Biotin Reagents for Antibody Pretargeting. 5. Additional Studies of

D. Scott Wilbur,* Donald K. Hamlin, Ming-Kuan Chyan, Brian B. Kegley, and Pradip M. Pathare. Department of Radiation Oncology, University of Washingto...
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Bioconjugate Chem. 2001, 12, 616−623

Biotin Reagents for Antibody Pretargeting. 5. Additional Studies of Biotin Conjugate Design To Provide Biotinidase Stability D. Scott Wilbur,* Donald K. Hamlin, Ming-Kuan Chyan, Brian B. Kegley, and Pradip M. Pathare Department of Radiation Oncology, University of Washington, Seattle, Washington 98195. Received January 31, 2001; Revised Manuscript Received April 27, 2001

An investigation was conducted in which the stabilities of four structurally different biotin derivatives were assessed with regard to biotinamide bond hydrolysis by the enzyme biotinidase. The biotin derivatives studied contained an extra methylene in the valeric acid chain of biotin (i.e., homobiotin), or contained conjugated amino acids having hydroxymethylene, carboxylate, or acetate functionalities on a methylene alpha to the biotinamide bond. The biotinidase hydrolysis assay was conducted on biotin derivatives that were radioiodinated at high specific activity, and then subjected to diluted human serum at 37 °C for 2 h. After incubation, assessment of biotinamide bond hydrolysis by biotinidase was readily achieved by measuring the percentage of radioactivity that did not bind with avidin. As controls, an unsubstituted biotin derivative which is rapidly cleaved by biotinidase and an N-methyl-substituted biotin derivative which is stable to biotinidase cleavage were included in the study. The results indicate that increasing the distance from the biotin ring structure to the biotinamide bond by one methylene only decreases the rate of biotinidase cleavage, but does not block it. The data obtained also indicate that placing a hydroxymethylene, carboxylate, or acetate alpha to the biotinamide bond is effective in blocking the biotinamide hydrolysis reaction. These data, in combination with data previously obtained, which indicate that biotin derivatives containing hydroxymethylene or carboxylate moieties retain the slow dissociation rate of biotin from avidin and streptavidin [Wilbur, D. S., et al. (2000) Bioconjugate Chem. 11, 569-583], strongly support incorporation of these structural features into biotin derivatives being used for in vivo targeting applications.

INTRODUCTION

Radiolabeled derivatives of biotin are being developed for application to “pretargeting” in targeted radiotherapy of cancer (1-5). A design criterion for radiolabeled biotin derivatives used in vivo is that they must be stabilized toward degradation by the enzyme biotinidase (6). Biotinidase is present in serum and tissues of both animals and humans in nanomolar concentrations (6). It is believed that the primary function of this enzyme is to cleave the biotinamide bond linking biotin (vitamin H) and lysine in biocytin (7, 8), such that the essential vitamin can be recycled. Biocytin is released after peptidase degradation of carboxylase enzymes containing biotin (9). An important finding is that biotinidase is not specific for cleaving the biotinamide bond in biocytin (10). This fact led us, and other investigators (11-14), to evaluate the effect of structural changes in biotin derivatives on blocking the biotinamide cleaving action of biotinidase. It was previously reported that an N-methylbiotinamide functionality, introduced by conjugation of biotin with N-methylglycine (sarcosine), completely blocked biotinidase cleavage of the biotinamide bond (12). Studies in our laboratory confirmed that biotin conjugated with sarcosine, and other N-methyl linker molecules, blocked the actions of biotinidase in mouse and human serum (15). In this investigation, we have extended our previous studies evaluating variations in functional groups in * Address correspondence to this author at the Department of Radiation Oncology, University of Washington, 2121 N. 35th St., Seattle, WA 98103-9103. Phone: 206-685-3085. FAX: 206685-9630. E-mail: [email protected].

biotin conjugates for blocking biotinidase activity. The studies were primarily directed at biotin conjugates which contain hydroxymethylene and carboxylate substituents alpha to the biotinamide bond (e.g., 4b and 5b). However, two other biotin derivatives were prepared to provide a better understanding of the structural features required for blocking biotinidase cleavage, and two more biotin derivatives were prepared as standards. Similar to the hydroxymethylene and carboxylate derivatives, one of the biotin derivatives tested contained an acetate substituent alpha to the biotinamide bond (6b). In another biotin derivative, there was an additional methylene in the valeric side chain of biotin (i.e., homobiotin derivative 3c). As standards for comparison, an unmodified biotin derivative that is rapidly cleaved by biotinidase (1c) and a N-methylbiotinamide derivative (2c) which is stable to cleavage by biotinidase were included in the study. As in previously studied biotin derivatives, 4,7,10-trioxatridecane-1,13-diamine was incorporated as a linker to aid in water solubilization (16). All of the biotin derivatives tested were radioiodinated to high specific activity to permit evaluation of the biotinidase cleavage. Reported herein are: (a) the syntheses of precursors to the radioiodinated biotin derivatives targeted for testing, (b) syntheses of iodinated HPLC standards, (c) radioiodination methods used, and (d) results obtained from the biotinidase assay. EXPERIMENTAL PROCEDURES

General. All chemicals purchased from commercial sources were analytical grade or better and were used without further purification unless noted. Most of the reagents used were obtained from Aldrich Chemical Co.

