18F-Fluorothiols: A New Approach To Label Peptides

Hammersmith Hospital, Hammersmith Imanet Ltd., Du Cane Road, London W12 0NN, U.K., and. Medicinal Chemistry, Medical Diagnostics, GE Healthcare ...
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Bioconjugate Chem. 2004, 15, 1447−1453

1447

18F-Fluorothiols:

A New Approach To Label Peptides Chemoselectively as Potential Tracers for Positron Emission Tomography†

Matthias Glaser,*,‡ Hege Karlsen,§ Magne Solbakken,§ Joseph Arukwe,§ Frank Brady,‡ Sajinder K. Luthra,‡ and Alan Cuthbertson§ Hammersmith Hospital, Hammersmith Imanet Ltd., Du Cane Road, London W12 0NN, U.K., and Medicinal Chemistry, Medical Diagnostics, GE Healthcare Bio-Sciences, P.O. Box 4220, Nydalen, N-0401 Oslo, Norway. Received May 26, 2004; Revised Manuscript Received August 31, 2004

[18F]Fluorothiols are a new generation of peptide labeling reagents. This article describes the preparation of suitable methanesulfonyl precursors and their use in no-carrier-added radiosyntheses of 18F-fluorothiols. The preparations of (3-[18F]fluoropropylsulfanyl)triphenylmethane, (2-{2-[2-(2-[18F]fluoroethoxy)ethoxy]ethoxy}ethylsulfanyl)triphenylmethane, and 4-[18F]fluoromethyl-N-[2-triphenylmethanesulfanyl)ethyl]benzamide starting from the corresponding methanesulfonyl precursors were investigated. Following the removal of the triphenylmethane protecting group, the 18F-fluorothiols were reacted with the N-terminal chloroacetylated model peptide ClCH2C(O)-LysGlyPheGlyLys. The corresponding radiochemical yields of 18F-labeled isolated model peptide, decay-corrected to 18F fluoride, were 10%, 32%, and 1%, respectively. These results indicate a considerable potential of 18F-fluorothiols for the chemoselective labeling of peptides as tracers for positron emission tomography (PET).

INTRODUCTION

Radiolabeled peptides are important imgaging agents for nuclear medicine in general and for positron emission tomography (PET) in particular. This is reflected by the number of review articles (1-5). The positron emitter fluorine-18 (T1/2 ) 110 min) as a widespread positron emitter has been widely used to radiolabel peptides and proteins of medical interest. Although there are reports on direct incorporation of 18F into peptides (6, 7), the most promising approach still relies on the use of prelabeled 18 F reagents. The majority of known 18F reagents for peptide and protein labeling are targeting primary amino groups such as the N-terminus or lysine side chains. This is achieved through active esters (8-10), aldehydes (11), imidate (12), or azido functionalities (2). Fluorine-18-labeled amine was also coupled to a peptide with a prelabeled Nsuccinimidyl moiety (13). However, these peptide-coupling reactions are often low yielding, which is usually due to hydrolysis side reactions of the activated 18F reagent. In addition, the synthesis of the 18F prosthetic groups usually requires the time-consuming preparation and purification of multiple radioactive intermediates, reducing the available amount of 18F at the end of synthesis. For instance, the production of the established N-succinimidyl p-[18F]fluorobenzoate comprises three steps dealing with synthesis and purification of radioactive intermediates (8, 14). More recently, efficient * Corresponding author. E-mail: [email protected]; Tel. +44(0) 20 8383 3162; Fax +44(0) 20 8383 3738. † A preliminary account of this work was presented at the 15th International Symposium on Radiopharmaceutical Chemistry in Sydney, Australia, August 10-14, 2003 [M. Glaser et al. (2003) J. Labelled Compd. Radiopharm. 46 S16]. ‡ Hammersmith Imanet Ltd. § GE Healthcare Bio-Sciences.

radiohalogenation was achieved by condensation of p-[18F]fluorobenzaldehyde with aminooxy-modified peptide (15). Alkylation reactions have only rarely been used for peptide labeling with fluorine-18-containing prosthetic groups. For example, one report mentions p-[18F]fluorophenacyl bromide to label several proteins (12). However, there was no control on the binding site of the labeling reagent. Alternatively, it is possible to take advantage of the high nucleophilicity of thiolates to couple an 18F containing molecule with activated peptides. Here, we wish to describe the preparation of 18F-prelabeled thiols and their chemoselective alkylation reaction with a chloroacetylmodified model peptide. RESULTS AND DISCUSSION

Precursors and [19F]Fluoro Standard Compounds. Methanesulfonic acid 3-tritylsulfanyl-propyl ester 1 was prepared from 3-mercapto-1-propanol by reacting with trityl chloride and mesyl chloride (Scheme 1). The Strityl-protected fluoro product 2 was obtained by fluoro demesylation of 1 using the KF-Kryptofix complex. The PEG derivative 3 was designed in order to have a prosthetic group with potentially better pharmacologic properties available. Tetra(ethylene glycol) was thiolated by reacting its monotosylate with thiourea followed by hydrolysis of the resulting uronium salt. Reaction with trityl chloride and mesyl chloride gave mesylate precursor 3. Reaction of 3 with tetrabutylammonium fluoride (TBAF) formed 4. The benzamide precursor 5 was devised as a reagent with improved UV-visiblity during chromatography. In the synthesis of 5, cysteamine was S-tritylated using triphenylmethanol in trifluoroacetic acid (TFA). After coupling with 4-hydroxymethylbenzoic acid pentafluorophenyl ester, the hydroxymethyl function was mesy-

10.1021/bc0498774 CCC: $27.50 © 2004 American Chemical Society Published on Web 10/21/2004

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Glaser et al.

