Radioiodination of Cyanocobalamin Conjugates ... - ACS Publications

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Bioconjugate Chem. 1999, 10, 912−920

Radioiodination of Cyanocobalamin Conjugates Containing Hydrophilic Linkers: Preparation of a Radioiodinated Cyanocobalamin Monomer and Two Dimers, and Assessment of Their Binding with Transcobalamin II D. Scott Wilbur,*,† Pradip M. Pathare,† Donald K. Hamlin,† Sheldon P. Rothenberg,‡ and Edward V. Quadros‡ Department of Radiation Oncology, University of Washington, Seattle, Washington 98195, and SUNY-Downstate Medical Center, Brooklyn, New York 11203. Received March 18, 1999; Revised Manuscript Received June 3, 1999

This report describes an investigation aimed at preparation of radioiodinated cyanocobalamin (CNCbl) monomers and dimers with improved water solubility and decreased nonspecific binding. In the investigation, synthesis and radioiodination reactions of one monomeric and two dimeric CN-Cbl derivatives were conducted. The initial step in the synthesis of the CN-Cbl derivatives was mild acid hydrolysis of CN-Cbl, 1, followed by separation of the resultant corrin ring b-, d-, and e-monocarboxylate isomers. The investigation was limited to preparation of conjugates of CN-Cbl-e-carboxylate, 2, as earlier studies had shown binding of that isomer with recombinant human transcobalamin II (rhTCII) was similar to CN-Cbl. In a second synthetic step, the hydrophilic linker moiety, 4,7,10-trioxa-1,13tridecandiamine, 3, was conjugated with 2 to form the adduct, 4. The synthesis of a monomeric CNCbl derivative, 6a, which can be used for radioiodination, was accomplished by reaction of 4 with p-tri-n-butylstannylbenzoate tetrafluorophenyl (TFP) ester, 5a. Two CN-Cbl dimers containing the arylstannane radioiodination moiety were also synthesized. The first dimer, 8a, was synthesized by cross-linking 4 with a stannylbenzoyl-aminoisophthalate di-TFP ester, 7a. The second dimer, 11a, was synthesized by reacting benzene tricarboxylate tri-TFP ester, 10, in a stepwise manner with 1 equiv of the adduct of 5a and 3 (forming 9a), followed by 2 equiv of 4. Iodobenzoate HPLC standards, 6b, 8b, and 11b, used in the radioiodination studies, were prepared in a manner similar to that of the stannylbenzoate derivatives. Radioiodinations were performed by reacting 6a, 8a, or 11a with N-chlorosuccinimide and Na[125I]I in methanol under neutral conditions. Radiochemical yields of 1742% were obtained. Evaluation of the binding properties of radiolabeled CN-Cbl conjugates with rhTCII showed that the dimer of CN-Cbl, 11b, bound more avidly than the monomer, 6b, and that the binding affinity of the dimer is essentially equivalent to that of unmodified CN-Cbl. Incubation of radioiodinated monomer, [125I]6b, and dimer, [125I]11b, with rhTCII followed by size-exclusion chromatographic analysis provided data that the monomer bound one rhTCII molecule whereas two rhTCII molecules were bound to approximately 30% of the dimer.

INTRODUCTION

Cobalamins (Cbl) function as coenzymes in two mammalian enzymes, methionine synthase and L-methylmalonyl-CoA mutase (1, 2). Although an essential nutrient, mammalian cells do not synthesize the basic Cbl structure, rather it is a vitamin (vitamin B12) obtained from microorganisms (2) and purified in its cyanocobalamin (CN-Cbl)1 form (3). The adsorption, transport, cellular uptake, and storage of cobalamins is highly regulated (48). Abnormalities in various aspects of this regulated process can result in hematological and neurologic disease (6, 9-11). Elucidation of the biochemical pathways and associated abnormalities of cobalamins have been aided greatly by the use of radiolabeled cobalamins (9, 12). The radiolabel widely used with cobalamins is 57Co, but other radionuclides of cobalt (e.g., 58Co and 60Co) and tritium have also been used (13-17). While the cobal* To whom correspondence should be addressed. Phone: 206-685-3085. Fax: 206-685-9630. E-mail: dswilbur@ u.washington.edu. † University of Washington. ‡ SUNY-Downstate Medical Center.

amins that contain 57Co have many applications, cobalamins labeled with other radionuclides may offer advantages in some applications. One important advantage that can be obtained with certain radionuclides is higher specific activities of CN-Cbl derivatives. Introduction of 57 Co into CN-Cbl is obtained by microbial synthesis, and the specific activity can be variable due to differences in growth conditions. Once incorporated, the 57Co-labeled product must be extracted and subjected to extensive purification. Tritium and 14C can be used but the specific activity of the product is inadequate for most applications. Recently, high specific activity 99mTc- and 111In1Abbreviations: BSA, bovine serum albumin; ca, carrieradded; Cbl, cobalamin; CN-Cbl, cyanocobalamin; cpm, counts per minute; DMIX, thioglycerol/dimethyl sulfoxide/trifluoroacetic acid mixture; DTPA, diethylenetriaminepentaacetic acid; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; HOAc, acetic acid; HSA, human serum albumin; ES, electrospray; Et3N, triethylamine; FAB, fast-atom bombardment; LC, liquid chromatography; NaOAc, sodium acetate; nca, no-carrier-added NCS, N-chlorosuccinimide; NHS, N-hydroxysuccinimide; PBS, phosphate-buffered saline; rt, room temperature; Rxn, reaction; TCII, transcobalamin II; rhTCII, recombinant human transcobalamin II; TFP, tetrafluorophenyl; TFP-OH, tetrafluorophenol.

