Bioconjugate Chem. 1998, 9, 765−772
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Protein and Peptide Labeling with (Cyclopentadienyl)tricarbonyl Rhenium and Technetium Todd W. Spradau and John A. Katzenellenbogen* Department of Chemistry, University of Illinois, 461 Roger Adams Laboratory, Box 37-5, 600 South Mathews Avenue, Urbana, Illinois 61801. Received April 13, 1998; Revised Manuscript Received July 24, 1998
A method for labeling proteins and peptides with (methoxycarbonyl cyclopentadienyl)tricarbonyl rhenium and technetium is described. The precursors used for this labeling are conveniently produced from perrhenate and pertechnetate, respectively, using a double ligand transfer reaction. For labeling the lysine residues of the model protein bovine serum albumin, the technetium methyl ester was saponified and then transformed into its N-hydroxysuccinimidyl ester. For the labeling of the model peptides leucine enkephalin, substance P, oxytocin, and the tumor imaging/therapy candidate octreotide, the rhenium methyl ester was saponified and activated using either 1-hydoxybenzotriazole or 1-hydroxy-7-azabenzotriazole. The activation and peptide-coupling reactions were followed using reversed-phase (C18) HPLC and yields averaged approximately 70%.
INTRODUCTION
Scheme 1. Double Ligand Transfer (DLT) Reaction
We recently reported a novel double ligand transfer (DLT) reaction that permits the rapid synthesis of substituted (cyclopentadienyl)tricarbonylrhenium (CpTR) complex 1 from KReO4 (Scheme 1) (1). The chemical stability and the ease of Cp-ring derivatization of these compounds make them attractive candidates for the preparation of technetium and rhenium-based radiopharmaceuticals, agents that are widely used in diagnostic imaging (99mTc) and radiotherapy (186Re, 188Re) (25). Complexes derived from 1 have not as yet been exploited for such applications, however, because they are difficult to prepare from ReO4-, the only available chemical form of 186Re and 188Re (6-8). 99mTc is also available only as 99mTcO4-, and as a result, most rhenium and technetium radiopharmaceuticals are inorganic complexes with the metals in a +5 oxidation state. If the scope of the DLT reaction could be extended to include the synthesis of the technetium analogues of the rhenium complex 1 [i.e., (cyclopentadienyl)tricarbonyl technetium (CpTT) complexes], a variety of new organometallic radiopharmaceuticals could be developed. One simple method which has been widely used for the creation of new radiopharmaceuticals involves the attachment of the radioisotope to a protein (antibody) or peptide that is known to localize in a desired region of the body (9, 10). The resulting adducts can then be used for imaging and/or therapy of that region if their biodistribution characteristics remain favorable. The goal of this study was to apply the DLT reaction to prepare precursors suitable for attaching CpTR and CpTT complexes to proteins and peptides. The many procedures that have been developed for incorporating technetium and rhenium into proteins and peptides can be grouped into three categories: (a) direct labeling, (b) indirect labeling, and (c) the preformed chelate approach. Each method has been used successfully in a variety of applications, but each has drawbacks and limitations (11). * Author to whom correspondence should be addressed. Phone: (217) 333-6310. Fax: (217) 333-7325. E-mail: jkatzene@ uiuc.edu.
In the direct-labeling approach, which is the simplest of the three, the sulfhydryl groups of the protein itself are exploited as chelation sites for the metal, and labeling is accomplished simply by incubation of the protein with ReO4- or TcO4- and a reducing agent (usually stannous ion) or by transchelation of the prereduced metal onto the protein from a donor ligand (12). The stability and incorporation level of the label can sometimes be enhanced by increasing the number of free thiols of the native protein by reducing disulfide bonds or by adding exogenous sulfhydryl groups by prior derivatization of the protein (13). Such procedures, however, can alter the structure and, consequently, the biological properties of the protein or peptide (14). Furthermore, the directlabeling approach is often plagued by ambiguities regarding the sites of attachment and oxidation states of the bound metals, as well as by instability of the label (13-17). Indirect labeling involves the attachment of strong chelators to the protein or peptide prior to direct labeling. A wide variety of tightly binding chelates have also been attached to proteins and peptides and labeled successfully [e.g., hydrazino nicotinamide (HYNIC) (18, 19), diaminedithiol (DADT) (20), bis(aminoethanethiol) (BAT) (21), and short peptide sequences (22-24)]. However, even though these strategies often result in increased complexation yields and label stability, the shortcomings of the direct-labeling strategies (site of attachment and oxidation state ambiguity, label instability) still remain. The preformed chelate approach seeks to eliminate the drawbacks associated with direct and indirect labeling. By this approach, the radiometal is first securely bound inside a chelate system, and then, in a subsequent step, the derivatized metal is firmly attached to the protein or peptide. The resulting product possesses a label that
