Trifunctional Conjugation Reagents. Reagents That Contain a Biotin

Bioconjugate Chem. 2002, 13, 1079−1092

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Trifunctional Conjugation Reagents. Reagents That Contain a Biotin and a Radiometal Chelation Moiety for Application to Extracorporeal Affinity Adsorption of Radiolabeled Antibodies D. Scott Wilbur,*,† Ming-Kuan Chyan,† Donald K. Hamlin,† Brian B. Kegley,† Rune Nilsson,‡ Bengt E. B. Sandberg,‡ and Martin Brechbiel§ Department of Radiation Oncology, University of Washington, Seattle, Washington 98195, Mitra Medical Technology, Lund, Sweden, and National Cancer Institute, Bethesda, Maryland. Received April 1, 2002; Revised Manuscript Received May 21, 2002

A method of removing radiolabeled monoclonal antibodies (mAbs) from blood using a device external to the body, termed extracorporeal affinity-adsorption (EAA), is being evaluated as a means of decreasing irradiation of noncancerous tissues in therapy protocols. The EAA device uses an avidin column to capture biotinylated-radiolabeled mAbs from circulated blood. In this investigation, three trifunctional reagents have been developed to minimize the potential deleterious effect on antigen binding brought about by the combination of radiolabeling and biotinylation of mAbs required in the EAA approach. The studies focused on radiolabeling with 111In and 90Y, so the chelates CHX-A′′DTPA and DOTA, which form stable attachments to these radionuclides, were incorporated in the trifunctional reagents. The first trifunctional reagent prepared did not incorporate a group to block the biotin cleaving enzyme biotinidase, but the two subsequent reagents coupled aspartic acid to the biotin carboxylate for that purpose. All three reagents used 4,7,10-trioxa-1,13-tridecanediamine as water-soluble spacers between an aminoisophthalate core and the biotin or chelation group. The mAb conjugates were radioiodinated to evaluate cell binding as a function of substitution. Radioiodination was used so that a direct comparison with unmodified mAb could be made. Evaluation of the number of conjugates per antibody versus cell binding immunoreactivities indicated that minimizing the number of conjugates was best. Interestingly, a decrease of radioiodination yield as a function of the number of isothiocyanate containing conjugates per mAb was noted. The decreased yields were presumably due to the presence of thiourea functionality formed in the conjugation reaction. Radiolabeling with 111In and 90Y was facile at room temperature for conjugates containing the CHXA′′, but elevated temperature (e.g., 45 °C) was required to obtain good yields with the DOTA chelate. Stability of 90Y labeled mAb in serum, and when challenged with 10 mM EDTA, was high. However, challenging the 90Y labeled mAb with 10 mM DTPA demonstrated high stability for the DOTA containing conjugate, but low stability for the CHX-A′′ containing conjugate. Thus, the choice between these two chelating moieties might be made on requirements for facile and gentle labeling versus very high in vivo stability. Application of the trifunctional biotinylation reagents to the blood clearance of labeled antibodies in EAA is under investigation. The new reagents may also be useful for other applications.

INTRODUCTION

The high-affinity binding pairs biotin/avidin (Av1) and biotin/streptavidin (SAv) have become important chemical tools in capture processes for a large number of diverse applications, both in vitro and in vivo. The most widely used in vitro detection systems employ biotinylated receptor binding biomolecules, particularly monoclonal antibodies (mAbs), in capture processes with avidin or streptavidin (2-5). An in vivo application of this capture process uses the biotin/streptavidin binding pair as conjugates of mAbs in an approach, termed “pretargeting”, to selectively localize radionuclides on cancer cells for therapy (6-8). The pretargeting approach to * Corresponding author. Address: Department of Radiation Oncology, University of Washington, 2121 N. 35th Street, Seattle, WA 98103-9103. Phone: 206-685-3085. FAX: 206-6859630. E-mail: [email protected]. † University of Washington. ‡ Mitra Medical Technology. § National Cancer Institute.

“endoradiotherapy”2 is being investigated due to the inherent problems associated with using directly radio1 Abbreviations: Av, avidin; FAB+, fast atom bombardment; BCA, bicinchoninic acid; BSA, bovine serum albumin; DOTA, 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid; DTPA, diethylenetriaminepentaacetic acid; EAA, extracorporeal affinity adsorption; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; ES+, electrospray; HABA, 4′-hydroxyazobenene2-carboxylic acid; HRMS, high-resolution mass spectrometry; LRMS, low-resolution mass spectrometry; mAb, monoclonal antibody; IEF, isoelectric focusing electrophoresis; NHS-LCbiotin, N-hydroxysuccinimidyl biotinyl-6-aminocaproic acid; rt, room temperature; SAv, streptavidin; (strept)avidin, signifies either avidin or streptavidin; t-Boc, tert-butoxycarbonyl; TCDI, thiocarbonyldiimidazole; TFA, trifluoroacetic acid; TFP, tetrafluorophenyl; TFP-OH, tetrafluorophenol; TFP-OTFA, tetrafluorophenyl trifluoroacetate. 2 “Endoradiotherapy” is a term used to describe an approach to radiotherapy in which a radiopharmaceutical, composed of a radionuclide coupled with a cancer cell targeting carrier molecule, is injected into a patient for cancer therapy. This approach is also termed “targeted radiotherapy”.

