Preparation of 18F-Human Serum Albumin: A Simple and Efficient

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Bioconjugate Chem. 2005, 16, 1329−1333

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Preparation of 18F-Human Serum Albumin: A Simple and Efficient Protein Labeling Method with 18F Using a Hydrazone-Formation Method Young Soo Chang,†,‡,§ Jae Min Jeong,†,‡,* Yun-Sang Lee,† Hyung Woo Kim,† Ganesha B. Rai,† Seung Jin Lee,§ Dong Soo Lee,† June-Key Chung,†,‡ and Myung Chul Lee† Department of Nuclear Medicine, Seoul National University College of Medicine, Seoul, Korea, Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea, and Department of Pharmacy, Ewha Womans University, Seoul, Korea. Received March 21, 2005; Revised Manuscript Received July 16, 2005

18 F-labeling of proteins and peptides is important for positron emission tomography (PET). Although there are many methods for the labeling of proteins with 18F, most of these are characterized by complicated procedures or low yields. Here, we report a novel and simple method which includes the preparation of [18F]fluorobenzaldehyde ([18F]FBA) and successive conjugation with hydrazinonicotinic acid-human serum albumin conjugate (HYNIC-HSA) via hydrazone formation. HYNIC-HSA, which can also be used for labeling with 99mTc, was prepared via reaction with N-hydroxysuccinimide (NHS) or tetrafluorophenyl (TFP) esters of HYNIC with HSA. No-carrier-added [18F]FBA was prepared by the nucleophilic substitution of [18F]fluoride to 4-trimethylammonium benzaldehyde triflate in the presence of tetrabutylammonium bicarbonate. [18F]FBA was purified by passing ion exchange cartridges (IC-H and QMA) and was adsorbed to a C18 Sep-Pak cartridge. The adsorbed [18F]FBA was then eluted with 50% ethanol. HYNIC-HSA was added to the solution and conjugated with [18F]FBA via hydrazone formation. 18F-HSA was purified with a PD10 column. Biodistribution of 18F-HSA, 99mTc-HSA, and [18F]FBA in mice were investigated at 10, 20, and 60 min after intravenous injection. The number of conjugated HYNIC molecules per HSA ranged from 5.2 to 23.2 depending on the reaction conditions. The labeling efficiency of 18F-FBA was 67 ( 15.7%. The radiochemical purity after purification was over 99%. The conjugation efficiency of HYNIC-HSA with [18F]FBA was between 25% and 90%. The conjugation efficiency was observed to increase with increases in the number of conjugated HYNIC, the HYNIC-HSA concentration, or temperature. 18F-HSA exhibited a biodistribution pattern similar to that of 99mTc-HSA while [18F]FBA showed much lower blood activity than that of 18F-HSA and 99m Tc-HSA. We concluded that 18F-HSA was successfully labeled using a novel method which involves hydrazone formation between [18F]FBA and HYNIC-HSA. This method can be applied to the 18F-labeling of other proteins or peptides.

INTRODUCTION

The application of bioactive proteins labeled with radionuclides for diagnostic imaging has emerged as a useful and interesting field in nuclear medicine (1-4). In particular, 18F-labeled proteins or peptides bear a great deal of clinical and research potential due primarily to their favorable nuclear characteristics with regard to positron emission tomography (PET) that has high resolution and sensitivity (5-7). Specially designed synthons are essential for the 18F labeling of proteins or peptides because the biomolecules cannot be directly labeled by nucleophilic [18F]fluorination. A variety of 18F-labeled synthons have been developed and successfully applied to a host of peptides and proteins for 18F labeling (8-12). With a few exceptions (8), the most effective procedures for the synthesis * Corresponding author: Jae Min Jeong, Ph.D., Department of Nuclear Medicine, Seoul National University Hospital, 28 Yungun-dong, Jongro-gu, Seoul 110-744, Korea. Tel: 82-2-20723805. Fax: 82-2-745-7690. E-mail address: [email protected]. † Department of Nuclear Medicine, Seoul National University College of Medicine. ‡ Cancer Research Institute, Seoul National University College of Medicine. § Ewha Womans University.