10.1021/bc980043t CCC: $15.00 © 1998 American Chemical Society Published on Web 09/03/1998
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Figure 1. Reagents used to couple CpTR to proteins. Scheme 2. General Strategy for Protein or Peptide Labeling with the Cyclopentadienyl Tricarbonyl Technetium (CpTT) Unit
Figure 2. Incorporation of [99gTc]CpTT into bovine serum albumin (BSA). Sephadex G-25 size exclusion chromatographic analysis of the labeling process. [99gTc]CpTT-labeled BSA elutes in the void volume (fraction 1). Unreacted active ester 8 or hydrolysis product 3 elutes in fractions 4-8. Further details are given in the Experimental Section. Table 1. Extension of the Double Ligand Transfer (DLT) Reaction from Rhenium to [99gTc]Technetium
is chemically well-defined and stable, but this approach comes at the cost of additional synthetic steps. Some “bifunctional” chelates that have been successfully employed in this strategy include tetradentate N2S2 (25, 26) and N3S (27, 28) system, and boronic acid adducts of technetium dioximes (BATOs) (29). The labeling strategy we have pursued is outlined in Scheme 2. The key step is the formation of methyl ester 2 from pertechnetate via the double ligand transfer (DLT) reaction. The subsequent steps involve simple modifications of the Cp ring side chain to make it suitable for coupling to the amino groups of proteins and peptides. Thus, this method can be classified as a preformed chelate approach, with active ester 4 being an organometallic analogue of the inorganic bifunctional chelates mentioned above. Most proteins and peptides labeled with technetium or rhenium contain inorganic derivatives of the metals, but a few examples of organometallic variations exist. Alberto and co-workers reported the conjugation of the Re(CO)3 fragment to proteins in a direct-labeling approach using [Re(OH2)3(CO)3]+ as the donor molecule (30), and, in an investigation similar to our own, Jaouen and co-workers described the coupling of CpTR to proteins using a preformed chelate approach with the active esters 5 and 6 (Figure 1) (31). In a subsequent study, pyrilium salt 7 was used as well (32). Our study expands upon this earlier work. RESULTS
Protein Labeling. The first step in the execution of our approach was to demonstrate that the DLT reaction could be extended from perrhenate to pertechnetate. Table 1 shows that the transformation did in fact work just as well with technetium as it did with rhenium. Notably, the desired methyl ester 2 could be prepared in very high yield. As shown in Scheme 3, the methyl ester of 2 could be rapidly and quantitatively saponified in a mixture of 1:1 NaOH (2M):dioxane to provide carboxylic acid 3. When cosolvents other than dioxane (e.g., THF, acetonitrile, and methanol) were tried, longer times (15-120 min) were
R
R′
% yield
COCH3 COCH3 CO2CH3
H COCH3 CO2CH3
73 81 89
Scheme 3. Demonstration of Protein Labeling with [99gTc]CpTT, Using Bovine Serum Albumin
required for the reaction to go to completion. The acid was then treated with a uronium salt described by Knorr and co-workers to provide the N-hydoxysuccinimidyl (NHS) ester 8 (33). We chose N-hydroxysuccinimide as the activating group for our protein coupling experiments because of the stability of the resultant ester in aqueous solution and its high reactivity toward the -amino groups of lysine residues (34). Coupling experiments were performed with the model protein bovine serum albumin (BSA), which contains 59 lysine residues and one N-terminal amino group for a total of 60 possible covalent attachment sites. The reactions were found to be rapid (complete in less than 5 min), with yields that varied depending on the relative amount of BSA to active ester 8 that was used. Figure 2 is a graphical representation of the elution profiles obtained upon size-exclusion column chromatographic separation (Sephadex G-25) of the products from three reactions with varying ratios of BSA to 8. The void volume of the column, which contained the labeled and unlabeled proteins, comprised the first 20-25 drops
Protein and Peptide Labeling
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Scheme 4. Labeling of Leucine Enkephalin with CpTT Using an NHS Ester
Figure 3. HPLC traces of the reaction of carboxylic acid 10 with HOBt (panel A) and HABt (panel B). The structures of compounds 10, 11a, 11b, and 12 are given in Scheme 5. The vertical arrow indicates the time of injection. HPLC conditions are given in the Experimental Section. Scheme 5. Peptide Labeling with CpTT Using Esters Activated by HOBt or HABt
Figure 4. Structures of HABt and HOBt.
