Bioconjugate Chem. 1997, 8, 442−446
442
TECHNICAL NOTES Conventional and High-Yield Synthesis of DTPA-Conjugated Peptides: Application of a Monoreactive DTPA to DTPA-D-Phe1-octreotide Synthesis† Yasushi Arano,*,‡ Hiromichi Akizawa,‡ Takashi Uezono,‡ Kenichi Akaji,§ Masahiro Ono,‡ Susumu Funakoshi,‡ Mitsuru Koizumi,| Akira Yokoyama,‡ Yoshiaki Kiso,§ and Hideo Saji‡ Department of Radiopharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan; Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607, Japan; and Cancer Institute Hospital, Toshima-ku, Tokyo 170, Japan. Received October 31, 1996X
Successful imaging of somatostatin receptor-positive tumors with 111In-DTPA-D-Phe1-octreotide has stimulated development of peptide radiopharmaceuticals using DTPA as the chelating agent. However, use of cyclic DTPA dianhydride (cDTPA) resulted in low synthetic yields of DTPA-peptide by either solution or solid-phase syntheses. This paper reports a novel high-yield synthetic procedure for DTPAD-Phe1-octreotide that is applicable to other peptides of interest using a monoreactive DTPA derivative. A monoreactive DTPA that possesses one free terminal carboxylic acid along with four carboxylates protected with tert-butyl ester (mDTPA) was synthesized. Fmoc-Thr(tBu)-ol, prepared from FmocThr(tBu)-OH, was loaded onto 2-chlorotrityl chloride resin. After construction of the peptide chains by Fmoc chemistry, mDTPA was coupled to the R amine group of the peptide on the resin in the presence of 1,3-diisopropylcarbodiimide and 1-hydroxybenzotriazole. Treatment of the mDTPApeptide-resin with trifluoroacetic acid-thioanisole removed the protecting groups and liberated [Cys(Acm)2,7]-octreotide-D-Phe1-DTPA from the resin. Iodine oxidation of the DTPA-peptide, followed by the reversed-phase HPLC purification, produced DTPA-D-Phe1-octreotide in overall 31.8% yield based on the starting Fmoc-Thr(tBu)-ol-resin. The final product gave a single peak on analytical HPLC, and amino acid analysis and mass spectrometry confirmed the integrity of the product. 111In radiolabeling of the product provided 111In-DTPA-D-Phe1-octreotide with >95% radiochemical yield, as confirmed by analytical reversed-phase HPLC, TLC, and CAE. These findings indicated that use of mDTPA during solid-phase peptide synthesis greatly increased the synthetic yield of DTPA- DPhe1-octreotide, due to the absence of nonselective reactions that are unavoidable when cDTPA is used. These results also suggested that mDTPA would be a versatile reagent to introduce DTPA with high yield into peptides of interest.
INTRODUCTION
Low molecular weight peptides such as octreotide (1, 2), chemotactic peptides (3, 4), RGD-peptides (5, 6), and vasoactive intestinal peptide (7, 8) have attracted strong attention as new vehicles to deliver radioactivity to target tissues in diagnostic nuclear medicine, due to their reduced immunogenicity and rapid distribution pharmacokinetics when compared with murine antibodies. Since localization of these peptides involves specific binding to receptors expressed on the target cells, these peptides should be radiolabeled at high specific activities for clinical application (4). Radiolabeling of these peptides with metallic radionuclides such as technetium-99m (99mTc) and indium-111 (111In) is important for clinical application of radiopharmaceuticals. * Author to whom correspondence should be addressed [telephone 81-75-753-4566; fax 81-75-753-4568; e-mail arano@ pharm.kyoto-u.ac.jp]. † This work is dedicated to the late Dr. Funakoshi. ‡ Kyoto University. § Kyoto Pharmaceutical University. | Cancer Institute Hospital. X Abstract published in Advance ACS Abstracts, April 15, 1997.