of 18F-labeled synthons are all multistep procedures. Among these synthons, N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) remains the most widely applied agent for many bioactive molecules with regard to in vivo stability and radiolabeling yields. However, [18F]SFB can be obtained via a time-consuming three-step synthesis method (5, 6). Therefore, it is necessary to develop simpler and more efficient 18F labeling methods with proteins or peptides for clinical routine use. Here, we report a novel and simple method for the preparation of 18F-labeled proteins. In this paper, we evaluated the efficacy of this method using human serum albumin (HSA) as a test protein. The resulting product, 18 F-HSA, might be used in blood pool imaging. The whole procedure is shown in Scheme 1. First, hydrazinonicotinic acid (HYNIC) was conjugated with HSA yielding HYNICHSA. [18F]fluorobenzaldehyde ([18F]FBA) was then prepared via the nucleophilic substitution of [18F]fluoride. Finally, [18F]FBA was conjugated to HYNIC-HSA via hydrazone formation. HYNIC-HSA might be used not only for 18F-labeling but also for 99mTc-labeling. EXPERIMENTAL PROCEDURES

Materials and Instrumentations. NMR spectroscopy was conducted with a Gemini 300 NMR spectrometer obtained from the Varian Company. Mass spectra

10.1021/bc050086r CCC: $30.25 © 2005 American Chemical Society Published on Web 08/25/2005

1330 Bioconjugate Chem., Vol. 16, No. 5, 2005 Scheme 1. Synthesis Formation of Hydrazone

of

18

F-Labeled

Technical Notes HSA

via

were obtained with a Applied Biosystems/MDS Sciex API 3000 LC/MS/MS System. A DU650 Spectrophotometer (Beckman Coulter) was used for absorbance measurements. A NaI well counter (Packard, Canberra Co.) and dose calibrator (Atomlab 100, Medical Systems Inc.) were used to measure the low and high levels of radioactivity, respectively. A Bio-Scan System 2000 imaging scanner was used to perform the Radio-TLC scan. For TLC, we purchased aluminum-backed silica gel 60 F254 from the E. Merck Company (Germany) and ITLC-SG plates from the Gelman Company. PD10 size-exclusion columns were from Amersham Biosciences (Sweden), C18 and QMA Sep-Pak cartridges were obtained from Waters (Milford, MA), IC-H Maxi-Clean cartridges were purchased from Alltech (Deerfield, IL), and BCA kit for the protein assay was from Pierce (Rockford, IL). [18F]Fluoride was produced by the 18O (p, n) 18F reaction on 18O-enriched (95%) water using a 13 MeV proton beam generated by a TR-13 cyclotron (Ebco Technologies, Vancouver, Canada). The 99Mo/99mTc-generator was purchased from the DuPont Company. HSA kit for 99mTc-labeling was obtained from Daiichi Radioisotope Laboratories, LTD (Japan). All other reagents and solvents, if not specified, were purchased from Aldrich, Sigma, or Fluka and were used with no further purification. Synthesis of HYNIC and 4-N,N,N-Trimethylammonium Benzaldehyde Triflate. N-Hydroxysuccinimide ester of hydrazinonicotinic acid (NHS-HYNIC) and tetrafluorophenyl ester of hydrazinonicotinic acid (TFPHYNIC) compounds were synthesized according to previously reported methods (14, 15). 4-N,N,N-Trimethylammonium benzaldehyde triflate was also synthesized according to a method published in the literature (16). Briefly, N,N-dimethylalininobenzaldehyde (100 mg, 0.44 mmol) was added to an evacuated and argon-purged 25 mL sidearm flask with a stirring bar. Methylene chloride (7 mL) was added to the flask with stirring, which produced a clear slightly greenish-yellow solution. Methyl trifluoromethanesulfonate (53.75 µL, 0.475 mmol) was then added, which resulted in an immediate color change to intense yellow. After stirring overnight, the crude product was obtained by addition of diethyl ether as a white powder (0.14 g). Recrystallizaiton from methylene