collected from the 5 cm column, while the small molecules included within the pores of the gel [i.e., unreacted 8 and 3 (from ester hydrolysis)], began to elute at approximately 40-45 drops. As the amount of BSA was increased from 0.01 to 1.0 equiv of active ester (8), the incorporation yields increased from 5% to greater than 70%. Since radiolabeling experiments using 99mTc, 186Re, and 188Re are typically done in the presence of a large excess of protein, the potential for the tracer-level labeling of proteins by this approach appears to be great. Peptide Labeling. The strategy outlined in Scheme 2 was also adopted for the labeling of selected peptides, with two changes from the protein-labeling protocol. First, rhenium was used instead of technetium, and second, activating groups other than NHS were employed. Scheme 4 shows that the conjugation of the CpTR NHS ester 5 to the model peptide leucine enkephalin was much slower than the corresponding reaction of the corresponding CpTT NHS ester 8 with BSA, most likely because of a relative increase in steric hindrance near the N-terminal amino group of the peptide compared to that of the numerous and more accessible -amino groups of the lysine residues of the protein. Reaction sequences with short-lived radioisotopes need to be as rapid as possible to maximize the specific activities of the final products. For this reason, we investigated alternative activating agents, bases, and solvents for the coupling reactions of rhenium carboxylate 10 (Scheme 5) with the model peptides leucine enkephalin (Tyr-Gly-Gly-Phe-Leu), substance P (Arg-Pro-Lys-Pro-
Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2), and oxytocin (CysTyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2, disulfide bridge: 1-6). Leucine enkephalin was included because of its simple structure and affordability, substance P because it possesses a lysine residue and thus two potential sites for labeling, and oxytocin because of its intramolecular disulfide bond. The disulfide bond was important to us because the fourth peptide we chose for this study, octreotide, also contains a cystine bridge and is, moreover, an attractive candidate for radiolabeling with Tc-99m (35, 36). Leucine Enkephalin, Oxytocin, and Substance P. We found that the related reagents 1-hydoxybenzotriazole (HOBt) and 1-hydroxy-7-azabenzotriazole (HABt) could both be used to provide intermediates that were capable of coupling with the model peptides (Scheme 5). The activation/coupling sequences were complete in less than 20 min for each of the peptides studied, and both steps could be followed using reversed-phase (C18) HPLC. Interestingly, the reaction of acid 10 with HOBt in the presence of DCC produced three distinct intermediates that were visible by HPLC analysis, each of which disappeared upon addition of peptide, whereas DCC/ HABt produced two intermediates (Figure 3). The product that was produced by both reagents, which eluted at 12.6 min under these conditions (Figure 3), was tentatively assigned structure 12, a reactive anhydride dimer of the starting carboxylic acid 10. The compound that eluted at 10 min in the DCC/HABt reaction was most likely the HABt adduct 11b (panel A), while that at 10.7 min in the DCC/HOBt reaction is probably its slightly more hydrophobic analogue 11a (panel B). The polar compound at 7.2 min may be a condensation product of acid 10 with DCC (or its urea), and its absence from the HABt reaction can be explained by the higher reactivity (nucleophilicity) of HABt vs HOBt toward this compound, due to its zwitterionic tautomer (Figure 4). The activation reactions were complete within 1 min in CH2Cl2, and the active intermediates produced were stable for at least 24 h in that solvent. When the same reactions were performed in DMF, no intermediates could be detected by HPLC, and starting acid 10 was the only peak observed (8.4 min; see a trace of 10 in Figure 3, panel B). Representative traces of the reactions of the active intermediates (Figure 3) with each of the three model peptides are shown in Figure 5. The peptides themselves eluted at approximately 5.9 min in the solvent gradient
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Figure 6. Structures of unprotected and Boc protected octreotide (14a and 14b) and their CpTR and CpTT labeled derivatives 15a, b and 16a, b.
Figure 5. HPLC traces of the reactions of CpTR with leucine enkephalin (panel A), oxytocin (panel B), and substance P (panel C). The structures of compounds 10 and 13a-c are shown in Scheme 5. The vertical arrow indicates the time of injection. HPLC conditions are given in the Experimental Section.
