Biotin Reagents for Antibody Pretargeting. 4. Selection of Biotin

Bioconjugate Chem. 2000, 11, 569−583

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Biotin Reagents for Antibody Pretargeting. 4. Selection of Biotin Conjugates for in Vivo Application Based on Their Dissociation Rate from Avidin and Streptavidin D. Scott Wilbur,* Ming-Kuan Chyan, Pradip M. Pathare, Donald K. Hamlin, Milah B. Frownfelter, and Brian B. Kegley Department of Radiation Oncology, University of Washington, 2121 North 35th Street, Seattle, Washington 98195. Received March 6, 2000; Revised Manuscript Received May 10, 2000

An investigation was conducted to determine the affect of structural variation of biotin conjugates on their dissociation rates from Av and SAv. This information was sought to help identify optimal biotin derivatives for in vivo applications. Fifteen biotin derivatives were conjugated with a cyanocobalamin (CN-Cbl) derivative for evaluation of their “relative” dissociation rates by size exclusion HPLC analysis. Two biotin-CN-Cbl conjugates, one containing unaltered biotin and the other containing iminobiotin, were prepared as reference compounds for comparison purposes. The first structural variations studied involved modification of the biotinamide bond with a N-methyl moiety (i.e., sarcosine conjugate), lengthening the valeric acid side chain by a methylene unit (i.e., homobiotin), and replacing the biotinamide bond with thiourea bonds in two conjugates. The rate of dissociation of the biotinCN-Cbl derivative from Av and SAv was significantly increased for biotin derivatives containing those structural features. Nine additional biotin conjugates were obtained by coupling amino acids or functional group protected amino acids to the biotin moiety. In the conjugates, the biotin moiety and biotinamide bond were not altered, but substituents of various sizes were introduced R to the biotinamide bond. The results obtained from HPLC analyses indicated that the rate of dissociation from Av or SAv was not affected by small substituents R to the biotinamide (e.g., methyl, hydroxymethyl, and carboxylate groups), but was significantly increased when larger functional groups were present. On the basis of the results obtained, it appears that biotin conjugates which retain an unmodified biotin moiety and have a linker molecule conjugated to it that has a small functional group (e.g., hydroxymethylene or carboxylate) R to the biotinamide bond are excellent candidates for in vivo applications. These structural features are obtained in the biotin amino acid conjugates: biotinserine, biotin-aspartate, biotin-lysine, and biotin-cysteine. Importantly, these biotin derivatives can be readily conjugated with other molecules for specific in vivo applications. In our studies, these derivatives will be used in the design of new biotin conjugates to carry radionuclides for cancer therapy using the pretargeting approach.

INTRODUCTION

The very high binding affinities of biotin with the proteins avidin (1) and streptavidin (2) have made combinations of these reagents of particular interest to investigators developing chemical assays for a large variety of applications (3-6). Most of the assays that have been developed, or are under development, are conducted in vitro. However, some applications are being developed which utilize the high affinity of these chemicals for selective binding in vivo. One example of an in vivo application is an antibody-based multistep approach to delivering therapeutic radionuclides to cancer cells in patients, termed “pretargeting” (7-17). In the pretargeting studies, monoclonal antibody (mAb)1 conjugates, streptavidin (SAv) and/or avidin (Av), and biotin conjugates are being evaluated in various combinations to maximize the amount of radionuclide delivered to cancer cells while minimizing the radiation dose to nontarget tissues in patients. Our group is developing new reagents that are optimized for use in the pretargeting approach to cancer therapy in patients. As part of that reagent development, * To whom correspondence should be addressed. Phone: (206) 685-3085. Fax: (206) 685-9630. E-mail: [email protected]. edu.

we have focused an effort on determining which design factors make biotin conjugates optimal for in vivo use. In our assessment, one of the most important design factors for biotin derivatives used in Targeted Radiotherapy is the requirement that the radionuclides bound to cancer cells be retained on those cells during the period of radioactive decay. In the pretargeting approach, retention of a radiolabeled biotin derivative on cancer cells is dependent on the dissociation rate of the mAb-SAv conjugate from its target antigen and on the dissociation rate of the biotin derivative from the mAb-SAv conjugate. The rate of dissociation of the biotin derivative containing the radionuclide is particularly important in vivo as endogenous biotin is always present (18-20). Administration of a large excess of avidin or streptavidin reagents can deplete endogenous biotin to allow binding of radiolabeled biotin with (strept)avidin on tumor cells. How1 Abbreviations: Av, avidin; CN-Cbl, cyanocobalamin; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; mAb, monoclonal antibody; r-SAv, recombinant streptavidin; rt, room temperature; (S)Av or (strept)avidin, denotes avidin or streptavidin; Sav, streptavidin; TCDI, 1,1-thiocarbonyldiimidazole; TFA, trifluoroacetic acid; TFP, tetrafluorophenyl; TFP-OH, tetrafluorophenol; TFP-OTFA, tetrafluorophenyl trifluoroacetate; TsCl, toluenesulfonyl chloride.

10.1021/bc000024v CCC: $19.00 © 2000 American Chemical Society Published on Web 06/29/2000

570 Bioconjugate Chem., Vol. 11, No. 4, 2000

ever, endogenous biotin is replenished and, therefore, will be available to bind in the place of any dissociated biotin derivative, effectively blocking its reassociation with the cancer cell. A decrease in quantity of radionuclides bound to cancer cells is problematic as it decreases the probability of killing those cells. Thus, in our studies to optimize biotin reagents for use in mAb-based pretargeting, it is important to have some idea of the dissociation rate of the biotin derivatives used relative to unmodified biotin. To assess the relative dissociation rates of biotin derivatives, we have recently investigated the use of biotin-dyes in an HPLC assay (21). From those studies, it was found that biotin derivatives which were conjugated with cyanocobalamin (CN-Cbl) could be used in a size exclusion HPLC analysis to determine their relative dissociation rates. In this investigation, we have prepared twelve new biotin derivatives, conjugated them with CN-Cbl, and determined their relative dissociation rates from Av and SAv. The biotin derivatives chosen for evaluation were modified in locations that we believed would retain strong binding with Av and SAv. The structures of the biotin derivatives chosen for study were also based on our belief that they were likely to be stable to degradation by serum biotinidase (22, 23). The biotin derivatives investigated contained modifications (1) in the length of side chain, (2) in the nature of bond between the linker and biotin moiety, and (3) in substituents on the methylene R to the biotinamide bond obtained by conjugation of biotin with a series of amino acids. As part of the investigation, three biotin-CN-Cbl derivatives previously tested (21) were included. The results obtained from the HPLC analyses conducted clearly indicate that certain structural modifications of biotin conjugates dramatically decrease their binding with (S)Av while other modifications have much less of an affect. Reported herein is the rationale for the design of new biotin conjugates, their synthesis, results of relative dissociation rates as measured by HPLC analysis, and a discussion of the implications of those results with regards to the choice of biotin conjugates to be used for in vivo applications. 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. Vitamin B12 (CN-Cbl); d-biotin, 4; 4,7,10-trioxa-1,13-tridecanediamine; 2,3,5,6-tetrafluorophenol; toluenesulfonyl chloride; L-serine, 21; and most other chemicals were purchased from Aldrich Chemical Co. (Milwaukee, WI). O-tert-Butyl-L-serine methyl ester hydrochloride, 19; L-aspartic acid β-tert-butyl ester, 23; and L-aspartic acid R-tert-butyl ester, 16, were obtained from Calbiochem-Novabiochem Corp. (San Diego, CA). Iminobiotin NHS ester, 2b, and recombinant streptavidin (r-SAv) were obtained from Sigma (St. Louis, MO). Avidin was purchased from Pierce (Rockford, IL). 2,3,5,6-Tetrafluorophenyl trifluoroacetate was prepared as previously described (24). 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).

Wilbur et al.

Spectral Analyses. 1H NMR spectra were obtained on either a Bruker AC-200 (200 MHz), a Bruker AC-500 (500 MHz), or a Bruker AC-750 (750 MHz) instrument. Proton chemical shifts are expressed as parts per million using tetramethylsilane as internal standard (δ ) 0.0 ppm). IR data were obtained on a Perkin-Elmer 1420 infrared spectrophotometer. Some mass spectral data were obtained on 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). Other mass spectral data (ES+) were obtained on a PE Biosystems Mariner Electrospray Time-of-Flight Mass Spectrometer. Reversed-Phase Chromatography. HPLC separations of biotin derivatives and biotin-CN-Cbl conjugates were obtained on Hewlett-Packard quaternary 1050 gradient pumping system with a variable wavelength UV detector (254 and 360 nm) and a Varex ELSD MKIII evaporative light-scattering detector. Analysis of the HPLC data were conducted on Hewlett-Packard 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) 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, then held at 100% MeOH for 8 min. Retention times (tR) are provided with the compound experimental. Size Exclusion Chromatography. Size exclusion HPLC analyses were conducted on a system consisting of a Hewlett-Packard 1050 Multiple Wavelength Detector (280 nm), isocratic pump, and a Spherogel TSK 5000PW column (7.5 mm × 300 mm, 10 µm; stainless steel, Altex). 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 0.75 mL/min was used. Preparation of 1. Preparation of 1 was accomplished by reaction of the c-lactone of CN-Cbl (25) with (neat) 4,7,10-trioxa-1,13-tridecanediamine at 50-55 °C for 1 h as previously described (21). Preparation of 2a. Preparation of the TFP ester of biotin, 2a, was accomplished as previously described (26). Preparation of 2c. Preparation of the TFP ester of the biotin-sarcosine adduct, 2c, was accomplished in situ as previously described (23). Preparation of 2d. To a solution of 0.4 g (1.6 mmol) homobiotin, 9, in 20 mL of DMF was added 0.23 mL (1.6 mmol) of Et3N, followed by 484 mg (1.86 mmol) of TFPOTFA. The reaction was stirred at room temperature for 30 min and the DMF was removed under vacuum. The residue was dissolved in CHCl3, washed with H2O and dried over anhydrous Na2SO4. The CHCl3 was removed to yield a 550 mg (87%) of 2d as a colorless solid. mp > 240 °C. 1H NMR (CDCl3, 500 MHz) δ 1.4 (m, 2H), 1.61.8 (m, 4H), 2.6 (t, J ) 3.6 Hz, 2H), 2.7 (d, J ) 6.8 Hz, 1H), 2.8 (dd, J ) 2.5, 6.4 Hz, 1H), 3.1 (m, 1H), 4.3 (m, 1H), 4.5 (m, 1H), 5.73 (s, 1H), 6.02 (s, 1H), 6.9 (m, 1H). IR (KBr) 3240, 2920, 2840, 1780, 1700, 1520, 1480, 1080 cm-1. HRMS mass calcd for C17H19N2O3SF4 (M + H)+: 407.1052. Found: 407.1038. HPLC: tR ) 13.1 min. Preparation of 2e. To a 115 mg (0.535 mmol) quantity of norbiotinamine, 12, dissolved in 5 mL of DMF was added 95 mg (0.535 mmol) of 1,1-thiocarbonyldiimi-