10.1021/bc9900340 CCC: $18.00 © 1999 American Chemical Society Published on Web 07/23/1999

Technical Notes

labeled cobalamins have been described for in vivo imaging of the transcobalamin II (TCII) receptors (18). In those studies, the chelating agent DTPA was conjugated to the corrin ring via a short lipophilic linker molecule. We have also described a method for labeling cobalamins with iodine radionuclides utilizing a pendant group attached to the corrin ring (19). While the attachment of a pendant group to cobalamins for radiolabeling alters the structure, if the attachment is made in specific locations, the binding with TCII is only minimally affected (20). The radioiodinated CN-Cbl derivatives prepared bound with TCII nearly as avidly as unaltered CN-Cbl and had specific activities nearly three times that of commercially available 57Co-labeled CN-Cbl (19). Several CN-Cbl derivatives previously prepared in our laboratory are under investigation as new anti-neoplastic therapeutics for AIDS related lymphoma (19-21). The cobalamin derivatives initially prepared contained the lipophilic linker moiety 1,12-diaminododecane. This linker was used as a 16 Å spacer between the CN-Cbl moiety and conjugated molecules to provide adequate distance such that binding with carrier proteins (e.g., TCII) is not affected. To obtain radioiodinated CN-Cbl derivatives, an arylstannyl moiety, p-tri-n-butylstannylhippuric acid, which serves as a penultimate precursor to radioiodination (22), was conjugated using the lipophilic diaminododecane linker. Radioiodination of the resultant cobalamin-stannylhippurate conjugates produced compounds with high specific activities, but the compounds bound nonspecifically to glassware and syringe needles. Although the nonspecific binding was reduced by addition of bovine serum albumin (BSA), this property made the radioiodinated CN-Cbls of limited use. Dimers of CN-Cbl were also produced using similar chemistry; however, attempted iodination and radioiodination of the CN-Cbl dimers containing arylstannyl moieties were unsuccessful. Our desire to decrease the nonspecific binding of monomeric radioiodinated CN-Cbl derivatives and to develop a method for radioiodination of CN-Cbl dimers led to an investigation of CN-Cbl derivatives that incorporate a more hydrophilic linker, 4,7,10-trioxa-1,13tridecandiamine, of similar length (i.e., 17 Å) in the place of 1,12-diaminododecane. Only the corrin ring e-monocarboxylate conjugates were prepared as that regioisomer had been previously shown to bind more avidly than conjugates of the other regioisomeric corrin ring carboxylates (20). The CN-Cbl derivatives were prepared in sets of two similar compounds, the arylstannane used for radioiodination and the corresponding aryliodide. Thus, two monomeric CN-Cbl derivatives and four dimeric CNCbl derivatives were prepared. The primary difference in the CN-Cbl dimers was that in one set of compounds two trioxatridecanediamines were incorporated whereas three of the same linkers were incorporated into the second set of dimers. Once prepared, the CN-Cbl conjugates containing arylstannane moieties were evaluated in radioiodination reactions, and their binding with recombinant human transcobalamin II (rhTCII) was assessed. EXPERIMENTAL PROCEDURES

Materials. All chemicals purchased from commercial sources were of analytical grade or better and were used without further purification. Cyanocobalamin (vitamin B12) was purchased from Sigma Chemical Co. (St. Louis, MO). N-Hydroxysuccinimide was purchased from Lancaster Synthesis Inc. (Windham, NH). All other reagents

Bioconjugate Chem., Vol. 10, No. 5, 1999 913

were obtained from Aldrich Chemical Co. (Milwaukee, WI). Solvents for HPLC analysis were obtained as HPLC grade and were filtered (0.2 µm) prior to use. Human serum albumin was obtained from Miles, Inc. (Elkhart, IN). Strongly basic anion exchange matrix, 2% crosslinked Dowex 1 chloride form, 200-400 mesh, Amberlite XAD-2 nonionic polymeric adsorbent, and octadecyl functionalized silica gel for column chromatography were obtained from Sigma-Aldrich (St Louis, MO). Bio-Sil NH2 (aminopropyl bonded silica) (40-63 µm) for column packing was purchased from Bio-Rad Laboratories (Hercules, CA). Radioactivity. [57Co]Cyanocobalamin was obtained from Amersham Corp. (Amersham, U.K.) at a specific activity of 200 µCi/µg. Na[125I] was purchased from NEN/ Dupont (Billerica, MA) as high concentration/high specific activity radioiodide in 0.1 N NaOH. Samples containing 57Co were measured in a Beckman 300 gamma counter or Wallac 1480 gamma counter, and those containing 125I were measured in a Capintec CRC-15R or a Capintec CRC-6A radioisotope calibrator. Procedures for safe handling of radioactive materials were followed in the manner previously described (19). Spectroscopic Analysis. 1H NMR spectra were obtained on a Bruker AC-500 (500 MHz) or a Bruker AC750 (750 MHz) instrument. The chemical shifts are expressed as parts per million using tetramethylsilane as an internal standard (δ ) 0.0 ppm). IR data were obtained on a Perkin-Elmer 1420 infrared spectrophotometer. Mass spectral data were obtained on a VG 70SEQ mass spectrometer with 11250J data system. Fast-atom bombardment (FAB) mass spectral data were obtained at 8 kV using a matrix of MeOH/DMIX (thioglycerol/DMSO/TFAA, 90/9/1). Electrospray (ES) mass spectra were acquired on a Sciex API III triple quadrapole spectrometer fitted with a nebulization assisted electrospray source (PE/Sciex, Thornhill, Ontario). Chromatography. Separation of compounds (nonradioactive) by HPLC was done on a Hewlett-Packard quaternary 1050 gradient pumping system with a variable wavelength UV detector (360 nm) and a Varex ELSD MKIII evaporative light-scattering detector. The HPLC data were analyzed using Hewlett-Packard HPLC ChemStation software. The compounds were separated by reversed-phase HPLC on an Alltech Altima C-18 column (5 µm, 4.6 × 250 mm) using one of two different gradient solvent systems at a flow rate of 1 mL/min. Gradient I used methanol as solvent A and aqueous 1% HOAc as solvent B. Starting with 40% A, the initial solvent mixture was held for 2 min, then the gradient was increased to 100% A over the next 10 min and then held at 100% A for 5 min. Retention times, tR, under these conditions were (min) 1 ) 2.7; 2 ) 5.1; 4 ) 2.3; 6a ) 15.7; 6b ) 10.8; 8a ) 14.8; 8b ) 10.5; 9a ) 18.4; 9b ) 10.5; 11a ) 14.8; 11b ) 10.3 . The starting solvent mixture for gradient II was 70% A, and all other parameters were the same as for gradient I. Retention times, tR, under these conditions were (min) 5a ) 13.8; 5b ) 20.7. Preparative scale purifications of CN-Cbl conjugates 6a, 8a, and 11a for use in radioiodination reactions were conducted with a gradient of MeOH/H2O (no HOAc). Chromatographic separation of radiolabeled cobalamins was carried out on a system consisting of two Beckman model 110B pumps, a Beckman 420 controller, a Beckman model 153 UV detector (254 nm), and a Beckman model 170 radioisotope detector. The compounds were separated on a Hamilton PRP-1 column (10 µm, 4.6 mm × 250 mm; Alltech) using one of two gradient