used, and varying amounts of the unreacted peptides can be seen in each trace. Yields averaged 70% for each reaction, based upon integration of the peaks corresponding to the coupled products 13a, 13b, and 13c (Scheme 5), which appeared at 9.0 and 7.3 min in the leucine enkephalin and oxytocin reactions, respectively (Figure 5, panels A and B), and at 7.8 and 9.4 min for the two conjugates obtained in the reaction with substance P (Figure 5, panel C). The CpTR derivative of leucine enkephalin (13a) was isolated and characterized by 1H NMR and FAB mass spectrometry. A peak at 8.4 min corresponding to starting acid 10 was a prominent feature in all of the HPLC traces (Figure 5) and indicates that there is a competition between amide bond formation with the peptide and hydrolysis of the activated intermediates in the coupling reactions. Although the intermediates were stable in pure CH2Cl2, their hydrolysis was found to be catalyzed by the base used in the reaction, with the rate of hydrolysis increasing in the order diisopropylethylamine < triethylamine , (dimethylamino)pyridine (DMAP). The yields of the labeled peptides produced in the presence of those bases decreased in the same order, and diisopropylethylamine, the most sterically hindered base that engendered the highest yields, was thereafter used in all of the reactions. Solvents other than methylene chloride, such as acetonitrile and DMF, were unsuitable for the preparation of the active esters; we found that if the active esters were first prepared in CH2Cl2, then DMF could subsequently be added to form a mixed solvent system in which the active intermediates were moderately stable. The addition of DMF was important for the coupling reactions with all of the peptides except leucine enkephalin, because while the latter reacted rapidly in CH2Cl2, the others were insoluble, and therefore unreactive, in that solvent. Octreotide Labeling. Octreotide (13a, Figure 6) interested us because of its known affinity for a wide range of somatostatin receptor-containing neuroendocrine tumors, including those of the lung, pancreas, pituitary, breast, and prostate (37). If labeled with
Figure 7. HPLC traces of reaction for the formation of Bocprotected CpTR-labeled octreotide 15a (panel A) and from CpTR-labeled octreotide 15b (panel B). The structures of compounds 10, 11b, 15a, and 15b are shown in Scheme 5 and Figure 6. The vertical arrow indicates the time of injection. HPLC conditions are given in the Experimental Section.
Tc-99m or Re-186/Re-188, it could potentially be used to detect and/or treat cancer in those tissues. Furthermore, it is an exceptionally challenging target for Tc/Re labeling, since its essential disulfide linkage is sensitive to the reducing conditions generally employed in labeling with those metals (38). Like oxytocin and substance P, octreotide was insoluble in CH2Cl2, and DMF was therefore required to promote its conjugation reactions. These reactions were typically performed by adding a methylene chloride solution of the freshly prepared active ester 11b to a vial containing solid, Boc-protected octreotide (14b). After removal of the CH2Cl2 under a nitrogen stream, DMF and diisopropylethylamine were added to initiate the coupling. Again, all attempts to do this sequence from start to finish in DMF were unsuccessful. Figure 7 shows representative reversed-phase (C18) HPLC traces for the formation of the CpTR-N-terminally labeled Boc-octreotide 15a (panel A) and its deprotection to CpTR-octreotide 15b (panel B). Starting acid 10 and its active ester 11b were visible at 4.6 and 8.1 min, respectively, in this gradient (panel A), while the Boc-octreotide (14b), which eluted at 6.2 min, had already been completely consumed. The coupled product (15a, 14.7 min) was isolated and characterized by its MALDI and FAB mass spectra, and its yield typically ranged 7080%. The final, deprotected product (15b, 6.1 min) (panel B) was generated in 95% trifluoroacetic acid, and it also was isolated and identified by its MALDI, FAB (Figure 8), and high-resolution FAB mass spectra. The same reaction sequence was finally repeated with technetium-99g-labeled acid 3 to provide the corresponding Boc-protected (16a) and deprotected (16b) [99Tc]CpTT-octreotide conjugates, which also were characterized by their FAB mass spectra. To our knowledge, these syntheses represent the first successful preparations of Re and Tc-99g-labeled octreotide, although Tc-99mlabeled octreotide derivatives have been reported (38-
Protein and Peptide Labeling
Figure 8. FAB mass spectrum of the CpTR-octreotide conjugate 15b. The peaks at m/z 1401.8 and 1403.7 correspond to the (M + Na)+ ion with 185Re and 187Re, respectfully.