10.1021/bc025535r CCC: $22.00 © 2002 American Chemical Society Published on Web 07/13/2002

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labeled mAbs in this application (9). Those problems include long blood residence times, low concentrations of radioactivity in the tumor, and heterogeneous deposition of radioactivity in tumors. While encouraging results have been obtained using pretargeting protocols (10, 11), that approach has problems as well, including complexity of protocols, immunogenicity of SAv, and interference by endogenous biotin (12, 13). The difficulty of circumventing the problems associated with pretargeting protocols has led investigators to consider other approaches to improve the in vivo characteristics of directly radiolabeled mAbs. One approach being investigated, termed “extracorporeal affinity-adsorption” (EAA3), uses a device external to the body to remove radiolabeled mAbs from the patient’s blood (14). If efficient removal of radiolabeled mAbs from blood is obtained, use of such a device may permit administration of higher doses of radiolabeled mAbs. This is important, as higher doses could increase the total amount of radioactivity in the tumor and decrease the heterogeneity of radiolabeled mAb deposition in the tumor. The biotin/avidin capture system can be used in the EAA approach to remove directly labeled mAbs from blood (15-17). To achieve its removal in the EAA approach, the radiolabeled mAb must be biotinylated. Thus, the procedure involves administration of a biotinylated and radiolabeled mAb, which after sufficient time for tumor localization is removed from a patient’s blood by passing it through a device that contains a high flow rate, high capacity avidin column. Using the EAA approach, complete removal of the radiolabeled antibodies will not be obtained unless every antibody has at least one biotin moiety conjugated. Unfortunately, biotinylation in combination with radiolabeling of mAbs can result in a heterogeneous mixture of products. This is particularly true when radionuclides other than radioiodine are used because radionuclide chelation moieties must also be conjugated to the mAb. While multiple conjugates of radiometal chelates and biotin can improve radiolabeling efficiency and capture by avidin columns, they can also result in a dramatic decrease in mAb binding to the target antigen. As a means of circumventing this problem and the issue of high conjugation heterogeneity, we have investigated the design and synthesis of trifunctional reagents that contain a biotin moiety, a radiometal chelation moiety, and a functional group for protein conjugation. By combining the biotin moiety and the radiolabeling moiety into a single molecule, every mAb that is radiolabeled will also have a biotin on it. Further, heterogeneity of radiolabeled mAb is diminished, as the same number of biotin molecules as chelates is always present, and a minimum number of conjugation reagents or moieties can be used. In the studies, three trifunctional reagents were synthesized, conjugated with mAbs, tested for radiolabeling with 111In and 90Y, and evaluated for efficiency for capture by an avidin column. The results of the investigation are described herein. EXPERIMENTAL PROCEDURES

General. Solvents and chemicals obtained from commercial sources were analytical grade or better and were used without further purification. Solvents for HPLC analysis were obtained as HPLC grade and were filtered (0.2 µm) prior to use. d-Biotin, 4,7,10-trioxa-1,13-tridec3 The terms extracorporeal immunoadsorption (ECIA) and whole blood immunoadsorption (WBIA) have also been used for this approach.

Wilbur et al.