chloride/diethyl ether produced a fine white crystalline powder. mp 105-107 °C; 1H NMR (DMSO-d6) δ: 3.64 (s, 9H, 3CH3), 8.18 (q, 4H, J ) 6.8 Hz, aromatic), 10.1 (s, 1H, CHO); 13C NMR (DMSO-d6) δ: 55.3, 120.7, 129.9, 135.8, 150.1, 191.2; ESI-MS (m/z): 165.1 [M + H]+. Preparation of HYNIC-HSA. HYNIC-HSA was prepared by reacting HSA with various amounts of NHS-HYNIC or TFP-HYNIC, according to the published method with some modifications (17). To 1 mL of 2% HSA solution in 0.05 M phosphate buffer (pH 7.5) we added 20 µL of NHS-HYNIC or TFP-HYNIC solution in dimethyl sulfoxide (DMSO). The molar ratios of HYNIC to HSA varied ranging from 8:1 to 32:1. After incubation for 2 h at room temperature, the unreacted HYNIC was removed with a PD10 size-exclusion column. HYNIC-HSA was eluted with 0.05 M phosphate buffer (pH 7.5). The protein concentration of purified HYNICHSA was determined using a BCA protein assay kit. The amount of conjugated HYNIC was determined by measuring optical density at 385 nm after conjugation with 0.5 mM p-nitrobenzaldehyde (extinction coefficient: 2.53 × 104 L mol-1 cm-1) for 5 h at room temperature thereby forming hydrazone (15, 18). Preparation of [18F]FBA. An aqueous solution of [18F]fluoride in 18O-enriched water was captured on a QMA light Sep-Pak cartridge which had been preconditioned with 5 mL of 0.5 M potassium bicarbonate and washed with 10 mL of deionized water. The [18F]fluoride on the cartridge was eluted with 1 mL of 2.3% tetrabutylammonium bicarbonate in 83.8% acetonitrile. Azeotropic distillation was conducted twice, each at 90 °C after addition of 1 mL acetonitrile under a gentle stream of argon gas. [18F]FBA was prepared via the nucleophilic substitution of [18F]fluoride to 4-N,N,N-trimethylaminebenzaldehyde triflate (16). To the above dried [18F]fluoride in tetrabutylammonium bicarbonate (16 mg, 52.7 µmol) was added 5 mg of 4-N,N,N-trimethylammonium benzaldehyde triflate in DMSO (0.5 mL) and reacted for 6 min at 100 °C. The reaction mixture was diluted with deionized water and passed through a set of ion exchange cartridges (two IC-H Maxi-Clean cartridges for cation exchanger and a QMA Sep-Pak cartridge for anion exchanger) to remove ionic components. The labeled product was then adsorbed to a C18 Sep-Pak light cartridge. The adsorbed [18F]FBA was eluted with 50% ethanol. Labeling efficiency was measured by a thin-layer chromatography with a Radio-TLC scanning (silica gel TLC/ethyl acetate:hexane ) 1:3; Rf values: [18F]fluoride ion ) 0.0 and [18F]FBA ) 0.7). Preparation of 18F-HSA. HYNIC-HSA was conjugated with [18F]FBA via hydrazone formation (Scheme 1). Purified [18F]FBA solution (0.3 mL) was added to the HYNIC-HSA solution (the concentration of HSA was 3 mg/1.2 mL 0.05 M phosphate buffer (pH 7.5)), and the solution was incubated for 30 min at room temperature, 50 °C or 60 °C. The labeled 18F-HSA was then purified with a PD10 column. Radiochemical purity was verified using ITLC-SG and normal saline or acetone. (ITLC-SG/normal saline; Rf values: 18F-HSA ) 0.0 and [18F]FBA ) 1.0; ITLC-SG/acetone; Rf values: 18F-HSA ) 0.0 and [18F]FBA ) 1.0). Stability Test of 18F-HSA. The labeled and purified 18F-HSA was then stored at room temperature, and the radiochemical purity was determined by chromatography as described in the Preparation of 18F-HSA section. The radiochemical purity was determined at 10 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h.