Relative Dissociation Rates of Biotin Derivatives

dazole. The reaction mixture was stirred at room temperature for 45 min, and the DMF was removed under vacuum. The resultant residue was dissolved in 0.5 mL of MeOH, and H2O was added until a precipitate formed. The white precipitate was filtered, washed with H2O, and dried under vacuum to give 76 mg (55%) of 2e. mp 168170 °C. 1H NMR (DMSO-d6, 200 MHz) δ 1.2-1.8 (m, 6H), 2.55 (d, J ) 6.4 Hz, 1H), 2.9 (dd, J ) 6.2, 2.5 Hz, 1H), 3.1 (m, 1H), 4.1 (m, 1H), 4.3 (m, 1H), 6.4 (d, J ) 9 Hz, 2H). IR (KBr) 3240, 2920, 2840, 2200, 2120, 1700, 1470, 1340, 1270, 1160, 1100 cm-1. HRMS mass calcd for C10H16N3OS2 (M + H)+: 258.0735. Found: 258.0739. HPLC: tR ) 11.0 min. Preparation of 2f. To a solution containing 100 mg of (0.437 mmol) biotinamine, 14, in 15 mL of DMF was added 78 mg of (0.437 mmol) 1,1-thiocarbonyldiimidazole. The reaction mixture was stirred at room temperature for 2 h, and the DMF was removed under vacuum. The residue was dissolved in 5 mL of MeOH, and H2O was added until a precipitate formed (3 vol). After storing the precipitate at 4 °C overnight, it was filtered, dried under vacuum, and triturated with MeOH to give 88 mg (74%) of biotin isothiocyanate, 2f, as colorless solid. mp 122124 °C. 1H NMR (DMSO-d6, 200 MHz) δ 1.2-1.8 (m, 8H), 2.55 (d, J ) 6.2 Hz, 1H), 2.8 (dd, J ) 6.2, 2.3 Hz, 1H), 3.1 (m, 1H), 3.64 (t, J ) 3.2 Hz, 2H), 4.1 (m, 1H), 4.3 (m, 1H), 6.4 (d, J ) 8.9 Hz, 2H). IR (KBr) 3220, 2910, 2840, 2160, 2080, 1690, 1460, 1210, 1060 cm-1. HRMS mass calcd for C11H18N3OS2 (M + H)+: 272.0891. Found: 272.0893. HPLC: tR ) 11.8 min. Preparation of 2j. To a solution of N-biotinyl-β-tertbutyl ester-L-aspartic acid, 27 (2.20 g, 5.29 mmol), and tetrafluorophenol (0.88 g, 5.29 mmol) in 50 mL of anhydrous DMF was added EDC (1.52 g, 7.93 mmol) at room temperature. The reaction mixture was stirred at room temperature for 48 h, then the solution was triturated with water (200 mL), filtered, washed with water completely to give the crude N-biotinyl-β-tert-butyl ester-L-aspartic acid TFP ester, 2j. The crude 2j was purified by silica gel column chromatography (50 g) eluting with 30% EtOAc/Hex mixture to afford 1.74 g (58%) of a colorless solid. mp 85.2-86.4 °C dec 1H NMR (CDCl3, 200 MHz) δ 1.46 (s, 9 H), 1.43 (d, 2H), 1.72 (m, 4 H), 2.32 (t, 2 H), 2.72 (d, 2H), 2.87 (m, 1 H), 2.94 (m, 1H), 3.12 (m, 3H), 4.32 (m, 1H), 4.48 (m, 1H), 5.23 (m, 1H), 5.51 (s, 1H), 6.43 (d, 1H), 7.03 (m, 1H), 7.31 (d, 1H). HRMS calcd for C24H30F4N3O6S (M + H)+: 564.1791. Found: 564.1793. HPLC: tR ) 13.1 min. Preparation of 2n. To a solution of N-Biotinyl-N-tert-Boc-L-Lysine, 28 (1.92 g, 4.06 mmol) and tetrafluorophenol (0.67 g, 4.06 mmol) in 50 mL of DMF was added EDC (0.86 g, 4.47 mmol). The reaction mixture was stirred at room temperature for 48 h, then the product was triturated with water (200 mL), filtered, washed with water thoroughly to give the crude product. The crude product was purified on a silica gel column (60 g) eluting with 30% EtOAc/hexane mixture to afford 1.14 g (45%) of the biotin-lysine-N-tBoc adduct, 2n, as a colorless solid. mp 77.9-79.4 °C (dec). 1H NMR (CDCl3, 200 MHz) δ 1.44 (s, 9 H), 1.54 (d, 4H), 1.71 (m, 4H), 1.98 (m, 2H), 2.32 (t, J ) 7.7 Hz, 2H), 2.71 (d, J ) 12.8 Hz, 1H), 2.90 (m, 1H), 3.14 (d, 3H), 3.29-3.34 (m, 2 H), 4.33 (m, 1H), 4.50 (m, 1H), 4.86 (m, 1H), 5.71 (s, 1H), 6.52 (s, 1H), 6.72 (s, 1H), 7.02 (m, 1H), 7.44 (t, 1H). HRMS calcd for C27H37F4N4O6S (M + H)+: 621.2370. Found: 621.2369. HPLC: tR ) 13.2 min. Preparation of 3a. Preparation of the biotin-cyanocobalamin conjugate 3a was accomplished by reaction of biotin TFP ester, 2a, with 1 as previously described (21).

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Preparation of 3b. Preparation of the iminobiotincyanocobalamin conjugate 3b was accomplished by reaction of iminobiotin NHS ester, 2b, with 1 as previously described (21). Preparation of 3c. Preparation of the biotin-sarcosine-cyanocobalamin conjugate 3c was accomplished by reaction of biotin-sarcosine TFP ester, 2c, with 1 as previously described (21). Preparation of 3d. To a solution of 100 mg of (0.063 mmol) CN-Cbl-c-lactone conjugate 1 dissolved in 10 mL of DMF was added 20 mg of (0.063 mmol) homobiotin TFP ester, 2d, then 33 µL of (0.082 mmol) Et3N. The reaction was stirred at room temperature for 4 h and the DMF was removed under vacuum. The residue was triturated in acetone and filtered to yield 108 mg (95%) of 3d as a red solid, mp 198-200 °C (dec). 1H NMR (CD3OD, 750 MHz) δ 1.20 (s, 3H), 1.25 (d, J ) 6.8 Hz, 3H), 1.37 (s, 3H), 1.39 (s, 3H), 1.43 (m, 6H), 1.46 (s, 3H), 1.62 (m, 6H), 1.75 (m, 6H), 1.88 (s, 3H), 1.94 (m, 2H), 2.05 (m, 4H), 2.18 (t, 4H), 2.25 (s, 3H), 2.26 (s, 3H), 2.36 (m, 1H), 2.47 (s, 3H), 2.48 (m, 2H), 2.55 (m, 2H), 2.60 (s, 3H), 2.67 (m, 4H), 2.88 (m, 4H), 3.11 (m, 1H), 3.25 (m, 4H), 3.30 (s, 3H), 3.33 (m, 2H), 3.40 (t, 2H), 3.52 (m, 4H), 3.59 (m, 8H), 3.76 (m, 1H), 3.90 (m, 1H), 4.08 (m, 1H), 4.17 (m, 1H), 4.30 (m, 1H), 4.48 (m, 1H), 4.66 (m, 1H), 6.04 (d, 1H), 6.28 (d, 1H), 6.50 (s, 1H), 7.14 (s, 1H), 7.25 (s, 1H). IR (KBr) 3340, 2920, 2860, 1660, 1570, 1500, 1360, 1210, 1140, 1080 cm-1. MS (FAB+) mass calcd for C84H126N17O20CoPS: 1814.82. Found: 1814.9 (M+). HPLC: tR ) 8.6 min. Preparation of 3e. To a solution of 150 mg of (0.095 mmol) CN-Cbl-c-lactone conjugate 1 dissolved in 10 mL of DMF was added 32 mg of (0.123 mmol) norbiotin isothiocyanate, 2e, then 20 µL of (0.095 mmol) Et3N. The reaction was stirred at room temperature for 48 h and the DMF was removed under vacuum. The residue was triturated in acetone and filtered to yield 163 mg (94%) of 3e as a red solid. mp 205-207 °C (dec). 1H NMR (CD3OD, 750 MHz) δ 0.45 (s, 3H), 1.20 (s, 3H), 1.25 (d, J ) 6.0 Hz, 3H), 1.37 (s, 3H), 1.39 (s, 3H), 1.44 (m, 2H), 1.46 (s, 3H), 1.62 (m, 6H), 1.75 (m, 3H), 1.81 (m, 3H), 1.88 (s, 3H), 1.95 (m, 2H), 2.05 (m, 3H), 2.15 (m, 4H), 2.25 (s, 3H), 2.26 (s, 3H), 2.37 (m, 1H), 2.47 (s, 3H), 2.45 (m, 2H), 2.54 (m, 2H), 2.60 (s, 3H), 2.66 (m, 4H), 2.85 (m, 2H), 2.92 (dd, 2H), 3.11 (m, 1H), 3.23 (m, 4H), 3.30 (s, 3H), 3.33 (m, 2H), 3.40 (t, 2H), 3.54 (m, 4H), 3.60 (m, 6H), 3.67 (m, 2H), 3.76 (m, 1H), 3.90 (m, 1H), 4.08 (m, 1H), 4.17 (m, 1H), 4.31 (m, 1H), 4.49 (m, 1H), 4.65 (m, 1H), 6.04 (d, 1H), 6.28 (d, 1H), 6.50 (s, 1H), 7.14 (s, 1H), 7.25 (s, 1H). IR (KBr) 3310, 2920, 2860, 2120, 1670, 1570, 1500, 1210, 1140, 1080 cm-1. MS (FAB+) mass calcd for C83H125N18O19CoPS2: 1831.79. Found: 1831.67 (M+). HPLC: tR ) 8.7 min. Preparation of 3f. To a solution of 150 mg of (0.095 mmol) CN-Cbl-c-lactone conjugate 1 dissolved in 10 mL of DMF was added 34 mg of (0.123 mmol) biotin isothiocyanate, 2f, then 20 µL of (0.095 mmol) Et3N. The reaction was stirred at room temperature for 24 h, and the DMF was removed under vacuum. The residue was triturated in acetone and filtered to yield 140 mg (80%) of 3f as a red solid. mp 200-202 °C (dec). 1H NMR (CD3OD, 750 MHz) δ 0.46 (s, 3H), 1.20 (s, 3H), 1.25 (d, J ) 6.0 Hz, 3H), 1.37 (s, 3H), 1.39 (s, 3H), 1.43 (m, 3H), 1.46 (s, 3H), 1.59 (m, 4H), 1.64 (m, 2H), 1.74 (m, 2H), 1.81 (m, 4H), 1.88 (s, 3H), 1.91 (m, 1H), 1.96 (d, J ) 13.7 Hz, 1H), 2.05 (m, 4H), 2.15 (m, 4H), 2.26 (s, 6H), 2.37 (d, J ) 3.4 Hz, 1H), 2.45 (m, 2H), 2.47 (s, 3H), 2.55 (m, 2H), 2.60 (s, 3H), 2.64 (m, 2H), 2.70 (d, J ) 12.8 Hz, 2H), 2.85 (m, 2H), 2.92 (dd, J ) 5.1, 12.8 Hz, 2H), 3.10 (m, 1H),


