Bioconjugate Chem. 2006, 17, 1017−1021
1017
Solid-Phase Synthesis of 2-[18F]Fluoropropionyl Peptides Jan Marik, Sven H. Hausner, Lauren A. Fix, M. Karen J. Gagnon, and Julie L. Sutcliffe* Department of Biomedical Engineering, University of California, Davis, California 95616. Received March 6, 2006; Revised Manuscript Received June 9, 2006
The 2-[18F]fluoropropionic (2-[18F]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-[18F]fluoropropionyl peptides. [18F]FPA was prepared and conjugated to the peptides attached to the solid phase support. The 18F-labeled peptides were obtained in 175 min with decay corrected yields of 10% (related to [18F]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.
INTRODUCTION Radiotracers labeled with fluorine-18 (18F, β+ t1/2 ) 109.7min) are extensively used for positron emission tomography (PET) imaging of various physiological and pathological processes (1). The use of biomolecules such as peptides, proteins, or nucleic acids labeled with 18F as agents for PET is a rapidly growing field (2). Examples of labeled peptides include octreotide (3), vasoactive intestinal peptide (4, 5), integrin specific peptides (6-10), N-(γ-glutamyl)lysine (11), neurotensin analogues (12), human C-peptide (13), and insulin (14). Incorporation of 18F into biomolecules, including peptides, is exclusively carried out by employing various prosthetic groups. A large number of prosthetic groups bearing 18F have been developed. These include acylation agents (3, 15-20), amidation agents (14), imidation agents (21-23), alkylation agents (21), and species for photochemical conjugation (20). Undoubtedly, 4-[18F]fluorobenzoic acid ([18F]FBA) is the most common prosthetic group used for labeling peptides since it can be relatively easily prepared, activated as succinimidyl fluorobenzoate ([18F]SFB), and subsequently conjugated to the purified peptide in aqueous media. The synthesis of [18F]SFB has been automated using the GE TracerLab synthesizer (24). However, the in-solution conjugation of [18F]SFB to the peptide is limited to sequences bearing only one acylation-prone nucleophilic group; otherwise a complex mixture of nonselectively radiolabeled products is obtained (5). The slight difference between the pKa of a backbone NR amino group and a N of lysine can be utilized, to some extent, to control the selectivity of the acylation process (25). To overcome this drawback, we previously developed a solid-phase approach for the incorporation of [18F]FBA into the protected peptide sequence attached to the solid support, followed by removal of the protecting groups and cleavage from the solid phase (8, 26). Our approach brings into the radiochemistry field all the advantages of solid-phase organic chemistry which has been extensively developed in the last 20 years. Particularly, it significantly simplifies the handling and purification of the desired product. In 1996, Wester et al. reported a study comparing various prosthetic groups for incorporation of 18F into proteins (20). * Corresponding author: Julie Sutcliffe, Ph.D., Department of Biomedical Engineering, 451 East Health Sciences Dr., University of California, Davis, Davis, CA 95616-5294. Tel: 530 754-7107. Fax: 530 754 5739. e-mail:
[email protected].
Among these reagents 2-[18F]fluoropropionc acid ([18F]FPA) (3, 18) offers an interesting alternative to the widely used [18F]FBA. Compared to the [18F]FBA, it was suggested that the [18F]FPA would not significantly alter the properties of the peptide required for the biological activity such as size and lipophilicity which may result, for example, in reduction of hepatobiliary excretion of the radiolabeled species (20). Previously, [18F]FPA was successfully attached to D-Phe1-octreotide and used for imaging of the somatostatin receptor (3) and was also used to radiolabel an RGD-containing glycopeptide (9, 10, 27, 28). The radiolabeled glycopeptide showed specificity for the Rvβ3 integrin during in vitro binding assays and for the Rvβ3 integrin expressing tumors in vivo. In both cases, the [18F]FPA was activated as a 4-nitrophenyl ester and conjugated to the peptide precursor in solution. The glycopeptide contained only one amino group; hence, the labeling using 4-nitrophenyl 2-[18F]fluoropropionate was accomplished in a single step. However, D-Phe1-octreotide contains Lys in position 5 and the acylation of the amino group in its side chain would result in loss of the high affinity to the somatostatin receptor. Therefore, the prosthetic group was attached to the N-terminal amino group of the -Boc-Lys5 protected octreotide followed by removal of the Boc protecting group using trifluoroacetic acid. In this paper, we extend our previously published procedure for radiolabeling of peptides on the solid-phase support, and the method for radiolabeling of peptides with [18F]FPA is described. Various coupling conditions and solid supports were investigated for the conjugation of the [18F]FPA to the peptides assembled on solid support.