Technical Notes

Figure 1. Conjugation of HYNIC to albumin as a function of the molar ratio of active HYNIC ester and HSA; NHS-HYNIC ([) or TFP-HYNIC (9). Protein concentration of HYNIC-HSA was determined using a BCA protein assay kit, and the amount of conjugated HYNIC was determined by measuring OD385 after conjugation with p-nitrobenzaldehyde (extinction coefficient: 2.53 × 104 L mol-1 cm-1).

Preparation of 99mTc-HSA. 99mTc-HSA was prepared and tested according to the protocols provided by the supplier. Biodistribution Study in Mice. All animal studies were conducted in compliance with the local regulations. Male ICR mice (n ) 3 or 4/group, 28 ( 1.9 g) were injected with 37 kBq (100 µL) of 18F-HSA, 99mTc-HSA, or [18F]FBA through the tail vein. The mice were subsequently sacrificed by cervical dislocation after 10, 20, and 60 min, respectively. The weight and radioactivity of the blood, muscle, fat, heart, lung, liver, spleen, stomach, intestine, kidney, brain, and bone from the sacrificed mice were then measured with electronic balance and gamma scintillation counter, respectively. Results were expressed as a percentage of injected dose per gram of tissue (% ID/g). RESULTS

Preparation of HYNIC-HSA. The reaction of HSA with NHS-HYNIC or TFP-HYNIC resulted in the introduction of a number of hydrazinonicotinyl sidechains. The amount of conjugated HYNIC increased with increases in the molar ratio of the active ester of HYNIC to HSA (Figure 1). The amount ranged from 5.2 to 23.2 according to the reaction conditions utilized. When TFPHYNIC was used, the amount of conjugated HYNIC molecules per HSA was slightly higher than that observed when NHS-HYNIC was used, but this difference was not significant (Figure 1). Preparation of [18F]FBA. [18F]FBA was synthesized via the nucleophilic substitution of [18F]fluoride to N,N,Ntrimethylammonium benzaldehyde in the presence of tetrabutylammonium bicarbonate. The labeling efficiency of [18F]FBA was about 67 ( 15.7%. The radiochemical purity of [18F]FBA after purification was more than 99%. Preparation of 18F-HSA. HYNIC-HSA was conjugated with the purified [18F]FBA via hydrazone formation, and the labeling efficiency was verified by an ITLCSG chromatogram. As the HYNIC-HSA concentration increased, the radiochemical yield also increased (Figure 2). The conjugation yield of HYNIC-HSA with [18F]FBA ranged from 25% to 90%. The more highly hydrazinonicotinylated HYNIC-HSA exhibited a higher conjugation yield. The radiochemical yield of the control HSA was less than 10% (Figure 3). The reaction temperature also influenced the radiochemical yield. This was determined to increase at higher temperatures (Figure 4).

Bioconjugate Chem., Vol. 16, No. 5, 2005 1331

Figure 2. Conjugation efficiency of HYNIC-HSA with [18F]FBA as a function of time. The protein concentrations are 3 mg/1.5 mL (b), 2 mg/1.5 mL (2), 1 mg/1.5 mL (9), and 0.5 mg/1.5 mL ([).

Figure 3. Conjugation efficiency of HYNIC-HSA with [18F]FBA as a function of time. The number of conjugated HYNIC per HSA on used HYNIC-HSA are 0 as a control (*), 5 ([), 12 (9), and 23 (b).