3.23 (m, 4H), 3.30 (s, 3H), 3.34 (m, 2H), 3.40 (t, 2H), 3.54 (m, 4H), 3.60 (m, 6H), 3,67 (d, J ) 14.7 Hz, 2H), 3.76 (m, 1H), 3.90 (m, 1H), 4.08 (m, 1H), 4.17 (m, 1H), 4.30 (m, 1H), 4.48 (dd, 1H), 4.65 (m, 1H), 6.04 (d, 1H), 6.28 (d, 1H), 6.50 (s, 1H), 7.14 (s, 1H), 7.25 (s, 1H). IR (KBr) 3340, 2920, 1660, 1570, 1490, 1210, 1110, 1060 cm-1. MS (FAB+) mass calcd for C84H127N18O19CoPS2: 1845.80. Found: 1845.95 (M +). HPLC: tR ) 9.2 min. Preparation of 2g and 3g. To a solution containing 43 mg of (0.130 mmol) biotin-R-methylbutyrate, 17, and 27 µL of (0.130 mmol) Et3N in 10 mL of DMF was added 51 mg of (0.195 mmol) TFP-OTFA. The reaction was stirred at room temperature for 30 min to form the TFP ester of biotin-R-methylbutyrate, 2g. To that solution was added a solution of 150 mg (0.095 mmol) of 1 and 10 µL of Et3N in 10 mL of DMF. The reaction mixture was stirred at room-temperature overnight. The DMF was removed under vacuum, and the residue was triturated with acetone to give 140 mg (78%) of 3g after removal of solvent. mp 194-196 °C (dec). 1H NMR (CD3OD, 750 MHz) δ 0.46 (s, 3H), 1.16, 1.25 (2xd, J ) 6.8 Hz, 3H), 1.20 (s, 3H), 1.37 (s, 3H), 1.39 (s, 3H), 1.43 (m, 3H), 1.46 (s, 3H), 1.62 (m, 6H), 1.74 (m, 4H), 1.88 (s, 3H), 1.93 (m, 2H), 2.05 (m, 4H), 2.17 (t, 4H), 2.25 (s, 3H), 2.26 (s, 3H), 2.37 (m, 4H), 2.45 (m, 2H), 2.47 (s, 3H), 2.55 (m, 2H), 2.60 (s, 3H), 2.66 (m, 4H), 2.85 (m, 2H), 2.92 (dd, J ) 4.3, 12.8 Hz, 2H), 3.10 (m, 1H), 3.22 (m, 4H), 3.30 (s, 3H), 3.33 (d, J ) 11.1 Hz, 2H), 3.40 (t, J ) 6.8 Hz, 2H), 3.51 (m, 4H), 3.59 (m, 6H), 3.64 (m, 2H), 3.76 (m, 1H), 3.90 (m, 1H), 4.08 (m, 1H), 4.17 (m, 1H), 4.23 (m, 1H), 4.30 (m, 2H), 4.48 (m, 1H), 4.65 (m, 2H), 6.04 (d, 1H), 6.28 (d, 1H), 6.50 (s, 1H), 7.14 (s, 1H), 7.25 (s, 1H). IR (KBr) 3340, 2920, 1660, 1570, 1500, 1400, 1210, 1110, 1080 cm-1. MS (FAB+) mass calcd for C87H130N18O21CoPS: 1885. Found: 1885 (M+). HPLC: tR ) 8.2 min. Preparation of 2h and 3h. TFP-OTFA (0.12 mL, 0.67 mmol) was added slowly to a mixture containing N-biotinyl-O-tert-butyl-serine, 25 (0.13 g, 0.34 mmol), Et3N (0.14 mL, 1.01 mmol), and anhydrous CH3CN (4.0 mL). The mixture was stirred at room temperature for 1 h, and then the volatile materials were evaporated under vacuum to give oily crude TFP ester, 2h. The crude 2h was redissolved in 3.0 mL of anhydrous DMF, then 1 (34.9 mg, 0.043 mmol) and Et3N (0.10 mL, 0.72 mmol) were added at room temperature, respectively. The reaction mixture was stirred at room temperature for 16 h. The mixture was triturated with 20 mL of EtOAc and filtered to yield 77 mg (92%) of 3h as a red solid. mp 187190 °C (dec). 1H NMR (CD3OD, 200 MHz) δ 0.46 (s, 3H), 1.12 (s, 9H), 1.25 (m, 8H), 1.38 (d, 6H), 1.46 (s, 3H), 1.69 (m, 8H), 1.89 (s, 2H), 2.02 (m, 8H), 2.26 (s, 6H), 2.222.29 (m, 2 H), 2.38 (s, 1H), 2.47 (s, 3H), 2.52 (m, 2H), 2.60 (s, 3H), 2.66-3.27 (m, 11H), 3.30 (m, 16H), 3.41 (m, 4H), 3.52 (m, 4H), 3.59 (m, 4H), 3.63 (m, 2H), 3.70-3.99 (m, 2H), 4.09 (m, 1H), 4.18 (t, 1H), 4.32 (m, 1 H), 4.48 (m, 1H), 4.65 (m, 1H), 6.05 (s, 1H), 6.28 (d, 1H), 6.51 (s, 1H), 7.14 (s, 1H), 7.25 (s, 1H), 7.98 (s, 1H); MS (ES+) mass calcd for C90H137CoN18O22PS (M + H)+: 1944. Found: 1944. HPLC: tR ) 9.8 min. Preparation of 2i and 3i. TFP-OTFA (0.13 mL, 0.74 mmol) was dropped slowly to a solution of N-biotinylserine, 26 (0.37 mmol), and Et3N (0.15 mL, 1.1 mmol) in anhydrous CH3CN (2 mL) at room temperature. The solution was stirred at room temperature for 30 min, and then volatile materials were evaporated under vacuum to afford crude TFP ester, 2i. The crude 2i was redissolved in a solution of anhydrous DMF (2 mL) and Et3N (0.15 mL, 1.1 mmol), then 1 (31.8 mg, 0.020 mmol) was added and stirred at room temperature for 16 h. The

Wilbur et al.

solution was triturated with 20 mL of ethyl acetate and filtered to give 36.2 mg (96%) of 3i as a red solid. mp 196-199 °C dec 1H NMR (CD3OD, 200 MHz) δ 0.45 (s, 3H), 1.23 (m, 8H), 1.37 (d, 6H), 1.46 (s, 3H), 1.69 (m, 8H), 1.88 (s, 2H), 2.02 (m, 8H), 2.26 (s, 6H), 2.22-2.29 (m, 2 H), 2.37 (s, 1H), 2.47 (s, 3H), 2.53 (m, 2H), 2.60 (s, 3H), 2.66-3.27 (m, 11H), 3.30 (m, 16H), 3.41 (m, 4H), 3.52 (m, 4H), 3.59 (s, 4H), 3.63 (m, 2H), 3.71-3.95 (m, 2H), 4.09 (m, 1H), 4.18 (t, 1H), 4.30 (m, 1 H), 4.48 (m, 1H), 4.65 (m, 1H), 6.05 (s, 1H), 6.28 (d, 1H), 6.51 (s, 1H), 7.14 (s, 1H), 7.25 (s, 1H); MS (ES+) mass calcd for C86H128CoN18O22PSNa (M + Na)+: 1911. Found: 1911. HPLC: tR ) 8.1 min. Preparation of 3j. N-Biotinyl-β-tert-butyl-L-aspartate TFP ester, 2j (8.0 mg, 14.15 µmol), was added to a solution of 1 (15.0 mg, 9.43 µmol) and Et3N (2.6 µL, 18.86 µmol) in 1.5 mL of anhydrous DMF at room temperature. The reaction mixture was stirred at room temperature for 1 h, then 20 mL of ethyl acetate was added and stirred for an additional 10 min. The precipitate was filtered and washed with EtOAc to give 17.6 mg (95%) of 3j as a red solid. mp 191-194 °C dec. 1H NMR (CD3OD, 200 MHz) δ 0.46 (s, 3H), 1.25 (m, 8H), 1.38 (d, 6H), 1.44 (s, 9 H), 1.46 (s, 3H), 1.69 (m, 8H), 1.89 (s, 2H), 2.02 (m, 8H), 2.26 (s, 6H), 2.22-2.29 (m, 2 H), 2.38 (s, 1H), 2.47 (s, 3H), 2.52 (m, 2H), 2.60 (s, 3H), 2.66-3.27 (m, 11H), 3.30 (m, 16H), 3.41 (m, 4H), 3.52 (m, 4H), 3.59 (m, 4H), 3.63 (m, 2H), 3.69-3.97 (m, 2H), 4.09 (m, 1H), 4.18 (t, 1H), 4.30 (dd, 1 H), 4.49 (dd, 1H), 4.65 (m, 1H), 6.05 (s, 1H), 6.28 (d, 1H), 6.51 (s, 1H), 7.14 (s, 1H), 7.25 (s, 1H); MS (ES+) mass calcd for C91H137CoN18O23PS (M + H)+: 1973. Found: 1973. HPLC: tR ) 9.8 min. Preparation of 2l and 3l. TFP-OTFA (0.085 mL, 0.49 mmol) was added dropwise to a solution of Nbiotinyl-L-aspartic acid R-tert-butyl ester, 18 (0.17 g, 0.41 mmol) and Et3N (0.17 mL, 1.23 mmol) in 4.0 mL of anhydrous CH3CN at room temperature. The mixture was stirred at room temperature for 20 min, and then volatile materials were evaporated under vacuum to give the TFP ester 2l as light-yellow oil. The crude 2l was redissolved in 3.0 mL of anhydrous DMF, then 1 (0.0813 g, 0.0511 mmol) and Et3N (0.17 mL, 1.23 mmol) were added. The reaction mixture was stirred at room temperature for 1 h, then 40 mL of ethyl acetate was added and stirred for an additional 10 min. The precipitate was filtered, washed with ethyl acetate, and evaporated to dryness to give 95 mg (94%) of 3l as a red solid. mp 178182 °C (dec). 1H NMR (CD3OD, 200 MHz) δ 0.46 (s, 3H), 1.24 (m, 8H), 1.38 (d, 6H), 1.45 (s, 9 H), 1.46 (s, 3H), 1.69 (m, 8H), 1.88 (s, 2H), 2.02 (m, 8H), 2.26 (s, 6H), 2.222.29 (m, 2 H), 2.37 (s, 1H), 2.47 (s, 3H), 2.52 (m, 2H), 2.60 (s, 3H), 2.66-3.27 (m, 11H), 3.30 (m, 16H), 3.41 (m, 4H), 3.52 (m, 4H), 3.58 (s, 4H), 3.63 (m, 2H), 3.68-3.96 (m, 2H), 4.09 (m, 1H), 4.18 (t, 1H), 4.31 (m, 1 H), 4.49 (m, 1H), 4.63 (m, 2H), 6.04 (s, 1H), 6.28 (d, 1H), 6.51 (s, 1H), 7.14 (s, 1H), 7.24 (s, 1H); MS (ES+) mass calcd for C91H137CoN18O23PS (M + H)+: 1972. Found: 1972. HPLC: tR ) 9.6 min. Preparation of 3n. N-Biotinyl-N--tert-Boc-L-lysine tetrafluorophenyl ester, 2n (8.8 mg, 14.15 µmol), was added to a solution of 1 (15.0 mg, 9.43 µmol) and Et3N (18.86 µmol) in 1.5 mL of anhydrous DMF at room temperature. The reaction mixture was stirred at room temperature for 1 h, then 20 mL of EtOAc was added and stirred for 10 min. The precipitate was filtered and washed with EtOAc to afford 18.4 mg (96%) of pure 3n as a red solid. mp 185-187 °C (dec). 1H NMR (CD3OD, 200 MHz) δ 0.46 (s, 3H), 1.26 (m, 8H), 1.38 (d, 6H), 1.43 (s, 9 H), 1.46 (s, 3H), 1.69 (m, 12H), 1.89 (s, 2H), 2.02 (m,