10.1021/bc0100096 CCC: $20.00 © 2001 American Chemical Society Published on Web 06/07/2001

Biotinidase-Stabilized Biotin Derivatives

(Milwaukee, WI). Biotin was obtained from Sigma Chemicals (St. Louis, MO). 4,7,10-Trioxa-1,13-tridecanediamine and sarcosine methyl ester were obtained from Fluka (St. Louis, MO). 2,3,5,6-Tetrafluorophenyl trifluoroacetate (TFP-OTFA)1 was prepared as previously described (17). O-tert-Butyl-L-serine methyl ester hydrochloride, L-aspartic acid β-tert-butyl ester, and L-aspartic acid R-tert-butyl ester were obtained from CalbiochemNovabiochem Corp. (San Diego, CA). Solvents for HPLC analysis were obtained as HPLC grade and were filtered (0.2 µm) prior to use. Silica gel chromatography was conducted with 70-230 mesh 60 Å silica gel (Aldrich Chemical Co.). Microcon-30 centrifugation concentrators were obtained from Amicon (Beverly, MA). 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. Radioactive Materials. Standard methods for safely using radionuclides of iodine were employed (18). Na125I was purchased from NEN/Dupont (Billerica, MA) as highpH, high-concentration solutions in 0.1 N NaOH. Measurement of 125I was accomplished on the Capintec CRC15R or a Capintec CRC-6A Radioisotope Calibrator. Radioactive samples were counted in a LOGIC Model 111B Gamma Counter (Abbott Laboratories, Chicago, IL). Spectral Analyses. 1H NMR were obtained on either a Bruker AC-200 (200 MHz) or a Bruker AC-500 (500 MHz) instrument. Chemical shifts are expressed as ppm using tetramethylsilane as internal standard (δ ) 0.0 ppm). IR data were obtained on a Perkin-Elmer 1420 infrared spectrophotometer. Mass spectral data (both low resolution and high resolution) of most compounds prepared in this study were obtained on a PerSeptive Biosystems Mariner Electrospray Time-of-Flight Mass Spectrometer (ESI-TOF). For analysis, the samples were dissolved in 50:50 MeOH/H2O and were introduced by an integral syringe infusion pump. Some mass spectral data (low resolution) were obtained on a VG Analytical (Manchester, England) VG-70 SEQ mass spectrometer with associated 11250J Data System. These mass spectral data were obtained by fast atom bombardment (FAB+) in a matrix of 2-hydroxyethyl disulfide (2-HEDS) and poly(ethylene glycol) 300 or 600 containing thioglycolate. High-resolution mass spectral data were used for compound identification. Compounds containing Sn isotopes had five matching isotopic masses, but only one is provided as identification. 1H NMR and HPLC were used to assess the purity of nonradioactive compounds (these data are provided in the Supporting Information). Chromatography. HPLC separations of nonradioactive compounds were obtained on a 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 HewlettPackard HPLC ChemStation software. All reactions were monitored by HPLC. Reversed-phase HPLC chromatography was carried out using an Alltech Altima C-18 column (5 µm, 250 mm × 4.6 mm) using a gradient solvent system at a flow rate of 1 mL/min. A gradient of 1 Abbreviations: Av, avidin; BSA, bovine serum albumin; cpm, counts per minute; DIP, direct insertion probe; EI, electron impact; 2-HEDS, 2-hydroxyethyl sulfide; HOHgBz, hydroxymercuribenzoic acid; nca, no-carrier-added; NCS, N-chlorosuccinimide; NHS, N-hydroxysuccinimide; PBS, phosphate-buffered saline; rt, room temperature; SAv, streptavidin; t-Boc, tertbutyloxycarbonyl; TFA, trifluoroacetic acid; TFP, tetrafluorophenyl; TFP-OTFA, tetrafluorophenyl trifluoroacetate.