Figure 4. The effects of reaction temperature on conjugation efficiency of HYNIC-HSA with [18F]FBA. The reaction temperatures are room temperature ([), 50 °C (9), and 60 °C (2).

Stability Test of 18F-HSA. 18F-HSA was very stable for 6 h both in 0.05 M phosphate buffer (pH ) 7.4) at room temperature and in human serum at 37 °C (Figure 5). Biodistribution Study in Mice. Biodistribution experiments using 18F-HSA, 99mTc-HSA and [18F]FBA were performed on normal mice (Figure 6). 18F-HSA and 99m Tc-HSA exhibited similar blood activities, while [18F]FBA exhibited much lower blood activity. The bone

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Figure 5. Stability test of 18F-HSA in 0.05 M phosphate buffer (pH ) 7.4) at room temperature (O) and in human serum at 37 °C (2).

Figure 6. Biodistribution of (A) 18F-HSA, (B) 99mTc-HSA, and (C) [18F]FBA. The labeled agents were injected through the tail veins of mice.

uptake of 18F-HSA was not increasing by time, which indicated that defluorination did not occur from 18F-HSA in vivo. In the other organs, 18F uptake was similar to or less than those observed with 99mTc-HSA. DISCUSSION

HYNIC is a famous bifunctional chelating agent, which is used for the labeling of proteins with 99mTc (1, 2, 13, 15, 17). A variety of functional groups, including active esters, isothiocyanates, maleimides, hydrazides, and R-haloamides, have been developed in order to conjugate bifunctional chelating agents to proteins (2). Hydrazides have been demonstrated to react with aldehyde groups via the formation of stable hydrazone bonds with high yields. Thus, the hydrazone formation reaction for the coupling of aldehyde and/or hydrazide-containing proteins and/or bifunctional chelating agents has been applied to the preparation of protein-protein or proteinbifunctional chelating agent conjugates (18, 19). To determine the number of conjugated HYNIC per HYNIC-conjugated proteins, p-nitrobenzaldehyde is used, as it reacts with HYNIC-protein quantitatively with generation of a specific hydrazone, which exhibits a characteristic absorption at 385 nm. Actually, the protocols of this experiment are inspired from the quanti-

Technical Notes

fication method used for HYNIC-protein. We used [18F]FBA instead of p-nitrobenzaldehyde in this novel and simple method for the labeling of proteins or peptides with 18F. This method proved to significantly reduce radiolabeling steps and reaction time relative to the other reported methods (5, 8). [18F]FBA is a famous synthon which is used for the synthesis and 18F-labeling of a variety of radiopharmaceuticals (16, 20). In general, either N,N,N-trimethylammonium benzaldehyde or nitrobenzaldehyde can be used as a precursor for the synthesis of [18F]FBA. We used N,N,N-trimethylammonium benzaldehyde because the positively charged quaternary ammonium can easily be removed from the neutral hydrophobic product using a combination of ion exchange and reverse phase cartridges. In this manner, [18F]FBA can be produced in a no-carrier-added state which provides high specific activity. Tetrabutylammonium bicarbonate, rather than crown ether such as Kryptofix 2.2.2, was used as a base to activate [18F]fluoride in a nucleophilic substitution reaction in order to facilitate the next purification step in this experiment. Crown ether is difficult to remove as both it and [18F]FBA are hydrophobic whereas the tetrabutylammonium ion can easily be removed by ion exchange cartridges together with precursor. The prepared 18F-HSA exhibited a high labeling efficiency and stability. Biodistribution studies in mice revealed that introduction of the HYNIC into HSA had no significant effects on the biological properties of HSA or on its in vivo stability as compared to 99mTc-HSA a verified and commercialized blood pool imaging agent. A recent report reveals a chemoselective formation of oxime between unprotected aminooxy-functionalized peptides and [18F]FBA with a high yield (21). They asserted that this method allowed for the fast and straightforward large-scale production of 18F-labeled peptides for clinical routine PET application. Their method and our HYNIC method have a mechanism in commonsnamely, that some ammonia derivatives including hydroxylamine, hydrazine, phenylhydrazine, or semicarbazide form stable conjugates with either aldehydes or ketones. In our lab, the application of the HYNIC method proved to be more favorable than the oxime method as HYNIC has been already verified with regard to its application as a bifunctional chelating agent for the labeling of proteins or peptides with 99mTc. In addition, HYNIC-protein can be applied directly not only for 18F labeling but also for 99m Tc lableing. There are some 18F-labeling synthons those react with thiols (22) or contain thiols (23). Although these thiolrelated synthons show high labeling efficiencies, highly reactive and unstable thiols can be a cause of problems in storage or preparation. Our biodistribution study confirmed that 18F-HSA labeled by this technique can be used for blood pool imaging, as it exhibited biodistribution pattern similar to that seen with 99mTc-HSA, which is an approved blood pool imaging agent, in the majority of the organs. [18F]FBA, a small hydrophobic molecule, exhibited a very different biodistribution pattern as expected. CONCLUSION