anediamine, 2,3,5,6-tetrafluorophenol, aminoisophthalate, di-tert-butyl dicarbonate, thiophosgene, 1,1′-thiocarbonyl-diimidazole, and most other chemicals were purchased from Aldrich Chemical Co. (Milwaukee, WI). L-Aspartic acid R-tert-butyl ester was obtained from Calbiochem-Novabiochem Corp. (San Diego, CA) or BACHEM California, Inc. (Torrance, CA). p-Isothiocyanatobenzyl-DOTA, 18, was purchased from Macrocyclics (www.macrocyclics.com, Dallas, TX). N-Hydroxysuccinimidyl biotinyl-6-aminocaproic acid (NHS-LC-biotin) and immobilized Avidin were purchased from Pierce (Rockford, IL). 2,3,5,6-Tetrafluorophenyl trifluoroacetate was prepared as previously described (18). High-purity, lowmetal HCl (Ultrex II) was obtained from J.T. Baker (Phillipsburg, NJ). High-purity NaOAc was obtained from Alfa Aesar (Ward Hill, MA). High-purity water was obtained from a Barnstead Easypure-Ultrapure water system. Microcon centrifugal filter devices, Microcon YM30, were obtained from Amicon/Millipore Corporation (Bedford, MA). Size exclusion columns, PD-10, were obtained from Amersham Pharmacia Biotech AB (Lund, Sweden). Silica gel chromatography was conducted with 70-230 mesh 60 Å silica gel (Aldrich Chemical Co.). Melting points were obtained in open capillary tubes on a Mel-Temp II apparatus with a Fluke 51 K/J electronic thermometer and are uncorrected. 1H NMR data, MS data, and HPLC chromatography were used to assess compound identity and purity (Data provided in the Supporting Information). Radioactive Materials. All reactions involving radioactive materials were conducted in a charcoal filteredPlexiglas enclosure within a fume hood. All radioactive materials were opened, handled, and stored in the Plexiglas enclosure until used in studies or removed as radiation waste. Standard radiation safety procedures were used. Radiation monitoring followed approved procedures. 111In and 90Y were obtained in 0.05 M HCl from NEN/ Dupont (now-Perkin-Elmer Life Sciences, Inc, Billerica, MA) in high specific activity. Na[125I]I was purchased from NEN/Dupont (Billerica, MA) as high-concentration/ high-specific-activity radioiodide in 0.1 N NaOH. Measurement of radioactivity was accomplished in a Capintec CRC-15R or a Logic Model 111B gamma counter (Abbott Labs, Chicago, IL). Spectral Analyses. All 1H NMR were obtained on either a Bruker AC-200 (200 MHz) or Bruker AF-301 (300 MHz) instrument. Proton chemical shifts are expressed as ppm using tetramethylsilane or 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, sodium salt as an internal standard (δ ) 0.0 ppm). Mass spectral data were obtained on either (1) a VG Analytical (Manchester, England) VG-70SEQ mass spectrometer with associated 11250J Data System using fast atom bombardment (FAB+) at 8 keV in a matrix of MeOH/DMIX (Thioglycerol/DMSO/TFAA: 90/9/1) or 3NBA (3-nitrobenzyl alcohol) or (2) a PerSeptive Biosystems Mariner Electrospray Time of Flight Mass Spectrometer (ESI-TOF). For ES+ analysis, the samples were dissolved in 50/50 MeOH/H2O and were introduced by an integral syringe infusion pump. Analytical Chromatography. Small Molecules. Reaction progress and purity of compounds were evaluated by reversed-phase HPLC analysis. HPLC separations were obtained on Hewlett-Packard quaternary 1050 gradient pumping system with a variable-wavelength UV detector (254 nm) and a Varex ELSD MKIII evaporative light-scattering detector. Analyses of the HPLC data were conducted on Hewlett-Packard HPLC ChemStation soft-