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MeOH and H2O/0.1% HOAc was used. Starting with 40% MeOH, the initial solvent mixture was held for 2 min; then the gradient was increased to 100% MeOH over the next 10 min, then held at 100% MeOH for 8 min. Retention times (tR) under these conditions for biotin conjugates are provided with the compound experimental data. HPLC separations of radioiodinated biotin derivatives were conducted using a gradient system which consisted 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 separations were obtained using HPLC conditions described for gradient I, above, except water was used in place of H2O/0.1% HOAc. The following compounds were prepared as previously described: 1a, 1b, [125I]1c, 2a, 2b, [125I]2c, (15); 7 (19); 8a, 8b (15); 10, 11, 12, 13 (19); 20 (20); 27 (21). BiotinNHS ester, 9, was provided as a gift from Quanta BioDesign (Powell, OH). N-(13-(p-Tri-n-butylstannylbenzoylamino)-4,7,10trioxatridecanyl)homobiotinamide, 3a. To a 0.203 g (0.50 mmol) quantity of the TFP ester of homobiotin, 7, dissolved in 10 mL of DMF was added 70 µL (50.8 mg, 0.50 mmol) of Et3N, then 0.307 g (0.51 mmol) of 8a. The reaction was stirred at room temperature for 1 h, and the DMF was removed under vacuum. The crude residue was loaded onto a silica column and eluted with EtOAc, and then mixtures of EtOAc/MeOH. The product eluted with 4:1 EtOAc/MeOH. Evaporation of the solvent provided 0.380 g (0.44 mmol, 89%) of 3a as a colorless solid, mp 106-107 °C. 1H NMR 500 MHz (CD3OD): 0.88 (t, J ) 7.0 Hz, 9H), 1.10 (t, J ) 8.1 Hz, 6H), 1.30-1.45 (m, 12H), 1.53-1.63 (m, 10H), 1.68-1.75 (m, 2H), 1.85-1.90 (m, 2H), 2.16 (t, J ) 7.3 Hz, 2H), 2.69 (d, J ) 13.3 Hz, 1H), 2.90 (dd, J ) 5.1, 12.5 Hz, 1H), 3.15-3.19 (m, 1H), 3.23 (t, J ) 6.6 Hz, 2H), 3.47 (t, J ) 6.6 Hz, 4H), 3.523.54 (m, 2H), 3.57-3.64 (m, 6H), 4.27 (dd, J ) 4.4, 8.1 Hz, 1H), 4.46 (dd, J ) 4.4, 8.1 Hz, 1H), 7.54 (d, J ) 8.1 Hz, 2H), 7.74 (d, J ) 8.1 Hz, 2H). ΗRMS calcd for C40H71N4O6SSn (M + H)+: 853.4090. Found: 853.4112 (Sn-118). HPLC: tR ) 16.9 min. N-(13-(p-Iodobenzoylamino)-4,7,10-trioxatridecanyl)homobiotinamide, 3b. To a 0.244 g (0.60 mmol) quantity of the TFP ester of homobiotin, 7, dissolved in 10 mL of DMF was added 84 µL (61 mg, 0.60 mmol) of Et3N, then 0.268 g (0.61 mmol) of 8b. The reaction was stirred at room temperature for 1 h, and the DMF was removed under vacuum. The crude residue was loaded onto a silica column and eluted with EtOAc, then mixtures of EtOAc/MeOH. The product eluted with 4:1 EtOAc/MeOH. Evaporation of the solvent provided 0.30 g (0.43 mmol, 72%) of 3b as a colorless solid, mp 124125 °C. 1H NMR 500 MHz (CD3OD): δ 1.33-1.45 (m, 6H), 1.54-1.62 (m, 4H), 1.73 (t, J ) 6.6 Hz, 2H), 1.87 (t, J ) 6.3 Hz, 2H), 2.16 (t, J ) 7.3 Hz, 2H), 2.69 (d, J ) 13.2 Hz, 1H), 2.91 (dd, J ) 5.2, 12.5 Hz, 1H), 3.15-3.18 (m, 1H), 3.23 (t, J ) 7.0 Hz, 2H), 3.44-3.49 (m, 4H), 3.53-3.63 (m, 8H), 4.28 (dd, J ) 4.4, 8.1 Hz, 1H), 4.47 (dd, J ) 4.4, 8.1 Hz, 1H), 7.56 (d, J ) 8.8 Hz, 2H), 7.83 (d, J ) 8.1 Hz, 2H). HRMS calcd for C28H44N4O6SI (M + H)+: 691.2026. Found: 691.2051. HPLC: tR ) 12.3 min. Preparation of Biotin Derivatives 17-19 and 2123. A 0.24 mmol quantity of a biotin-amino acid derivative (11, 12, or 13) was combined with 50 µL (0.36 mmol) of Et3N in 5 mL of DMF. The mixture was cooled to 4 °C, 69 mg (0.27 mmol) of TFP-OTFA was added, and then the mixture was allowed to stir for 30 min at 4 °C. An additional 40 µL (0.29 mmol) of Et3N was added, followed