We developed a novel and simple method for the preparation of 18F-HSA using a hydrazone formation mechanism between [18F]FBA and HYNIC-HSA. The generated 18F-HSA exhibited excellent in vitro and in vivo properties as a blood-pool imaging agent. This method

Bioconjugate Chem., Vol. 16, No. 5, 2005 1333

Technical Notes

might prove quite applicable to the labeling of other proteins with 18F as well. ACKNOWLEDGMENT

This study was supported by a grant from the KOSEF. Supporting Information Available: 1H NMR spectrum of N,N,N-trimethylammonium benzaldehyde, 13C NMR spectrum of N,N,N-trimethylammmonium benzaldehyde, mass spectrum of N,N,N-trimethylammonium benzaldehyde, UV spectrum of p-nitrobenzaldehyde, UV spectrum of HSA-HYNIC, UV spectrum of HSA-HYNIC + p-nitrobenzaldehyde, and UV spectrum of HSA + p-nitrobenzaldehyde. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Fichna, J., and Janecka, A. (2003) Synthesis of targetspecific radiolabeled peptides for diagnostic imaging. Bioconjugate Chem. 14, 3-17. (2) Weiner, R. E., and Thakur, M. L. (2002) Radiolabeled peptides in the diagnosis and therapy of oncological diseases. Appl. Radiat. Isot. 57, 749-763. (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) Jeong, J. M., Hong, M. K., Lee, J., Son, M., So, Y., Lee, D. S., Chung, J.-K., and Lee, M. C. (2004) 99mTc-Neolactosylated human serum albumin for imaging the hepatic asialoglycoprotein receptor. Bioconjugate Chem. 15, 850-855. (5) Okarvi, S. M. (2001) Recent progress in fluorine-18 labeled peptide radiopharmaceuticals. Eur. J. Nucl. Med. 28, 929938. (6) 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. (7) Varagnolo, L., Stokkel, M. P. M., Mazzi, U., and Pauwels, E. K. J. (2000) 18F-labeled radiopharmaceuticals for PET in oncology, excluding FDG. Nucl. Med. Biol. 27, 103-112. (8) Wester, H. J., Hamacher, K., and Stocklin, 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. (9) Vries, E. F. J., Vroegh, J., Elsinga. P. H., and Vaalburg, W. (2003) Evaluation of fluorine-18-labeled alkylating agents as potential synthons for the labeling of oligonucleotides. Appl. Radiat. Isot. 58, 469-476. (10) Vaidyanathan, G., and Zalutsky, M. R. (1992) Labeling proteins with fluorine-18 Using N-succinimidyl 4-[18F]fluorobenzoate. Nucl. Med. Biol. 19, 275-281. (11) Downer, J. B., McCarthy, T. J., Edwards, W. B., Anderson, C. J., and Welch, M. J. (1997) Reactivity of p-[18F]Fluorophenacyl bromide for radiolabeling of proteins and peptides. Appl. Radiat. Isot. 48, 907-916.