S1043-1802(97)00023-2 CCC: $14.00
For radiolabeling with 111In, diethylenetriaminepentaacetic acid (DTPA) is usually attached to peptide molecules to provide an 111In chelating site. The resulting 111 In-labeled peptides possess specific activities and in vivo stabilities sufficient for clinical application (2). Indeed, recent clinical studies have demonstrated high target-to-nontarget ratios of radioactivity even at early postinjection times (9, 10). Furthermore, attachment of hydrophilic 111In-DTPA chelate to peptides altered the excretion pathway from hepatobiliary to urinary excretion, which reduced abdominal radioactivity levels, as has been successfully observed with 111In-DTPA-D-Phe1-octreotide (Figure 1) (1, 9, 11). These findings indicate that DTPA would be a suitable chelating agent for 111In radiolabeling of peptides for receptor scintigraphy. For incorporation of a DTPA chelating site into peptides, cyclic DTPA dianhydride (cDTPA; Figure 2A) is used as the bifunctional chelating agent of choice, due to its simple conjugation reactions with peptides and ready availability from commercial sources. However, since cDTPA possesses two anhydride groups, formations of inter- and intramolecular cross-linkings are inevitable in its conjugation reactions with peptides (2, 3). Formation of ester bonds between DTPA and tyrosine residues © 1997 American Chemical Society
Technical Notes
Figure 1. Chemical structure of DTPA-D-Phe1-octreotide.
Figure 2. Chemical structures of cyclic DTPA dianhydride (A) and mDTPA (B).
of peptides has also been reported (12). Such undesirable side reactions along with a requirement of repeated purification result in low synthetic yield of DTPAconjugated peptides when cDTPA is used as the bifunctional chelating agent. Since radiolabeling of peptides with 111In-DTPA is useful not only for diagnostic applications but also for screening peptides for use in nuclear medicine, new synthetic procedures are warranted for preparation of DTPA-conjugated peptides with high synthetic yields. Recently, we developed a monocarboxylic acid derivative of DTPA, with the rest of the four carboxylates being protected with acid-removable tert-butyl esters (mDTPA) as illustrated in Figure 2B (13). Since mDTPA possesses only one free carboxylic acid, formation of inter- and intramolecular cross-linking would be prevented during conjugation reactions with peptides. mDTPA also provides a coordination geometry similar to that of cDTPA when cDTPA is conjugated with peptides without any side reactions. The high solubilities of mDTPA in various organic solvents make this reagent versatile for either liquid- or solid-phase syntheses. In the present study, a simple and high-yield synthetic procedure for DTPAconjugated peptides was designed with mDTPA using an octapeptide, octreotide, as a model. MATERIALS AND METHODS
General. Amino acid analyses were performed with a Hitachi L8500 amino acid analyzer (Hitachi Co. Ltd., Tokyo, Japan) utilizing postcolumn ninhydrin detection. Optical rotations were measured with SEPA-300 (Horiba Co. Ltd., Kyoto, Japan). Analytical HPLC characterization of DTPA-conjugated peptides was performed on a YMC AM302 column (4.6 × 150 mm; YMC Co. Ltd., Kyoto, Japan), eluted with a linear gradient of acetonitrile (20-80%, 30 min) in 0.1% aqueous trifluoroacetic acid (TFA) at a flow rate of 0.9 mL/min. The eluent was monitored by measuring the UV absorption at both 230 and 254 nm. FPLC (Pharmacia, Uppsala, Sweden) was carried out with a YMC ODS-AQ300 column (1.5 × 50 cm) eluted with a linear gradient of 60% acetonitrile0.1% aqueous TFA (0-100%, 400 min) in 0.1% aqueous TFA at a flow rate of 3 mL/min. The eluent was monitored by measuring the UV absorption at 254 nm.