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by addition of 0.24 mmol of 8b or 20. The reaction mixture was allowed to come to room temperature and was stirred for an additional hour at that temperature. The mixture was concentrated under vacuum, and purified over a silica gel column (2.5 cm × 22 cm). The column was first eluted with EtOAc, then with increasing amounts (i.e., 10-40%) of methanol in EtOAc. 2-(N-Biotinoyl)-1-(N′-(13′-p-iodobenzoylamino)4′,7′,10′-trioxatridecaneamino)- L -serine-O-tertbutyl Ether, 17. This compound was obtained as a colorless oil in 45% yield. 1H NMR 200 MHz (CD3OD): δ 1.17 (s, 9H), 1.44 (m, 4H), 1.67 (m, 6H), 1.87 (m, 2H), 2.29 (t, J ) 7.3 Hz, 2H), 2.69 (d, 2H), 2.90 (dd, J ) 5.1, 12.5 Hz, 1H), 3.30 (m, 6H), 3.55 (m 10H), 4.30 (m, 1H), 4.39 (m, 1H), 4.48 (m, 1H), 7.56 (d, J ) 8.4 Hz, 2H), 7.83 (d, J ) 8.8 Hz, 2H). HRMS calcd for C34H55IN5O8S (M + H)+: 820.2816. Found: 820.2806. HPLC: tR )13.2 min. 2-(N-Biotinoyl)-1-(N′-(13′-p-iodobenzoylamino)4′,7′,10′-trioxatridecaneamino)-L-aspartate-4-O-tertbutyl Ester, 18. This compound was obtained as a colorless solid in 65% yield, mp 165-167 °C. 1H NMR 200 MHz (CD3OD): δ 1.43 (s, 9H), 1.72 (m, 3H), 1.87 (m, 3H), 2.26 (t, J ) 6.9 Hz, 1H), 2.59 (m, 2H), 2.79 (d, J ) 6.2 Hz, 2H), 2.89 (d, J ) 4.7 Hz, 1H), 2.95 (d, J ) 4.7 Hz, 1H), 3.31 (m, 8H), 3.51 (m, 4H), 3.61 (m, 8H), 4.30 (m, 1H), 4.48 (m, 1H), 4.66 (m, 1H), 7.56 (d, J ) 8.4 Hz, 2H), 7.83 (d, J ) 8.4 Hz, 2H). 1HRMS calcd for C35H55IN5O9S (M + H)+: 848.2765. Found: 848.2742. HPLC: tR ) 12.9 min. 2-(N-Biotinoyl)-4-(N′-(13′-p-iodobenzoylamino)4′,7′,10′-trioxatridecaneamino)-L-aspartate-1-O-tertbutyl Ester, 19. This compound was obtained as a colorless oil in 35% yield. 1H NMR 200 MHz (CD3OD): δ 1.44 (s, 9H), 1.71 (m, 3H), 1.89 (m, 3H), 2.24 (t, J ) 7.3 Hz, 1H), 2.68 (m, 2H), 2.86 (d, J ) 3.3 Hz, 1H), 2.99 (d, J ) 3.3 Hz, 1H), 3.30 (m, 8H), 3.49 (m, 4H), 3.62 (m, 8H), 4.30 (m, 1H), 4.41 (m, 1H), 4.63 (m, 1H), 7.57 (d, J ) 8.8 Hz, 2H), 7.84 (d, J ) 8.8 Hz, 2H). HRMS calcd for C35H55IN5O9S (M + H)+: 848.2765. Found: 848.2767. HPLC: tR ) 12.8 min. 2-(N-Biotinoyl)-1-(N′-(13′-tert-butoxycarbonylamino)-4′,7′,10′-trioxatridecaneamino)-L-serine-O-tertbutyl Ether, 21. This compound was obtained as a colorless oil in 82% yield. 1H NMR 200 MHz (CD3OD): δ 1.18 (s, 9H), 1.43 (s, 9H), 1.72 (m, 10H), 2.30 (t, J ) 6.9 Hz, 2H), 2.70 (d, 1H), 2.94 (dd, J ) 5.1, 12.5 Hz, 1H), 3.12 (t, J ) 6.9 Hz, 2H), 3.22 (m, 1H), 3.30 (m, 4H), 3.35 (s, 2H), 3.52 (m, 4H), 3.61 (m, 6H), 4.30 (m, 1H), 4.40 (t, J ) 5.5 Hz, 1H), 4.49 (m, 1 H). HRMS calcd for C32H60N5O9S (M+H)+: 690.4112. Found: 690.4117. HPLC: tR ) 12.5 min. 2-(N-Biotinoyl)-1-(N′-(13′-tert-butoxycarbonylamino)-4′,7′,10′-trioxatridecaneamino)-L-aspartate-4-Otert-butyl Ester, 22. This compound was obtained as a colorless oil in 52% yield. 1H NMR 200 MHz (CD3OD): δ 1.44 (s, 18H), 1.74 (m, 8H), 2.09 (t, 2H), 2.54 (m, 2H), 2.79 (d, J ) 6.2 Hz, 1H), 2.89 (d, J ) 4.7 Hz, 1H), 2.95 (d, J ) 4.7 Hz, 1H), 3.11(t, J ) 6.9 Hz, 2H), 3.31 (m, 8H), 3.51 (t, J ) 6.2 Hz, 3H), 3.61 (m, 8H), 4.30 (m, 1H), 4.48 (m, 1H), 4.66 (m, 1H). HRMS calcd for C33H60N5O10S (M + H)+: 718.4061. Found: 718.4045. HPLC: tR ) 12.5 min. 2-(N-Biotinoyl)-4-(N′-(13′-tert-butoxycarbonylamino)-4′,7′,10′-trioxatridecaneamino)-L-aspartate-1-Otert-butyl Ester, 23. This compound was obtained as a colorless oil in 59% yield. 1H NMR 200 MHz (DMSO-d6): δ 1.37 (s, 18H), 1.50 (m, 2H), 1.58 (m, 8H), 2.09 (t, 2H), 2.44 (m, 4H), 2.54 (m, 2H), 2.82 (m, 1H), 2.95 (m, 2H), 3.07 (m, 2H), 3.48 (m, 10H), 4.13 (m, 1 H), 4.31 (m, 1 H),

Wilbur et al.