Metal-Chelating Biotinylation Reagents

ware. Reversed-phase HPLC chromatography was carried out using an Alltech Altima C-18 column (5 µm, 250 mm × 4.6 mm) with a gradient solvent system at a flow rate of 1 mL/min. The gradient mixture was composed of MeOH (solvent A) and 0.1% aqueous HOAc (solvent B). Starting with 40% MeOH, the initial solvent mixture was held for 2 min, increased to 100% MeOH over the next 10 min, and then held at 100% MeOH for 8 min. Retention times (tR) are provided with the compound experimental. Proteins. All antibody conjugates were analyzed by size exclusion HPLC analysis. Size exclusion HPLC analyses were conducted on a system consisting of a HewlettPackard 1050 Multiple Wavelength Detector (280 nm), isocratic pump, and a Protein-Pak glass 300SW column (7.5 mm × 300 mm, 10 µm; Waters Corp., Milford, MA). The mobile phase was an aqueous solution containing 50 mM potassium phosphate (pH 6.8), 300 mM NaCl, 1 mM EDTA, and 1 mM NaN3. A flow rate of 1.0 mL/min was used. Retention time for intact mAb is 8.4-8.6 min using this system. Radio-HPLC. Radiolabeled antibodies were analyzed by size exclusion HPLC. HPLC separations of radiolabeled conjugated antibodies were conducted on a system consisting of a Waters 510 pump, a Waters Lambda Max model 481 UV detector (280 nm), and a Beckman model 170 radioisotope detector. The radiolabeled mAbs were separated by size exclusion chromatography on a Bioselect 250-5 column (8 mm × 300 mm; 10 µm; Bio-Rad, Hercules, CA). The mobile phase was an aqueous solution containing 50 mM potassium phosphate (pH 6.8), 300 mM NaCl, 1 mM EDTA, and 1 mM NaN3. A flow rate of 1.0 mL/min was used. The retention time for the radiolabeled proteins (gamma trace) was approximately 10.8 min using this system. 5-N-tert-Boc-3-((15′-Biotinyl)-4′,7′,10′-Trioxapentadecanylamino)Isophthalamic Acid 2,3,5,6-Tetrafluorophenyl Ester, 5. N-(13-Amino-4,7,10-trioxatridecanyl)biotinamide (19), 4, (54 mg, 0.12 mmol) in anhydrous DMF (2.0 mL) was added over 20 min to a solution of N-tert-Boc-5-aminoisophthalate bis-tetrafluorophenyl ester (20), 3 (100 mg, 0.17 mmol), and Et3N (0.048 mL, 0.34 mmol) in anhydrous DMF (8 mL) at room temperature. After the mixture was stirred at room temperature for 1 h, volatile materials were removed under vacuum. The residue was purified by silica gel column (60 g) eluting with 10% MeOH/EtOAc to afford 51 mg (49%) of 5 as a colorless solid, mp 180-183 °C. 1H NMR (CD3OD, 200 MHz): δ 1.43 (m, 1H), 1.54 (s, 9 H), 1.67 (m, 3H), 1.90 (m, 2H), 2.18 (m, 2H), 2.69 (d, 2H), 2.92 (m, 1H), 4.28 (dd, 1H), 4.47 (dd, 1H), 7.49 (m, 1H), 7.83 (t, 1H), 7.97 (s, 2H); LRMS (ES+; M + H) 858.3. HRMS calcd for C39H52F4N5O10S (M + H)+: 858.3371. Found: 858.3374. HPLC: tR ) 14.0 min. 13-(Benzylthiourea-CHX-A′′)-4,7,10-Trioxatridecane-Diamine, 6. A quantity (10.4 mg, 0.018 mmol) of isothiocyanatobenzyl-CHX-A′′ DTPA (21), 17, in 2 mL of anhydrous DMF was added over 10 min to a solution of 4,7,10-trioxa-1,13-tridecanediamine (39 mg, 0.18 mmol) in 2 mL of anhydrous DMF at room temperature. After the addition was completed, the reaction mixture was stirred at room temperature for 30 min and then triturated with 40 mL of EtOAc. The product was filtered and washed with EtOAc/CHCl3 to afford 9.6 mg (67%) of 6 as a tacky material. 1H NMR (D2O, 200 MHz): δ 1.26 (t, J ) 7.3 Hz, 4H), 1.92 (m, 8H), 2.17 (m, 2H), 3.03 (t, 8H), 3.40 (m, 4H), 3.65 (m, 9H), 3.69 (s, 10H), 7.24 (d, J ) 8.0