Bioconjugate Chem., Vol. 8, No. 3, 1997 443
Preparative HPLC purification was carried out on a YMC SH-343-5AM column (20 × 250 mm), which was eluted with a linear gradient of acetonitrile (20-80%, 60 min) in 0.1% aqueous TFA at a flow rate of 5 mL/min. The eluent was monitored by measuring the UV absorption at 254 nm. Fast atom bombardment mass spectra (FABMS) were obtained on a JMS-HX110 spectrometer equipped with a JMA-DA5000 (JEOL Ltd., Tokyo, Japan). Proton nuclear magnetic resonance (1H-NMR) spectra were recorded on an AC 300 (300 MHz) spectrometer (Bruker), and the chemical shifts are reported in parts per million downfield from an internal tetramethylsilane standard. Melting points are reported uncorrected. Analytical HPLC characterization of 111InDTPA-D-Phe1-octreotide was performed on a Cosmosil 5C18-MS column (4.6 × 150 mm, Nacalai Tesque, Kyoto, Japan), eluted with a linear gradient of 40-80% MeOH in 0.05 M acetate buffer (pH 5.5) in 20 min at a flow rate of 1 mL/min. The final solvent composition was maintained for another 5 min, as reported (2). Radiochemical purities of 111In-DTPA-D-Phe1-octreotide were also determined by TLC and cellulose acetate electrophoresis (CAE). TLC (Merck Art. 5553) was developed with a 10% aqueous solution of ammonium chloride-methyl alcohol (1:1), while CAE was run at an electrostatic field of 0.8 mA/cm for 40 min in veronal buffer (I ) 0.06, pH 8.6). 9-Fluorenylmethoxycarbonyl (Fmoc) amino acid derivatives and 2-chlorotrityl chloride resin were purchased from Nova Biochem (La¨ufelfingen, Switzerland). 111InCl3 was supplied by Nihon Medi-Physics Co. Ltd. (Tokyo, Japan). Preparation of mDTPA. mDTPA was synthesized as reported previously (13). Briefly, one terminal amine of diethylenetriamine was trifluoroacetylated with ethyl trifluoroacetate, and the unprotected amines were alkylated with tert-butyl bromoacetate. After alkylation of amide nitrogen by tert-butyl bromoacetate in the presence of sodium hydride, the trifluoroacetyl protecting group was removed with anhydrous hydrazine in tert-butyl alcohol. The resulting secondary amine was alkylated with benzyl bromoacetate in the presence of N,N-diisopropylethylamine. mDTPA was then obtained in an almost quantitative yield by catalytic hydrogenation with Pd/C in ethyl acetate. Fmoc-Thr(tBu)-ol. This compound was synthesized according to the procedure of Rodriguez et al. (14). To a chilled (-15 °C) solution of Fmoc-Thr(tBu)-OH (1.99 g, 5 mmol) in 5 mL of ethylene glycol dimethyl ether were added N-methylmorpholine (0.56 mL, 5 mmol) and isobutyl chloroformate (0.65 mL, 5 mmol) while the reaction temperature was maintained. After 1 min of stirring, the precipitate was removed, and a suspension of NaBH4 (0.57 g, 15 mmol) in 15 mL of water was added at the same temperature. After 30 s, 125 mL of water was then added. The reaction solution was extracted with ethyl acetate (50 mL × 3), and the combined organic layers were washed with 5% aqueous NaHCO3, followed by brine. The combined organic layers were dried over anhydrous Na2SO4, and the solvent was removed in vacuo. Fmoc-Thr(tBu)-ol was obtained as a white solid (1.36 g, 70.9%) with a mp of 42-45 °C after column chromatography on silica gel using chloroform as an eluent: 1H-NMR (CDCl3) δ 1.16 (3H, d, J ) 6.2 Hz, CHCH3), 1.20 (9H, s, tBu), 2.88 (1H, broad, OH), 3.61 (1H, broad, CHCH2OH), 3.66 (2H, broad, CHCH2OH), 3.94 (1H, m, CHCH3), 4.22 (1H, t, J ) 6.8 Hz, CHCH2CO), 4.40 (2H, m, CHCH2CO), 5.28 (1H, d, J ) 7.5 Hz, NH), 7.30 (2H, d, J ) 7.4 Hz, aromatics), 7.38 (2H, t, J ) 7.2 Hz, aromatics), 7.59 (2H, d, J ) 7.4 Hz, aromatics), 7.74 (2H, d, J ) 7.4 Hz, aromatics); FAB-MAS calcd for C23H29-
444 Bioconjugate Chem., Vol. 8, No. 3, 1997
Arano et al.
Figure 3. Analytical HPLC profile of DTPA-D-Phe-Cys(Acm)Phe-D-Trp-Lys-Thr-Cys(Acm)-Thr-ol. Figure 4. Analytical HPLC profile of DTPA-D-Phe1-octreotide.