40). The in vivo properties of those compounds were not ideal, however. The results of our own Tc-99m labeling of octreotide using the chemistry described in this report and the tissue distribution of this radiopharmaceutical will be reported elsewhere (41). CONCLUSION
A double ligand transfer reaction that was previously developed for the rapid synthesis of substituted (cyclopentadienyl)tricarbonylrhenium complexes from KReO4 was found to be effective for the preparation of the correponding technetium compounds from NH499gTcO4 as well. One of the products of this reaction, namely the methyl ester substituted derivative 2, was found to be a useful precursor for the 99gTc labeling of protein lysine residues. The protocol included transformation of methyl ester 2 into NHS ester 8, and then treatment with the model protein bovine serum albumin in pH 9 buffer. These reactions were complete in less than 5 min, and incorporation yields varied depending upon the relative amounts of BSA to active ester used, but exceeded 70% in the reactions performed with 1 equiv of each coupling partner. In like manner, the model peptides leucine enkephalin, substance P, and oxytocin were labeled using the HOBt and/or HABt active ester derivatives of rhenium acid 10. The activation and peptide-coupling reactions were followed using reversed-phase (C18) HPLC, and it was found that although DMF was required as a solvent in the couplings of two of the three peptides, it was an unsuitable medium for the activation step, which worked best in CH2Cl2. Finally, the octapeptide octreotide, which is an attractive candidate for radiolabeling with 99mTc, 186Re, and/or 188 Re, was labeled for the first time with nonradioactive rhenium and Tc-99g, using the reaction protocol optimized for the model peptides. It is hoped that the chemistry described in this paper will prove useful for the preparation of a variety of 99mTc, 186Re, and 188Relabeled proteins and peptides, including octreotide. EXPERIMENTAL SECTION
General Comments. The following reagents were purchased from the commercial sources indicated and used as received: acetylferrocene, 1,1′-diacetylferrocene, O-(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate, 1-hydoxybenzotriazole, 1-hydroxy-7-azabenzotriazole, dicyclohexylcarbodiimide, pH 9 buffer (Hydrion), from Aldrich; chromium hexacarbonyl from Alfa; chromium(III) chloride from Strem; ammonium pertechnetate from Alan Davison, MIT; and bovine serum albumin, leucine enkephalin, substance P, oxytocin, and
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Sephadex G-25 superfine gel from Sigma. Diisopropylethylamine and dimethylformamide were purchased from Aldrich and distilled from CaH2 and MgSO4, respectively. Boc-protected octreotide was provided by M. J. Welch, Washington University. 1,1′-Bis(carbomethoxy)ferrocene was prepared as described elsewhere (1). General Procedure for Double Ligand Transfer Reactions. Cautionary Note. Pressure tubes were placed within a specially made solid aluminum block containing holes drilled deep enough to admit the tubes to about three-fourths of their height and wide enough to allow room for the addition of some mineral oil to ensure good thermal contact. The tubes and aluminum base were covered with a matching hollow aluminum screw cap, equipped with a small hole aligned with one drilled in the base (to the same depth as those for the pressure tubes) to hold a metal thermometer. This containment device minimized the potential danger of explosions during heating, allowed for the efficient stirring of the reactions, and enabled the reaction temperature to be monitored readily. All manipulation of sealed reaction tubes during and after the reactions were done with suitable protective equipment: blast shield, full-face visor, heavy gloves. Details of the procedure are described elsewhere (1). Briefly, NH499gTcO4 (0.0106 g, 0.0586 mmol), 1,1′-bis(methoxycarbonyl)ferrocene (0.1034 g, 0.342 mmol), Cr(CO)6 (0.0923 g, 0.419 mmol), and CrCl3 (0.0249 g, 0.157 mmol) were combined in a 4 mL pressure tube containing a magnetic stir bar. Dry methanol (1 mL) was added, and the tube was sealed, heated to 165 °C over the course of an hour, and then cooled to -78 °C. The reaction mixture was transferred to a 15 mL disposable vial with CH2Cl2 and the solvent was evaporated under a stream of N2 at 30 °C. The mixture was redissolved in a minimum of CH2Cl2 and purified by flash column chromatography (1:1 hexane: benzene) to give 0.0159 g (89%) of tricarbonyl(methoxycarbonylcyclopentadienyl)technetium (2). 