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Hz, 2H), 7.39 (d, J ) 8.0 Hz, 2H); LRMS (FAB+; M + H) 815. HRMS calcd for C36H59N6O13S (M + H)+: 815.3861. Found: 815.3853. HPLC: tR ) 2.6 min. 5-N-tert-Boc-3-((15′-Biotinyl)-4′,7′,10′-Trioxapentadecanylamino)-1-((13-(Benzylthiourea-CHX-A′′)-4,7,10-Trioxatridecanediamino)Aminoisophthalic Acid, 7. A 15.3 mg quantity of 5 (0.018 mmol) in 1.0 mL of anhydrous DMF was added to a solution of 6 (9.6 mg, 0.012 mmol) and Et3N (5.0 µL, 0.036 mmol) in 1.5 mL of anhydrous DMF at room temperature. The solution was stirred at room temperature for 4 h and then triturated with 40 mL of EtOAc/CHCl3 to give 11.0 mg (62%) of 7 as an oil, which was used in the subsequent reaction without purification. 1H NMR (CD3OD, 200 MHz): δ 1.43 (s, 9H), 1.58 (m, 8H), 1.79 (m, 6H), 2.08 (t, 4H), 2.83 (m, 4H), 3.15 (t, 4H), 3.21 (m, 8H), 3.25 (m, 6H), 3.41 (m, 20H), 3.53 (m, 16H), 4.18 (dd, 1H), 4.38 (dd, 1H), 4.65 (m, 1H), 7.11 (m, 4H), 7.83 (m, 1H), 7.89 (m, 1H), 7.99 (m, 1H). LRMS (ES+) mass calcd for C69H107N11O22S2 (M + Na)+: 1528. Found: 1528. HPLC: tR ) 10.9 min. 5-Isothiocyanato-3-((15′-Biotinyl)-4′,7′,10′-Trioxapentadecanylamino)-1-((13-(Benzylthiourea-CHXA′′)-4,7,10-Trioxatridecanediamino)Aminoisophthalic Acid, 8. A solution containing 11.0 mg, (0.0073 mmol) of 7 in 0.5 mL TFA was stirred at room temperature for 30 min; then the volatile materials were removed under vacuum. The residue was redissolved in 2.0 mL of H2O; then thiophosgene (1.7 µL, 0.022 mmol) in 2.0 mL of CHCl3 was added, and that mixture was stirred at room temperature for 1 h. The excess thiophosgene and CHCl3 were evaporated in fume hood under a stream of argon. The remaining aqueous phase was evaporated to dryness under vacuum to afford the 9.2 mg (87%) of 8 as a colorless tacky material. 1H NMR (DMSO-d6, 200 MHz): δ 1.69 (m, 8H), 1.93 (m, 6H), 2.20 (m, 4H), 2.73 (t, 4H), 3.15 (t, 4H), 3.25 (m, 8H), 3.35 (m, 6H), 3.54 (m, 20H), 3.65 (m, 16H), 4.33 (m, 1H), 4.51 (m, 1H), 4.70 (m, 1H), 7.36 (m, 4H), 7.93 (m, 1H), 7.96 (m, 1H), 8.02 (m, 1H). LRMS (FAB+) mass calcd for C65H98N11O20S3K (M + K)+: 1487. Found: 1487. HPLC: tR ) 11.5 min. 1-((N-Biotinyl)-L-Aspartyl)-4,7,10-Trioxatridecane1,13-Diamine TFA Salt, 9. This compound was synthesized as previously reported (22). In the final synthetic step, a 396 mg quantity (0.55 mmol) of 13-N-tert-Boc-1((N-biotinyl)-L-aspartyl-R-tert-butyl ester)-4,7,10-trioxatridecane-1,13-diamine in 2 mL TFA was stirred at room temperature for 30 min, and then the volatile materials were removed under vacuum. The isolated 9 was used directly in the next reaction. 5-N-tert-Boc-3-((15′-((N-Biotinyl)-β-L-Aspartyl)-4′,7′,10′-Trioxapentadecanylamino)-Isophthalamic Acid 2,3,5,6-Tetrafluorophenyl Ester, 10. A solution of 9 (310 mg, 0.55 mmol), Et3N (0.23 mL, 1.65 mmol), and anhydrous DMF (5 mL) was dripped slowly into a solution of 3 (320 mg, 0.55 mmol) in anhydrous DMF (3 mL) at room temperature. The reaction mixture was stirred at room temperature for 30 min, and then volatile materials were removed under vacuum. The residue was purified by silica gel column (50 g) eluting with 10% MeOH/EtOAc to afford 190 mg (31%) of 10 as a colorless tacky material. 1HNMR (CD3OD, 200 MHz): δ 1.45 (m, 2H), 1.54 (s, 9 H), 1.70 (m, 8H), 1.91 (m, 2H), 2.25 (m, 2H), 2.59-3.01 (m, 2H), 3.11-3.33 (m, 6H), 3.45-3.70 (m, 11H), 4.32 (dd, J ) 4.4, 7.7 Hz, 1H), 4.50 (dd, J ) 4.4, 7.7 Hz, 1H), 4.65 (t, J ) 6.2 Hz, 1H), 7.51 (m, 1H), 8.25 (m, 2H), 8.48 (t, J ) 1.8 Hz, 1H); LRMS (ES+; M + Na) 995.4. HRMS calcd for C43H56F4N6O13SNa (M + Na)+: 995.3460. Found: 995.3393. HPLC: tR ) 13.8 min.