4.42 (m, 1 H), 6.39 (d, J ) 10.6 Hz, 2H), 6.75 (t, 5.9 Hz, 1H), 7.88 (t, J ) 5.5 Hz, 1H), 8.04 (d, J ) 7.7 Hz, 1H). HRMS calcd for C33H60N5O10S (M + H)+: 718.4061. Found: 718.4060. HPLC: tR ) 12.3 min. Preparation of 4a, 5a, and 6a. For each of the p-iodo compounds 17, 18, and 19, 2 mL of neat TFA was added and stirred for 10 min (the serine compound 17 was stirred for 30 min). The mixture was evaporated and dried on high vacuum overnight (yield ∼99%). The isolated biotin derivatives were washed several times with ether to remove any remaining TFA. 2-(N-Biotinoyl)-1-(N′-(13′-p-iodobenzoylamino)4′,7′,10′-trioxatridecaneamino)-L-serine, 4a. This compound was obtained in nearly quantitative yield as a colorless oil. 1H NMR 200 MHz (CD3OD): δ 1.65 (s, 2H), 1.44 (m, 4H), 1.67 (m, 6H), 1.87 (m, 2H), 2.29 (t, J ) 7.3 Hz, 2H), 2.69 (d, J ) 12.5 Hz, 2H), 2.90 (dd, J ) 4.7, 12.5 Hz, 1H), 3.01 (d, 1H), 3.30 (m, 6H), 3.55 (m, 10H), 4.30 (m, 1H), 4.39 (m, 1H), 4.48 (m, 1H), 7.56 (d, J ) 8.8 Hz, 2H), 7.83 (d, J ) 8.8 Hz, 2H). HRMS calcd for C30H47IN5NaO8S (M + H)+: 764.2190. Found: 764.2179. HPLC: tR ) 11.4 min. 2-(N-Biotinoyl)-1-(N′-(13′-p-iodobenzoylamino)4′,7′,10′-trioxatridecaneamino)-L-aspartate, 5a. This compound was obtained in nearly quantitative yield as a colorless oil. 1H NMR 200 MHz (CD3OD): δ 1.72 (m, 3H), 1.87 (m, 3H), 2.26 (t, J ) 7.3 Hz, 1H), 2.59 (m, 2H), 2.79 (m, 2H), 2.89 (d, J ) 3.3 Hz, 1H), 2.95 (d, J ) 3.3 Hz, 1H), 3.31 (m, 8H), 3.51 (t, J ) 6.2 Hz, 4H), 3.61 (m, 8H), 4.30 (m, 1H), 4.48 (m, 1H), 4.66 (m, 1H), 7.56 (d, J ) 8.4 Hz, 2H), 7.83 (d, J ) 8.4 Hz, 2H). HRMS calcd for C31H46IN5NaO9S (M + Na)+: 814.1958. Found: 814.1937. HPLC: tR ) 11.6 min. 2-(N-Biotinoyl)-4-(N′-(13′-p-iodobenzoylamino)4′,7′,10′-trioxatridecaneamino)-L-aspartate, 6a. This compound was obtained in 99% yield as a colorless oil. 1 H NMR 200 MHz (CD3OD): δ 1.28 (s, 2H), 1.71 (m, 3H), 1.89 (m, 3H), 2.24 (t, 2H), 2.68 (m, 1H), 2.86 (d, J ) 4.7 Hz, 1H), 2.99 (d, J ) 4.7 Hz 1H), 3.30 (m, 8H), 3.49 (m, 4H), 3.62 (m, 8H), 4.30 (m, 1H), 4.41 (m, 1H), 4.63 (m, 1H), 7.57 (d, J ) 8.7 Hz, 2H), 7.84 (d, J ) 8.7 Hz, 2H). HRMS calcd for C31H46IN5NaO9S (M + Na)+: 814.1958. Found: 814.1957. HPLC: tR ) 11.5 min. Preparation of [125I]3c. To 50 µL of a 1 mg/mL solution of 3a was added 1-5 µL of Na125I in 0.1 N NaOH, followed by 10 µL of a 1 mg/mL solution of NCS in MeOH. After 2 min, 10 µL of a 1 mg/mL aqueous sodium metabisulfite solution was added. The reaction mixture was then drawn into a syringe for purification by HPLC. The radioiodinated [125I]3c was obtained from the HPLC effluent in 13% yield. Preparation of [125I]4b, [125I]5b, and [125I]6b using N-Hydroxysuccinimidyl p-[125I]Iodobenzoate, 27. The syntheses were accomplished from the N-t-Boc derivatives 21, 22, and 23 as depicted in Scheme 3. The steps involved are as follows. Removal of t-Boc Protecting Group in Situ To Prepare 24, 25, and 26. A 100 µg quantity of biotin derivative 21, 22, or 23 in 100 µL of MeOH was evaporated to dryness, and 100 µL TFA was added. This solution was stirred for 30 min, and the TFA was evaporated under a stream of argon to yield 24, 25, and 26 as TFA salts. The TFA salt residues were diluted with 30 µL of DMF and 20 µL of triethylamine. Preparation of [125I]27. To 50 µL of HPLC-purified p-trin-butylstannylbenzoate N-hydroxysuccinimide ester (21) in MeOH/5% HOAc was added 3 µL (1.68 mCi) of Na125I followed by 10 µL of 1 mg/mL NCS in MeOH. After 10 min at room temperature, 10 µL of a 1 mg/mL solution

Biotinidase-Stabilized Biotin Derivatives

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Table 1. Biotin Derivatives Prepared

of sodium metabisulfite was added to quench the reaction. The mixture was then reduced to dryness under a stream of argon, and 50 µL of DMF was added. Radiolabeling of Biotin Derivatives. To the basic solution containing 24, 25, or 26 in DMF was added 15 µL of [125I]27. After 30 min at room temperature, the entire reaction mixture was injected on the HPLC column and eluted. The major radioactivity peaks were collected. The percent of radioactivity in the peak corresponding to the iodo standards (4a, 5a, 6a) on the HPLC chromatogram was noted ([125I]4b, 43%; [125I]5b, 44%; [125I]6b, 46%). The isolated radiochemical yields were as follows: [125I]4b, 25%; [125I]5b, 29%; [125I]6b, 31%. Measurement of Biotinidase Cleavage. The same experimental conditions were used as previously reported (15). Briefly, the radiolabeled biotin derivative was diluted to obtain approximately 5000 cpm per 10 µL in a well counter. To a 0.5 mL aliquot of diluted serum (1:10 with 20 mM phosphate buffer, pH 6.8) was added 10 µL of the HPLC-purified radiolabeled biotin derivative in MeOH. Each tube was lightly vortexed and placed in a 37 °C heating block. After the desired time (e.g., 2 h), the vials were removed, and 40 µL of 2 mM 4-(hydroxymercuri)benzoic acid (HOHgBz), sodium salt in H2O, was added to inhibit biotinidase. To that solution was added an excess of avidin (40 µL; 0.24 mg), and it was incubated at 37 °C for another 30 min. The solution was split into two 200 µL aliquots, and the samples were transferred