NO4 [M + H]+ m/z 384.2175, found 384.2159; [R]26 D ) +0.017 (5.08 mg/mL, CH3OH). Loading of 2-Chlorotrityl Chloride Resin with Fmoc-Thr(tBu)-ol. Fmoc-Thr(tBu)-ol was linked to 2-chlorotrityl chloride resin as described (15). 2-Chlorotrityl chloride resin (393 mg, 1.5 mmol/g), Fmoc-Thr(tBu)ol (679 mg, 1.77 mmol), and pyridine (0.286 mL, 3.54 mmol) were stirred for 21 h in a mixed solution of dichloromethane (2.89 mL) and dimethylformamide (DMF, 2.89 mL). After the resin was washed with DMF, methanol (5 mL) was added and the mixture was stirred for 30 min to remove any remaining reactive chloro functionality. The loaded resin was successively washed with DMF and dichloromethane and desiccated to provide a substitution level of 0.287 mmol/g of resin as determined according to the method of Meienhofer (16). Elongation. All amino acids were protected with NRFmoc. Side-chain protecting groups were Cys(Acm), Lys(Boc), Thr(tBu) (where Acm is acetamidomethyl). The peptide chain was constructed manually according to the published cycle consisting of (I) 20 min of deprotection with 20% piperidine-DMF and (II) 2 h of coupling of the Fmoc amino acid derivative (2.5 equiv) with 1,3-diisopropylcarbodiimide (DIPCDI, 2.5 equiv) and 1-hydroxybenzotriazole hydrate (HOBt, 2.5 equiv) in DMF (17). The coupling reaction was repeated when the resin became positive to the Kaiser test (18). The satisfactory incorporation of the respective amino acids was further confirmed by amino acid analysis after acid hydrolysis of the assembled peptide resin. mDTPA Conjugation. After construction of the peptide chain on the resin, the Fmoc protecting group was removed by treating with 20% piperidine-DMF, and a mixture of mDTPA, DIPCDI, and HOBt (2.5 equiv each) in DMF was added and reacted for 2 h, as described above. Cleavage of Peptide from the Resin and Deprotection. Thioanisole (0.5 mL) and TFA (5 mL) were added to fully protected peptide resin [mDTPA-D-PheCys(Acm)-Phe-D-Trp-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr(tBu)-ol-resin, 200 mg] at 0 °C, and the mixture was stirred at room temperature for 2 h. After the mixture had cooled to 0 °C, dry ether was then added to precipitate crude peptide, and the peptide was extracted with 6 M aqueous guanidine hydrochloride (5 mL). After the resin was removed by filtration, the crude product was purified by FPLC. Fractions containing the peptide were collected, and the solvent was removed by lyophilization to afford 51.89 mg [58.8% from Fmoc-Thr(tBu)-ol-resin] of DTPA-D-Phe-Cys(Acm)-Phe-D-Trp-Lys-Thr-Cys(Acm)Thr-ol as a white powder. The purified peptide gave a single peak at a retention time of 11.95 min on HPLC, as shown in Figure 3. Amino acid ratios of the peptide after 6 N HCl hydrolysis: Thr × 1, 0.90; Phe × 2, 2.00; Lys × 1, 1.07; FAB-MAS calcd for C69H100N15O21S2 [M + H]+, m/z 1538.6660, found 1538.6713.
Disulfide Bond Formation. The S-protected peptide (10 mg, 6.5 µmol) was dissolved in 80% aqueous methanol (10 mL). To this solution was added 217.6 µL of 20% iodine in methanol in one portion, and the mixture was stirred at room temperature for 1 h. The excess iodine was reduced with 1 M ascorbic acid in water. This solution was subjected to FPLC and HPLC purifications, and fractions containing the desired product were collected and lyophilized to afford 4.90 mg (54.1%) of DTPAD-Phe1-octreotide as a white powder. This peptide showed a single peak at a retention time of 12.50 min on HPLC, as shown in Figure 4. Amino acid ratios after 6 N HCl hydrolysis: Thr × 1, 0.87; Phe × 2, 2.00; Lys × 1, 1.15; FAB-MAS calcd for C63H88N13O19S2 [M + H]+, m/z 1394.5761, found 1394.5789. Radiolabeling of DTPA-D-Phe1-octreotide with 111 In. 111In radiolabeling of DTPA-D-Phe1-octreotide was performed according to the procedure of Bakker (2) with slight modifications as follows: To 20 µL of DTPA-D-Phe1octreotide (0.05 mg/mL) in 0.1 M acetic acid was added 58 µL of a solution of 111InCl3 (74 MBq/mL) in 0.02 M HCl. The reaction mixture was then incubated at room temperature for 30 min. RESULTS AND DISCUSSION