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3-((15′-(N-Biotinyl)-β-L-Aspartyl)-4′,7′,10′-Trioxapentadecanylamino)-1-((13-(Benzylthiourea-CHXA′′)-4,7,10-Trioxatridecanediamine)-Aminoisophthalic Acid, 11. Preparation A (Scheme 2). A solution of 6 (64 mg, 78.7 µmol) and Et3N (19 µL, 137 µmol) in anhydrous DMF (3 mL) was added dropwise to a solution of 10 (76 mg, 78.7 µmol) in anhydrous DMF (2 mL) at room temperature. The reaction mixture was stirred at room temperature for 1h; then DMF was removed under vacuum. The residue was redissolved in 2 mL of neat TFA and stirred at room temperature for 10 min. After that time, TFA was removed under vacuum, and the crude product was purified on a Sephadex G-15 column eluting with water. This provided 100 mg (83%) of 11 as a colorless tacky material. 1H NMR (D2O, 200 MHz): δ 0.86 (t, J ) 6.6 Hz, 4H), 1.25 (m, 8H), 1.41-1.83 (m, 16H), 2.08 (m, 4H), 2.56 (m, 4H), 2.80 (m, 1H), 3.07 (m, 6H), 3.17-3.93 (m, 34H), 4.14 (m, 2H), 4.28 (m, 1H), 6.38 (m, 2H), 6.52 (m, 2H), 7.13 (s, 2H), 7.40 (m, 1H), 8.40 (m, 2H). LRMS (FAB+) mass calcd for C68H105N12O23S2 (M + H)+: 1521. Found: 1521. HPLC: tR ) 7.9 min. Preparation B (Scheme 3). A quantity (200 mg, 0.18 mmol) of 15 was dissolved in 2 mL of neat TFA, and the solution was stirred for 10 min at room temperature. The TFA was removed under vacuum, and the residue was dissolved in H2O (4 mL). To that solution was added Na2CO3 (140 mg, 1.30 mmol), 17 (80 mg, 0.13 mmol), H2O (4 mL), and DMF (4 mL). The reaction mixture was stirred at room temperature for 4 h and then neutralized with HOAc. The neutralized solution was placed on a gel filtration column (Sephadex G-15, 2.5 × 50 cm) and eluted with H2O to provide 157 mg (77%) of 11 with physical and spectral properties identical to those reported above. 1-Isothiocyanato-3-((15′-(N-Biotinyl)-β-L-Aspartyl)4′,7′,10′-Trioxapenta-Decanylamino)-1-((13-(Benzylthiourea-CHX-A′′)-4,7,10-Trioxatridecanediamine)Aminoisophthalic Acid, 12. Thiophosgene (25 µL, 0.33 mmol) in 3 mL of CHCl3 was added to a 3 mL aqueous solution of 11 (100 mg, 66 µmol) at room temperature. The mixture was stirred at room temperature for 1 h; then excess thiophosgene and CHCl3 were evaporated in fume hood under a stream of argon. The aqueous phase was evaporated to dryness under vacuum to afford the 84 mg (82%) of 12 as a light yellow tacky material. 1H NMR (D2O, 200 MHz): δ 0.86 (t, J ) 6.6 Hz, 4H), 1.25 (m, 8H), 1.44-1.82 (m, 16H), 2.09 (m, 4H), 2.54 (m, 4H), 2.81 (2d, J ) 5.1 Hz, 1H), 3.09 (m, 6H), 3.27-3.83 (m, 34H), 4.12 (m, 1H), 4.30 (m, 1H), 4.49 (m, 1H), 7.31 (d, 2H), 7.44 (d, 2H), 7.87 (t, 1H), 7.97 (s, 2H), 8.03 (d, J ) 7.0 Hz, 1H), 8.30 (s, 1H), 8.72 (t, J ) 5.5 Hz, 2H). LRMS (FAB+) mass calcd for C69H103N12O23S3 (M + H)+: 1563. Found: 1563. HPLC: tR ) 11.7 min. 3-(13′-N-tert-Boc)-Trioxatridecanediamine-5-Ntert-Boc-Aminoisophthalate TFP Ester, 14. N-tertBoc-trioxadiamine (23), 13 (0.44 g, 1.39 mmol), and Et3N (0.29 mL, 2.08 mmol) in anhydrous DMF (5 mL) were added dropwise over 3 h to a solution of N-tert-Bocaminoisophthalate di-TFP ester, 3 (0.80 g, 1.39 mmol), in anhydrous DMF (5 mL) at room temperature. After the addition was completed, the mixture was stirred at room temperature for another 30 min, and then the volatile materials were evaporated under vacuum. The product was purified by silica gel column (40 g) eluting with 50% EtOAc/Hexane to afford 0.74 g (73%) of 14 as a colorless oil. 1H NMR (CDCl3, 200 MHz): δ 1.42 (s, 9H), 1.54 (s, 9H), 1.69 (m, 2H), 1.77 (s, 1H), 1.91 (m, 2H), 2.92 (d, J ) 14.7 Hz, 1H), 3.17 (m, 2H), 3.42-3.51 (m, 4H), 3.56-3.72 (m, 8H), 4.89 (t, J ) 5.1 Hz, 1H), 6.97-7.14

Wilbur et al.