(12) Iwata, R., Pascali, C., Bogni, A., Horvath, G., Kovacs, Z., Yanai, K., and Ido, T. (2000) A new, convenient method for the preparation of 4-[18F]fluorobenzyl halides. Appl. Radiat. Isot. 52, 87-92. (13) Abrams, M. J., Juweid, M., TenKate, C. I., Schwartz, D. A., Hauser, M. M., Gaul, F. E., Fuccello, A. J., Rubin, R. H., Strauss, H. W., and Fischman, A. J. (1990) Technetium-99mhuman polyclonal IgG radiolabeled via the hydrazino nicotinamide derivative for imaging focal sites of infection in rats. J. Nucl. Med. 31, 2022-2028. (14) Ono, M., Arano, Y., Mukai, T., Uehara, T., Fujioka, Y., Ogawa, K., Uehara, T., Saga, T., Konishi, J., and Saji, H. (2001) 99mTc-HYNIC-derivatized ternary ligand complexes for 99mTc-labeled polypeptides with low in vivo protein binding. Nucl. Med. Biol. 28, 215-224. (15) Schwartz, D. A., Abrams, M. J., Hauser, M. M., Gaul, F. E., Larsen, S. K., Rauh, D., and Zubieta, J. A. (1991) Preparation of hydrazine-modified proteins and their use for the synthesis of 99mTc-protein conjugates. Bioconjugate Chem. 2, 333-338. (16) Haka, M. S., Kilbourn, M. R., Watkins, G. L., and Toorongian, S. A. (1989) Aryltrimethylammonium trifluoromethanesulfonates as precursors to aryl [18F]fluorides: imporved synthesis of [18F]GBR-13119. J. Labelled Compd. Radiopharm. 27, 823-838. (17) Verbeke, K., Hjelstuen, O., Debrock, E., Cleynhens, B., Roo, M., and Verbruggen, A. (1995) Comparative evaluation of 99Tcm-Hynic-HSA and 99Tcm-MAG3-HSA as possible blood pool agents. Nucl. Med. Commun. 16, 942-957. (18) King, T. P., Zhao, S. W., and Lam T. (1986) Preparation of protein conjugates via intermolecular hydrazone linkage. Biochemistry 25, 5774-5779. (19) Jeong, J. M., Lee, J., Paik, C. H., Kim, D. K., Lee, D. S., Chung, J.-K., and Lee, M. C. (2004) Site-specific 99mTc-labeling of antibody using dihydrazinophthalazine (DHZ) conjugation to Fc region of heavy chain. Arch. Pharm. Res. 27, 961-967. (20) Choe, Y. S., Oh, S. J., Shim, I., Naruto, S., Chi, D. Y., Kim, S. E., Lee, K. H., Choi, Y., and Kim, B. T. (2000) Syntheses and biological evaluation of 18F-labeled-3-(1-benzyl-piperidin4-yl)-1-(1-methyl-1H-indol-3-yl)propan-1-ones for In vivo mapping of acetylcholinesterase. Nucl. Med. Biol. 27, 263-267. (21) 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 octerotide analogs. J. Nucl. Med. 45, 892-902. (22) Toyokuni, T., Walsh, J. C., Dominguez, A., Phelps, M. E., Barrio, J. R., Gambhir, S. S., and Satyamurthy, N. (2003) Synthesis of a new heterobifunctional linker, N-[4-(aminooxy)butyl]maleimide, for facile access to a thiol-reactive 18Flabeling agent. Bioconjugate Chem. 14, 1253-1259. (23) Glaser, M., Karlsen, H., Solbakken, M., Arukwe, J., Brady, F., Luthra, S. K., and Cuthbertson, A. (2004) 18F-Fluorothiols: a new approach to label peptides chemiselectively as potential tracers for positron emission tomography. Bioconjugate Chem. 15, 1447-1453.

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