914 Bioconjugate Chem., Vol. 10, No. 5, 1999

solvent systems at a flow rate of 1 mL/min. The first solvent system consisted of 50% MeOH as solvent A and 50% water as solvent B. The initial solvent mixture was held for 2 min, then the gradient was increased to 100% A over the next 10 min, and 100% A was held for 5 min. Retention times, tR, under these conditions were 6b ) 13.8; 11b ) 13.4. In the second gradient, the starting solvent mixture was 60% A, the initial solvent mixture was held for 2 min, then the gradient was increased to 100% A over next 13 min and held there for 10 min. Retention time, tR, under these conditions was 8b ) 12.8 min. A preparative LC system containing a Rainin Rabbitplus peristaltic pumping system, a Dynamax (model UV1) UV-vis absorbance detector, and a Dynamax model FC1 fraction collector were used to obtain pure samples of 6a, 6b, 8a, 8b, 11a, and 11b. A glass column (150 psi, C18, 25 mm × 500 mm) packed with either Bio-Sil NH2 (for separation of CN-Cbl-carboxylates) or reversed-phase C18 was used. The eluant solvents and gradients were the same as above. Fractions were collected and evaluated by analytical HPLC before combining those containing only the desired compound. Synthesis of CN-Cbl-e-carboxylic acid, 2. The CNCbl corrin ring e-carboxylate, 2, was prepared by acid hydrolysis of CN-Cbl, 1, and purified by preparative LC on Bio-Sil NH2 as previously described (20). Synthesis of 4. Conjugation of the CN-Cbl-e-carboxylate, 2, with trioxatridecanediamine, 3, to form the adduct 4 was accomplished as previously described (21). Synthesis of p-Tri-n-butylstannylbenzoyltrioxatridecanediamine-CN-Cbl Conjugate, 6a. To a solution containing 0.3 g (0.193 mmol) of 4 in 20 mL of DMF and 27 µL of Et3N was added 0.108 g (0.193 mmol) of p-tri-n-butylstannylbenzoate TFP ester, 5a, over a 5-10 min period. The reaction mixture was stirred at room temperature for 2 h (HPLC monitored) and then evaporated to dryness. The resultant solid residue was dissolved in 10 mL of 60% methanol/H2O, loaded on a preparative C18 reversed-phase LC column, and eluted with the same solvent mixture as used for loading the column. The fractions containing the final product were evaporated to dryness to yield 42% of 6a, mp 170-172 °C. 1H NMR (MeOH-d4, 750 MHz): δ 0.35 (s, 2H); 0.72.6 (m, 80H); 2.76 (m, 2H); 3.0-3.65 (m, 38H); 3.8 (m, 1H); 3.95-4.1 (m, 3H); 4.2 (m, 1H); 4.4 (m, 1H); 4.6 (m, 1H); 5.95 (s, 1H); 6.18 (s, 1H); 6.47 (s, 1H); 7.02 (s, 1H); 7.16 (s, 1H); 7.45 (m, 2H); 7.65 (m, 2H). MS (FAB+) mass calcd for C92H139N15O18CoPSn: 1952. Found: 1953 (M + H). IR (KBr): 3300, 3200, 2900, 1640, 1570, 1200, 1070. Synthesis of p-Iodobenzoyltrioxatridecanediamine-CN-Cbl Conjugate, 6b. The same procedure as described for the synthesis of 6a was used, except 0.076 g (0.193 mmol) of p-iodobenzoate TFP ester, 5b, was added in place of 5a. The product was isolated in 35% yield, mp 161-163 °C. 1H NMR (MeOH-d4, 750 MHz): δ 0.44 (s, 2H); 1.05-1.5 (m, 13H); 1.65-2.7 (m, 40H); 2.84 (m, 2H); 3.15-3.75 (m, 40H); 3.88 (m, 1H); 4.05-4.2 (m, 2H); 4.3 (m, 1H); 4.54 (m, 1H); 4.65 (m, 1H); 6.04 (s, 1H); 6.27 (s, 1H); 6.56 (s, 1H); 7.11 (s, 1H); 7.25 (s, 1H); 7.56 (m, 2H); 7.82 (m, 2H). MS (FAB+) mass calcd for C80H112N15O18CoPI: 1787. Found: 1788 (M + H). IR (KBr): 3300, 3200, 2900, 1640, 1570, 1200, 1070. 4′-(Tri-n-butylstannyl)benzoyl-5-aminoisophthalate DiTFP Ester, 7a, and 4′-Iodobenzoyl-5-aminoisophthalate DiTFP Ester, 7b. These compounds were synthesized as described previously (21). Synthesis of 8a. To a solution containing 0.3 g (0.193 mmol) of 4 in 20 mL of DMF was added 30 µL of Et3N

Wilbur et al.