8H), 2.26 (s, 6H), 2.22-2.29 (m, 2 H), 2.38 (s, 1H), 2.47 (s, 3H), 2.52 (m, 2H), 2.60 (s, 3H), 2.66-3.27 (m, 13H), 3.30 (m, 16H), 3.41 (m, 4H), 3.52 (m, 4H), 3.59 (m, 4H), 3.63 (m, 2H), 3.69-3.97 (m, 2H), 4.09 (m, 1H), 4.18 (t, 1H), 4.30 (m, 1 H), 4.49 (m, 1H), 4.63 (m, 1H), 6.05 (s, 1H), 6.28 (d, 1H), 6.51 (s, 1H), 7.14 (s, 1H), 7.25 (s, 1H); MS (ES+) mass calcd for C94H144CoN19O23PS (M+): 2029. Found: 2029. HPLC: tR ) 10.1 min. Preparation of 3k, 3m, and 3o. Removal of t-Bu esters. The quantity of the t-Bu ester or N-tBoc protected biotin-CN-Cbl conjugate (3j, 3l, or 3n) was dissolved in neat trifluoroacetic acid and stirred at room temperature for 15 min. TFA was evaporated under vacuum. The crude product was redissolved in methanol (1 mL) and triturated with ethyl acetate (20-30 mL). The solid was filtered and dried under vacuum. 3k. A 20 mg quantity (10 µmol) dissolved in 1 mL of TFA gave 19 mg (98%) of a red solid after workup. mp 208-212 °C (dec). 1H NMR (CD3OD, 200 MHz) δ 0.45 (s, 3H), 1.24 (m, 8H), 1.38 (d, 6H), 1.46 (s, 3H), 1.69 (m, 8H), 1.89 (s, 2H), 2.02 (m, 8H), 2.26 (s, 6H), 2.22-2.29 (m, 2 H), 2.38 (s, 1H), 2.47 (s, 3H), 2.52 (m, 2H), 2.60 (s, 3H), 2.66-3.27 (m, 11H), 3.30 (m, 16H), 3.41 (m, 4H), 3.52 (m, 4H), 3.59 (m, 4H), 3.63 (m, 2H), 3.69-3.95 (m, 2H), 4.09 (m, 1H), 4.18 (t, 1H), 4.30 (dd, 1 H), 4.48 (dd, 1H), 4.64 (m, 1H), 6.04 (s, 1H), 6.28 (d, 1H), 6.51 (s, 1H), 7.14 (s, 1H), 7.25 (s, 1H); MS (ES+) mass calcd for C87H129CoN18O23PS (M + H)+: 1916. Found: 1916. HPLC: tR ) 8.4 min. 3m. A 9 mg quantity (4.6 µmol) dissolved in 0.5 mL of TFA gave 7.9 mg (90%) of a red solid after workup. mp 204-207 °C (dec). 1H NMR (CD3OD) δ 0.46 (s, 3H), 1.25 (m, 8H), 1.38 (d, 6H), 1.46 (s, 3H), 1.69 (m, 8H), 1.89 (s, 2H), 2.02 (m, 8H), 2.26 (s, 6H), 2.22-2.29 (m, 2 H), 2.38 (s, 1H), 2.47 (s, 3H), 2.52 (m, 2H), 2.60 (s, 3H), 2.663.27 (m, 11H), 3.30 (m, 16H), 3.41 (m, 4H), 3.52 (m, 4H), 3.59 (m, 4H), 3.63 (m, 2H), 3.69-3.95 (m, 2H), 4.09 (m, 1H), 4.18 (t, 1H), 4.29 (dd, 1 H), 4.49 (dd, 1H), 4.64 (m, 1H), 6.04 (s, 1H), 6.28 (d, 1H), 6.51 (s, 1H), 7.14 (s, 1H), 7.25 (s, 1H); MS (ES+) mass calcd for C87H126CoN18O22PS (M - H2O): 1898. Found: 1898. HPLC: tR ) 7.7 min. 3o. A 9 mg quantity (4.5 µmol) dissolved in 0.5 mL of TFA gave 8 mg (93%) of a red solid after workup. mp 198-201 °C (dec). 1H NMR (CD3OD, 200 MHz) δ 0.45 (s, 3H), 1.24 (m, 8H), 1.38 (d, 6H), 1.46 (s, 3H), 1.69 (m, 12H), 1.89 (s, 2H), 2.04 (m, 8H), 2.26 (s, 6H), 2.25 (m, 2 H), 2.38 (s, 1H), 2.47 (s, 3H), 2.52 (m, 2H), 2.60 (s, 3H), 2.95 (m, 13H), 3.30 (m, 16H), 3.41 (m, 4H), 3.52 (m, 4H), 3.59 (m, 4H), 3.63 (m, 2H), 3.82 (m, 2H), 4.09 (m, 1H), 4.18 (t, 1H), 4.30 (m, 1 H), 4.48 (m, 1H), 4.64 (m, 1H), 6.05 (s, 1H), 6.28 (d, 1H), 6.51 (s, 1H), 7.14 (s, 1H), 7.25 (s, 1H); MS (ES+) mass calcd for C89H136CoN19O21PS (M + H)+: 1930. Found: 1930. HPLC: tR ) 2.3 min. Biotin Methyl Ester, 5. This compound was prepared by reaction of biotin, 4, in methanolic HCl as previously described (27) to give a nearly quantitative yield of 5 as a colorless solid. mp 116-118 °C. HPLC: tR ) 8.31 min. Biotinol, 6. This compound was prepared by reduction of a 1 g (3.88 mmol) of 5 with LiAlH4 as previously described (28) to give 0.89 g (100%) of 6 as a colorless solid. mp 164-168 °C. HPLC: tR ) 6.52 min. Biotinol-p-toluene Sulfonate, 7. A 0.7 g (3 mmol) quantity of biotinol, 6, was dissolved in pyridine at 0 °C, and 1.14 g (6 mmol) toluenesulfonyl chloride was slowly added. The reaction mixture was stirred at 0 °C for 5 h and poured over crushed ice. The aqueous layer was extracted with CH2Cl2 (3 × 100 mL). The CH2Cl2 solution was washed with 2% aqueous HOAc then with aqueous saturated NaCl. The CH2Cl2 solution was then dried over


anhydrous Na2SO4, filtered, and evaporated to yield 1.0 g (2.6 mmol, 87%) of 7 as a colorless solid. mp 145-147 °C. 1H NMR (CDCl3, 200 MHz) δ 1.3 (m, 2H), 1.6 (m, 2H), 2.4 (s, 3H), 2.65 (d, 1H), 2.85 (dd, 1H), 3.05 (m, 1H), 3.95 (m, 2H), 4.2 (m, 1H), 4.4 (m, 1H), 5.3 (s, 1H), 5.9 (s, 1H), 7.3 (d, 2H), 7.7 (d, 2H); IR (KBr) 3240, 2920, 1700, 1460, 1360, 1270, 1170, 1080 cm-1. HRMS mass calcd for C17H25N2O4S2 (M + H)+: 385.1256. Found: 385.1232. HPLC: tR ) 11.9 min. Biotin Nitrile, 8. This compound was prepared by reaction of 0.8 g (2.1 mmol) of 7 and NaCN in absolute alcohol as previously described (29) to give 0.30 g (60%) of 8 as a colorless solid. mp 153-154 °C. HPLC: tR ) 5.7 min. Homobiotin, 9. A 230 mg (0.96 mmol) quantity of 8 in 15 mL of 1 N NaOH was heated to reflux for 3 h. The reaction mixture was acidified with concentrated HCl and a white precipitate formed. The precipitate was filtered, washed with water, and dried under vacuum to give a near quantitative yield of 9 as a colorless solid. mp > 240 °C. 1H NMR (MeOH-d4, 500 MHz) δ 1.4 (m, 2H), 1.55-1.8 (m, 4H), 2.2 (t, J ) 3.3 Hz, 2H), 2.8 (d, J ) 6.8 Hz, 1H), 3.0 (dd, J ) 2.5, 6.6 Hz, 1H), 3.35 (m, 1H), 4.4 (m, 1H), 4.6 (m, 1H). IR (KBr) 3380, 3260, 2920, 1700, 1620, 1480, 1450, 1420, 1140, 1280, 1080 cm-1. HRMS mass calcd for C11H19N2O3S (M + H)+: 259.1116; Found 259.1115. HPLC: tR ) 7.4 min. Norbiotinamine N-tBoc, 11. This compound was prepared as previously described (30). Briefly, reaction of TFP ester, 2a, with azide to form the acyl azide 10, then rearrangement and trapping of the amine to give the N-tBoc protected norbiotinamine, 11. HPLC: tR ) 10.7 min. Norbiotinamine, 12. This compound was prepared by treatment of 11 with trifluoroacetic acid as previously described (30). 1H NMR (MeOH-d4, 200 MHz) δ 1.4-1.9 (m, 6H), 2.7 (d, J ) 6.5 Hz, 1H), 2.8-3.0 (m, 3H), 3.13.3 (m, 1H), 4.3 (m, 1H), 4.5 (m, 1H). HRMS mass calcd for C9H18N3OS (M + H)+: 216.1171; Found 216.1171. HPLC: tR ) 2.8 min. Biotinamide, 13. To an ice-bath cooled solution containing 700 mg (1.8 mmol) of biotin TFP ester, 2a, in 5 mL of DMF was added 30 mL of concentrated NH4OH solution dropwise. After the addition, the reaction mixture was stirred in the ice bath for 5 min. Then the ice bath was removed and the reaction mixture was allowed to come to room temperature while stirring for 2 h. The white solid was filtered, washed with water and ether, and dried under vacuum to give a near quantitative yield of 13 as a white solid. mp 235-237 °C (dec). 1H NMR (DMSO-d6, 200 MHz) δ 1.1-1.8 (m, 6H), 2.0 (t, J ) 3.6 Hz, 2H), 2.6 (m, 2H), 2.8 (dd, J ) 6.2, 2.4 Hz, 1H), 3.1 (m, 1H), 4.1 (m, 1H), 4.3 (m, 1H), 6.4 (d, J ) 5.7 Hz, 2H), 6.7 (s, 1H), 7.4 (s, 1H). IR (KBr) 3340, 3200, 2920, 1680, 1470, 1410, 1270, 1160, 1080 cm-1. HRMS mass calcd for C10H18N3O2S (M + H)+: 244.1119. Found: 244.1111. HPLC: tR ) 6.5 min. Biotinamine, 14. To a 300 mg (1.24 mmol) quantity of biotinamide, 13, in 20 mL of THF was added 225 mg of (5.7 mmol) LiAlH4. The suspension was refluxed for 24 h. Following this, the reaction mixture was cooled with an ice bath, and H2O was added dropwise to destroy the excess LiAlH4. The product was extracted with CHCl3 (3 × 100 mL). The CHCl3 solution was washed with H2O then with aqueous saturated NaCl. The CHCl3 solution was then dried over anhydrous Na2SO4, filtered, and evaporated to yield 280 mg (98%) of 14 as a colorless solid. mp 209-211 °C. 1H NMR (MeOH-d4, 500 MHz) δ 1.3-1.8 (m, 10H), 2.66 (t, J ) 3.5 Hz, 2H), 2.74 (d, J )


Wilbur et al.