DTPA-D-Phe1-octreotide was originally synthesized by protecting the N-amine group of the lysine residue of octreotide with a Boc group, followed by condensation of the unprotected NR-amine group with cDTPA (2). However, the synthetic yield of the final product was rather low, which had been caused by the nonselective reaction procedure. Since the reaction of octreotide with (Boc)2O forms a mixture of NR-Boc-, N-Boc-, and NR,N-di-Bococtreotide as well as unreactive octreotide (19), separation of the desired N-protected product from the mixture was required before conjugation reaction with cDTPA. Furthermore, since cDTPA possesses two anhydride moieties, formation of intermolecular cross-linking is unavoidable during the conjugation reaction, as also observed with cDTPA conjugation of chemotactic peptides (3). Recently, Edwards et al. used solid-phase peptide synthesis to prepare DTPA-D-Phe1-octreotide (20). They reacted cDTPA with protected octreotide precursor on resin before aminolysis with threoninol, followed by deprotection of Boc groups of D-Trp4, Lys5, and tBu group of Thr with TFA. The overall synthetic yield of DTPAD-Phe1-octreotide by this protocol was 5%, although this procedure excluded one of the two nonselective reactions (Boc protection reaction) of the original procedure. They concluded that the rather low synthetic yield of the final product, DTPA-D-Phe1-octreotide, is attributed to formations of intermolecular cross-linking during the cDTPA conjugation reaction. These findings strongly suggested that DTPA-D-Phe1octreotide would be synthesized in higher yields if the
Bioconjugate Chem., Vol. 8, No. 3, 1997 445
Technical Notes
Scheme 1. Synthetic Procedure for DTPA-D-Phe1-octreotide Using a Monoreactive DTPA Derivative, mDTPA
DTPA conjugation reaction could be performed without inducing intermolecular cross-linking. Thus, we designed a new synthetic procedure for DTPA-conjugated peptides using a monoreactive derivative of DTPA, mDTPA, as outlined in Scheme 1. Fmoc-Thr(tBu)-ol, prepared according to the method of Rodriguez (14), was loaded on the 2-chlorotrityl chloride resin with a substitution level of 0.29 mmol/g of resin. After the remaining chloride function was inactivated with MeOH, the combination of piperidine treatment and DIPCDI plus HOBt procedure (17) served to elongate the peptide chain manually to prepare D-Phe-Cys(Acm)-Phe-D-Trp-Lys(Boc)-Thr(tBu)Cys(Acm)-Thr(tBu)-ol-resin. mDTPA was then coupled to the NR-amine group of the peptide in the presence of HOBt and DIPCDI, and mDTPA-conjugated peptideresin was treated with TFA-thioanisole to liberate the peptide from the resin. During this treatment, the Boc and tBu protecting groups of the peptide and DTPA were simultaneously removed. After HPLC purification, DTPAD-Phe-Cys(Acm)-Phe-D-Trp-Lys-Thr-Cys(Acm)-Thr-ol was obtained as a white solid with an overall yield of 58.8% based on the starting Fmoc-Thr(tBu)-ol-resin. The product gave a single peak on analytical HPLC (Figure 3). Integrity of the purified product was further determined by amino acid analyses and FAB-MS. Formation of disulfide bonds in the S-protected DTPApeptide was then performed in the presence of iodine. After the reaction was quenched with ascorbic acid, DTPA-D-Phe1-octreotide was obtained with a yield of 54.1% after FPLC and HPLC purifications. The final product showed a single peak on analytical HPLC (Figure 4). Although DTPA-D-Phe1-octreotide had a retention time very close to that of its precursor, DTPA-D-Phe-Cys(Acm)-Phe-D-Trp-Lys-Thr-Cys(Acm)-Thr-ol, on the analytical HPLC (12.50 vs 11.95 min), as shown in Figures 3 and 4, coelution of the two compounds indicated two separated peaks on the same HPLC system (data not shown). A similar phenomenon was observed in the previous study (21). Amino acid analyses and FAB-MS further confirmed the integrity of the final product. The low synthetic yield in this step might have been due to the interference of Trp residue during the formation of the intramolecular disulfide bonds (20). Despite this, the overall synthetic yield of DTPA-D-Phe1-octreotide based on the starting Fmoc-Thr(tBu)-ol-resin was 31.8%, which is much higher than those of previously reported procedures. The integrity of the DTPA chelating group of DTPA-D-Phe1-octreotide was confirmed by the 111In ra-
Figure 5. Radioactivity profiles of 111In-DTPA-D-Phe1-octreotide on analytical HPLC (A), TLC (B), and CAE (C).