followed by 0.084 g (0.096 mmol) of 7a. The reaction mixture was stirred at room temperature for 3 h and then evaporated to dryness. The resultant solid residue was dissolved in 10 mL of 80% methanol/H2O, loaded on a preparative C18 reversed-phase LC column, and eluted with the same solvent mixture as used for loading the column. The fractions containing the final product were evaporated to dryness to yield 43% of 8a, mp 238-241 °C (dec). 1H NMR (MeOH-d4, 500 MHz): δ 0.45 (s, 4H); 0.9-2.7 (m, 140H); 2.8-2.9 (m, 5H); 3.1-3.8 (m, 64H); 3.88 (m, 4H); 4.08-4.2 (m, 6H); 4.34 (m, 3H); 4.48 (m, 4H); 4.68 (m, 4H); 6.05 (s, 2H); 6.28 (s, 2H); 6.57 (s, 2H); 7.14 (s, 2H); 7.26 (s, 2H); 7.6-7.8 (m, 3H); 7.9-8.01 (m, 3H); 8.33 (s, 1H). MS (ES) mass calcd for C173H251N31O37Co2P2Sn: 3654. Found: 3655 (M + H). IR (KBr): 3300, 3200, 2900, 1640, 1570, 1200, 1070. Synthesis of 8b. The same procedure was used as described for the synthesis of 8a, except 0.067 g (0.096 mmol) of 7b was added in place of 7a. The product was isolated in 45% yield, mp 210-213 °C (dec). 1H NMR (MeOH-d4, 500 MHz): δ 0.44 (s, 4H); 1.0-2.65 (m, 115H); 2.8-2.9 (m, 5H); 3.1-3.8 (m, 62H); 3.88 (m, 4H); 4.044.2 (m, 6H); 4.34 (m, 3H); 4.48 (m, 4H); 4.67 (m, 4H); 6.04 (d, J ) 5.5 Hz, 2H); 6.26 (s, 2H); 6.56 (d, J ) 6.5 Hz, 2H); 7.13 (d, J ) 4 Hz, 2H); 7.24 (d, J ) 3.5 Hz, 2H); 7.73-7.79 (m, 3H); 7.88-8.01 (m, 3H); 8.3 (s, 1H). MS (ES) mass calcd for C161H224N31O37Co2P2I: 3491. Found: 3492 (M + H). IR (KBr): 3300, 3200, 2900, 1640, 1570, 1200, 1070. N-(13′-Amino-4′,7′,10′-trioxatridecanyl)-4-tributylstannylbenzamide, 9a, and N-(13′-Amino-4′,7′,10′trioxatridecanyl)-4-iodobenzamide, 9b. Compounds 9a and 9b were synthesized as described previously (23). Synthesis of 11a. A solution of 9a (0.058 g, 0.095 mmol) in 25 mL of anhydrous DMF was added dropwise over 1 h to a solution of 10 (0.062 g, 0.095 mmol) and Et3N (40 µL) in 15 mL of DMF. The reaction was stirred at room temperature for 30 min and a solution of 4 (0.190 mmol, 0.296 g) in 10 mL of DMF was added. The reaction mixture was stirred at room temperature for 24 h and then evaporated to dryness. The resultant solid residue was dissolved in 10 mL of 70% methanol/H2O, loaded on a preparative C18 reversed-phase LC column, and eluted with the same solvent mixture as used for loading the column. The fractions containing the product were evaporated to dryness to obtain 11a in 30% yield, mp 285290 °C. 1H NMR (MeOH-d4, 750 MHz): δ 0.44 (s, 4H); 0.85-2.7 (m, 138H); 2.85 (m, 5H); 3.15-3.8 (m, 88H); 3.87 (m, 3H); 4.05-4.2 (m, 6H); 4.3 (m, 3H); 4.45-4.7 (m, 10H); 6.03 (s, 2H); 6.27 (s, 2H); 6.56 (s, 2H); 7.11 (s, 2H); 7.25 (s, 2H); 7.54 (d, J ) 7.5 Hz, 2H); 7.73 (d, J ) 8.5 Hz, 2H); 8.39 (s, 3H). MS (ES) mass calcd for C184H274N32O41Co2P2Sn: 3887. Found: 3886 (M - H). IR (KBr): 3300, 3200, 2900, 1640, 1570, 1200, 1070. Synthesis of 11b. The same procedure was used as described for the synthesis of 11a, except 0.043 g (0.095 mmol) of 9b, which was added in place of 9a. The product was isolated in 26% yield, mp 215-218 °C (dec). 1H NMR (MeOH-d4, 750 MHz): δ 0.49 (s, 4H); 1.15-1.55 (m, 8H); 1.7-2.75 (m, 80H); 2.9 (m, 5H); 3.2-3.85 (m, 112H); 3.96 (m, 4H); 4.1-4.27 (m, 6H); 4.4 (m, 3H); 4.55-4.75 (m, 8H); 6.1 (s, 2H); 6.34 (s, 2H); 6.63 (s, 2H); 7.18 (s, 2H); 7.31 (s, 2H); 7.6 (d, J ) 8.0 Hz, 2H); 7.87 (d, J ) 8.5 Hz, 2H); 8.45 (s, 3H). MS (ES) mass calcd for C172H247N32O41Co2P2I: 3724. Found: 3723 (M - H). IR (KBr): 3300, 3200, 2900, 1640, 1570, 1200, 1070.