EXPERIMENTAL PROCEDURES Materials. Solvents and chemicals were purchased from Aldrich (Milwaukee, WI) if not stated otherwise. Racemic (()2-fluoropropionic acid was obtained from Matrix Scientific (Columbia, SC). The protected amino acids and coupling reagents were purchased from NovaBiochem (San Diego, CA) or GL Biochem (Shanghai, China). Resins for solid-phase peptide synthesis were obtained from the following vendors: NovaGel HL (A) and Amino PEGA (F) from NovaBiochem (San Diego, CA), TentaGel S (G) from Rapp Polymere GmbH (Tubingen, Germany), PL-PEGA (E) from Polymer Laboratories (Amherst, MA), CLEAR Amide (C) from Peptides International, Inc. (Louisville, KY), Rink Amide-MBHA (B) from Tianjin Nankai Hecheng Sci. and Tech. Co. Ltd. (Tianjin, China), and
10.1021/bc0600564 CCC: $33.50 © 2006 American Chemical Society Published on Web 07/06/2006
1018 Bioconjugate Chem., Vol. 17, No. 4, 2006
Marik et al.
Table 1. Decay Corrected Yields of [18F]FPA Coupling to the Different Peptides on NovaGel HL Resin and Yields of the Product Cleavagea compd 1 2 3 4 a
sequence [18F]FPA-YGGFL [18F]FPA-NAVPNLRGDLQVLAQKVART [18F]FPA-AGDLHVLR AGDLHVLR-Ebes-Lys([18F]FPA)
coupling
cleavage
total
purity
16 6 10 14
36 43 40 44
6 3 4 6
90 76 99 98
Yields and purity are given in %.
PAL-PEG-PS (D) from Applied Biosystems (Warrington, UK). Protected hydrophilic ethylene glycol-based linker 2,2′-(ethylenedioxy)bis(ethylamine)monosuccinamide (Fmoc-Ebes-OH) was synthesized according to the previously published procedure (29), and 9-methylanthranyl 2-bromopropionate was prepared according to the published procedure (18). Mass spectrometry analysis was performed using a Finnigan LCQ instrument. RPHPLC was used to analyze and purify the products. Analytical reverse-phase HPLC system A: Phenomenex Jupiter 4 µm Proteo 90A (250 × 4.6 mm, 4 µm), linear gradient 0.05% TFAacetonitrile 10-90% in 30 min, flow rate 1 mL/min. Semipreparative reverse-phase HPLC system B: Phenomenex Jupiter 10 µm Proteo 90A (250 × 10 mm, 10 µm), linear gradient 0.05% TFA-90% acetonitrile 10-90% in 30 min, flow rate 3 mL/min; system C: Phenomenex Jupiter 10 µm C18 300A (250 × 10 mm, 10µm), isocratic 60% acetonitrile - 0.05% TFA, flow rate 3 mL/min. 18F was produced from the 18O(p,n)18F nuclear reaction on [18O]H2O (Marshall Isotopes Ltd. (Tel Aviv, Israel)) using the CTI RDS 111 negative ion cyclotron. SepPak SPE cartridges were obtained from Waters (Milford, MA). Peptide Synthesis. The peptides 1-4 (Table 1) were synthesized on resin A. The synthesis was performed using a Pioneer continuous flow synthesizer ABI (Foster City, CA) using 4-fold excess of amino acid and DIC/HOBt activation and a 2 h coupling cycle followed by a 10 min Fmoc deprotection with 20% piperidine in DMF. Peptide 1 synthesized on resins B-G was prepared manually using 3-fold excess of amino acid and using HATU/DIEA activation for 30 min followed by Fmoc deprotection with 20% piperidine-DMF (2 × 10 min). The hydrophilic linker was incorporated into the peptide chain using a Fmoc-protected analogue (Fmoc-EbesOH) and HATU/DIEA activation. The N-terminal amino group in peptide 4 was protected by incubation of the resin with 3-fold excess of di-tert.-butyl dicarbonate and DIEA for 30 min to yield N-Boc-protected peptide. The allyloxycarbonyl (Alloc) protecting group in peptide 4 was removed using 0.2 equiv of Pd[PPh3]4 and 20 equiv of phenylsilane 2 × 30 min (30). The peptide resins were washed with DMF and methanol and dried in vacuo. Synthesis of (()-2-Fluoropropionyl Peptides. 