Scheme 1. Preparation of the Trityl-Protected Fluorothiols 2, 4, and 6 from the Corresponding Mesylates 1, 3, and 5a

a Reagents and conditions: (i) 1. TrCl, NEt , 6 h; 2. MsCl, 3 NEt3, 30 min; (ii) KF, Kryptofix, 80 °C, 90 min; (iii) 1. TsCl, NEt3, 17 h; 2. thiourea, EtOH, reflux, 24 h; 3. NaOH, reflux, 2.5 h; 4. TrCl, NEt3, 7 h; 5. MsCl, NEt3, 2 h; (iv) TBAF, 90 °C, 30 min; (v) 1. TrOH, TFA, NEt3, 30 min; 2. 4-hydroxymethylbenzoic acid pentafluorophenyl ester, NMM, 36 h; 3. MsCl, NMP, 48 h; (vi) KF, Kryptofix, 65 °C, 10 min.

Figure 1. HPLC analysis of a reaction mixture of an 18F-2 preparation showing 18F-2 (b) (coeluted with standard 2), and unreacted [18F]fluoride (a).

lated. The fluoro standard 6 was prepared by reaction of 5 with KF-Kryptofix. [18F]Fluorothiols. The mesylate 1 gave only moderate yields of 18F-2 (24%) after a 5 min reaction with [18F]KFKryptofix carbonate in acetonitrile at 80 °C as analyzed by HPLC. A plateau of 60% was reached after 30 min. DMF as solvent under identical conditions showed an improvement (72% after 30 min). However, DMSO proved to be the optimal solvent resulting in 79 ( 10% (n ) 6) after already 5 min reaction time (Figures 1 and 2a). The PEG-type mesylate 3 did react in DMSO somewhat slower using the above conditions. The maximum incorporation was observed after 15 min (77% of 18F-4, Figure 2b). The yields in acetonitrile and DMF after 30 min were relatively low with 30% and 41%, respectively. The slower kinetics might be explained by the larger size of mesylate 3 compared with the small propyl compound 1. The benzamide mesylate 5 was tested using the experimental settings described. Here, the best yields of 18 F-6 were obtained after reacting in acetonitrile for 5 min (25%, Figure 2c). The use of DMF and DMSO resulted only in yields of 7% and 17%, respectively (after 5 min), suggesting that the choice of solvent may have little impact on labeling efficiency. The benzyl mesylate

Figure 2. Radiolabeling yields of (a) 18F-2, (b) 18F-4, and (c) 18F-6 in DMSO (2), DMF ([), or MeCN (9) as obtained from HPLC analysis.

group is highly activated toward nucleophilic displacements as it was already observed during the 19F labeling work. In those studies the mesylate 5 HPLC signal quickly degraded as a new peak appeared instead. This compound was identified as 4-hydroxymethyl-N-[2-(tritylsulfanyl)ethyl]benzamide. A similar behavior could be seen in the synthesis of 18F-6. At the present this effect is not fully understood as the reaction was carried out in a strictly anhydrous environment. The 18F-fluorodemesylation of 5 was also investigated at lower temperatures. The reaction occurred already at room temperature forming 22% of 18F-6 after 7 min. After that time, HPLC analysis revealed complete consumption of 5. Cooling to -17 °C followed by warming up to room temperature gave no 18F-6 but only the hydrolysis product mentioned above. In an experiment without potassium carbonate neither hydroxy product or 18F-6 were detected after 5 min at room temperature and heating for 15 min at 80 °C. Obviously, the yields of 18F-6 might be still improved either by testing some alternative bases with the 18F-Kryptofix complex or by using higher substrate concentrations of 5. Further, our experiments indicated some degradation of already formed 18F-6 in all three solvent systems after

New 18F-Peptide Labeling Reagents

Bioconjugate Chem., Vol. 15, No. 6, 2004 1449

Table 1. Radiochemical Yields (rcy) of [18F]Fluoropeptides 8, 10, and 12 Including Intermediates mesylate precursor S-Tr protected

1 79 (

18F-reagenta

18F-fluorothiola

10%c (18F-2) 88% (18F-7) 93% (18F-8) 10% (18F-8)

18F-fluoropeptidea

rcy of 18F-fluoropeptideb

3 (18F-4)

77% 95% (18F-9) 41% (18F-10) 32% (18F-10)

5 21% (18F-6) 74% (18F-11) 86% (18F-12) 1% (18F-12)

a

Yields based on analytical HPLC, referring to individual steps. b Yields of isolated 18F-fluoropeptide, based on [18F]fluoride and corrected for decay. c n ) 6.

Scheme 2. Preparation [18F]Fluorothiols 7, 9, and 11 as Labeling Reagents for the Corresponding [18F]Fluoropeptides 8, 10, and 12a

a Reagents and conditions: (i) 1. tC18-SepPak-plus; 2. TFA/ TIS/H2O; (ii) ClCH2C(O)-KGFGK-OH, pH 9-10, 70-80 °C, 30 min.

Figure 4. HPLC analysis of the reaction mixture of the labeled peptide 18F-8 (a) with spiked standard 8 (b).

Figure 3. HPLC analysis of the reaction mixtures of the deprotection product 18F-7 (a). 18

5-15 min heating. In fact, the stability of F-fluorobenzyl derivatives has been recently studied by others (16). Peptide Labeling with [18F]Fluorothiols. After its preparation, the S-trityl compound 18F-2 could be quantitatively transferred from DMSO into acetonitrile using a C18 SepPak cartridge. The radiochemical purity of 18F-2 was >99%. The trityl protecting group was removed with TFA in the presence of triisopropylsilane (TIS) to form 7 in good yield (Scheme 2, Figure 3, Table 1). The N-chloroacetylated model peptide ClCH2C(O)-KGFGKOH could be labeled in excellent yield. An analytical HPLC trace of the labeling mixture is shown in Figure 4. Following identical protocols, the [18F]fluorothiol compounds 18F-4 and 18F-6 were deprotected to react with the chloroacetyl peptide to form 9 and 11, respectively. The efficiencies of the single steps are summarized in Table 1. An investigation to test the stability of deprotected thiol 7 was carried out. This experiment suggested poor stability of the 18F-thiol at pH 9-10 at room temperature.