Technical Notes

Synthesis of CN-Cbl-p-[125I]Iodobenzamide Derivatives [125I]6b, [125I]8b, and [125I]11b. Purification of 6a, 8a, and 11a by HPLC. A saturated solution of a CN-Cbl derivative was prepared in methanol. This solution was passed through a 45 µm filter and three to five 20 µL aliquots were injected on the HPLC (C18 column). The product peak from each injection was collected, and the collected fractions were combined. The concentration of the cobalamin derivative in the combined fractions was calculated from the UV absorbance at 360 nm. The cobalamin-containing solution was then divided into aliquots containing 0.5 mg each, dried under a stream of argon, and stored at 4 °C. Preparation of nca [125I]6b. The HPLC-purified 6a was dissolved in methanol at a concentration of 1 mg/mL and 50 µL of that solution was placed in a small vial. To the solution was added 1 µL (92 µCi) of Na[125I]I in 0.1 N NaOH, followed by addition of 2 µL of 0.1 N HOAc. To the resulting solution was added 25 µL of a 1 mg/mL solution of NCS in MeOH. After 15 min, 25 µL of a 1.0 mg/mL solution of sodium metabisulfite in H2O was added to quench the reaction. A total of 87 µCi in approximately 75 µL of solution was removed from the reaction vessel by syringe and injected on an HPLC column (PRP-1 column). The iodinated product was collected from the HPLC effluent (13.8 min), yielding 24 µCi (28%) of [125I]6b. The radioiodinated product was evaluated by HPLC and stored at 4 °C. Preparation of nca [125I]8b. A 3.5 µL (1.0 mCi) aliquot of Na[125I]I in 0.1 N NaOH was added to 50 µL of a 1 mg/mL solution of 8a, followed by the addition of 4.0 µL of 0.1 N HOAc. To the resulting solution was added 10 µL of a solution containing 1 mg/mL NCS in methanol. After 15 min, the entire reaction mixture (990 µCi) was drawn into a syringe and injected onto an HPLC column (PRP-1 column). The collected fraction (12.7 min) yielded 168 µCi (17%) of 8b. Preparation of ca [125I]11b. A 1 µL (253 µCi) aliquot of Na[125I]I in 0.1 N NaOH was added to 50 µL of a 1 mg/ mL solution of 11a, followed by addition of 2 µL of 0.095 mg/mL solution of NaI. To the resulting solution was added 10 µL of solution containing 1 mg/mL NCS in methanol. After 15 min, the entire reaction mixture (245 µCi) was drawn into a syringe and injected onto an HPLC column (PRP-1). The collected fraction (13.5 min) yielded 103 µCi (42%) of 11b. Determination of Specific Activity of [125I]6b and [125I]11b. Specific activities of the [125I]6b and [125I]11b were determined by calculation (theoretical) and by TCIIbinding assay. The calculation of specific activities was adjusted for time between preparation and when binding assays were run (45 days; 59% remaining) and adjusted for added NaI (ca reaction only). In the TCII-binding assay, increasing amounts of radiolabeled CN-Cbl were incubated for 1 h with aliquots of human serum which had a predetermined Cbl-binding capacity. Protein bound and free radiolabeled compound were separated by adsorption of unbound radiolabeled CN-Cbl to hemoglobin-coated charcoal. The cpm for saturation of TCII with radiolabeled CN-Cbl provided cpm per picogram of compound. The cpm were corrected for efficiency of the counter (80%), and the values were converted into microcurie to obtain a specific activity in microcurie per microgram. Binding of CN-Cbl-[125I]iodobenzoate Derivatives with Recombinant Human Transcobalamin II (rhTCII). rhTCII was produced and partially purified as previously reported (24). The rhTCII was diluted in 0.025% HSA/PBS/0.1% Triton x-100 to bind approxi-


mately 1 pmol of CN-Cbl/100 µL. The [125I]6b and [125I]11b were diluted respectively to 100 000 and 60 000 cpm/ mL in 0.05% HSA/PBS/0.1% Triton x-100. The binding of 6b and 11b to rhTCII was measured in a competitive binding assay in which 100 µL of each 125I-labeled or 57Co-labeled CN-Cbl was mixed with 0.1-8 pmol of unlabeled CN-Cbl, followed by addition of 100 µL of rhTCII solution to each tube. The volume was adjusted to 1 mL with PBS and incubated for 1 h at room temperature. The protein-bound fraction was determined by adsorption of the free fraction to hemoglobin-coated charcoal (2.5% charcoal/0.25% hemoglobin in PBS). The amount of compound bound as a function of concentration in the reaction was calculated and plotted. Size-Exclusion Chromatography of 125I-Labeled 6b and 11b Bound to rhTCII. The quantities of 1 pmol of [125I]6b and 2 pmol of [125I]11b were separately incubated for 1 h at room temperature with a 10-fold molar excess of rhTCII in a 0.75 mL solution containing 20 mM tris/200 mM NaCl/0.1% Triton x-100. Blue dextran (0.25 mL, 10 mg/mL) was added to each sample to mark the void volume of the column. The sample was layered on a 1.5 × 90 cm Sephacryl S-200 column. The column was equilibrated and eluted in the same buffer at a flow rate of 1 mL/min, and 2 mL fractions were collected. The radioactivity in each fraction was determined in a gamma counter, and the elution profile of TCII-bound compound was plotted. RESULTS

Synthesis of CN-Cbl Conjugates. The CN-Cbl corrin ring e-carboxylate, 2, was obtained by preparative LC separation of the mixture of CN-Cbl monocarboxylic acids produced by acid hydrolysis of CN-Cbl, 1 (20). The isolated CN-Cbl-e-carboxylate, 2, was conjugated with 4,7,10-trioxa-1,13-tridecandiamine, 3, by reaction with a carbodiimide (EDC) in water containing NaCN/NHS to yield compound 4 as shown in Scheme 1. The stannylbenzoate conjugate, 6a, and iodobenzoate conjugate, 6b, were prepared by the reaction of 4 with either the TFP ester of p-tri-n-butylstannylbenzoic acid, 5a, or the TFP ester of p-iodobenzoic acid, 5b. Compounds 6a and 6b were isolated pure from the conjugation reaction in 42 and 35% yield, respectively. The approaches taken to synthesize the hydrophilic CN-Cbl dimers are shown in Schemes 2 and 3. As in previous syntheses of CN-Cbl dimers (21), each synthesis utilized a trifunctional cross-linking reagent to couple two CN-Cbl moieties. In one synthesis (Scheme 2), the reagent used to cross-link the two CN-Cbl moieties contained an arylstannane or aryliodide moiety such that the CN-Cbl dimer could be formed in one step. Thus, reaction of 4 with either the di-TFP ester of p-(tri-nbutylstannyl)benzoylaminoisophthalate, 7a, or the diTFP ester of p-iodobenzoyl-aminoisophthalate, 7b, provided the desired CN-Cbl dimers, 8a and 8b in 43 and 45% yields, respectively, after purification. The requisite aminoisophthaloyl derivative, 7b, was prepared by conjugation of p-iodobenzoyl chloride with 5-aminoisophthalic acid followed by preparation of the di-TFP ester using EDC and TFP-OH in ethyl acetate (21). Conversion of the aryl iodide, 7b, into the aryl stannane, 7a, was accomplished in 62% yield using bis(tributyl)tin and palladium catalyst. The synthesis of the second set of CN-Cbl dimers, 11a and 11b, was accomplished by the reaction shown in Scheme 3. In a stepwise manner, 1 equiv of either the trioxatridecanediamine-conjugated arylstannane, 9a, or


Wilbur et al.