Scheme 1. General Scheme for Preparation of Biotin-CN-Cbl Derivatives, 3a-o

a

DMF, Et3N, rt, 1-16 h, 80-96%. b3j, 3l, or 3n,TFA, rt, 15 min, 90-98%.

6.3 Hz, 1H), 2.96 (dd, J ) 6.5, 2.5 Hz, 1H), 3.3 (m, 1H), 4.3 (m, 1H), 4.5 (m, 1H). IR (KBr) 3220, 2920, 2840, 1690, 1570, 1470, 1320, 1250, 1160, 1090 cm-1. HRMS mass calcd for C10H20N3OS (M + H)+: 230.1327; Found 230.1318. HPLC: tR ) 2.8 min. Preparation of 17. This compound was prepared by reaction of the biotin TFP ester 2a with 3-aminobutyric acid, 15, as previously described (23). Preparation of 18. TFP-OTFA (0.14 mL, 0.82 mmol) was added slowly to a mixture containing d-biotin, 4, (0.10 g, 0.41 mmol), Et3N (0.17 mL, 1.23 mmol), and anhydrous DMF (12 mL) at room temperature. The mixture was stirred at room temperature for 10 min and then mixed with a solution of water (3 mL), acetone (5 mL), sodium hydrogen carbonate (0.172 g, 2.05 mmol), and R-tert-butyl-L-aspartic acid, 16, (0.0924 g, 0.41 mmol). After the reaction mixture was stirred at room temperature for 6 h, volatile materials were evaporated under vacuum. The residue was washed with 20 mL of ethyl acetate and neutralized with 22 mL of 0.1 N HCl. After setting the precipitate at 5 °C for overnight, it was filtered, washed with water, and dried under high vacuum to yield 0.14 g (82%) of 18 as a colorless solid. mp 158-160 °C. 1H NMR (CD3OD, 200 MHz) δ 1.46 (s, 9 H), 1.49 (m, 2H), 1.66 (m, 4H), 2.27 (t, 2H), 2.78 (m, 2H), 2.97 (m, 1 H), 3.31 (m, 2H), 4.41 (dd, 1 H), 4.59 (dd, J ) 5.0 Hz, 1 H), 4.75 (m, 1 H). HRMS calcd for C18H29N3NaO6S (M + Na)+: 438.1675. Found: 438.1675. HPLC: tR ) 9.2 min. Preparation of 24. Biotin tetrafluorophenyl ester, 2a (0.404 g, 1.03) was added slowly to a solution of O-tert-

butyl-serine methyl ester 19 (0.218 g, 1.03 mmol) and Et3N (0.43 mL, 3.09 mmol) in anhydrous DMF (5 mL) at room temperature. The reaction mixture was stirred at room temperature for 1 h and then was evaporated to dryness. The residue was purified by silica gel column chromatography (40 g) eluting with 5% MeOH/EtOAc to give 0.37 g (89%) of 24 as an oily solid. 1H NMR (CDCl3, 200 MHz) δ 1.14 (s, 9H), 1.48-1.84 (m, 6H), 2.31 (t, J ) 7.0 Hz, 2H), 2.72-2.94 (m, 2H), 3.56 (dd, J ) 3.3 Hz, 1H), 3.81 (m, 2H), (dd, J ) 4.4 Hz, 1H), 4.53 (dd, J ) 4.4 Hz, 1H), 4.72 (m, 1H), 5.19 (s, 1H), 5.84 (s, 1H). HRMS calcd for C18H32N3O5S (M + H)+: 402.2063. Found: 402.2061. HPLC: tR ) 10.5 min. Preparation of 25. A solution containing potassium hydroxide (0.203 g, 3.62 mmol) N-biotinyl-O-tert-butylserine methyl ester, 24 (0.413 g, 1.03 mmol), methanol (5 mL), and water (5 mL) was stirred at 50-55 °C for 1 h. After the solution was cooled to room temperature, pH of the solution was adjusted to 4-5. Solvents were evaporated under vacuum, then the residue was taken up by methanol and evaporated to dryness. The crude product was purified by silica gel column chromatography (30 g) eluting with 20% MeOH/EtOAc to give 0.26 g (65%) of 25 as a colorless solid. mp 164-166 °C. 1H NMR (DMSO-d6, 200 MHz) δ 1.07 (s, 9H), 1.23-1.68 (m, 6H), 2.11 (t, J ) 7.0 Hz, 2H), 2.43-2.60 (m, 2H), 2.82 (dd, J ) 4.8 Hz, 1H), 3.08 (m, 1H), 3.41 (m, 1H), 3.99 (m, 1H), 4.12 (dd, J ) 4.4 Hz, 1H), 4.30 (dd, J ) 5.1 Hz, 1H), 6.40 (s, 1H), 6.55 (s, 1H), 7.35 (d, J ) 7.3 Hz, 1H), 8.43 (s, 1H). HRMS calcd for C17H30N3O5S (M + H)+: 388.1906. Found: 388.1912. HPLC: tR ) 11.8 min.

Chart 1. Biotin Conjugates of CN-Cbl-c-lactone Used as Standards



Chart 2. Biotin Conjugates of CN-Cbl-c-lactone Which Are Altered in Biotinamide Functionality

Preparation of 26. A mixture containing N-biotinylO-tert-butyl-serine methyl ester, 24 (0.15 g, 0.37 mmol), potassium hydroxide (0.062 g, 1.11 mmol), methanol (2 mL), and water (2 mL) was stirred at 55 °C for 30 min. The solution was taken to dryness under vacuum, then 1.5 mL of trifluoroacetic acid was added and stirred at room temperature for 16 h. Volatile materials were evaporated under vacuum, then the residue was purified by silica gel column chromatography (30 g) eluting with 50% MeOH/EtOAc to give 0.11 g (90%) of 26 as a colorless solid. mp 182-184 °C (dec). 1H NMR (CD3OD, 200 MHz) δ 1.49 (m, 2H), 1.68 (m, 4H), 2.31 (m, 2H), 2.70 (d, 1H), 2.94 (dd, 1H), 3.22 (m, 1H), 3.31 (m, 2H), 3.91 (d, 1H), 4.33 (m, 2H), 4.49 (dd, 1 H). HRMS calcd for C13H20N3Na2O5S (M - H + 2Na)+: 376.0919. Found: 376.0924. HPLC: tR ) 3.7 min. Preparation of 27. Biotin tetrafluorophenyl ester, 2a (2.12 g, 5.39 mmol) was added to a solution containing L-aspartic acid β-tert-butyl ester, 22 (1.0 g, 5.29 mmol), sodium bicarbonate (1.47 g, 17.44 mmol), water (40 mL), and acetone (40 mL) at room temperature. The reaction mixture was stirred at room temperature for 16 h and then evaporated to dryness. The residue was washed with

60 mL of ethyl acetate and neutralized with 19.0 mL of 1.0 N HCl. After setting at 5 °C for overnight, the precipitate was filtered, washed with water and dried under high vacuum to yield 1.91 g (87%) of the biotinaspartate-β-O-tBu ester adduct, 27 as a colorless solid. mp 167.6-168.5 °C. 1H NMR (CD3OD, 200 MHz) δ 1.45 (s, 9 H), 1.48-1.90 (m, 6 H), 2.22-2.30 (m, 2 H), 2.602.78 (m, 2 H), 2.83-2.97 (m, 1 H), 3.15-3.24 (m, 1 H), 3.29-3.32 (m, 1 H), 4.30 (dd, J ) 4.4 Hz, 1 H), 4.48 (dd, J ) 5.0 Hz, 1 H), 4.73 (dd, J ) 5.5 Hz, 1 H). HRMS calcd for C18H29N3O6S (M + H)+: 416.1855. Found: 416.1868. HPLC: tR ) 9.6 min. Preparation of 28. Biotin tetrafluorophenyl ester, 2a (1.64 g, 4.18 mmol) was added to a solution of N--tertBoc-L-lysine, 23 (1.0 g, 4.06 mmol), sodium bicarbonate (1.13 g, 13.40 mmol), water (40 mL), and acetone (40 mL) at room temperature. The reaction mixture was stirred at room temperature for 16 h, then volatile materials were evaporated under vacuum. The residue was washed with 60 mL of ethyl acetate and neutralized with 14.0 mL of 1.0 N HCl. After the product set at 5 °C overnight, the product was filtered, washed with cold water, and dried under vacuum to yield 1.91 g (87%) of the biotin-

Chart 3. Biotin Conjugates of CN-Cbl-c-lactone Which Have Substituents Alpha to Biotinamide Functionality


Wilbur et al.

Figure 1. Distances from biotin ring structure to the carbonyl or thiocarbonyl functionality in biotin conjugates having altered biotinamide functionalities. Biotin is included for comparison. The distances shown were obtained from structural models in Chem3D (CambridgeSoft Corp., Cambridge, MA) after structural and energy minimization.

lysine-N-tBoc adduct, 28 as a colorless solid. mp 156.8157.5 °C. 1H NMR (CD3OD, 200 MHz) δ 1.43 (s, 9 H), 1.48-1.90 (m, 10 H), 2.26 (t, J ) 7.2 Hz, 2 H), 2.69 (d, J ) 12.8 Hz, 1 H), 2.89-3.05 (m, 3 H), 3.16-3.26 (m, 1 H), 3.29-3.34 (m, 2 H), 4.26-4.34 (m, 2 H), 4.49 (dd, J ) 4.8 Hz, 1 H). HRMS calcd for C21H36N4O6S (M + Na)+: 495.2253. Found: 495.2240. HPLC: tR ) 10.3 min. Relative Dissociation Rate Analysis. To 300 µL of a 1 mg/mL solution of avidin (300 µg; 4.4 nmol) (or r-SAv) in a plastic microcentrifuge vial was added 4 equiv (18 nmol) of a biotin conjugate (3a-o) dissolved in 30 µL of a 10% aqueous DMSO solution. The avidin/biotin derivative mixture was incubated for 1 h at room temperature, and 100 µL of the solution was injected into the HPLC to assay binding. To the remaining avidin/biotin derivative solution was added 40 µL of a 1 mg/mL solution of biotin (40 µg; 164 nmol) in 10% aqueous DMSO. That solution was incubated for 1 h and 100 µL was removed for analysis by HPLC. The remaining solution was incubated for an additional 2 h, then another 100 µL was removed and analyzed by HPLC.