After 1 h the HPLC signal of 7 was completely degraded. The use tris(2-carboxyethyl)phosphine (TCEP) as stabilizer was considered. TCEP as a selective reductant for disulfides has been described to be superior to dithiothreitol (17, 18). Thiol 7 showed high stability in the presence of TCEP. Therefore, an additive of this stabilizer was used in the subsequent peptide labeling work. The isolated radiochemical yields of [18F]fluoropeptides 8, 10, and 12 are listed in Table 1. Both the reduced thermal stability of the 18F-fluorobenzyl group and loss of radiolabeled peptide during the final HPLC purification possibly led to a decreased radiochemical yield of peptide 12. Only low levels of radioactivity of 18F have been used for this study, and therefore, specific radioactivities have not been measured.The total synthesis time was 2-3 h. CONCLUSIONS

[18F]Fluorothiols have been introduced as a new group of no-carrier-added 18F-reagents to label peptides as tracers for PET. The chloroacetyl function was used to chemoselectively enable the binding of the prosthetic 18Fgroup to a model peptide. The protocol simplifies peptide labeling because the reagent is prepared in only two steps that involved handling of 18F. In addition, there was also no need to purify the 18F-compound. The radiochemistry should be easily amenable to automation. MATERIALS AND METHODS

Preparation of Precursors and Standards. Chemicals. Fmoc amino acids, the Fmoc-Lys(Boc)-Sasrin resin, and peptide coupling reagents were purchased from Novabiochem, Bachem, and Applied Biosystems, respectively. 4-Hydroxymethylbenzoic acid pentafluorophenyl ester was obtained from MilliGen. All other reagents and solvents were purchased from Acros, Aldrich, or Fluka. Mass spectra were recorded on a Finnigan LCQ system using electrospray ionization (ESI). MALDI-TOF mass spectra were measured on a Finnigan Lasermat 2000

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instrument. Accurate mass spectra (HRMS) were measured on a Micromass Q-Tof Ultima machine coupled with a nano-LC system using Mass Lynx 3.5 software. NMR spectra were obtained from a Varian Unity Inova 500 instrument at 25 °C using a 5 mm 1H{15N-31P} indirect detection pfg probe. Analytical and semipreparative reversed-phase HPLC using different gradients [solvent A: water (0.1% TFA), solvent B: acetonitrile (0.1% TFA)], and UV detection at 214 and 254 nm were performed on Shimadzu, Beckman, or Waters chromatography systems. Cl-Ac-Lys-Gly-Phe-Gly-Lys-OH. Assembly of the amino acid sequence Lys-Gly-Phe-Gly-Lys-OH was done using fully automated synthesis (ABI 433A synthesis machine) with Fmoc-Lys(Boc)-Sasrin resin (0.25 mmol) using the Fastmoc Single Couple procedure with HBTU activation. Chloroacetic acid was coupled manually using chloroacetic anhydride (1.25 mmol). Simultaneous removal of the peptide from the resin and deprotection of side-chain protecting groups were carried out in trifluoroacetic acid containing triisopropylsilane and water (95: 2.5:2.5 v/v/v). After filtration, the solution was concentrated under reduced pressure and the residue was washed with diethyl ether. The crude product was purified by reversed-phase preparative chromatography (Phenomenex Luna C18(2) column, 250 × 50 mm, 10 µm; gradient 0-30% solvent B over 60 min; flow rate 50 mL/ minute), affording 128 mg (83%) of pure compound. The product was analyzed by HPLC [Phenomenex Luna C18(2), 50 × 4.6 mm, 5 µm; gradient 0-30% solvent B over 10 min; flow rate 2 mL/min; tR ) 5.1 min] and MS, m/z ) 612.8 (M + H)+; HRMS: found m/z ) 612.2936 (M + H)+ for C27H42N7O7Cl calcd m/z ) 612.2907. Methanesulfonic Acid 3-Tritylsulfanylpropyl Ester 1. A solution of triphenylmethanol (391 mg, 1.50 mmol) in trifluoroacetic acid (10 mL) was added dropwise to a stirred solution of 3-mercaptopropyl alcohol (127 µL, 1.50 mmol) in trifluoroacetic acid (10 mL). The trifluoroacetic acid was evaporated under reduced pressure immediately after mixing of the reagents. The crude product was purified by reversed-phase preparative chromatography (Phenomenex Luna C18(2) column, 250 × 50 mm, 10 µm; gradient 70-80% solvent B over 60 min; flow rate 50 mL/min), affording 372 mg (74%) of pure compound (mp 122-125 °C). 1H NMR (chloroformd, 500 MHz): 7.40-7.44 (m, 6H, trityl o-H); 7.26-7.31 (m, 6H, trityl m-H); 7.19-7.24 (m, 3H, trityl p-H); 3.56 (t, 2H, 3JHH ) 6.2 Hz, CH2O); 2.28 (t, 2H, 3JHH ) 7.2 Hz, CH2S); 1.62 (t of t, 2H, 3JHH ) 6.2 Hz, 3JHH ) 7.2 Hz, OCH2CH2CH2S). To a solution of 3-tritylsulfanylpropan-1-ol (372 mg, 1.11 mmol) in tetrahydrofuran (10 mL) were added triethylamine (209 µL, 1.50 mmol) and methanesulfonyl chloride (117 µL, 1.50 mmol). The precipitate was filtered off after stirring at room temperature for 1 h. Tetrahydrofuran was evaporated under reduced pressure, and the crude product was purified by reversed-phase preparative chromatography (Phenomenex Luna C18(2) column, 250 × 50 mm, 10 µm; gradient 80-100% solvent B over 60 min; flow rate 50 mL/min), affording 318 mg (69%) of pure compound (mp 122-125 °C). 1H NMR (chloroform-d, 500 MHz): 7.39-7.44 (m, 6H, trityl o-H); 7.26-7.32 (m, 6H, trityl m-H); 7.19-7.24 (m, 3H, trityl p-H); 4.13 (t, 2H, 3JHH ) 6.4 Hz, CH2O); 2.92 (s, 3H, CH3S); 2.29 (t, 2H, 3JHH ) 7.1 Hz, CH2S); 1.71 (t of t, 2H, 3J 3 HH ) 6.4 Hz, JHH ) 7.1 Hz, OCH2CH2CH2S). (3-Fluoropropylsulfanyl)triphenylmethane 2. Potassium fluoride (1.4 mg, 0.024 mmol) and Kryptofix (9 mg, 0.024 mmol) were dissolved in acetonitrile (0.2 mL)