Scheme 1. Synthetic Route Used to Prepare CN-Cbl Conjugates 6a and 6b

a

NHS/NaCN/H2O/3/HCl/EDC/rt/4 days. bDMF/Et3N/5a or 5b/rt/2 h

aryliodide, 9b, was reacted with the tri-TFP ester of benzenetricarboxylic acid, 10, followed by addition of 2 equiv of the trioxatridecanediamine adduct of CN-Cble-carboxylate, 4. The progress of the reaction after each addition was followed by HPLC analysis. Preparation of 9a and 9b was accomplished by reaction of the TFP ester of p-tri-n-butylstannylbenzoic acid or the TFP ester of p-iodobenzoic acid with trioxatridecanediamine, 3, as previously described (23). Following this procedure, and purification by HPLC, the CN-Cbl dimers 11a and 11b were obtained in 30 and 26% yields, respectively. Iodination Reactions. Although the isolated arylstannanes 6a, 8a, and 11a were >95% pure by HPLC analysis, the starting material was further purified by preparative HPLC prior to radioiodination to ensure that reactive minor impurities would not compete in ncaradioiodinations. Initial radioiodination of 6a was carried out in MeOH employing NCS/Na[125I]I. The HPLC retention time for the new radioiodinated species indicated that the arylstannane was not substituted by iodine, suggesting that the radioiodide may have undergone another reaction, such as displacement of the cyano group on the cobalt. Radioiodination in aqueous 1% AcOH provided the desired radioiodinated product, but the yield was low (20-30%), and an additional radioiodinated species which was similar in lipophilicity to the starting arylstannane was also formed. Addition of just enough HOAc to neutralize the NaOH in the added Na[125I]I gave only the desired radioiodinated product, albeit again in low yields (17-42%). Although there are a number of possible causes for the low radiochemical yields, one

explanation for the low yields is that recovery of the radioiodinated compound from the HPLC column did not reflect the actual yields. This statement is made since it was observed that a large proportion of the radioactivity was retained on the HPLC column even after extended elution times. Radioiodination of 11a under the same conditions as used to prepare [125I]6b and [125I]8b failed to give the desired nca product, [125I]11b. However, when the reaction was carried out with a small quantity of carrier NaI, the radioiodinated ca product was obtained in 42% yield. The in vitro stability of [125I]6b, [125I]8b (20), and [125I]11b was evaluated by reexamining the radioiodinated CN-Cbl conjugates after storage at 4 °C for over 3 months. Radiochromatographic peaks of the isolated products were unchanged (peak areas were corrected for decay) after storage for 1 h, 24 h, and 3 months. Specific activities for [125I]6b and [125I]11b were estimated by calculation and by measurement of the quantity of radioactivity that it takes to saturate TCII in serum which has a known binding capacity for CN-Cbl. The specific activity value for [125I]6b obtained by calculation is 720 µCi/µg and that obtained by the binding assay was 625 µCi/µg. These numbers are very close, but the lower specific activity measured by binding is most likely closer to the actual value as one would expect some decrease in specific activity (from theoretical) due to the introduction of stable iodide in solvents and reagents. The specific activity for the carrier added [125I]11b was calculated to be 12 µCi/µg. In contrast, the specific activity measured by TCII binding in serum was 38 µCi/µg. It is not known why there is a 3 times difference in these numbers, but

Technical Notes



a

2 equiv of 4/DMF/Et3N/7a or 7b/rt/3 h.

they are fairly close considering the two different methods of obtaining them. Competitive Binding with rhTCII. The CN-Cbl monomer [125I]6b and CN-Cbl dimer [125I]11b were evaluated for their competitive binding to (partially purified) rhTCII relative to CN-Cbl. The assay was set up such that a quantity of rhTCII that would bind approximately 1 pmol of [57Co]CN-Cbl was used in each evaluation. Trace quantities of [57Co]CN-Cbl, [125I]6b, or [125I]11b were mixed with 0.1-8 pmol of unlabeled CNCbl, then mixed with rhTCII. The percent radioactivity bound relative to the picomoles of unlabeled CN-Cbl was measured. The results obtained are provided in Figure 1 and show that the binding of radioiodinated monomer 6b as well as the dimer 11b to rhTCII is essentially unaltered and is similar to that of CN-Cbl. The initial flat portion of the graph representing 100% binding is due to having less than saturating quantities CN-Cbl present. Evaluation of [125I]6b and [125I]11b Binding to rhTCII. A 1 pmol quantity of monomer [125I]6b and 2 pmol of dimer [125I]11b were incubated with 10-fold molar excess of rhTCII at room temperature, and the resulting adducts were examined by size-exclusion gel chromatography (Sephacryl S-200). Fractions were collected from the column and the radioactivity in each fraction was

determined. The elution profile of the binding of [125I]6b, [125I]11b, and [57Co]CN-Cbl with rhTCII is plotted in Figure 2. The [57Co]CN-Cbl, the [125I]6b monomer, and approximately 70% of the[125I]11b dimer eluted from the column as a single peak corresponding to ∼40 kDa, the size of the monomeric rhTCII. A second peak of ∼80 kDa was only observed with the dimer 11b and accounted for ∼30% of the rhTCII-bound radiolabel. DISCUSSION

This investigation was undertaken because our previous attempts to synthesize radioiodinated CN-Cbl derivatives produced compounds that were not optimal for our needs due to their poor solubility in aqueous medium and their propensity to adhere to glass and metal (needle) surfaces. Furthermore, we were unsuccessful in preparing a radioiodinated CN-Cbl dimer. Although the cause of the nonspecific binding is uncertain, we thought that this property could be due, at least in part, to the hydrophobic linker molecule, 1,12-diaminododecane. It was apparent that the hydrophobic linker made the CNCbl conjugates very lipophilic and resulted in their having low water solubility. Also, NMR data from the CN-Cbl dimers suggested that the tri-n-butylstannylbenzoate moiety was affected by the lipophilic nature of the linker, perhaps resulting in shielding it from reacting


Wilbur et al.