to two Microcon-30 filters and centrifuged at 3000g for 10 min. The material remaining on the filter was washed with 4 × 100 µL of phosphate buffer, concentrating by centrifugation each time. The top and bottom (containing filtrate and washes) of the Microcon-30 were counted in a well counter. The ratio of nonbound counts (bottom) to the total counts (top and bottom) was calculated to determine the percent of the biotin derivative that had been cleaved. As a control, this procedure was repeated as above except that the 4-(hydroxymercuri)benzoic acid was added prior to the radiolabeled biotin derivative to inhibit biotinidase activity. RESULTS

Synthesis and Radioiodination of Biotin Derivatives. The biotin derivatives synthesized for the study are shown in Table 1. Radioiodinated derivatives ([125I]1c, [125I]2c, [125I]3c, [125I]4b, [125I]5b, and [125I]6b) were prepared for evaluation in the biotinidase assay. The arylstannane derivatives (1a, 2a, 3a) were prepared as precursors for radioiodination, and nonradioactive iodophenyl derivatives (1b, 2b, 3b, 4a, 5a, and 6a) were prepared for use as HPLC standards. Syntheses of homobiotin-stannylbenzoate derivative 3a and the homobiotiniodobenzoate derivative 3b were accomplished using the same synthetic approach as previously described for 1a, 2a, 1b, and 2b (15). The synthetic route employed is shown in Scheme 1. In the syntheses, homobiotin tetra-

Scheme 1. Synthesis of Homobiotin-Trioxatridecanediamine-Benzoyl Derivatives 3a, 3b, and [125I]3c

a

DMF, Et3N, rt, 1 h, 8a (89%), 8b (72%). bMeOH, 1% HOAc, NCS, Na125I, rt, 5 min, 13% (radiochemical yield).

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Scheme 2. Synthesis of Biotin-Amino Acid-Trioxatridecanediamine-p-iodobenzoyl Derivatives 4a, 5a, 6a

a,b

Reference 19. cDMF, Et3N, 4 °C, TFP-OTFA, 30 min. dEt3N, 8b, rt, 1 h (35-65%). eTFA.

fluorophenyl (TFP) ester, 7, was generated in situ by reaction of homobiotin with TFP-OTFA (19), and was subsequently reacted with one of the substituted benzoyldiaminotrioxatridecane adducts, 8a or 8b, to provide 3a and 3b in 89% and 72% overall yield, respectively. Syntheses of iodo-HPLC standards 4a, 5a, and 6a were accomplished as shown in Scheme 2. The biotin-amino acid adducts 10-13 were prepared from reaction of the N-hydroxysuccinimide ester of biotin 9 with the corresponding t-Bu-protected amino acids, followed by in situ preparation of the tetrafluorophenyl (TFP) esters 1416 as previously described (19). The p-iodobenzoyl adduct of trioxadiamine, 8b, was then reacted with the TFP esters to provide the adducts 17, 18, and 19 in 45%, 65%, and 35% yield, respectively. Subsequent conversion of tert-butyl ether/ester adducts to the desired 4a, 5a, and 6a was accomplished in neat trifluoroacetic acid (TFA) in nearly quantitative yield. High specific activity radioiodination was required for the biotinidase assay. Radioiodinated derivatives 1c, 2c, and 3c were obtained by reaction of HPLC-purified stannylbenzoyl derivatives 1a, 2a, and 3a with nocarrier-added (nca) Na125I in MeOH/1% HOAc using N-chlorosuccinimide (NCS) as the oxidant (15). After radiolabeling, the radioiodinated biotin derivatives [125I]1c, [125I]2c, and [125I]3c were purified by HPLC to rid them of radiochemical contaminants that might interfere with interpretation of the biotinidase assay results. In an effort to improve on the initial studies, which employed two HPLC purification steps in the preparation of [125I]1c, [125I]2c, and [125I]3c, an alternate method that had only one HPLC purification step was used to prepare the subsequent radioiodinated derivatives [125I]4b, [125I]5b, and [125I]6b. In the alternate method (Scheme 3), radioiodination was accomplished by preparing p-[125I]iodobenzoate N-hydroxysuccinimide (NHS) ester, [125I]27, from the stannylbenzoate NHS ester (21), followed by reaction with the amino-biotin derivatives 24-26. The amino-biotin derivatives 24-26 were prepared in situ by removal of the t-Boc groups from 21-23 using neat TFA. Although it was anticipated that the desired products would be obtained in high yield using the alternate