Glaser et al.

with heating. A solution of mesylate 1 (5 mg, 0.012 mmol) in acetonitrile (0.2 mL) was added. The mixture was heated at 80 °C for 90 min. The crude product was purified by HPLC (Vydac 218TP column, C18, 250 × 22 mm, 10 µm; gradient 40-90% B in 40 min; flow rate 10 mL/min; λ ) 254 nm). The yield of 2 was 2 mg (50%). The product was analyzed by HPLC [Phenomenex Luna C18(2) column, 50 × 4.6 mm, 3 µm; gradient 40-80% solvent B in 10 min; flow rate 1 mL/min; tR ) 7.12 min, λ ) 254 and 214 nm] and 1H NMR (chloroform-d, 500 MHz): 7.39-7.44 (m, 6H, trityl o-H); 7.26-7.31 (m, 6H, trityl m-H); 7.19-7.24 (m, 3H, trityl p-H); 4.36 (d of t, 2H, 2JHF ) 47.2 Hz, 3JHH ) 5.9 Hz, CH2F); 2.28 (t, 2H, 3 JHH ) 7.3 Hz, CH2S); 1.72 (d of t of t, 2H, 3JHF ) 25.4 Hz, 3JHH ) 5.9 Hz, 3JHH ) 7.3 Hz, FCH2CH2CH2S). Methanesulfonic Acid 2-{2-[2-(2-Tritylsulfanylethoxy)ethoxy]ethoxy}ethyl Ester 3. Triethylamine (13 mL, 93 mmol) was added to a stirred solution of ptoluenesulfonyl chloride (9.82 g, 51.5 mmol) in tetra(ethylene glycol) (20.0 g, 103 mmol) at 0 °C. The suspension was stirred at 0 °C for 1 h and at room temperature for 16 h. After adding of dichloromethane (350 mL), the solution was extracted with hydrochloric acid (1 M, 2 × 100 mL) and water (200 mL). The organic phase was dried (MgSO4). After concentration, the product was purified by flash chromatography (ethyl acetate, first fraction: tetra(ethylene glycol) ditosylate) to give ptoluenesulfonic acid 2-{2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}ethyl ester as a colorless oil (8.9 g, 49%). 1H NMR (chloroform-d, 500 MHz): 7.78-7.83 (m, 2H, CH3C6H4S); 7.32-7.37 (m, 2H, CH3C6H4S); 4.15-4.19 (m, 2H, SOCH2CH2); 3.58-3.74 (m, 14H, OCH2CH2); 2.45 (s, 3H, CH3C6H4S). A solution of thiourea (0.94 g, 12 mmol) and p-toluenesulfonic acid 2-{2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}ethyl ester (4.09 g, 11.8 mmol) in absolute ethanol (32 mL) was heated at reflux for 24 h under argon. The reaction mixture was cooled and a solution of sodium hydroxide (1.23 g, 30.8 mmol) in ethanol/water (9:1 v/v, 15 mL) was added. The mixture was refluxed under argon for 2.5 h, cooled to room temperature and acidified to pH 2 (concentrated HCl). After concentration the residue was purified by flash chromatography (ethyl acetate/ethanol, 10:1 v/v) to give 2-{2-[2-(2-mercaptoethoxy)ethoxy]ethoxy}ethanol as a colorless oil (1.6 g, 66%). 1H NMR (chloroform-d, 500 MHz): 3.72-3.76 (m, 2H, OCH2CH2O); 3.60-3.71 (m, 12H, OCH2CH2); 2.71 (d of t, 2H, 3JHH ) 8.1 Hz, 3JHH ) 6.6 Hz, HSCH2CH2O); 2.41 (broad s, 1H, HOCH2CH2O); 1.63 (t, 1H, 3JHH ) 8.1 Hz, HSCH2CH2O). To 2-{2-[2-(2-mercaptoethoxy)ethoxy]ethoxy}ethanol (310 mg, 1.47 mmol) and trityl chloride (452 mg, 1.62 mmol) under argon was added simultaneously triethylamine (2.1 mL, 14.7 mmol) and tetrahydrofuran (25 mL). After stirring for 7 h, methanol (6 mL) was added and stirring was continued for 5 min. The product was isolated by flash chromatography (ethyl acetate) to give 2-{2-[2-(2-tritylsulfanylethoxy)ethoxy]ethoxy}ethanol as a colorless oil (550 mg, 83%). 1H NMR (chloroform-d, 500 MHz): 7.39-7.43 (m, 6H, trityl o-H); 7.25-7.30 (m, 6H, trityl m-H); 7.18-7.23 (m, 3H, trityl p-H); 3.68-3.72 (broad m, 2H, CH2OH); 3.61-3.67 (m, 4H, OCH2CH2O); 3.57-3.60 (m, 2H, OCH2CH2OH); 3.56-3.59 (m, 2H, OCH2CH2O); 3.43-3.47 (m, 2H, OCH2CH2O); 3.30 (t, 2H, 3J 3 HH ) 6.9 Hz, OCH2CH2S); 2.44 (t, 2H, JHH ) 6.9 Hz, 3 OCH2CH2S); 2.40 (broad t, 1H, JHH ) 5.8 Hz, OCH2CH2OH). Methanesulfonyl chloride (47 µL, 0.60 mmol) was added to a stirred solution of tetrahydrofuran (12 mL),