RESULTS

The primary goal of this research effort was to obtain information about how chemical modification of biotin conjugates affects their dissociation rate from Av and SAv. To attain that goal, several biotin derivatives were synthesized, conjugated with a cyanocobalamin (CN-Cbl) derivative (1), and their relative dissociation rates were measured by size exclusion HPLC analysis. Compound Synthesis. A general synthetic route to preparing the biotin-CN-Cbl derivatives is shown in Scheme 1. The synthesis of the CN-Cbl derivative 1, chosen for preparing the conjugates, is easily accomplished in high yield (>80%) by reacting the readily prepared c-lactone of CN-Cbl (25, 31, 32) with 4,7,10trioxa-1,13-tridecanediamine at 55 °C for 1 h (21). This compound was conjugated with the modified biotin derivatives for evaluation in the HPLC analysis. Modifications of biotin included increasing the biotin side chain length (Chart 2), changing the type of bond used to conjugate the biotin moiety with a linker (Chart 2), and conjugation with amino acids to introduce substit-

Scheme 2. Synthesis of Homobiotin TFP Ester, 2d

a MeOH, HCl, 99%. bLiAlH , 100%. cPyridine, TsCl, 0 °C, 5 h, 87%. dNaCN, EtOH, 60%. e1 N NaOH, ∆, 3 h, 99%. fDMF, Et N, 4 3 TFP-OTFA, rt, 30 min, 87%.



Scheme 3. Synthesis of Norbiotin Isothiocyanate, 2e

a

NaN3. btBuOH, ∆. cTFA. dDMF, TCDI, rt, 45 min, 55%.

uents on the methylene group R to the biotinamide functionality (Chart 3). Biotin-CN-Cbl Conjugates Used as Reference Compounds (3a, 3b, and 3c; Chart 1). Our previous studies demonstrated that the biotin-CN-Cbl conjugate, 3a, which has no alteration in the biotin moiety or linker, has a very slow dissociation rate from Av and SAv, similar to that of biotin (21). Because of the very slow dissociation rate, 3a was prepared for use as a reference compound. Similarly, our previous studies demonstrated that no binding was obtained with iminobiotin-CN-Cbl conjugate, 3b, so it was prepared as a reference compound for biotin derivatives that have no retention. The previous studies also demonstrated that a biotin derivative which is modified with a methyl substituent on the biotinamide nitrogen, 3c, had a measurable dissociation rate from Av and SAv over a 1-3 h period (21). Since we routinely incorporate the Nmethylglycine moiety into our new biotin derivatives, 3c was of particular interest as a reference compound for comparison purposes. Syntheses of 3a, 3b, and 3c were accomplished in high yield by reaction of biotin TFP ester, 2a, iminobiotin NHS ester, 2b, or biotin-sarcosine TFP ester, 2c with CN-Cbl derivative, 1 at room temperature for 4 h (21). Biotin-CN-Cbl Conjugates That Have Altered Biotinamide Bonds (3d-f; Chart 2). Most biotin conjugates are prepared by reacting an activated ester of biotin with an amine containing moiety. If a primary aliphatic amine is conjugated, high binding affinity with Av and SAv is obtained, but in vivo applications are

limited due to rapid cleavage of the biotinamide bond by biotinidase (33). Thus, our initial studies were focused on alteration of the biotinamide bond, either by moving it away from the biotin ring structure with insertion of a methylene group (Figure 1, panel B, increase of 1.2 Å) or by changing the nature of the biotinamide bond. Although it was uncertain whether addition of an extra methylene in the side chain of biotin, as is found in homobiotin (34), would be stabile to biotinidase cleavage, it was of interest to know if the rate of dissociation from Av and SAv would be affected by this change. Therefore, homobiotin tetrafluorophenyl ester, 2d, was prepared as shown in Scheme 2. Reaction of 2d with the CN-Cbl derivative, 1, provided the homobiotin-CN-Cbl conjugate, 3d, in 95% yield. In a second approach, the biotinamide bond was replaced with a thiourea bond to assess how that modification affected the dissociation rate. We chose to prepare two biotin conjugates, 3e and 3f, containing thiourea bonds for the study. Not only was the nature of the biotinamide bond changed in biotin conjugates prepared, but the distance from the biotin ring structure to the hydrolyzable thiocarbonyl was increased 1.2-2.5 Å over that of the normal biotinamide bond (Figure 1, panels C and D). This fact, we believed, would help to stabilize the bonds toward biotinidase cleavage. One derivative, 3e, was prepared from norbiotin isothiocyanate, 2e, and the other derivative, 3f, was prepared from biotin isothiocyanate, 2f. The syntheses of 2e and 2f are shown in Schemes 3 and 4, respectively. Conjugations of 1 with norbiotin isothiocyanate,

Scheme 4. Synthesis of Biotin Isothiocyanate, 2f

a

DMF, concentrated NH4OH, 0 °C to rt, 2 h, 99%. bTHF, LiAlH4, ∆, 24 h, 98%. cDMF, TCDI, rt, 2 h, 74%.


Wilbur et al.

Scheme 5. Synthesis of Biotin-β-Amino Acid TFP Esters, 2g and 2l

a

15, DMF, Et3N, rt, 24 h, 28%; 16, H2O, acetone, NaHCO3, rt, 6 h, 82%. bTFP-OTFA, DMF, Et3N, rt, 30 min.

2e, and biotin isothiocyanate 2f, provided the CN-Cbl conjugates 3e and 3f in 94% and 80% yields, respectively. Biotin-CN-Cbl Conjugates That Have Substituents Alpha to the Biotinamide Functionality (3g-o, Chart 3). Biotin conjugates which have functional groups of varying size R to the biotinamide bond were targeted for synthesis because previous studies had indicated that this modification might retain the high affinity binding and additionally result in stabilization toward biotinidase cleavage (23). The R substituents were obtained by incorporating amino acids or functional group protected amino acids between biotin and the hydrophilic linker. Three amino acid conjugates, 3g, 3l, and 3m (Chart 3), were prepared from amino acids that had carboxylates beta to the amine (3-aminobutyric acid, 15, and L-aspartic acid-R-tBu ester, 16). The syntheses of these derivatives is shown in Scheme 5. Reaction of 15 or 16 with the TFP ester of biotin, 2a, provided biotin adducts 17 and 18. The TFP esters 2g and 2l were prepared in situ from 17 and 18, then conjugated withCN-Cbl derivative 1 to provide 3g and 3l in 78 and 94% yields, respectively. Removal of the tBu ester from 2l was accomplished in neat TFA to provide 3m in 90% yield. Three additional amino acid derivatives (a serine derivative, 19; an aspartate derivative, 22; and a lysine derivative, 23) were conjugated with biotin TFP ester, 2a as shown in Scheme 6. Some difficulties with the conjugations of biotin and serine, 21, led to the use of the diprotected, methyl ester/tBu ether containing, derivative 19. Reaction of biotin TFP ester, 2a, with 19 provided the biotin serine methyl ester derivative 24 in 89% yield. Hydrolysis of the crude biotin conjugate, 24,

with KOH gave the free carboxylate 25 in 65% isolated yield. When the crude hydrolysis product was treated with TFA without isolation, a 90% yield of the fully deprotected biotin-serine conjugate, 26, was obtained. To evaluate the difference in dissociation rates due to steric encumbrance, preparation of CN-Cbl conjugates of both 25 and 26 was desired. Conjugation of 25 with CN-Cbl derivative 1 was accomplished by in situ generation of the TFP ester, 2h, followed by reaction with 1. This reaction sequence provided the desired conjugate 3h in 92% isolated yield. Conjugation of 26 was accomplished in the same manner, where in situ generated TFP ester 2i was reacted with 1 to provide 3i in 96% isolated yield. Conjugation of L-aspartic acid, which has the betacarboxylate protected as a t-Bu ester, 22, results in a different biotin conjugate than conjugation of the R-carboxylate protected aspartate derivative, 18. Reaction of biotin-TFP ester, 2a, with 22 provided the conjugate 27 in 87% yield. Activation of 27 for conjugation was accomplished by preparing the TFP ester, 2j, in 58% isolated yield. The biotin-aspartate TFP ester, 2j, was subsequently conjugated with 1 to give 3j in 95% isolated yield. The tBu ether moiety was removed from 3j by reaction in TFA for 15 min to provide a 98% isolated yield of 3k. A conjugate containing biotin and lysine, 3o, was prepared in which a free amine was present. Biotin conjugates containing lysine in that configuration were particularly attractive as they appeared to have the potential for conjugation of an additional moiety, provided the dissociation rate was not increased. Conjugation of the -N-tBoc protected L-lysine, 23, with biotin TFP ester, 2a, provided 28 in 87% yield. Conversion of 28 to its TFP ester was accomplished using EDC and

Scheme 6. Synthesis of Biotin-r-Amino Acid TFP Esters, 2h-j and 2n

a 19, DMF, Et N, rt, 1 h, 89%; 20, KOH, MeOH, H O, 1 h, 55 °C, 65%; 21, TFA, 16 h, 90%; 22 or 23,H O, acetone, NaHCO , rt, 3 2 2 3 16 h, 87%. b25 or 26,TFP-OTFA, Et3N, CH3CN, rt, 1 h; 27 or 28, TFP-OH, DMF, EDC, rt, 48 h.



Table 1. Percentage of Biotin-CN-Cbl Dyes Bounda with Av or SAv at 1 and 3 h after Being Challenged by a Large Excess of Biotinb biotin -CN-Cbl derivative binding with Av 3a 3b 3c 3d 3e 3f 3g 3h 3i 3j 3k 3l 3m 3n 3o binding with SAv 3a 3c 3e 3g 3k

1h

3h

>98 0 98 8 73 49 98 29 98 0 0 98 30 >98 98 30 50 >98 70

>98 98 36

a

The quantity bound was measured by the peak areas of (S)Avbound dye vs free dye at 360 nm. Retention times of bound and free dye were determined by standards. In some of the chromatograms, an additional small peak between the bound and free was noted. It is not clear what that peak is composed of. In those cases, the area of that peak was not included in the percentages shown. bA 53 M quantity of biotin was added to a solution containing 4 M equiv of biotin-CN-Cbl derivative bound with Av or SAv.