(m, 1H), 7.51 (s, 1H), 7.57 (s, 1H), 8.01 (s, 1H), 8.28 (t, J ) 1.5 Hz, 1H), 8.63 (s, 1H); LRMS (ES+; M + Na) 754.3. HRMS calcd for C34H45F4N3O10Na (M + Na)+: 754.2939. Found: 754.2941. HPLC: tR ) 14.8 min. 3-(13′-N-tert-Boc)Trioxatridecanediamine-1-(13′′Biotin-Asp-OH)Trioxadiamine-5-N-tert-Boc-Aminoisophthalate, 15. Biotin-Asp-OH-trioxadiamine TFA salt, 9 (0.32 g, 0.48 mmol), was added to a solution containing 14 (0.35 g, 0.48 mmol) and Et3N (0.15 mL, 1.05 mmol) in anhydrous DMF (5 mL). The resultant mixture was stirred at room temperature for 1 h. Volatile materials were removed under vacuum and the residue was purified by silica gel column (40 g, 50% MeOH/ EtOAc) to yield 0.45 g (83%) of 15 as a tacky oil. 1H NMR (CD3OD, 200 MHz): δ 1.41 (s, 9H), 1.53 (s, 9H), 1.631.74 (m, 6H), 1.86-1.92 (m, 4H), 2.18-2.29 (m, 2H), 2.58 (dd, J ) 8.0, 14.8 Hz, 1H), 2.68 (d, J ) 12.4 Hz, 1H), 2.74 (dd, J ) 4.9, 14.8 Hz, 1H), 2.91 (dd, J ) 4.9, 13.0 Hz, 1H), 3.09 (t, J ) 6.8 Hz, 2H), 3.16-3.20 (m, 1H), 3.22 (t, J ) 6.8 Hz, 2H), 3.30-3.31 (m, 4H), 3.44-3.54 (m, 12H), 3.59-3.66 (m, 16H), 4.30 (dd, J ) 4.3, 8.0 Hz, 1H), 4.48 (dd, J ) 4.3, 8.0 Hz, 1H), 4.55 (dd, J ) 4.9, 7.4 Hz, 1H), 7.84 (t, J ) 1.2 Hz, 1H), 7.99 (t, J ) 1.9 Hz, 1H), 8.02 (t, J ) 1.9 Hz, 1H). LRMS (ES+) calcd for C52H87N8O17S (M + H)+: 1128. Found: 1128. HPLC: tR ) 11.4 min. 3-(13′-ThioureabenzylDOTA)Trioxadiamine-1-(13′′Biotin-Asp-OH)Trioxadiamine-5-Aminoisophthalate, 19. A 34 mg quantity (0.03 mmol) of 15 was dissolved in 1 mL of neat TFA and stirred at room temperature for 10 min. The TFA was removed under a stream of Ar, and the residue was redissolved in H2O (1 mL). To that solution was added sodium carbonate (31.8 mg, 0.3 mmol) and DOTA-benzyl-NCS, 18 (20 mg, 0.036 mmol) in DMF (1 mL). The reaction mixture was stirred at room temperature for 4 h, neutralized with HOAc, and purified by gel filtration (Sephadex G-15) eluting with water to give 33 mg (74%) of 19. 1H NMR (D2O): δ 1.241.45 (m, 3H), 1.47-1.99 (m, 14H), 2.26 (t, J ) 7.1 Hz, 3H), 2.52 (dd, J ) 10.0, 14.9 Hz, 1H), 2.69-2.82 (m, 1H), 2.95 (dd, J ) 4.4, 13.2 Hz, 1H), 3.11-3.30 (m, 17H), 3.423.71 (m, 38H), 4.37 (dd, J ) 4.0, 7.7 Hz, 1H), 4.48-4.60 (m, 2H), 7.18-7.31 (m, 5H), 7.38 (s, 1H), 7.45 (t, J ) 1.5 Hz, 1H). LRMS (ES+) calcd for C66H104N13O21S2 (M + H)+: 1479. Found: 1479. LRMS calcd for C66H103N13NaO21S2 (M + Na)+: 1501. Found: 1501. HPLC: tR ) 8.2 min. 3-(13′-ThioureabenzylDOTA)Trioxadiamine-1-(13′′Biotin-Asp-OH)Trioxadiamine-5-Isothiocyanato-Aminoisophthalate, 20. 1,1′-Thiocarbonyldiimidazole (3.6 mg, 0.02 mmol) was added to a solution of 19 (25 mg, 0.017 mmol) in anhydrous DMF (1 mL). The reaction mixture was stirred at room temperature for 1 h; then 30 mL of ether was added and stirred for another 20 min. Solvents were carefully decanted, and the residue was washed with ether (2 × 20 mL) to afford the 21 mg (82%) of 20 as light-yellow tacky solid. 1H NMR (D2O): δ 1.251.44 (m, 3H), 1.50-1.99 (m, 14H), 2.20-2.31 (m, 3H), 2.53 (dd, J ) 9.9, 14.7 Hz, 1H), 2.66-2.83 (m, 1H), 2.91-3.00 (m, 4H), 3.13-3.34 (m, 14H), 3.46-3.71 (m, 38H), 4.39 (dd, J ) 4.7, 8.1 Hz, 1H), 4.48-4.61 (m, 2H), 7.18-7.38 (m, 4H), 7.47 (s, 1H), 7.80 (s, 1H), 8.02 (s, 1H). LRMS calcd for C67H102N13O21S3 (M + H)+: 1521. Found: 1521. LRMS calcd for C67H101N13NaO21S3 (M + Na)+: 1543. Found: 1543. HPLC: tR ) 11.0 min. Antibody Demetalation and Conjugation. The following procedure is an example; other quantities of the two antibodies (the antirenal cell antibody A6H (24) and the anti-PSMA antibody 107-1A4 (25)) and conjugation