1H NMR (200 MHz, CDCl3) δ ppm: 5.96 [t, J ) 2.38 Hz, 2H, -COCp-H2(R)], 5.27 [t, J ) 2.16 Hz, 2H, -CO-Cp-H2(β)], 3.79 (s, 3H, CpCOOCH3). MS (EI): m/z 306 (M+). Anal. Calcd for C10H7O5Tc: C, 39.23; H, 2.30. Found: C, 39.35; H, 2.27. Tricarbonyl(acetylcyclopentadienyl)technetium. The reactions were performed according to the general procedure and purified by flash column chromatography (4:1 hexane:ethyl acetate) to give 0.040 g (81%) of tricarbonyl(acetylcyclopentadienyl)technetium from diacetylferrocene, and 0.033 g (73%) from acetylferrocene. 1 H NMR (500 MHz, CDCl3) δ ppm: 5.94 [t, J ) 2.31 Hz, 2H, -CO-Cp-H2(R)], 5.31 [t, J ) 2.30 Hz, 2H, -COCp-H2(β)], 2.33 (s, 3H, CpCOCH3). 13C NMR (125 MHz, CDCl3) δ ppm: 193.36 (COCH3), 90.50, 87.12 (C5H4), 90.31 (C ipso), 29.68 (Tc(CO)3), 26.53 (COCH3). MS (EI): m/z 290 (M+). General Procedure for the Labeling of Bovine Serum Albumin. To a stirred solution of tricarbonyl(methoxycarbonylcyclopentadienyl)technetium (2) (1.4 mg, 0.0046 mmol) in dioxane (0.5 mL) was added an aqueous solution of NaOH (2 M, 0.5 mL). After 5 min, concentrated HCl was added dropwise until the reaction was acidic to litmus paper. The solvent was evaporated at 40 °C under a gentle stream of nitrogen, and the solid residue was redissolved in DMF (400 µL). Triethylamine (2 µL, 0.014 mmol) and O-(N-succinimidyl)-N,N,N′,N′tetramethyluronium tetrafluoroborate (5 mg, 0.017 mmol) were added, and the solution was stirred for 5 min. The reaction mixture, containing NHS active ester 8, was then added to a solution of bovine serum albumin (38
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mg, 0.000 55 mmol) in pH 9 buffer (6 mL), and the reaction vial was further rinsed with DMF (200 µL) and pH 9 buffer (1.4 mL) to provide a final solution of 7.5% DMF in the pH 9 buffer. After stirring for 10 min, 10 µL of the reaction mixture was purified by passing it through 5 cm of Sephadex G-25 Superfine gel in a disposable Pasteur pipet and eluting with pH 9 buffer. Sixty drops of eluent was collected in 10-drop fractions, with the first two fractions (20 drops) roughly corresponding to the column void volume as determined by the elution profile of Blue Dextran. The amount of radioactivity in each fraction was determined by scintillation counting of the fractions in solutions of Aquasol (12 mL) scintillation fluid. An incorporation yield of at least 50% was determined by comparing the total activity in all fractions to the activity in the first three fractions. Reactions performed according to the above procedure showed an incorporation yield of 5% from 1.34 mg (0.004 38 mmol) tricarbonyl(methoxycarbonylcyclopentadienyl)technetium and 3.81 mg (0.0552 µmol) BSA and at least 70% from 1.16 mg (0.0038 mmol) tricarbonyl(methoxycarbonyl cyclopentadienyl)technetium and 309 mg (0.00447 mmol) BSA. A graphical representation of the elution profiles is shown in Figure 2. Analytical Methods. Reversed-phase (C18) HPLC analyses were performed using a Vydac C18 column (4.6 mm × 25 cm) at a flow rate of 1.5 mL/min. Gradients were performed with mobile phases A (0.1% TFA in H2O) and B [0.1% TFA in acetonitrile/0.1% TFA in H2O (10:1)] under conditions described in the individual reactions below. UV detection was performed at either 254 or 280 nm. General Peptide Labeling Procedure. To a stirred solution of tricarbonyl(methoxycarbonyl cyclopentadienyl)rhenium (0.25 mg, 0.06 µmol) in dioxane (0.5 mL) was added an aqueous solution of NaOH (2 M, 0.5 mL). After 5 min, concentrated HCl was added dropwise until the reaction was acidic to litmus paper. The solvent was evaporated at 40 °C under a gentle stream of nitrogen, and the solid residue was redissolved in CH2Cl2 (300 µL). 