diolabeling of DTPA-D-Phe1-octreotide, where 111In-DTPAwas obtained with >95% radiochemical yield at a specific activity of 4.3 MBq/µg when analyzed by HPLC, TLC, and CAE (Figure 5). D-Phe1-octreotide
CONCLUSIONS
The findings of this study indicated that an application of mDTPA to solid-phase peptide synthesis of octreotide provides DTPA-D-Phe1-octreotide with a high overall synthetic yield, due to the absence of nonselective reac-
446 Bioconjugate Chem., Vol. 8, No. 3, 1997
tions during synthesis. Furthermore, the present procedure can be applied to the incorporation of a DTPA chelating moiety into various peptides of interest. Thus, use of mDTPA during solid-phase peptide syntheses is a convenient method for preparing DTPA-conjugated peptides in high yield. ACKNOWLEDGMENT
We thank Nihon Medi-Physics Co. Ltd. for their kind gift of 111InCl3. This work was supported in part by Grants-in-Aid for Developing Scientific Research (07672419 and 08557135) from the Ministry of Education, Science and Culture of Japan. LITERATURE CITED (1) Bakker, W. H., Krenning, E. P., Breeman, W. A., Koper, J. W., Kooij, P. P., Reubi, J.-C., Klijn, J. G., Visser, T. J., Docter, R., and Lamberts, S. W. (1990) Receptor scintigraphy with a radioiodinated somatostatin analogue: radiolabeling, purification, biologic activity, and in vivo application in animals. J. Nucl. Med. 31, 1501-1509. (2) Bakker, W. H., Albert, R., Bruns, C., Breeman, W. A. P., Hofland, L. J., Marbach, P., Pless, J., Pralet, D., Stolz, B., Koper, J. W., Lamberts, S. W. J., Visser, T. J., and Krenning, E. P. (1991) [111In-DTPA-D-Phe1]-octreotide, a potential radiopharmaceutical for imaging of somatostatin receptorpositive tumors: synthesis, radiolabeling and in vitro validation. Life Sci. 49, 1583-1591. (3) Fischman, A. J., Pike, M. C., Kroon, D., Fucello, A. J., Rexinger, D., tenKate, C., Wilkinson, R., Rubin, R. H., and Strauss, H. W. (1991) Imaging focal sites of bacterial infection in rats with indium-111-labeled chemotactic peptide analogs. J. Nucl. Med. 32, 483-491. (4) Fischman, A. J., Babich, J. W., and Strauss, H. W. (1993) A ticket to ride: peptide radiopharmaceuticals. J. Nucl. Med. 34, 2253-2263. (5) Knight, L. C., Radcliffe, R., Maurer, A. H., Rodwell, J. D., and Alvarez, V. L. (1994) Thrombus imaging with technetium99m synthetic peptides based upon the binding domain of a monoclonal antibody to activated platelets. J. Nucl. Med. 35, 282-288. (6) Lister-James, J., Knight, L. C., Maurer, A. H., Bush, L. R., Moyer, B. R., and Dean, R. T. (1996) Thrombus imaging with a technetium-99m-labeled, activated platelet receptor-binding peptide. J. Nucl. Med. 37, 775-781. (7) Virgolini, I., Kurtaran, A., Raderer, M., Leimer, M., Angelberger, P., Havlik, E., Li, S., Scheithauer, W., Niederle, B., Valent, P., and Eichler, H. G. (1995) Vasoactive intestinal peptide receptor scintigraphy. J. Nucl. Med. 36, 1732-1739. (8) Virgolini, I., Raderer, M., Kurtaran, A., Angelberger, P., Banyai, S., Yang, Q., Li, S., Banyai, M., Pidlich, J., Niederle, B., Scheithauer, W., and Valent, P. (1994) Vasoactive intestinal peptide-receptor imaging for the localization of intestinal adenocarcinomas and endocrine tumors. N. Engl. J. Med. 331, 1116-1121. (9) Krenning, E. P., Bakker, W. H., Kooij, P. P. M., Breeman, W. A. P., Oei, H. Y., de Jong, M., Reubi, J. C., Visser, T. J., Bruns, C., Kwekkeboom, D. J., Reijs, A. E. M., van Hagen, P. M., Koper, J. W., and Lamberts, S. W. J. (1992) Somatostatin receptor scintigraphy with indium-111-DTPA-D-Phe1-octreotide in man: metabolism, dosimetry and comparison with iodine-123-Tyr-3-octreotide. J. Nucl. Med. 33, 652-658.