Metal-Chelating Biotinylation Reagents

reagents were also used in the studies. (a) Reagent Preparation. Preparation of low-metal reagents and buffers used was as previously described (26). (b) Demetalation. A 5 mg quantity of a monoclonal antibody was placed in a Slide-A-Lyzer 10K dialysis cassette (Pierce) and dialyzed against 1L metal free HEPES with a minimum of 5 buffer changes over 3 days at 4 °C. To each buffer change solution was added 5 g of Chelex-100 resin. The antibody was then removed from the dialysis cassette and placed in an acid washed microcentrifuge tube. Care was taken to avoid the introduction of metals to the final solution. The demetalated mAb was stored at 4-8 °C until used in conjugation reactions. (c) Conjugation with A6H. (1) A stock solution of 8 was prepared by dissolving 2 mg in 150 µL of 10% aqueous DMSO4. Aliquots of that solution were diluted with additional 10% aqueous DMSO to obtain 5, 10, 25, or 50 equiv in 15 µL. Then, 15 µL aliquot of the solutions containing the varying quantities of 8 were added to vials containing 150 µL of a 0.67 mg/mL (100 µg) of A6H in HEPES buffer at pH 8.5 for 16-18 h. After the reaction period, the A6H conjugates were purified by elution over a size exclusion column (Sephadex G-25, PD-10). The quantity of 8 conjugated with A6H was determined from the HABA and UV/BCA analyses as described in the following section. The analyses indicated that 0.4-11.5 conjugates of 8 were obtained per A6H molecule, depending on the amount reacted. (2) A stock solution of 8 was prepared by dissolving 1.3 mg in 58 µL of DMSO. Then 30 µL of the DMSO solution of 8 (15 equiv) was added to a vial containing 1.2 mL of a 3.87 mg/mL solution of A6H in HEPES buffer at pH 8.5. The reaction mixture was stirred at room temperature overnight. After conjugation, the reaction mixture was placed in a dialysis cassette and was dialyzed against 3 × 1L of metal free citrate buffer (50 mM Na citrate, 150 mM NaCl, 0.05% NaN3, pH 5.5) containing 5 g Chelex-100, followed by 2 × 1L metal free saline. The A6H conjugate had 2.9 conjugates of 8 per mAb. 107-1A4. Stock solutions of the trifunctional biotinylation reagents 12 and 20 were made in water. Aliquots of the stock solutions were diluted with additional water to obtain reagent solutions that contained 10, 25, or 50 equiv in 100 µL. Stock solutions of NHS-LC-biotin (1.15 mg in 38 µL), isothiocyanatobenzyl-CHX-A′′, 17 (1.15 mg in 29 µL), and isothiocyanatobenzyl-DOTA, 18 (1.15 mg in 34 µL), were prepared in DMSO. The conjugation reactions were conducted as follows: To 303 µL of a 6.6 mg/mL solution of demetalated 107-1A4 (2 mg, 13.3 nmol) was added the appropriate volume (2 µL/10 equiv; 5 µL/ 25 equiv; 10 µL/50 equiv) of a DMSO solution containing NHS-LC-biotin, 12, 17, 18, or 20. After conjugation, the reaction mixtures were placed in dialysis cassettes and were dialyzed against 3 × 1L of metal free citrate buffer containing 5 g Chelex-100, followed by 2 × 1L metal free saline. The demetalated conjugated antibody (in saline) was stored at 4-8 °C until used in radiolabeling experiments. 4 Due to their low water solubility, some of the reagents (i.e., 17, 18, and NHS-LC-biotin) were dissolved in DMSO prior to addition to the aqueous solutions. It should be noted that the trifunctional reagent 8 is soluble in water so the 10% aqueous DMSO does not have to be used. Addition of 8 in 10% aqueous DMSO solution for conjugation with A6H, and additions of 17, 18 and NHS-LC-biotin in neat DMSO for conjugation with 1071A4, resulted in conjugation solutions that had