a

9a or 9b/DMF/Et3N/rt/30 min; 2 equiv 4/rt/24 h

with radioiodination reagents. Therefore, we reasoned that using a more hydrophilic linker might provide CNCbl derivatives with more favorable characteristics. We have previously used the ether-containing linker 4,7,10-trioxa-1,13-tridecandiamine to increase water solubility of biotin derivatives (23, 25) and CN-Cbl dimers (21). This linker increases the solubility of the compound greatly over that of an aliphatic linker moiety. Accordingly, we observed a marked improvement in water solubility of the radioiodinated CN-Cbl derivatives and found that, in contrast to the more aliphatic CN-Cbl derivatives, these compounds did not adhere to glass or metal (syringe needle) surfaces. Previously prepared CNCbl dimers which were cross-linked using aliphatic diaminododecane linkers required dissolution in DMSO and dilution with water (2-10% DMSO solutions) to prepare 1 mg/mL solutions. In contrast, 1 mg of 8b or 11b was found to be soluble in only 10 µL of water, indicating that the compounds have a solubility of >100 mg/mL. The reason for including three trioxatridecanediamine linkers in the CN-Cbl dimer 11b was primarily

to evaluate whether providing a greater distance between the arylstannane moiety and the cross-linking aminoisophthalate moiety would improve the radiochemical yield. Unfortunately, it appears to have done just the opposite as no radioiodinated product was obtained when the reaction was conducted under nca conditions. Despite this, we have been successful in obtaining radioiodinated CN-Cbl dimers, and the new radioiodinated CN-Cbl conjugates can be formulated in aqueous solution without difficulty. To date there have been few evaluations to determine the in vitro and in vivo characteristics of the radioiodinated CN-Cbl derivatives. As in our previous studies, comparison of the binding of the corrin ring e-carboxylate conjugates (Figure 1) indicate that their binding with rhTCII is similar to that of unaltered CN-Cbl. It is interesting to note that the CN-Cbl monomer [125I]6b binds with a slightly lower avidity than CN-Cbl, but binding of [125I]11b appears to be essentially equivalent to CN-Cbl. The CN-Cbl dimers were designed to bind with two rhTCII moieties, which in turn may alter

Technical Notes

Figure 1. Competitive binding curves for [57Co]CN-Cbl, 1, and iodobenzoyl-CN-Cbl derivatives, 6b and 11b, with rhTCII. [57Co]CN-Cbl (0.01 pmol) was mixed with increasing concentrations of 1 or Cbl derivatives 6b or 11b. Diluted rhTCII (100 µL) with a binding capacity of approximately 1 pmol of 1 was then added to each tube. The volume was adjusted to 1 mL with PBS and incubated at room temperature for 1 h. Free and protein bound radioactivity was separated by adsorption to hemoglobin coated charcoal, and the radioactivity in each fraction was determined and plotted.

binding with the cell surface receptor for TCII-Cbl. We hypothesized that CN-Cbl dimer cross-linked with the aminoisophthalate moiety and two trioxatridecanediamine linkers would have adequate space between the two CN-Cbl moieties (41 Å linker, fully extended) to permit binding with two rhTCII molecules. In this study, the question of whether the CN-Cbl dimer can bind with two rhTCII molecules was addressed successfully for the first time. The results obtained by size-exclusion separa-


tion of complexes formed with the CN-Cbl monomer 6b and the CN-Cbl dimer 11b (Figure 2) appear to indicate that at least a fraction of the CN-Cbl dimer binds with two rhTCII molecules. Additional studies need to be conducted to determine if what appears to be an equilibrium mixture can be shifted to having mostly two TCII bound and to determine if there is a difference in binding affinity between the first TCII bound and the second TCII bound. It also remains to be determined if the two TCII molecules bound to the same CN-Cbl dimer can interact with two TCII-receptors on the cell surface, and if this should occur, what effect it would have on TCII-Cbl internalization. Summary. Three CN-Cbl conjugates containing arylstannane moieties for radioiodination have been synthesized. Use of the hydrophilic linker molecule, 4,7,10-trioxa-1,13-tridecandiamine, in the place of 1,12diaminododedane that was previously employed, resulted in radioiodinated CN-Cbl derivatives that are water soluble and do not have the propensity to adhere to glass and metal surfaces. From our earlier studies, it was demonstrated that the corrin ring e-monocarboxylate isomer bound more tightly with TCII than the other regioisomers. Therefore, in this investigation we used only e-carboxylate isomer. One Cbl monomer and two Cbl dimers were prepared using conjugates of CN-Cbl-ecarboxylate and the hydrophilic linker trioxatridecanediamine. Arylstannane CN-Cbl conjugates were prepared as reactive compounds for site-specific radioiodination, and the corresponding aryliodides were prepared as HPLC standards. Following the syntheses, attempts to develop optimal conditions for radioiodination of CN-Cblarylstannane conjugates were fraught with difficulties. In our earlier investigation we showed that good radiochemical yields (40-65%) could be obtained under neutral reaction conditions. In this study, the radiochemical yields ranged 17-42%. High specific activity was obtained in radioiodination of CN-Cbl monomer 6b and CNCbl dimer 8b. Radioiodination of dimer 11b with nca radioiodine failed, but ca radioiodination was successful. The binding of radioiodinated CN-Cbl derivatives to rhTCII was similar to that observed with unmodified CNCbl. The results obtained indicate that the dimer of CN-

Figure 2. Elution profile of radioactivity in fractions from size exclusion chromatography of [57Co]CN-Cbl, [125I]6b, and [125I]11b bound to rhTCII. In the experiment, 1 pmol of [125I]6b and 2 pmol of [125I]11b were each incubated with a 10-fold molar excess rhTCII for 1 h at room temperature. Blue dextran (0.25 mL, 10 mg/mL) was added to each sample to mark the void volume for the column. The sample was layered on a 1.5 × 90 cm Sephacryl S-200 column. The column was equilibrated and eluted in the same buffer at a flow rate of 1 mL/min and 2 mL fractions were collected. The radioactivity in each fraction was determined and plotted.