2-Fluoropropionic acid ([19F]FPA) was coupled to the peptide using 3-fold excess of [19F]FPA and DIC/HOBt activation for 30 min. The progress of the reaction was monitored by ninhydrin-based Kaiser test (31). The [19F]FPA-peptides 1-4 were cleaved from the solid support using trifluoroacetic acid/triisopropylsilane/ water 95:2.5:2.5 v/v/v. The crude peptides were precipitated from the cleavage mixture by the addition of an excess of chilled diethyl ether. The solvent was then removed, and the solid product was triturated three times with diethyl ether. The peptides were purified on semipreparative HPLC (system B) and characterized using mass spectrometry. Analytical data for peptides 1-4: 1 HPLC (system A) retention times 15.5, 15.6 min; MS ESI m/z 629.5 [M + H]+; 2 HPLC (system A) retention time 14.6 min; MS ESI m/z 2236.8 [M + H]+; 3 HPLC (system A) retention times 10.7, 11.0 min; MS ESI m/z 953.7 [M + H]+; 4 HPLC (system A) retention time 9.4 min; MS ESI m/z 1311.8 [M + H]+. Radiochemical Synthesis of (()-2-[18F]Fluoropropionic Acid. The fluoride anion was captured on a QMA Sep-Pak
Scheme 1. Labeling of Peptides with 2-[18F]fluoropropionic Acid ([18F]FPA) on Solid Phase
cartridge and eluted with 1 mL of 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane (K222) and potassium carbonate solution (10 mg of K222, 2 mg of K2CO3) in acetonitrile containing 6% water. The acetonitrile was evaporated at 100 °C under a gentle stream of nitrogen and dried by azeotropic distillation with 3 × 1 mL of acetonitrile. The solution of 9-methylanthranyl 2-bromopropionate in acetonitrile (2 mL) was added, and the mixture was heated in sealed vial to 100 °C for 15 min. The product was isolated on semipreparative HPLC (system C) retention time 18-20 min. The fraction containing the product was diluted with water to 10 mL, and the product was captured on a C-18 Sep-Pak cartridge. The cartridge was washed with 5 mL of water and dried with a stream of nitrogen. The 9-methylanthranyl 2-[18F]fluoropropionate was eluted with 3 mL of dichloromethane, and the solvent was evaporated under a stream of nitrogen. The methylanthranyl ester was hydrolyzed using 200 µL of the hydrolysis mixture (9.5 mL water, 0.5 mL triethylamine) and 300 µL of DMF in a closed vial at 100 °C for 10 min. The solvent was evaporated at 100 °C under a stream of nitrogen, and the product was dried using azeotropic distillation with acetonitrile (3 × 1 mL). The product was obtained with a decay corrected yield of 63% and a synthesis time of 82 min (Scheme 1). Radiochemical Synthesis of (()-2-[18F]Fluoropropionyl Peptides (1-4). The coupling of [18F]FPA to the peptide and the cleavage from the resin were carried out in a 1 mL syringe equipped with a frit at the bottom of the cylinder. The reagents and washing solvents were withdrawn and dispensed using the plunger. This procedure can be easily performed using robotic manipulators. The [18F]FPA in 50 µL of DMF was added to 1-2 mg of the peptide resin swollen in DMF, followed with HATU (5 mg) in 50 µL of DMF, and DIEA (10 µL) in 50 µL of DMF. The reaction mixture was incubated with shaking at 30 °C for 30 min. The solvent was removed, and the solid support was washed with DMF (3 × 0.5 mL) and methanol (3 × 0.5 mL). The TFA-based cleavage mixture (0.7 mL of trifluoroacetic acid/triisopropylsilane/water 95:2.5:2.5 v/v/v) was withdrawn into the syringe, and the reaction mixture was incubated at 30 °C for (3 × 10 min). The product was collected, and the resin was washed with dichloromethane (0.5 mL). The
Solid-Phase Synthesis of 2-[18F]Fluoropropionyl Peptides
cleavage solution was evaporated under a stream of nitrogen, and the crude product was analyzed on radio-HPLC (system A). Analytical data for peptides 1-4 (the radiochemical yields were decay corrected to the end of [18F]FPA preparation): 1 HPLC (system A) retention time 15.7 min; yield 6%; 2 HPLC (system A) retention time 14.7 min; yield 3%; 3 HPLC (system A) retention times 11.0, 11.2 min; yield 4%; 4 HPLC (system A) retention time 9.6 min; yield 6%. The synthesis time from EOB was 175 min.