New 18F-Peptide Labeling Reagents

2-{2-[2-(2-tritylsulfanylethoxy)ethoxy]ethoxy}ethanol (227 mg, 0.500 mmol), and triethylamine (139 µL, 1.00 mmol). After stirring for 2 h, the mixture was filtered and concentrated. The residue was purified by flash chromatography (ethyl acetate) affording 3 as a colorless oil (238 mg, 90%). 1H NMR (chloroform-d, 300 MHz): 7.38-7.45 (m, 6H, trityl o-H); 7.17-7.32 (m, 9H, trityl m-H and p-H); 4.33-4.37 (m, 2H, S(O)2OCH2CH2O); 3.71-3.77 (m, 2H, S(O)2OCH2CH2O); 3.58-3.68 (m, 4H, OCH2CH2O); 3.41-3.58 (m, 4H, OCH2CH2O); 3.3 (t, 2H, OCH2CH2S, 3JHH ) 6.9 Hz); 2.99 (s, 3H, CH3S); 2.42 (t, 2H, OCH2CH2S, 3JHH ) 6.9 Hz). (2-{2-[2-(2-Fluoroethoxy)ethoxy]ethoxy}ethyl)trityl Sulfide 4. A stirring solution of 3 (71.7 mg, 0.14 mmol) and tetrabutylammonium fluoride (1.1 M in tetrahydrofuran, 127 µL, 0.14 mmol) was heated at 90 °C for 30 min. The solvent was evaporated, and the residue was purified by flash chromatography (ethyl acetate/ hexane, 1/1) to give 4 as a colorless oil (45 mg, 70%). 1H NMR (chloroform-d, 500 MHz): 7.40-7.45 (m, 6H, trityl o-H); 7.25-7.31 (m, 6H, trityl m-H); 7.25-7.31 (m, 3H, trityl p-H); 4.47-4.60 (d of m, 2H, 2JHF ) 47.8 Hz, FCH2CH2O); 3.68-3.78 (d of m, 2H, 3JHF ) 29.6 Hz, FCH2CH2O); 3.62-3.68 (m, 4H, OCH2CH2O); 3.44-3.60 (m, 4H, OCH2CH2O); 3.31 (t, 2H, 3JHH ) 6.9 Hz, OCH2CH2S); 2.44 (t, 2H, 3JHH ) 6.9 Hz, OCH2CH2S); HRMS: found m/z ) 472.2333 (M + NH4)+ for C27H31O3SF calcd m/z ) 472.2316. Methanesulfonic Acid 4-[2-(Tritylsulfanyl)ethylcarbamoyl]benzyl Ester 5. To a stirred solution of cysteamine (3.85 g, 50.0 mmol) in trifluoroacetic acid (50 mL) was added triphenylmethanol (13.0 g, 50.0 mmol). The mixture was stirred at room temperature for 30 min and concentrated. After addition of diethyl ether (250 mL), the precipitate was filtered off and washed with diethyl ether (2 × 50 mL). The trifluoroacetic acid salt was partitioned between aqueous KOH solution (1 M, 150 mL) and diethyl ether (150 mL). The organic phase was dried (MgSO4) and S-tritylcysteamine was crystallized from diethyl ether/n-hexane to give a white solid (9.2 g, 58%). ESI-MS: m/z ) 243 (M - Ph)•+; 1H NMR (chloroform-d, 500 MHz): 7.41-7.44 (m, 6H, trityl o-H); 7.257.30 (m, 6H, trityl m-H); 7.19-7.23 (m, 3H, trityl p-H); 2.60 (t, 2H, 3JHH ) 6.6 Hz, CH2N); 2.32 (t, 2H, 3JHH ) 6.6 Hz, CH2S); 1.07 (broad s, 2H, NH2). To a solution of S-trityl cysteamine (1.6 g, 5.0 mmol) and 4-hydroxymethylbenzoic acid pentafluorophenyl ester (1.5 g., 5.0 mmol) in dichloromethane (40 mL) was added N-methylmorpholine (0.55 mL, 5.0 mmol). After stirring at room temperature for 36 h, the precipitate was filtered off, washed with dichloromethane (3 × 5 mL), and dried to give 4-hydroxymethyl-N-[2-(tritylsulfanyl)ethyl]benzamide as a white solid (1.3 g, 57%). ESI-MS: m/z ) 476 (M + Na+); 1H NMR (DMSO-d6, 500 MHz): 8.53 (t, 1H, 3JHH ) 5.7 Hz, NH); 7.75-7.79 (m, 2H, OCH2C6H4CO); 7.36-7.40 (m, 2H, OCH2C6H4CO); 7.297.35 (m, 12H, trityl o-H and p-H); 7.22-7.28 (m, 3H, trityl p-H); 5.29 (t, 1H, 3JHH ) 5.6 Hz, OH); 5.54 (d, 2H, 3JHH ) 5.6 Hz, CH2O); 3.23 (d of t, 2H, 3JHH ) 5.7 Hz, 3JHH ) 7.1 Hz, CH2N); 2.33 (t, 2H, 3JHH ) 7.1 Hz, CH2S). To a solution of 4-hydroxymethyl-N-[2-(tritylsulfanyl)ethyl]benzamide (226 mg, 0.50 mmol) in anhydrous tetrahydrofuran (8 mL) were added N-methyl-2-pyrrolidinone (61 µL, 0.55 mmol) and methanesulfonyl chloride (85 µL, 1.1 mmol). The reaction mixture was stirred for 48 h at room temperature and filtered through silica. Purification by column chromatography (silica, 1% methanol in chloroform) gave 5 as an oil that solidified slowly on storage (213 mg, 80%). 1H NMR (chloroform-d, 500