1,3-Dicyclohexylcarbodiimide (0.5 mg, 2.4 µmol) and 1-hydroxybenzotriazole (0.9 mg, 6.7 µmol) were added, and the solution was stirred for 5 min. Reversed-phase (C18) HPLC analyses were performed using the following gradient: 20% B for 1 min, 20% B to 30% B in 1 min, 30% B for 1 min, 30% B to 50% B in 2 min, and 50% B to 60% B in 10 min, which showed that the activation step to form the HOBT ester 11a was complete [see Figure 3 (panel A)]. Leucine enkephalin (0.7 mg, 1.1 µmol) and diisopropylethylamine (1 µL) were added, and the reaction was stirred for 20 min, at which time RP-HPLC indicated the formation of CpTR/leucine enkephalin coupled product (rt ) 9.0 min) in a yield of 70% [Figure 5 (panel A)]. The peak at 9.0 min, corresponding to the CPTR-labeled leucine enkephalin 13a, was collected and concentrated in vacuo. Comparison of the 1H NMR (300 MHz, CDCl3) of the isolated product with leucine enkephalin revealed the following new peaks (δ ppm): 6.26 and 6.11 [d, J ) 1.58 Hz, 2H, -CO-Cp-H2(R)], 5.54 and 5.51 [d, J ) 1.95 Hz, 2H, -CO-Cp-H2(β)]. MS (FAB): m/z 940 (M + Na)+, 962 (M + 2Na)+. CpTR Labeling of Oxytocin. The reaction was performed according to the general procedure, with two changes: (a) 1-hydroxy-7-azabenzotriazole (1 mg, 7.4 µmol) was used instead of 1-hydroxybenzotriazole (HOBt) (see Figure 3, Panel B for an HPLC trace of the HABT active ester 11b), and (b) DMF (50 µL) was added to the activated ester at the same time as the diisopropylethylamine (1 µL) and oxytoxcin (0.5 mL, 0.47 µmol). The
Spradau and Katzenellenbogen
reaction was analyzed by RP-HPLC with the gradient of the general procedure, which revealed a new peak attributed to N-terminally CpTR-labeled oxytocin 13b [70%, Figure 5 (panel B)] from tricarbonyl(methoxycarbonylcyclopentadienyl)rhenium (0.25 mg, 0.66 µmol). CpTR Labeling of Substance P. The reaction was performed according to the general procedure, with one change: DMF (50 µL) was added to the activated ester at the same time as the diisopropylethylamine (1 µL) and substance P (0.6 mg, 0.4 µmol). The reaction was analyzed by RP-HPLC using the gradient of the general procedure, which revealed two new peaks of equal intensity attributed to N-terminally and lysine CpTRlabeled substance P 13c [75%, Figure 5 (panel C)] from tricarbonyl(methoxycarbonylcyclopentadienyl)rhenium (0.25 mg, 0.66 µmol). CpTR Labeling of Boc-Octreotide. The reaction was performed according to the general procedure, with the following changes: (a) 1-hydroxy-7-azabenzotriazole (1 mg, 7.4 µmol) was used instead of 1-hydoxybenzotriazole (HOBt), and (b) a methylene chloride solution of the newly formed active ester 11b was added to a vial containing solid Boc-octreotide 14b (150 µg, 0.1 µmol), and the solvent was evaporated at 40 °C under a gentle stream of nitrogen. DMF (100 µL) and diisopropylethylamine (1 µL) were added, and the reaction was stirred for 20 min. The reaction was analyzed by RP-HPLC using a gradient of 50% B to 70% B over 20 min. A new peak attributed to N-terminally CpTR-labeled Boc-octreotide 15a [80%, Figure 7 (panel A)] was obtained from the original tricarbonyl(methoxycarbonylcyclopentadienyl)rhenium (0.125 mg, 0.33 µmol). The peak was collected and concentrated in vacuo. MS (MALDI): m/z 1503 (M + Na)+. Deprotection of CpTR-Boc-Octreotide. CpTRBoc-octreotide 15a (e150 µg, 0.1 µmol) was dissolved in a 95% solution of trifluoroacetic acid in H2O (100 µL) and stirred for 2 min. The reaction was then quenched with water (100 µL) and analyzed by RP-HPLC, again using a gradient of 50% B to 70% B in 20 min. A new peak attributed to N-terminally CpTR-labeled octreotide 15b [90%, Figure 7 (panel B)] was collected and concentrated in vacuo. HR-FAB-MS m/z calcd for C58H69N10O14S2NaRe: 1403.389151. Found: 1403.391400 [∆ -1.6 ppm, (M + Na)+]. CpTT Labeling of Boc-Octreotide. The reaction was performed according to the general procedure, with the following changes: (a) tricarbonyl(methoxycarbonylcyclopentadienyl)technetium (15 µg, 0.05 µmol) was used instead of tricarbonyl(methoxycarbonylcyclopentadienyl)rhenium as the starting material; (b) 1-hydroxy-7-azabenzotriazole (0.1 mg, 0.7 µmol) was used instead of 1-hydoxybenzotriazole (HOBt); and (c) a methylene chloride solution of the newly formed active ester 11b was added to a vial containing solid Boc-octreotide 14b (50 µg, 0.03 µmol), and the solvent was evaporated at 40 °C under a gentle stream of nitrogen. DMF (100 µL) and diisopropylethylamine (1 µL) were added, and the reaction was stirred for 30 min. The reaction was analyzed by RP-HPLC using a gradient of 45% B to 65% B in 20 min. A new peak attributed to N-terminally CpTT-labeled Boc-octreotide 16a (80%, 14.6 min) was collected and concentrated in vacuo. MS (FAB): m/z 1414 (M + Na)+, 1431 (M + K)+. Deprotection of CpTT-Boc-Octreotide. CpTTBoc-octreotide 16a (e50 µg, 0.03 µmol) was dissolved in a 95% solution of trifluoroacetic acid in H2O (100 µL) and stirred for 2 min. The reaction was then quenched with water (100 µL) and analyzed by RP-HPLC, again using