RESULTS AND DISCUSSION The procedure for the preparation of [18F]FPA from racemic 9-methylanthranyl 2-bromopropionate provides both enantiomers; thus, two diastereoisomers of the final labeled peptide are obtained. The function of the prosthetic group is to introduce the radionuclide into the target molecule without changing its biological properties; therefore, the presence of two diastereoisomers of the product is not expected to have any effect on its pharmacological properties. To address this problem and the effect of the prosthetic group in general, two approaches for the introduction of the prosthetic group are presented (Scheme 1). In the first approach the prosthetic group was attached to the amino terminus of the peptide sequence (Table 1, compounds 1-3) as previously reported (8, 26). This strategy is the most straightforward method to introduce the prosthetic group into the molecule and does not significantly change its final size. However, when the prosthetic group is attached directly to the peptide, it can potentially have a deteriorating effect on the biological properties of the peptide, especially in the case where a free amino terminus is required for receptor binding. In the latter approach, the prosthetic group was attached to the C-terminal part of the molecule via the N amino group of lysine. The ethylene glycol-based hydrophilic linker Ebes was inserted between the lysine bearing the prosthetic group and the peptide (Table 1, compound 4) (29). The suitability of the first or second labeling strategy should be investigated for each particular application. We slightly modified the previously published procedure for the preparation of [18F]FPA and its use for protein radiolabeling (18). The methylanthranyl 2-[18F]fluoropropionate was obtained from the 2-bromo precursor and purified on semipreparative HPLC followed by the hydrolysis of the 9-methylanthranyl ester using 5% aqueous triethylamine and DMF as a cosolvent. The obtained [18F]FPA was dried using azeotropic distillation with acetonitrile in the presence of DMF. We found that DMF prevented the undesired evaporation of the volatile product, and it did not have any significant effect on the subsequent coupling reaction as DMF is the reaction solvent. Three different carboxylic acid activating reagents were used in this study: N,N′-diisopropylcarbodiimide (DIC), 2-(7-aza1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), and diethyl cyanophosphonate (DECP). DIC activates the acid as the O-acyl diisopropyl urea derivative which reacts subsequently with the primary or secondary amine. HATU is a superior reagent used for coupling of “difficult” sequences in the peptide synthesis. The 7-aza-1-hydroxybenzotriazolyl ester is formed in the presence of base, and it reacts readily with nucleophiles such as a primary or secondary amine. DECP forms a mixed anhydride of 2-fluoropropionic acid and diethyl phosphonate which subsequently reacts with a primary or secondary amine in the presence of base (32). We found that HATU-mediated activation provided comparable results to the DECP activation for coupling [18F]FPA to the peptide 1; however, the DIC activation did not provide satisfactory results. The yield of the coupling reaction using HATU was 16%, and the purity of the crude product was 90%. In the case of DECP activation the yield of the conjugation was 9% and the purity
Bioconjugate Chem., Vol. 17, No. 4, 2006 1019
of the final product was 93%. The only radioactive material isolated from the activation mixture was unreacted [18F]FPA. [18F]FPA was conjugated to various peptides listed in Table 1. The products were obtained in yields ranging from 6 to 16%, and only a slight dependency of the yield on the size and complexity of the peptide was observed. The radiochemical purity was confirmed by coinjection of the corresponding [19F]FPA-labeled peptides. The standards were prepared by DIC mediated coupling of [19F]FPA to the N-terminus or NH of lysine and analyzed using HPLC and mass spectrometry. The cleavage was performed at 30 °C, and the purity of the crude material was in the range of 90-99% in most cases. In solidphase peptide synthesis, the purity of the crude products decreases with increasing length of the peptide chain; this accounts for the lower purity of the 20 amino acid long peptide 2. The “no-carrier-added” (n.c.a) radiolabeled peptides were obtained with a specific activity greater than 37 GBq/µmol. In the standard peptide synthesis strategy, 3-fold excess of the amino acid and coupling reagent and 6-fold excess of the base are used to accomplish the successful attachment of the amino acid to the free amino terminus of the peptide chain attached to the resin (33). Such conditions cannot be satisfied in the case of coupling of the n.c.a. prosthetic groups such as [18F]FPA, and as a result, the amount of the coupling reagent is in huge excess if compared to the amount