Bioconjugate Chem., Vol. 15, No. 6, 2004 1451

MHz): 7.72-7.76 (m, 2H, OCH2C6H4CO); 7.46-7.50 (m, 2H, OCH2C6H4CO); 7.40-7.44 (m, 6H, trityl o-H); 7.247.30 (m, 6H, trityl m-H); 7.19-7.24 (m, 3H, trityl p-H); 6.23 (broad t, 1H, 3JHH ) 5.7 Hz, NH); 5.28 (s, 2H, CH2O); 3.30 (d of t, 2H, 3JHH ) 5.7 Hz, 3JHH ) 6.2 Hz, CH2N); 2.55 (t, 2H, 3JHH ) 6.2 Hz, CH2S); 2.97 (s, 3H, CH3S). 4-Fluoromethyl-N-[2-(tritylsulfanyl)ethyl]benzamide 6. A solution of Kryptofix (15 mg, 40 µmol) in anhydrous acetonitrile (0.2 mL) was added to solid potassium fluoride (2.3 mg, 80 µmol). The mixture was shaken for 5 min and added to 5 (21 mg, 40 µmol) in acetonitrile (0.4 mL) followed by heating at 65 °C for 10 min. Product 6 was isolated by preparative HPLC [Phenomenex Luna C18 (2) column, 250 × 21.2 mm, 5 µm; gradient 40-80% solvent B over 10 min; flow rate 10 mL/ min] as a white solid (4.5 mg, 25%). 1H NMR (chloroformd, 500 MHz): 7.71-7.75 (m, 2H, FCH2C6H4CO); 7.427.44 (m, 2H, FCH2C6H4CO); 7.40-7.44 (m, 6H, trityl o-H); 7.24-7.29 (m, 6H, trityl m-H); 7.19-7.23 (m, 3H, trityl p-H); 6.23 (broad t, 1H, 3JHH ) 5.8 Hz, NH); 5.44 (d, 2H, 2J 3 HF ) 47.4 Hz, CH2F); 3.65 (d of t, 2H, JHH ) 5.8 Hz, 3J 3 HH ) 6.5 Hz, CH2N); 2.55 (t, 2H, JHH ) 6.5 Hz, CH2S); HRMS: found m/z ) 478.1624 (M + Na)+ for C29H26OSF calcd m/z ) 478.1611. 6-Amino-2-{2-[2-(2-{6-amino-2-[2-(3-fluoropropylsulfanyl)acetylamino]hexanoylamino}acetylamino)3-phenylpropionylamino]acetylamino}hexanoic Acid (19F-8). The trityl compound 2 (1 mg, 3 µmol) was deprotected with a mixture of trifluoroacetic acid (25µL), triisopropylsilane (5 µL), and water (5µL) at room temperature for 5 min. The mixture was cooled to 0 °C, a solution of ClAc-KGFGK-OH (3.6 mg, 6 µmol) in water (1 mL) added, followed by adjusting the pH to 9 (25% ammonia), and heating for 30 min at 60 °C. Product 19F-8 was isolated by preparative HPLC [Phenomenex C18(2) column, 250 × 10 mm, 10 µm; gradient 0-30% solvent B in 30 min; flow rate 5 mL/min; λ ) 254 nm] with 0.5 mg (20%) yield. The labeled peptide was characterized by LC-MS [Phenomenex Luna C18(2) column, 50 × 4.6 mm, 3 µm, gradient 0-30% solvent B in 30 min; flow rate 1 mL/min; λ ) 214 and 254 nm, Rf ) 6.88 min, m/z ) 670.35 (M + H)+]; HRMS: found m/z ) 670.3410 (M + H)+ for C30H48N7O7FS calcd m/z ) 670.3393. 6-Amino-2-{2-[2-(2-{6-amino-2-[2-(2-{2-[2-(2-fluoroethoxy)ethoxy]ethoxy}ethylsulfanyl)acetylamino]hexanoylamino}acetylamino)-3-phenylpropionylamino]acetylamino}hexanoic Acid (19F-10). To a vial containing trityl compound 19F-4 (1.2 µmol) was added a trifluoroacetic acid/triisopropylsilane/water mixture (95: 2.5:2.5 v/v/v, 130 µL). After gentle agitating for 5 min, the mixture was concentrated in vacuo and sodium carbonate/ bicarbonate buffer (0.1 M, pH 9.1, 0.1 mL) was added followed by a solution of ClAc-KGFGK-OH (3.6 mg, 6.0 µmol) in sodium carbonate/bicarbonate buffer (0.1 M, pH 9.1, 0.15 mL). The coupling product 19F-10 was purified by preparative HPLC [Phenomenex Luna C18(2) column, 250 × 10 mm, 10 µm; gradient 40-80% solvent B over 30 min; flow rate 5 mL/min] giving a yield of 4.0 mg (42%). The labeled peptide 19F-10 was characterized by MALDIMS, m/z ) 788 (M + H)+; HRMS: found m/z ) 788.4061 (M + H)+ for C35H58N7O10FS calcd m/z ) 788.4023. 6-Amino-2-(2-{2-[2-(6-amino-2-{2-[2-(4-fluoromethylbenzoylamino)ethylsulfanyl]acetylamino}hexanoylamino)acetylamino]-3-phenylpropionylamino}acetylamino)hexanoic Acid (19F-12). The labeled peptide 19F-12 was synthesized using the protocol for the preparation of 19F-10. The coupling product 19F-12 was purified by preparative HPLC [Phenomenex Luna C18(2) column, 250 × 21.2