Protein and Peptide Labeling
a gradient of 45% B to 65% B in 20 min. A new peak attributed to N-terminally CpTT-labeled octreotide 16b (90%, 10.0 min) was collected and concentrated in vacuo. MS (MALDI): m/z 1294 (M + H)+. ACKNOWLEDGMENT
We are grateful for the support of this research through grants from the National Institutes of Health (PHS 5R01 CA25836) and the Department of Energy (DE FG02 86ER60401). We are grateful to A. Davison and M. J. Welch for providing us with research materials and to C. J. Anderson and M. Lanahan for providing Bococtreotide. LITERATURE CITED (1) Spradau, T. W., and Katzenellenbogen, J. A. (1998) Preparation of cyclopentadienyltricarbonylrhenium complexes using a double ligand transfer reaction. Organometallics (in press). (2) Schwochau, K. (1994) Technetium Radiopharmaceuticals- Fundamentals, Synthesis, Structure, and Development. Angew. Chem., Int. Ed. Engl. 33, 2258-2267. (3) Lisic, E. C., Mirzadeh, S., and Knapp, F. F., Jr. (1993) Synthesis of carrier-free rhenium-188(V)DMSA using triphenyl phosphine as a facile reducing agent. J. Labeled Compd. Radiopharm. 33, 65-75. (4) John, E., Thakur, M. L., DeFulvio, J., McDevitt, M. R., and Damjanov, I. (1993) Rhenium-186-Labeled Monoclonal Antibodies for Radioimmunotherapy: Preparation and Evaluation. J. Nucl. Med. 34, 260-267. (5) Goldenberg, D. M., and Griffiths, G. L. (1992) Radioimmunotherapy of Cancer: Arming the Missiles. J. Nucl. Med. 33, 1110-1112. (6) Crocker, L. S., Gould, G. L., and Heinekey, D. M. (1988) Improved synthesis of carbonylrhenium. J. Organomet. Chem. 342, 243-244. (7) Calderazzo, F., Mazzi, U., Pampaloni, G., Poli, R., Tisato, F., and Zanazzi, P. F. (1989) Reduction of Ammonium Pertechnetate and Ammonium Perrhenate with CO: Synthesis of M2(CO)10 (M ) Tc, Re) and crystal and molecular structure of the trinuclear cyano-bridged derivative Re3(CN)3(CO)12. Gazz. Chim. Ital. 119, 241-247. (8) Knight Castro, H. H., Meetsma, A., Teuben, J. H., Vaalburg, W., Panek, K., and Ensing, G. (1991) Synthesis, reactions and structure of Cp′Tc(CO)3 derivatives. J. Organomet. Chem. 410, 63-71. (9) Griffiths, G. L., Goldenberg, D. M., Jones, A. L., and Hansen, H. J. (1992) Radiolabeling of Monoclonal Antibodies and fragments with technetium and rhenium. Bioconjugate Chem. 3, 91-99. (10) Eckelman, W. C. (1995) Radiolabeling with technetium99m to study high-capacity and low-capacity biochemical systems. Eur. J. Nucl. Med. 22, 249-263. (11) Liu, S., Edwards, D. S., and Barrett, J. A. (1997) 99mTc labeling of highly potent small peptides. Bioconjugate Chem. 8, 621-636. (12) Rhodes, B. A. (1991) Direct labeling of proteins with Tc-99m. Nucl. Med. Biol. 18, 667-676. (13) Eckelman, W. C., and Steigman, J. (1991) Direct labeling with Tc-99m. Nucl. Med. Biol. 18, 3-7. (14) Hnatowich, D. J., Mardirossian, G., Rusckowski, M., Fogarasi, M., Virzi, F., and Winnard, J., P. (1993) Directly and indirectly technetium-99m-labeled anitbodies-A comparison of in vitro and animal in vivo properties. J. Nucl. Med. 34, 109-119. (15) Garron, J. Y., Moinereau, M., Pasqualini, R., and Saccavini, J. C. (1991) Direct Tc-99m labeling of monoclonal antibodies: radiolabeling and in vitro stability. Nucl. Med. Biol. 18, 695-703. (16) Hnatowich, D. J., Virzi, F., Winnard, P., Fogarasi, M., and Rusckowski, M. (1994) Investigations of ascorbate for direct labeling of antibodies with Tc-99m. J. Nucl. Med. 35, 127134.
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Spradau and Katzenellenbogen (38) Maina, T., Stolz, B., Albert, R., Bruns, C., Koch, P., and Ma¨cke, H. (1994) Synthesis, radiochemistry and biological evaluation of a new somatostatin analogue (SDZ 219-387) labeled with technetium-99m. Eur. J. Nucl. Med. 21, 437444. (39) Kolan, H., Li, J., and Thakur, M. L. (1996) Sandostatin labeled with Tc-99m: In vitro stability, in vivo validity and comparison with In-111-DTPA-octreotide. Pept. Res. 9, 144150. (40) Zamora, P. O., Stratesteffan, M., Guhlke, S., Sass, K. S., Cardillo, A., Bender, H., and Biersack, H. J. (1996) Radiotracer binding to brain microsomes determined by thinlayer chromatography. Nucl. Med. Biol. 23, 61-67. (41) Spradau, T. W., Edwards, W. B., Anderson, C. J., Welch, M. J., and Katzenellenbogen, J. A. Synthesis and biological evaluation of Tc-99m-cyclopentadienyltricarbonyltechnetiumlabeled octreotide. Nucl. Med. Biol. (in press).
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