TFP-OH to provide 2n in 45% isolated yield. Conjugation of 2n with 1 provided 3n in 96% yield. The tBoc protecting group was removed from 3n with TFA to give 3o in 93% yield. HPLC Analyses for Relative Dissociation Rates. The relative rates of dissociation from Av were estimated by HPLC analysis for all biotin-CN-Cbl derivatives prepared [Chart 1 (3a-c), Chart 2 (3d-f), and Chart 3 (3g-o)]. Relative rates of dissociation from SAv were also evaluated for a few of the biotin-CN-Cbl derivatives (3a, 3c, 3e, 3g, and 3k). In the analyses, 4 M equiv of a biotin-dye conjugate were added to 1 M equiv of Av or SAv, and the solution was incubated at room temperature for 1 h. At that time, an aliquot was removed for size exclusion HPLC analysis (t ) 0). Following that analysis, 53 M equiv of biotin was added to the Av/dye mixture, and aliquots were taken at 1 and 3 h post-biotin addition for HPLC analyses. HPLC peak areas for bound and free biotin-CN-Cbl dye were used to obtain percentages. The percent bound was calculated by dividing peak area for the protein-bound dye by the total peak area (bound and free) for dye (362 nm). Percentages of biotin-CN-Cbl conjugates bound with Av or SAv are tabulated in Table 1. A large variation in the percent of biotin-CN-Cbl dye bound with Av or SAv was noted within the 3 h assay period. With the exception of the iminobiotin derivative, 3b, all of the biotin-CN-Cbl derivatives were fully bound with Av and SAv when no biotin was present. As in our previous studies, unmodified biotin-CN-Cbl derivative 3a did not dissociate from either Av or SAv at a detectable rate in the assay period when a large excess of biotin was introduced. The homobiotin derivative, 3d, and the derivatives that contained thiourea bonds, 3e and 3f, dissociated rapidly when biotin was added. Biotin derivatives that have substituents R to the biotinamide

bond, e.g., the R-methyl derivative 3g, the R-hydroxymethylene derivative 3i, and R-carboxylate derivative 3m, all had no detectable dissociation in the presence of biotin within the 3 h test period. In contrast to this, biotin-CN-Cbl derivatives which contained the larger substituents, R-acetate, 3k, and R-butylamine, 3o, dissociated relatively rapidly. Interestingly, the biotin-CNCbl derivatives which had substituents R to the biotinamide containing the very bulky tBu ether, 3h, tBu esters 3j, 3l and tBoc group, 3n, appeared to be more tolerated than the derivatives that had changes in the biotinamide bond (3c-f). Another interesting observation is that removal of the bulky N-tBoc group in the protected lysine adduct, 3n, to give the free amine, 3o, resulted in a very rapid dissociation rate. This increase in the dissociation rate must reflect a difficulty in binding with the positive amine derivative. Since Av has a high pI [e.g., 10 (1)], the observed dissociation rate may be due to repulsion of charges. Although there were some differences in the dissociation rates of biotin-CN-Cbl derivatives when bound with SAv versus Av (Table 1), the trend for a biotin derivative to have either a slow dissociation or a more rapid dissociation was retained. DISCUSSION

The high affinity binding of biotin with Av (K ) 1015 M-1) (2, 35, 36) and SAv (K ) 1013-1014 M-1) (2, 37) makes these compounds particularly attractive for conjugates used in the cancer pretargeting approach. While the binding affinities might be expected to decrease when biotin is conjugated with another molecule, it seems reasonable that with the appropriately designed biotin conjugates, high affinity binding can be retained. This statement is based on the observation that many biotin conjugates have been prepared which have an apparent high affinity for Av and/or SAv and on the fact that in biotin derivatives, which have an unmodified bicyclic biotin ring structure, most of the “hydrogen-bonding network” is retained (38). The design of biotin conjugates for this application appeared to be quite simple in the initial examination, however, as studies have progressed it has become apparent that there are specific requirements that must be met in the design of new biotin conjugates for in vivo application. Those requirements include (1) high water solubility, (2) incorporation of a spacer between biotin and the molecule to which it is attached, and (3) incorporation of a functional group that blocks the biotinamide cleavage by biotinidase without significantly decreasing binding affinity with Av or SAv. Water solubility has not been a concern in biotin derivatives prepared to carry radiometals (39-44). However, our initial investigations to design biotin conjugates for pretargeting halogen radionuclides were hampered by the very low water solubility of biotin derivatives. This difficulty was overcome by introducing a hydrophilic molecule between the biotin moiety and the molecule that was being conjugated (23, 26, 45, 46). In addition to increasing water solubility, the hydrophilic linking molecule acts as a spacer between the biotin moiety and its conjugated molecule. A number of research efforts have demonstrated that introduction of a spacer moiety between biotin and certain molecules can improve binding with Av and/or SAv (47-53). Therefore, in our optimization of biotin derivatives, we generally include a hydrophilic linker molecule between biotin and the molecule with which it is to be conjugated. Our initial in vitro studies were also hampered by an instability of biotin conjugates. This instability appeared


to be brought about by cleavage of biotin by the enzyme biotinidase (22, 33, 54-56). Other investigators had indicated that modification of the biotinamide bond with a methyl group blocked this cleavage (57), and our own studies confirmed that finding (23). Since obtaining that result, we have made a number of biotin derivatives that contain the hydrophilic linker and also contain the N-methyl biotinamide functionality to provide in vivo stability. More recently, we examined the relative association and dissociation rates of some biotin derivatives, including the N-methyl derivative by HPLC analyses (21). While only small differences were observed in the association rates, significant increases in the rate of dissociation of biotin conjugates from Av and SAv were observed. Rapid dissociation rates of biotin derivatives from Av or SAv can be problematic for in vivo application, particularly in pretargeting of radionuclides for therapy. Although the rate at which radiolabeled biotin molecules are released from a tumor site is dependent on a number of factors, the rate of dissociation from Av or SAv is perhaps the most important factor. It is our belief that in an optimal therapy situation, the pretargeted mAbSAv conjugate is homogeneously distributed in the tumor and all of the available biotin binding sites are filled with radiolabeled biotin derivatives. When all of the available biotin binding sites are filled, a homogeneous distribution of the radionuclide is obtained in the tumor. If only a portion of the biotin binding sites on the tumor-bound mAb-SAv conjugate are filled, it unlikely that a homogeneous distribution will be obtained. The biotin binding sites on cells nearest to the blood vessels will be filled to a higher extent than those further from the blood vessels. This situation has previously been described for high affinity mAbs as having a “binding site barrier” (58). When some of the biotin binding sites are unfilled, the dissociation rate is not as important since avidity will play a role in keeping the radiolabeled biotin derivative in the tumor. However, when the biotin binding sites are filled, either by the initial radiolabeled biotin derivative or by endogenous biotin, then the dissociation rate of the biotin derivative is of paramount importance in tumor retention. Dissociation rates of biotin derivatives from Av and SAv have previously been accurately measured using [14C]biotin2 or [3H]biotin (37, 59). In our studies, an accurate value for the dissociation rates was not required, so we chose to use a rapid nonradioactive HPLC assay to measure relative dissociation rates of biotin derivatives over a 3-4 h period (21). In previous studies, it was demonstrated that relative dissociation rates could be obtained by challenging biotin-cyanocobalamin (biotinCN-Cbl) conjugates that were bound to Av or SAv with a large excess (e.g., >50 Μ equiv) of biotin. On the basis of those studies, we chose to prepare biotin derivatives conjugated with a CN-Cbl derivative, 1, as shown in Scheme 1. In our initial approach to biotin modification, we hypothesized that structural modification of the biotinamide bond might provide stability toward biotinidase cleavage while retaining the high binding affinity. The very simple modification of extending the side chain of biotin by one methylene unit to form the homobiotin derivative, 3d, was examined first. Surprisingly, the homobiotin derivative was found to have a rapid dissociation rate relative to an unmodified biotin derivative, 3a (Table 1). Our next approach to biotin modification was to change the type of bond connecting biotin and the linker from the biotinamide bond to a thiourea bond. Two

Wilbur et al.

thiourea bonded biotin derivatives, 3e and 3f, were prepared and evaluated. These derivatives demonstrated even faster dissociation rates than the homobiotin derivative. The results obtained in the initial studies indicate that the biotin moiety and the biotinamide bond should not be altered if retention of the very high affinity binding is desired. Biotin derivatives which have amino acids conjugated to biotin were also of interest in our binding study. The interest in biotin-amino acid derivatives was brought about by a previous literature report (59) and our own experiences, which indicated that biotinidase cleavage may be blocked if the conjugates has substituents R to the biotinamide bond. Rosebrough reported that after reaction of the sulfhydryl group, the biotin derivative formed from conjugation of cysteine and biotin was stable to biotinidase cleavage (60). The biotin derivative formed in that study has a carboxylate R to the biotinamide. In our own studies, it was noted that conjugation of biotin with 3-aminobutyric acid provided a substantial decrease in the rate of biotin cleavage by biotinidase (23). We were particularly interested in determining how the size and nature of functional group R to the biotinamide functionality affected the dissociation rate. Thus, a series of derivatives which contained substituents R to the biotinamide bond were prepared (Chart 3). The series included a methyl (3g), a hydroxymethylene (3i), an acetate (3k), a carboxylate (3m), and a butylamine (3o). The synthetic routes to the targeted biotin derivatives required that tBu ether, tBu ester, and N-tBoc protecting groups be used. The biotin derivatives which contained the protecting groups were also conjugated with the CN-Cbl moiety. While not of primary interest, evaluating those biotin derivatives (3h, 3j, 3l, and 3n) permitted an analysis of the effect of bulky protecting group on the dissociation rate. The results obtained from the HPLC analyses indicate that the smaller substituents, the methyl, hydroxymethylene and carboxylate groups do not increase the dissociation rate, whereas larger substituents such as an acetate or a butylamine group increase the dissociation rate significantly. As expected, a significant steric effect was also noted for biotin derivatives containing the tBu and tBoc protecting groups. The goal of this investigation was to obtain information on how modifications of biotin conjugates affected their dissociation rates from Av and SAv such that biotin derivatives which retained very slow dissociation rates could be identified. It seems likely that there are other modifications than those studied that could retain slow dissociation rates from Av and SAv and also possess biotinidase stability. However, once it was determined in these studies that the biotin moiety must remain unaltered to retain a slow dissociation rate, our efforts were directed solely at evaluating the effect of substituents R to the biotinamide bond. In contrast to biotin derivatives used for in vitro assays, such as previously reported radioiodinated biotin derivatives (61-65), we believe that structural modifications will be required in designing optimal biotin derivatives. Although some of the previously reported radiohalogenated (66-72) and metal binding biotin derivatives (13, 39, 40, 42, 43, 7377) have been successfully applied to pretargeting without incorporating all of the structural parameters of biotin conjugates listed above, full optimization of the biotin conjugate structure for in vivo use should provide a more efficacious reagent when applied to Targeted Radiotherapy. On the basis of previous studies, we have developed a number of radiolabeled biotin derivatives that contain a

Relative Dissociation Rates of Biotin Derivatives Chart 4. Preferred Biotin Derivatives for in Vivo Applications