of the acid. The excess of the uronium salt-based coupling reagents such as HATU could lead to the guanidylation of the N-terminus of the peptide (33). We investigated the effect of the amount of the HATU/DIEA in the reaction mixture, and we found that the yields of the coupling reaction were not changed when 1-10 mg of HATU was used together with 10 µL of DIEA in 200 µL of DMF. Lower amounts of HATU led to lower yields, and a lower amount of the base (DIEA) was not sufficient to adjust pH to the desired range 8-9. Unfortunately, the yield of the conjugation remained between 10 and 20%. Since the yield of the [18F]FPA coupling was comparable with that of DECP, which uses the mixed anhydride activation method, and the N-terminal guanidylation is not possible, the formation of the N-terminal guanidylation byproducts is therefore not a yieldlimiting effect. The presence of an auxiliary base required to prevent undesired evaporation of the [18F]FPA during azeotropic removal of the water from the mixture used for the hydrolysis of the precursor could affect the yield of the conjugation. The commonly used bases are tetralkylammonium hydroxides (20) or potassium carbonate K222 mixture (34). As mentioned above, we found that the high boiling point aprotic solvent DMF minimized the loss of the hydrolyzed acid during the azeotropic distillation. The presence of potassium carbonate K222 complex led to lower yields of the conjugation; however, the use of DMF provided the product with yields and purity identical to the previously reported tetramethylammonium hydroxide. To further investigate the factors limiting the coupling of the prosthetic groups to the resin-bound peptides, we compared various commercially available solid-phase peptide synthesis (SPPS) resins. The common SPPS resins are polymer beads with a diameter of 100-300 µm. The linker allows selective detachment of the product from the solid support and is covalently attached to the polymer matrix. There are two factors which affect the behavior of the solid support: the composition of the polymer matrix and the nature of the linker. The polymer matrix used for SPPS is based on various cross-linked polystyrene (PS), poly(ethylene glycol) (PEG), polyamide or polyacrylamide copolymers, or grafted polymers. The amount of the linker attached to the beads determines the loading of the particular resin. The nature of the linker determines the nature of the C-terminus of the final product as well as the procedure
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Table 2. Decay Corrected Yields of [18F]FPA Coupling to the Peptide 1 Attached to the Different Solid Supports and Yields of the Product Cleavagea resin
resin
coupling
cleavage
total
purity
A B C D E F G
NovaGel HL Rink Amide-MBHA CLEAR Amide PAL-PEG-PS PL-PEGA Amino-PEGA TentaGel S
16 15 9 13 9 14 14
36 76 75 89 70 71 81
6 11 7 11 7 10 11
90 99 98 100 92 95 92
a
Yields and purity are given in %.
required for cleavage. In this study, we focused on the preparation of the peptide amides; thus, the acid-sensitive linkers Rink and PAL were used. The results are summarized in Table 2. We did not find any significant effect of the resin nature on the coupling step, and the best obtained yields remained below 20% for all resins evaluated. However, we found some resins more suitable for this radiochemical application due to better yields obtained during the cleavage step. The cleavage of the product from the solid support and simultaneous side chain protecting group removal were accomplished using a mixture of trifluoroacetic acid and appropriate scavengers. We found that the incubation of the resin with the cleavage mixture for 30 min provided 75% of the product from the Rink linker decorated resins, except the high loading NovaGel resin where the cleavage yields were below 50%. The resin containing the PAL linker provided 89% of the product in 20 min incubation. The use of very acid-labile linkers such as XAL has been previously reported to provide similar cleavage reaction yields (8, 26). However, the stability of side chain protecting groups has to be considered in this case; when the cleavage time is short, some of them may not be fully removed. In conclusion, [18F]FPA was conjugated with various peptides. The 18F-labeled peptides were obtained in 175 min with decay corrected yields around 10% (related to [18F]fluoride) and with a purity of 76-99%. Several solid-phase synthesis supports were found suitable for this application.
ACKNOWLEDGMENT We would like to thank Salma Jivan (radiochemist, Center for Molecular and Genomic Imaging) and Dave Kukis (Cyclotron Facilities Manager, Center for Molecular and Genomic Imaging) for radionuclide production. Supporting Information Available: RP-HPLC chromatographs for compounds 1-4. The material is available free of charge via the Internet at http://pubs.acs.org.
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