Arano et al. (10) Krenning, E. P., Kwekkeboom, D. J., Bakker, W. H., Breeman, W. A. P., Kooij, P. P. M., Oei, H. Y., Hagen, M. v., Postema, P. T. E., de Jong, M., Reubi, J. C., Visser, T. J., Reijs, A. E. M., Hofland, L. J., Koper, J. W., and Lamberts, S. W. J. (1993). Somatostatin receptor scintigraphy with [111In-DTPA-D-Phe1]- and [123I-Tyr3]-octreotide: the Rotterdam experience with more than 1000 patients. Eur. J. Nucl. Med. 20, 716-731. (11) Marbach, P., Andres, H. A., Azria, M., Bauer, W., Briner, U., Buchheit, K.-H., Doepfner, W., Lemaire, M., Petcher, T. J., Pless, J., and Reubi, J.-C. (1987) Chemical structure, pharmacodynamic profile and pharmacokinetics of SMS 201995 (Sandstatin®). Sandostatin® in the Treatment of Acromegaly (S. W. J. Lamberts, Ed.) pp 53-60, Springer-Verlag, Berlin. (12) Maisano, F., Gozzini, L., and de Hae¨n, C. (1992) Coupling of DTPA to proteins: A critical analysis of the cyclic dianhydride method in the case of insulin modification. Bioconjugate Chem. 3, 212-217. (13) Arano, Y., Uezono, T., Akizawa, H., Ono, M., Wakisaka, K., Nakayama, M., Sakahara, H., Konishi, J., and Yokoyama, A. (1996) Reassessment of diethylenetriaminepentaacetic acid (DTPA) as a chelating agent for indium-111 labeling of polypeptides using a newly synthesized monoreactive DTPA derivative. J. Med. Chem. 39, 3451-3460. (14) Rodriguez, M., Llinares, M., Doulut, S., Heitz, A., and Martinez, J. (1991) A facile synthesis of chiral N-protected β-amino alcohols. Tetrahedron Lett. 32, 923-926. (15) Wenschuh, H., Beyermann, M., Haber, H., Seydel, J. K., Krause, E., and Bienert, M. (1995) Stepwise automated solid phase synthesis of naturally occurring peptaibols using Fmoc amino acid fluorides. J. Org. Chem. 60, 405-410. (16) Meienhofer, J., Waki, M., Heimer, E. P., Lambros, T. J., Makofske, R. C., and Chang, C.-D. (1979) Solid phase synthesis without repetitive acidolysis. Preparation of leucylalanyl-glycyl-valine using 9-fluorenylmethyloxycarbonylamino acids. Int. J. Pept. Protein Res. 13, 35-42. (17) Akaji, K., Fujii, N., Tokunaga, F., Miyata, T., Iwanaga, S., and Yajima, H. (1989) Studies on peptides. CLXVIII. Syntheses of three peptides isolated from horseshoe crab hemocytes, Tachyplesin I, Tachyplesin II, and Polyphemusin I. Chem. Pharm. Bull. 37, 2661-2664. (18) Kaiser, E., Colescott, R. L., Bossinger, C. D., and Cook, P. I. (1970) Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal. Biochem. 34, 595-598. (19) Smith-Jones, P. M., Stolz, B., Bruns, C., Albert, R., Reist, H. W., Fridrich, R., and Ma¨cke, H. R. (1994) Gallium-67/ gallium-68-[DFO]-octreotidesa potential radiopharmaceutical for PET imaging of somatostatin receptor-positive tumors: Synthesis and radiolabeling in vitro and preliminary in vivo studies. J. Nucl. Med. 35, 317-325. (20) Edwards, W. B., Fields, C. G., Anderson, C. J., Pajeau, T. S., Welch, M. J., and Fields, G. B. (1994) Generally applicable, convenient solid-phase synthesis and receptor affinities of octreotide analogs. J. Med. Chem. 37, 3749-3757. (21) Akaji, K., Fujino, K., Tatsumi, T., and Kiso, Y. (1993) Total synthesis of human insulin by regioselective disulfide formation using the silyl chloride-sulfoxide method. J. Am. Chem. Soc. 115, 11384-11392.
BC970023B