1452 Bioconjugate Chem., Vol. 15, No. 6, 2004

mm, 5 µm; gradient 5-60% acetonitrile over 60 min; flow rate 10 mL/min] yielding 2 mg (42%) of a white solid. The labeled peptide was characterized by LC-MS [Phenomenex Luna C18(2) column, 50 × 4.6 mm, 3 µm; gradient 5-60% acetonitrile over 10 min; flow rate 0.3 mL/min, tR ) 4.3 min, m/z ) 789.4 (M + H+)]; HRMS: found m/z ) 789.3743 (M + H)+ for C37H53N8O8SF calcd m/z ) 789.3764. Radiochemistry. Chemicals. Chemicals including anhydrous solvents were purchased from Sigma-Aldrich (Gillingham, UK). HPLC solvents were received from Fisher Scientific (Loughborough, UK). Production of 18F. Fluorine-18 was produced by a Scanditronix MC 40 cyclotron using the 18O(p,n)18F reaction. Enriched [18O]H2O (10-30% 18O) was irradiated by protons (19 MeV) with an integrated beam current of 5-10 µAh. HPLC. The HPLC system was a Beckman System Gold instrument equipped with a gamma detector (Bioscan Flow-count). A Phenomenex Luna C18(2) column (50 × 4.6 mm, 3 µm; flow rate 1 mL/min) was used for analytical HPLC. A semipreparative column [Phenomenex Luna C18(2), 250 × 10 mm, 5 µm; flow rate 4 mL/ min] was used for the final purification of labeled peptides. The following mobile phase/gradient systems were used: solvent A: water (0.1%) TFA, solvent B: acetonitrile (0.1% TFA). gradient 1: 1 min 40% B, 40-80% B in 15 min gradient 2: 1 min 30% B, 30-80% B in 10 min gradient 3: 1 min 30% B, 5-30% B in 20 min, 3080% B in 5 min gradient 4: 1 min 0% B, 0-30% B in 10 min 3-[18F]Fluoro-1-mercaptotritylpropane (18F-2). Fluorine-18 containing water (370 MBq, 1 mL) was added to a Wheaton vial (2 mL), charged with Kryptofix 222 (10 mg, 27 µmol), potassium carbonate (1 mg, 7 µmol, dissolved in 50 µL water), and acetonitrile (0.8 mL). The solvent was removed by heating at 110 °C for 1 h under a stream of nitrogen. Anhydrous acetonitrile (0.5 mL) was added and again evaporated as before. This step was repeated twice. The vial was cooled to room temperature followed by injecting a solution of 1 (1 mg, 2.4 µmol) in anhydrous DMSO (0.2 mL). The reaction mixture was stirred at 80 °C for 5 min and analyzed by HPLC (gradient 1, tR ) 16.2 min). The reaction mixture was diluted with DMSO/water (1:1 v/v, 0.15 mL) and loaded onto a SepPak-Plus cartridge (tC18, Waters) that had been conditioned (10 mL acetonitrile, 20 mL water). The cartridge was washed with water (10 mL) and the 18F-2 eluted using acetonitrile. 2-{2-[2-(2-[18F]Fluoroethoxy)ethoxy]ethoxy}mercaptotritylethane (18F-4). This preparation was carried out from 3 as described for 18F-2. The reaction mixture was analyzed by HPLC (gradient 2, tR ) 12.6 min). 3-[18F]Fluoromethyl-N-(2-mercaptotritylethyl)benzamide (18F-6). Again, 18F-6 was prepared as described for 18F-2. The mesylate precursor 5 was stirred at room temperature for 15 min to give 18F-6 as analyzed by HPLC (gradient 1, tR ) 13.6 min). 6-Amino-2-{2-[2-(2-{6-amino-2-[2-(3-[18F]fluoropropylsulfanyl)acetylamino]hexanoylamino}acetylamino)-3-phenylpropionylamino]acetylamino}hexanoic Acid (18F-8). A solution of 18F-2 in acetonitrile (72 MBq, 0.5 mL) was evaporated to dryness using a stream of nitrogen and heating at 100 °C. A mixture of trifluoroacetic acid (0.05 mL), triisopropylsilane (0.01 mL), and water (0.01 mL) was added followed by heating for 10 min at 80 °C. The resulting thiol 18F-7 was not isolated

Glaser et al.

(analytical HPLC: gradient 1, tR ) 2.5 min,). After the vial was cooled to 0 °C, ammonia (28% in water, 0.1 mL, Aldrich), TCEPxHCl (38 mg, 1.3 µmol, Aldrich) and peptide precursor ClAc-KFGFK-OH (2 mg, 3.2 µmol) in water (0.05 mL) were added. The mixture was stirred for 30 min at 80 °C. After diluting with 0.2 mL of water/ MeCN/TFA (95:5:0.1 v/v/v), the mixture was purified on a semipreparative HPLC (gradient 3, tR ) 19.2 min) and analyzed by analytical HPLC (gradient 4, tR ) 10.3 min). 6-Amino-2-{2-[2-(2-{6-amino-2-[2-(2-{2-[2-(2-[18F]fluoroethoxy)ethoxy]ethoxy}ethylsulfanyl)acetylamino]hexanoylamino}acetylamino)-3-phenylpropionylamino]acetylamino}hexanoic Acid (18F-10). The preparation of 18F-10 was carried out from 18F-4 following the method described for 18F-8. The labeled peptide 18F-10 was isolated by semipreparative HPLC (gradient 3, tR ) 19.8 min). 6-Amino-2-(2-{2-[2-(6-amino-2-{2-[2-(4-[18F]fluoromethylbenzoylamino)ethylsulfanyl]acetylamino}hexanoylamino)acetylamino]-3-phenylpropionylamino}acetylamino)hexanoic Acid (18F-12). The labeled peptide 18F-12 was synthesized from 18F-6 using the protocol for the preparation of 18F-8. The product 18F12 was obtained from semipreparative HPLC (gradient 3, tR ) 22.8 min). Stability Test of 18F-7. 18F-2 was deprotected as described above and dispensed in two conical vials A and B (1 MBq in 20 µL each). A yield of 70% deprotection product 18F-7 was established by HPLC analysis. A solution of TCEP × HCl (10 µL, 2.7 µmol) was added to vial A, followed by ammonia (50 µL, 28%). Ammonia (50 µL, 28%) was also added to vial B. After 1 h at room temperature, aliquots (10 µL) of both vials were analyzed by HPLC. Vial A showed 43% of thiol 18F-7 preserved with an increased peak at the solvent front. Numerous radioactive products but no signal for 18F-7 were found in vial B. ACKNOWLEDGMENT