Cbl 11b binds more avidly with rhTCII than the monomer of CN-Cbl, 6b, and its binding affinity is essentially equivalent to that of CN-Cbl. Size-exclusion chromatographic analysis of [125I]11b binding with TCII indicated that approximately 30% bound with two TCII molecules. ACKNOWLEDGMENT

We thank Dr. Ross Lawrence (Medicinal Chemistry Department, University of Washington) for efforts in obtaining mass spectral data. This work was supported in part by the Department of Radiation Oncology at the University of Washington and by an NIH grant (DK 28561) to S.P.R. Supporting Information Available: HPLC chromatograms, 1H NMR, and mass spectra for CN-Cbl derivatives 6a, 6b, 8a, 8b, 11a, and 11b are provided. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Banerjee, R. (1997) The Yin-Yang of Cobalamin Biochemistry. Chem. Biol. 4, 175-186. (2) Roth, J. R., Lawrence, J. G., and Bobik, T. A. (1996) Cobalamin (Coenzyme B12): Synthesis and Biological Significance. Annu. Rev. Microbiol. 50, 137-181. (3) Wagner, A. F., and Folkers, K. (1964) Cyanocobalamin and the Cobamide Coenzymes. Vitamins and Coenzymes, pp 194243, Interscience Publishers, New York. (4) Ellenbogen, L. (1975) Absorption and Transport of Cobalamin. Intrinsic Factor and the Transcobalamins. In Cobalamin. Biochemistry and Pathophysiology (B. M. Babior, Ed.) pp 215-286, Wiley-Interscience, New York. (5) Allen, R. H. (1975) Human Vitamin B12 Transport Proteins. Prog. Hematol. 9, 57-84. (6) Seetharam, B. (1994) Gastrointestinal Absorption and Transport of Cobalamin (Vitamin B12). In Physiology of the Gastrointestinal Tract. (L. R. Johnson, Ed.) pp 1997-2026, Raven Press, New York. (7) Rothenberg, S. P., and Quadros, E. V. (1995) Transcobalamin II and the membrane receptor for the transcobalamin II-cobalamin complex. Baillieres Clin. Haematol. 8, 499-514. (8) Nicolas, J. P., and Gueant, J. L. (1995) Gastric intrinsic factor and its receptor. Baillieres Clin. Haematol. 8, 515531. (9) Linnell, J. C. (1975) The Fate of Cobalamins in vivo. In Cobalamin. Biochemistry and Pathophysiology (B. M. Babior, Ed.) pp 287-333, Wiley-Interscience, New York. (10) Mahoney, M. J., and Rosenberg, L. E. (1975) Inborn Errors of Cobalamin Metabolism. In Cobalamin. Biochemistry and Pathophysiology (B. M. Babior, Ed.) pp 369-402, WileyInterscience, New York. (11) Beck, W. S. (1975) Metabolic Features of Cobalamin Deficiency in Man. In Cobalamin. Biochemistry and Pathophysiology (Babior, B. M., Ed.) pp 403-450, Wiley-Interscience, New York. (12) Quadros, E. V., Jackson, B., Joffbrand, A. V., and Linnell, J. C. (1979) Interconversion of Cobalamins in Human Lym-

Wilbur et al. phocytes in Vitro and the Influence of Nitrous Oxide on Synthesis of Cobalamin Coenzymes. In Vitamin B12 (B. Zagalak and W. Friedrich, Eds.) pp 1045-1054, Walter de Gruyter & Co., Berlin. (13) Smith, E. L., Hockenhull, D. J. D., and Quilter, A. R. J. (1952) Tracer Studies with the B12 Vitamins. Biochem. J. 52, 387-388. (14) Maddock, A. G., and Coelho, F. P. (1954) The Retention of Cobalt-60 in Vitamin B12. J. Chem. Soc. 4702-4704. (15) Bradley, J. E., Smith, L. E., Baker, S. J., and Mollin, D. L. (1954) The use of the radioactive isotope of Cobalt Co58 for the preparation of labeled vitamin B12. Lancet 267, 476-477. (16) Smith, E. L., Fantes, K. H., Ball, S., Waller, J. G., Emery, W. B., Anslow, W. K., and Walker, A. D. (1952) B12 Vitamins (Cobalamins) 1. Vitamins B12c and B12d. Biochem. J. 52, 389395. (17) Toraya, T., Ohashi, K., Ueno, H., and Fukui, S. (1975) Preparation, Properties and Biological Activities of Succinyl Derivatives of Vitamin B12. Bioinorg. Chem. 4, 245-255. (18) Collins, D. A., and Hogenkamp, H. P. C. (1996) Synthesis and Biological Activity of Radiolabeled DTPA-Cobalamin Analogues. J. Nucl. Med. 37, 350P. (19) Wilbur, D. S., Hamlin, D. K., Pathare, P. M., Heusser, S., Vessella, R. L., Buhler, K. R., Stray, J. E., Daniel, J., Quadros, E. V., McLoughlin, P., and Morgan, A. C. (1996) Synthesis and nca-radioiodination of arylstannylcobalamin conjugates. Evaluation of aryliodocobalamin conjugate binding to transcobalamin II and biodistribution in mice. Bioconjugate Chem. 7, 461-474. (20) Pathare, P. M., Wilbur, D. S., Heusser, S., Quadros, E. V., McLoughlin, P., and Morgan, A. C. (1996) Synthesis of cobalamin-biotin conjugates that vary in the position of cobalamin coupling. Evaluation of cobalamin derivative binding to transcobalamin II. Bioconjugate Chem. 7, 217-232. (21) Pathare, P. M., Wilbur, D. S., Hamlin, D. K., Heusser, S., Quadros, E. V., McLoughlin, P., and Morgan, A. C. (1997) Synthesis of cobalamin dimers using isophthalate crosslinking of corrin ring carboxylates and evaluation of their binding to transcobalamin II. Bioconjugate Chem 8, 161-172. (22) Wilbur, D. S. (1992) Radiohalogenation of Proteins: An Overview of Radionuclides, Labeling Methods, and Reagents for Conjugate Labeling. Bioconjugate Chem. 3, 433-470. (23) Wilbur, D. S., Hamlin, D. K., Pathare, P. M., and Weerawarna, S. A. (1997) Biotin Reagents for Antibody Pretargeting. Synthesis, Radioiodination and In Vitro Evaluation of Water Soluble, Biotinidase Resistant Biotin Derivatives. Bioconjugate Chem. 8, 572-584. (24) Quadros, E. V., Sai, P., and Rothenberg, S. P. (1993) Functional Human Transcobalamin II Isoproteins Are Secreted by Insect Cells Using the baculovirus Expression System. Blood 81, 1239-1245. (25) Wilbur, D. S., Hamlin, D. K., Pathare, P. M., and Weerawarna, S. A. (1997) Biotin Reagents for Antibody Pretargeting. 2. Synthesis and In Vitro Evaluation of Biotin Dimers and Trimers for Cross Linking of Streptavidin. Bioconjugate Chem. 8, 819-832.

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