approach, this was not found to be the case. Indeed, several products were obtained in the conjugation reactions, such that purification by HPLC gave only moderate (25-31%) yields of radiolabeled products. Identification of the desired radioiodinated products was obtained by HPLC retention time comparison of, and coelution with, the nonradioactive iodo standards. As an example, an HPLC radiochromatogram of the product mixture obtained in the reaction of 25 with [125I]27 is shown in Figure 1A. An HPLC chromatogram of the nonradioactive 5b using UV detection is shown in Figure 1B. Since the elution of samples through the radioactivity detector is approximately 0.2 min after the UV detector at the flow rate used, a good correlation of the standard and radiolabeled product was obtained. Co-injection of the iodo standard 5b further confirmed the identity. All isolated radiolabeled biotin derivatives were single peaks after reinjection on the radio-HPLC. Biotinidase Assay. The isolated radioiodinated biotin derivatives [125I]1c, [125I]2c, [125I]3c, [125I]4b, [125I]5b, and [125I]6b were evaluated in an assay which determined the amount of radioactivity that bound avidin after the samples were incubated in human serum. This biotinidase cleavage assay is based on the fact that hydrolysis of the biotinamide bond results in separation of the biotin moiety from the radioactivity, which can be measured as a lack of radioactivity binding with avidin. Since the active site of biotinidase contains a sulfhydryl moiety, the biotinidase enzymatic activity can be quenched with the addition of hydroxymercuribenzoic acid (HOHgBz) (22). Thus, a comparison of the amount of avidin binding in the absence or presence of HOHgBz provides confirmation that the lack of binding (i.e., free radioactivity) was due to enzymatic activity. In an initial evaluation (Table 2, assay 1), biotinidase cleavage of the homobiotin derivative [125I]3c was compared with a biotin derivative, [125I]1c, which had previously been shown to be rapidly degraded, and a second derivative, [125I]2c, which had been shown to be highly resistant to degradation (15). In a second evaluation (Table 2, assay 2), biotin derivatives having substituents alpha to the biotinamide bond ([125I]4b, [125I]5b, [125I]6b)

Biotinidase-Stabilized Biotin Derivatives

Bioconjugate Chem., Vol. 12, No. 4, 2001 621

Figure 1. (A) Radiochromatogram of crude product mixture from reaction of 25 with [125I]27 (Scheme 3). (B) UV chromatogram of purified nonradioactive 5b. Scheme 3. Synthesis of Radioiodinated Biotin-Amino Acid-Trioxatridecanediamine-p-iodobenzoyl Derivatives [125I]4b, [125I]5b, and [125I]6b

a

Et3N, 20, rt, 1 h (52-82%). bTFA, rt, 10-30 min, 99%. cDMF, Et3N, 30 min, rt, 25-31% (radiochemical yield).

were evaluated. In the analyses, radiolabeled biotin derivatives were incubated in diluted (10%) human serum for 2 h at 37 °C. Following that, HOHgBz was added to quench the reaction, and a phosphate-buffered saline (PBS) solution containing a large excess of avidin was added to that mixture. The biotin derivative was allowed to bind with avidin over a 30 min period at 37 °C. Assessment of the percent of radioactivity that did not bind with avidin was obtained by sizing ultrafiltration separation (Microcon-30). The radiolabeled biotin that was not hydrolyzed remained with the avidin in the top of the Microcon-30, whereas the cleaved material filtered through the membrane. In separate analyses, serum was treated with HOHgBz prior to incubation with the radioiodinated biotin derivative. Duplicate runs were conducted, except for the homobiotin derivative 3c, which was conducted in triplicate. DISCUSSION

We are pursuing the development of radiolabeled biotin derivatives for application to the diagnosis and therapy of cancer using the monoclonal antibody-based “pretargeting method”. During our investigations, and similar investigations of other research groups, it has become apparent that it is essential for application of biotin

derivatives in vivo that they be designed to block the enzymatic action of the enzyme biotinidase (8, 10, 23, 24). Although biotinidase stabilization is an essential design feature in new biotin reagents for in vivo application, it is also important that biotinidase cleavage be blocked without affecting the high binding affinity of biotin with avidin (Av) and streptavidin (SAv). Following the lead of other investigators (12) in our previous biotin design studies, we found that conversion of the biotinamide bond into a N-methylbiotinamide bond blocked cleavage of that bond (15). More recently, we developed a method for evaluating the relative rates of association and dissociation of biotin derivatives from Av and SAv using sizeexclusion HPLC (25). Using the HPLC analysis, we evaluated a number of biotin conjugates, including a biotin adduct that contained the N-methyl moiety, biotinsarcosine (19). Unfortunately, it was found that the derivative containing a N-methylbiotinamide structure had a more rapid dissociation rate from Av and SAv than biotin conjugates that lacked this structural feature. Not surprisingly, it was found that any alteration of the valeric acid side chain or the biotinamide bond in biotin derivatives resulted in an increased dissociation rate. The studies demonstrated that the dissociation rates were minimally affected when biotin conjugates contained an unaltered biotin moiety linked to molecules (e.g., amino

622 Bioconjugate Chem., Vol. 12, No. 4, 2001

Wilbur et al.

Table 2. Percentage of Radioiodinated Biotin Derivatives Filtered in the Presence of Avidin after being Incubated in Diluted Human Serum at 37 °C for 2ha radioiodinated biotin derivative assay 1 1c 2c 3c assay 2 1c 2c 4b 5b 6b

group 1 w/o HOHgBzb

group 2 with HOHgBzc

90.4 ( 0.8 5.5 ( 0.0 12.3 ( 0.1

3.8 ( 0.8 4.3 ( 0.0 3.4 ( 1.6

87.6 ( 1.9d 3.0 ( 0.1 2.9 ( 0.4 1.6 ( 0.0 1.9 ( 2.1

2.2e 1.9 ( 0.0 2.8 ( 0.1 3.2 ( 0.2 1.5 ( 0.1

a All values represent the percentage of radioactivity filtered through a Microcon-30 centrifugation unit (i.e., not retained by binding with avidin or nonspecifically bound). The activity that filtered through is considered as cleaved from biotin by biotinidase. The values are an average of duplicate assays ( the standard deviation, except for 3c, which is the average of triplicate assays. Human serum was obtained 1-2 days prior to the assay (refrigerated at 4 °C) and was diluted to 10% of its original composition with 0.9% sterile saline. b Assay 1 values represent activity filtered from diluted, but untreated (i.e., without addition of HOHgBz), serum after an excess of avidin was added. c Assay 2 values represent activity filtered from diluted and HOHgBz-treated serum after an excess of avidin was added. d A similar value (90%) was obtained when this assay was conducted without avidin. Thus, for 1c, it appears that as much as 10% is retained by nonspecific adherence. e One sample was tested.

