB using a comparable amount of anxA5 (100 µg) gave only a 10% radiochemical yield based upon [18F]SFB (58). Higher radiochemical yields of up to 70% for the conjugation step were only achievable when fairly high protein concentrations of up to 6 mg/mL were used (57, 60, 61). Moreover, conjugation reaction of anxA5 with [18F]SFB can rely on the availability of 22 lysine residues and the N-terminus in the protein as potential reaction sites. This leads to a highly random [18F]fluorobenzoylation of the available primary amines abundant in anxA5, thus avoiding a regioselective and chemoselective introduction of the 18F label as in the case of [18F]FDG-MHO-anxA5. Moreover, in some instances the use of the amine-reactive agent [18F]SFB for protein labeling can fail completely. As an example, steric hindrance of the N-terminal region of apoB100 due to oxidative modification of apoB-100 by myeloperoxidase or hypochlorous acid prevents radiolabeling with the acylation agent [18F]SFB (32). Starting from 20 MBq of [18F]FDG-MHO, the effective specific activity of purified 18F-labeled anxA5 was calculated to be 2-4 GBq/µmol at the end of synthesis (related to anxA5, Mr ) 36 000 Da). Purification of the reaction mixture by sizeexclusion chromatography gave radiochemically pure [18F]FDGMHO-anxA5, which could be completely separated from unreacted [18F]FDG-MHO and other radioactive byproduct not further identified. The quality of purified [18F]FDG-MHO-anxA5 was suitable to be used directly for subsequent small animal PET studies. Radiolabeling and purification of radiolabeled anxA5 was monitored by radio-SDS-PAGE. Figure 7 shows representative radio-SDS-PAGE patterns of the reaction mixture (lane A) and purified 18F-labeled anxA5 fraction (lane B).

Figure 8. Small animal PET images of [18F]FDG-MHO-anxA5 at 2 min (A) and 4 h (B,C,D) after tracer application. (B) Coronal section on the level of kidneys. (C) Coronal section on the level of xenographted tumor. (D) Coronal section as (C) but expanded scale intensity.


Wuest et al.

Figure 9. Representative time-activity curves from one animal after single intravenous application of [18F]FDG-MHO-anxA5. (A) Time-activity concentration (SUV) curves of the heart, tumor, and muscle (note the semilogarithmic scale). (B) Time-activity (%ID) curves of liver, intestine, gall bladder, kidney, and urinary bladder.

Small Animal PET Imaging of [18F]FDG-MHO-anxA5. Biodistribution and kinetics of [18F]FDG-MHO-anxA5 was evaluated in a pilot small animal PET study in a human colorectal adenocarcinoma (HT-29) murine xenograft model. The murine xenograft model was not treated with an apoptosis inducing agent. Figure 8 shows small animal PET images at 2 min and 4 h after injection of [18F]FDG-MHO-anxA5. Figure 9 depicts representative time-activity curves for heart (blood pool), tumor, and muscle (A), liver, intestine, gall bladder, kidney, and urinary bladder (B). After intravenous application of [18F]FDG-MHO-anxA5, the radiotracer was rapidly distributed in the blood pool. [18F]FDGMHO-anxA5 showed a blood clearance with a mean half-life of 6.5 min, which is slower in comparison to the 2.6 min halflife as determined for [18F]fluorobenzoylated anxA5 (R. Bergmann et al., unpublished results). Thus, chemoselective labeling of the cysteine residue in anxA5 with [18F]FDG-MHO leads to a 18F-labeled anxA5 derivative with a longer residence time in the blood pool resulting in a better availability of the radiotracer for in vivo imaging of apoptosis. Radiolabeled anxA5 was mainly accumulated in the kidney cortex, reaching 8.2%ID at 200 min p.i. The high activity accumulation in the renal excretory system is consistent with reports in the literature using 18F-labeled anxA5 derivatives in normal rats (59, 61). At the same time point, approximately 4%ID of the injected activity was found in the liver. A part of activity was eliminated into the bile (1.6%ID at 200 min p.i.) and intestine (2.6%ID at 200 min p.i.). The activity in the xenografted tumor increased over the time reaching a tumorto-muscle ratio of about 3 at 200 min p.i. Therefore, although the absolute activity in the HT-29 tumor was relatively low (SUV ) 0.12) the tumor could be clearly delineated in the PET images (Figure 9).

CONCLUSION We have developed a novel thiol-reactive prosthetic group ([18F]FDG-MHO) based upon the readily available PET radiotracer [18F]FDG. The straightforward access to [18F]FDG makes this procedure especially attractive, e.g., for research groups without a complete PET facility. The novel prosthetic group was used for the chemoselective 18F-labeling of glutathione as a thiol-containing model peptide and single cysteine residue containing anxA5 as a model protein. The use of [18F]FDG-MHO as thiol group reactive prosthetic group allows the exclusive labeling of cysteine 315 in anxA5 while providing excellent radiochemical yields for the conjugation step even at very low protein concentration. Moreover, [18F]FDG-MHO-

anxA5 represents the first example of a defined, chemoselectively 18F-labeled annexin derivative. Taken together, the ease of preparation and the excellent yields of the conjugation step with cysteine residues make [18F]FDG-MHO a very interesting and promising novel prosthetic group for chemoselective 18F labeling of thiol groupcontaining peptides and proteins.

ACKNOWLEDGMENT The authors wish to thank S. Preusche for radionuclide production, T. Krauss for technical assistance, K. Schumann, D. Pfitzmann, and R. Herrlich for assistance in animal experiments. We are grateful to Dr. N. Budisa, Max Planck Institute of Biochemistry, Molecular Biotechnology, for the kind gift of recombinant Annexin A5. The project was partly supported by the EU project BioCare No. 505785 and by a grant from the Deutsche Forschungsgemeinschaft (DFG; grant no. PI304/1-1).

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Synthesis and Application of [18F]FDG-Maleimidehexyloxime ([18F

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