We would like to thank Colin Steel and the cyclotron operators of Hammersmith Imanet Ltd. for providing us with fluorine-18. LITERATURE CITED (1) Okarvi, S. M. (2001) Recent progress in fluorine-18 labeled peptide pharmaceuticals. Eur. J. Nucl. Med. 28, 929-938. (2) Wester, H.-J., Hamacher, K., and Stoecklin, G. (1996) A comparative study of N. C. A. Fluorine-18 labeling of proteins via acylation and photochemical conjugation. Nucl. Med. Biol. 23, 365-372. (3) Wilbur, D. S. (1992) Radiohalogenation of proteins: An overview of radionuclides, labeling methods, and reagents for conjugate labeling. Bioconjugate Chem. 3, 433-470. (4) Lundqvist, H., and Tolmachev, V. (2002) Targeting peptides and positron emission tomography. Biopolymers (Pept. Sci.) 66, 381-392. (5) Heppeler, A., Froidevaux, S., Eberle, A. N., and Maecke, H. R. (2000) Receptor targeting for tumor localisation and therapy with radiopeptides. Curr. Med. Chem. 7, 971-994. (6) Jelinski, M., Hamacher, K., and Coenen, H. H. (2001) Direct 18F-substitution of hydroxy groups in peptides using nonofluorobutane-1-sulfonyl[18F]fluoride. J. Labelled Compd. Radiopharm. 44, S151-153. (7) Ogawa, M., H. K., Oishi, S., Miyake, Y., Kawasumi, Y., Ito, K., Fujii, N., and Iida, H. (2002) Direct single step 18F fluorination of peptides. J. Nucl. Med. (suppl.) 43, 137P. (8) Vaidyanathan, G., and Zalutsky, M. R. (1992) Labeling proteins with fluorine-18 using N-succinimidyl 4-[18F]fluorobenzoate. Nucl. Med. Biol. 19, 275-281.

New 18F-Peptide Labeling Reagents (9) Lang, L., and Eckelman, W. C. (1994) One-step synthesis of 18F labeled [18F]-N-succinimidyl 4-(fluoromethyl)benzoate for protein labeling. Appl. Radiat. Isot. 45, 1155-1163. (10) Guhlke, S., Wester, H.-J., Bruns, C., and Stoecklin, G. (1994) (2-[18F]Fluoropropionyl-(D)phe1)-octreotide, a potential radiopharmaceutical for quantitative somatostatin receptor imaging with PET: Synthesis, radiolabeling, In Vitro validation and biodistribution in mice. Nucl. Med. Biol. 21, 819825. (11) Herman, L. W., Fischman, A. J., Tompkins, R. G., Hanson, R. N., Byon, C., Strauss, H. W., and Elmaleh, D. R. (1994) The use of pentafluorophenyl derivatives for the 18F labelling of proteins. Nucl. Med., Biol. 21, 1005-1010. (12) Kilbourn, M. R., Dence, C. S., Welch, M. J., and Mathias, C. J. (1987) Fluorine-18 labeling of proteins. J. Nucl. Med. 28, 462-470. (13) Shai, Y., Kirk, K. L., Channing, M. A., Dunn, B. B., Lesniak, M. A., Eastman, R. C., Finn, D., Roth, J., and Jacobson, K. A. (1989) 18F-Labeled insulin: A prosthetic group methodology for incorporation of a positron emitter into peptides and proteins. Biochemistry 28, 4801-4806.

Bioconjugate Chem., Vol. 15, No. 6, 2004 1453 (14) Wu¨st, F., Hultsch, C., Bergmann, R., Johannsen, B., and Henle, T. (2003) Radiolabelling of isopeptide N--(γ-glutamyl)L-lysine by conjugation with N-succinimidyl-4-[18F]fluorobenzoate. Appl. Rad. Isot. 59, 43-48. (15) Poethko, T., Schottelius, M., Thumshirn, G., Hersel, U., Herz, M., Henriksen, G., Kessler, H., Schwaiger, M., and Wester, H.-J. (2004) Two-step methodology for high-yield routine radiohalogenation of peptides: 18F-labeled RGD and Octreotide analogs. J. Nucl. Med. 45, 892-902. (16) Magata, Y., Lang, L., Kiesewetter, D. O., Jagoda, E. M., Channing, M. A., and Eckelman, W. C. (2000) Biologically stable [18F]-labeled benzylfluoride derivatives. Nucl. Med., Biol. 27, 163-168. (17) Burmeister-Getz, E., Xiao, M., Chakrabarty, T., Cooke, R., and Selvin, P. R. (1999) A comparison between the sulfhydryl reductants tris(2-carboxyethyl)phosphine an dithiothreitol for use in protein biochemistry. Anal. Biochem. 273, 73-80. (18) Burns, J. A., Butler, J. C., Moran, J., and Whitesides, G. M. (1991) Selective reduction of disulfides by tris(2-carboxyethyl)phosphine. J. Org. Chem. 56, 2648-2650.

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