acids) that had a substituent on a methylene group alpha to the biotinamide bond. As might be expected, large substituents alpha to the biotinamide bond significantly increased dissociation rates, but less sterically bulky groups, e.g., methyl, hydroxymethylene, and carboxylate, had little affect on the dissociation rates. Importantly, previous studies demonstrated that having a methyl substituent alpha to the biotinamide bond decreased the rate of biotinidase cleavage, but did not block it (19). This investigation was primarily conducted to determine if biotin-amino acid adducts containing the small substituents carboxylate, hydroxymethylene, and acetate were stable to biotinidase cleavage. Prior to the study, we believed there would be a good likelihood of obtaining biotinidase stability. This belief was based on our previous studies and information in the literature relating to structural features of biotin conjugates and stability toward biotinidase cleavage (10, 11, 23, 26-30). Perhaps the most relevant data were reported by Rosebrough, which indicated that an R-carboxylate can block biotinidase activity (11). In that study, the stability toward biotinidase hydrolysis was examined in biotin adducts of deferoxamine, either directly coupled or containing N-lysyl and cysteinyl linkers. High stability to biotinidase hydrolysis was only demonstrated in the biotin derivative that contained a cysteine linker, which has a carboxylate alpha to the biotinamide bond. The data on biotinidase cleavage obtained in this study indicate that the substituents hydroxylmethylene, carboxylate, and acetate, when placed on a linker molecule alpha to the biotinamide bond, completely block biotinidase activity. These results correlate well with data previously published. The finding that homobiotin is hydrolyzed at a slow rate is consistent with the results reported by Pispa that alterations in the valeric acid chain “almost completely blocked biotinidase activity” (10). However, the increased dissociation rate demonstrated for homobiotin derivatives (19) eliminates it from being an optimal candidate for in vivo use. Foulon et al.

(14, 31) have described the use of norbiotinamine conjugates for blocking the biotinidase activity. Based on our studies with norbiotin derivatives (19), it might be expected that an unfavorable dissociation rate from avidin and SAv would be obtained with these conjugates, making them nonoptimal as well. In this investigation, we included a biotin derivative that contains an acetate moiety alpha to the biotinamide bond (6b) even though it was known that this structural feature increased the dissociation rate from Av and SAv, because it was of interest to determine if larger substituents blocked biotinidase. The fact that it completely blocked biotinidase activity suggests that any group larger than a methyl blocks biotinidase activity. The R-acetate might be considered to be isosteric with the o-aminobenzoic acid which had been previously shown to block biotinidase activity (26). Although we have not examined the dissociation rate of the biotin-o-aminobenzoic acid adduct, it might also be expected to have an increased dissociation rate based on our previous studies. There are likely to be other structural features of biotin derivatives that will both block biotinidase activity and exhibit the slow dissociation rate with Av and SAv. For example, a linker containing an ethyl group alpha to the biotinamide bond would be a good candidate. We did not evaluate that compound in this study as we felt it would be more lipophilic than desired for in vivo applications. In contrast to our studies, where the evaluation of biotinidase activity of biotin conjugates was based on preparation of targeted high specific activity radioiodinated biotin derivatives, it is likely that optimal structural features may be predictable in the future through molecular modeling techniques. Biotinidase has been isolated, and information on the nature of the binding pocket is being obtained (26, 32). The sequence and cDNA cloning of biotinidase have been completed (33), and more information about its binding pocket is being learned by homology data with other enzymes (34). At this time, we believe that biotin derivatives containing a carboxylate or hydroxymethylene moiety alpha to the biotinamide bond are the best candidates for developing reagents for in vivo application. ACKNOWLEDGMENT

We are grateful for the financial support provided by the Department of Energy, Medical Applications and Biophysical Research Division, Office of Biological and Environmental Research, under Grant DE-FG0398ER62572. We thank Quanta BioDesign for their generous gift of biotin-NHS ester used in the studies. Supporting Information Available: HPLC chromatograms, 1H NMR spectra, LRMS, and HRMS of the new compounds prepared (3a, 3b, 4a, 5a, 6a, 17, 18, 19, 21, 22, 23) in the research described in this paper (42 pages). This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Paganelli, G., Malcovati, M., and Fazio, F. (1991) Monoclonal Antibody pretargetting techniques for tumour localization: the avidin-biotin system. Nucl. Med. Commun. 12, 211-234. (2) Goodwin, D. A., and Meares, C. F. (1997) Pretargeting: General Principles. Cancer (Suppl.) 80, 2675-2680. (3) Yau, E. K., Theodore, L. J., and Gustavson, L. M. (1996) Pretargeting Methods and Compounds, United States Patent 5,541,287.

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