N-methyl biotin moiety (78). The data obtained in this study indicates that the dissociation rates from Av and SAv are significantly increased when biotin conjugates contain a biotinamide bond that has been modified as a N-methyl derivative. However, it must be emphasized that these data alone do not indicate that N-methyl biotinamide derivatives will not be useful for in vivo application. Indeed, Axworthy et al. (77) have demonstrated that a Y-90 labeled N-methyl biotin derivative can be successfully used in a pretargeting approach for cancer therapy in the mouse model. The dissociation rates are not the only factors in determining if a biotin derivative will be useful in vivo, and dissociation rates are not fully predictive of tumor retention in vivo as other factors, such as avidity, affect the retention of biotin derivatives in a tumor. Relative dissociation rates do, however, provide a parameter that can be optimized for in vivo use as most applications that utilize the biotin/ SAv binding pair do so because of the high affinity of these compounds. On the basis of the studies herein, biotin derivatives in which the structure of biotin is not modified but contains a linker molecule that has a small substituent, such as a hydroxymethylene or carboxylate group, R to the biotinamide bond should provide excellent candidates for in vivo applications. Incorporation of structural features that result in slow dissociation from Av or SAv alone does not result in compounds that are optimized. For example, although high binding affinity was obtained with the derivative containing an R methyl group (3g), our previous studies indicated that it was not completely stable to biotinidase degradation (23) making that configuration nonoptimal for in vivo use. Other biological factors, such as half-life in blood, metabolic pathway, and routes of excretion also play a role in obtaining the optimal biotin derivative for in vivo applications. Ultimately, optimization of biotin derivatives for in vivo applications must have these parameters included. Our present choice of biotin derivatives is shown in Chart 4. Coupling serine with biotin provides an R-hydroxymethylene containing derivative, 29. Coupling biotin with either an aspartate, lysine, or cysteine can provide an R-carboxylate as these adducts, 30, 31, and 32, can be conjugated to other molecules (R) through their carboxylate, amine, or sulfhydryl groups (respec-


tively). The biotin-serine conjugate 29 and biotin-aspartate 30 are shown with our preferred hydrophilic linker as part of the biotin derivative. The biotin-lysine conjugate, 31, and biotin-cysteine conjugate, 32, might be more effectively used for conjugation with molecules that are ionized or have an inherent high water solubility. Importantly, we have conducted studies with radioiodinated derivatives that have shown the R-hydroxymethylene and R-carboxylate functionalities effectively block cleavage by biotinidase (79). In Summary. Fifteen biotin derivatives were prepared and conjugated with a CN-Cbl derivative, 1. The relative dissociation rates of those compounds were measured by size exclusion HPLC. The HPLC analyses provided definitive data on how the various modifications in the biotin structure affected the dissociation rates from Av. Only the unmodified biotin conjugate, 3a, and the modified conjugates that contained a methyl (3g), hydroxymethylene (3i), or carboxylate (3m) retained the slow dissociation rate found with biotin. Slightly different values were obtained when SAv was used rather than Av, but the trends for slow dissociation were the same as found with Av. From these data, we propose that the best biotin conjugates for in vivo applications are those that have a hydroxymethylene or a carboxylate R to the biotinamide bond. Biotin derivatives with these structural features retain high binding affinities. In other studies, we have demonstrated that biotin derivatives with these structural features are also stable to biotinidase cleavage. ACKNOWLEDGMENT

We thank Dr. Ross Lawrence (Department of Medicinal Chemistry, University of Washington) for efforts in obtaining mass spectral data. We are grateful for the financial support provided by the Department of Energy, Medical Applications and Biophysical Research Division, Office of Health and Environmental Research under Grant DE-FG03-98ER62572. Supporting Information Available: HPLC chromatograms, NMR spectra, and mass spectra of all new or not fully characterized compounds (3d-o, 2e, 2f, 2j, 2n, 7, 9, 13, 18, 24-26, 28). This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Green, N. M. (1975) Avidin. Adv. Protein Chem. 29, 85133. (2) Green, N. M. (1990) Avidin and Streptavidin. Methods Enzymol. 184, 51-67. (3) Bayer, E. A., and Wilchek, M. (1980) The Use of the AvidinBiotin Complex as a Tool in Molecular Biology. Methods Biochem. Anal. 26, 1-45. (4) Bayer, E. A., and Wilchek, M. (1996) The Avidin-Biotin System. In Immunoassay (E. P. Diamandis, and T. K. Christopoulos, Eds.) pp 237-267, Academic Press, San Diego. (5) Diamandis, E. P., and Christopoulos, T. K. (1991) The Biotin-(Strept)Avidin System: Principles and Applications in Biotechnology. Clin. Chem. 37, 625-636. (6) Wilchek, M., and Bayer, E. A. (1990) Introduction to AvidinBiotin Technology. Methods Enzymol. 184, 5-45. (7) Hnatowich, D. J., Virzi, F., and Rusckowski, M. (1987) Investigations of Avidin and Biotin for Imaging Applications. J. Nucl. Med. 28, 1294-1302. (8) Paganelli, G., Riva, P., Deleide, G., Clivio, A., Chiolerio, F., Scassellati, G. A., Malcovati, M., and Siccardi, A. G. (1988) In Vivo Labeling of Biotinylated Monoclonal Antibodies by Radioactive Avidin: A Strategy to Increase Tumor Radiolocalization. Int. J. Cancer 2, 121-125.

582 Bioconjugate Chem., Vol. 11, No. 4, 2000 (9) Paganelli, G., Pervez, S., Siccardi, A. G., Rowlinson, G., Deleide, G., Chiolerio, F., Malcovati, M., Scassellati, G. A., and Epenetos, A. A. (1990) Intraperitoneal Radio-Localization of Tumors Pre-Targeted by Biotinylated Monoclonal Antibodies. Int. J. Cancer 45, 1184-1189. (10) Paganelli, G., Magnani, P., Zito, F., Villa, E., Sudati, F., Lopalco, L., Rossetti, C., Malcovati, M., Chiolerio, F., Seccamani, E., Siccardi, A. G., and Fazio, F. (1991) Three-step monoclonal antibody tumor targeting in carcinoembryonic antigen-positive patients. Cancer Res. 51, 5960-5966. (11) 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. (12) Fritzberg, A. R., Kasina, S., Rao, T. N., Vanderheyden, J.L., and Srinivasan, A. (1997) Metal-Radionuclide-Labeled Proteins and Glycoproteins for Diagnosis and Therapy, U.S. Patent 5,616,692. (13) Goodwin, D. A., and Meares, C. F. (1997) Pretargeting: General Principles. Cancer (Suppl.) 80, 2675-2680. (14) Goodwin, D. A., Meares, C. F., and Osen, M. (1998) Biological properties of biotin-chelate conjugates for pretargeted diagnosis and therapy with the avidin/biotin system. J. Nucl. Med. 39, 1813-1818. (15) Sakahara, H., and Saga, T. (1999) Avidin-biotin system for delivery of diagnostic agents. Adv. Drug Del. 37, 89-101. (16) Stoldt, H. S., Aftab, F., Chinol, M., Paganelli, G., Luca, F., Testori, A., and Geraghty, J. G. (1997) Pretargeting Strategies for Radio-immunoguided Tumour Localisation and Therapy. Eur. J. Cancer 33, 186-192. (17) Zhu, H., Jain, R. K., and Baxter, L. T. (1998) Tumor Pretargeting for Radioimmunodetection and Radioimmunotherapy. J. Nucl. Med. 39, 65-76. (18) Mock, D. M. (1999) Biotin status: which are valid indicators and how do we know? J. Nutr. 129, 498S-503S. (19) Zempleni, J., and Mock, D. M. (1999) Biotin biochemistry and human requirements. J. Nutr. Biochem. 10, 128-138. (20) Wang, H., and Pevsner, J. (1999) Detection of endogenous biotin in various tissues: novel functions in the hippocampus and implications for its use in avidin-biotin technology. Cell Tissue Res. 296, 511-516. (21) Wilbur, D. S., Pathare, P. M., Hamlin, D. K., Frownfelter, M. B., Kegley, B. B., Leung, W.-Y., and Gee, K. R. (2000) Evaluation of Biotin-Dye Conjugates for Use in an HPLC Assay to Assess Relative Binding of Biotin Derivatives with Avidin and Streptavidin. Bioconjugate Chem. 11, 584-598. (22) Hymes, J., Fleischhauer, K., and Wolf, B. (1997) Biotinidase in Serum and Tissues. Methods Enzymol. 279, 422-434. (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) Gamper, H. B., Reed, M. W., Cox, T., Virosco, J. S., Adams, A. D., Gall, A. A., Scholler, J. K., and Meyer, R. B. (1993) Facile Preparation of Nuclease Resistant 3′ Modified Oligodeoxynucleotides. Nucleic Acids Res. 21, 145-150. (25) 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. (26) Wilbur, D. S., Hamlin, D. K., Vessella, R. L., Stray, J. E., Buhler, K. R., Stayton, P. S., Klumb, L. A., Pathare, P. M., and Weerawarna, S. A. (1996) Antibody fragments in tumor pretargeting. Evaluation of biotinylated Fab′ colocalization with recombinant streptavidin and avidin. Bioconjugate Chem. 7, 689-702. (27) Sundberg, S. A., Barrett, R. W., Pirrung, M., Lu, A. L., Kiangsoontra, B., and Holmes, C. P. (1995) Spatially-Addressable Immobilization of Macromolecules on Solid Supports. J. Am. Chem. Soc. 117, 12050-12057. (28) Alves, A. M., Holland, D., and Edge, M. D. (1989) A Chemical Method of Labeling Oligodeoxyribonucleotides with Biotin: A Single Step Procedure Using a Solid-Phase Methodology. Tetrahedron Lett. 30, 3089-3092.

Wilbur et al. (29) Hofmann, K., Chen, C., Bridgwater, A., and Axelrod, A. E. (1947) Furan and Tetrahydrofuran Derivatives. VII. The Synthesis and Biological Activity of a Number of Oxybiotin Homologues. J. Am. Chem. Soc. 69, 191-195. (30) Szalecki, W. (1996) Synthesis of Norbiotinamine and Its Derivatives. Bioconjugate Chem. 7, 271-273. (31) Wagner, F. (1965) Reactions of the cyano and alkyl cobalamins. Proc. R. Soc. London, Ser. A 288, 344-347. (32) Lien, E. L., Ellenbogen, L., Law, P. Y., and Wood, J. M. (1974) Studies on the Mechanism of Cobalamin Binding to Hog Intrinsic Factor. J. Biol. Chem. 249, 890-894. (33) Pispa, J. (1965) Animal Biotinidase. Ann. Med. Exp. Biol. Fenniae 43, 3-40. (34) Ruis, H., Brady, R. N., McCormick, D. B., and Wright, L. D. (1968) Bacterial Degradation of Biotin. II. Catabolism of 14C-Homobiotin and 14C-Norbiotin. J. Biol. Chem. 243, 547551. (35) Green, N. M. (1963) Avidin. 3. The Nature of the BiotinBinding Site. Biochem. J. 89, 599-609. (36) Green, N. M. (1966) Thermodynamics of the Binding of Biotin and some Analogues of Avidin. Biochem. J. 101, 774780. (37) Chilkoti, A., and Stayton, P. S. (1995) Molecular Origins of the Slow Streptavidin-Biotin Dissociation Kinetics. J. Am. Chem. Soc. 117, 10622-10628. (38) Klumb, L. A., Chu, V., and Stayton, P. S. (1998) Energetic roles of hydrogen bonds at the ureido oxygen binding pocket in the streptavidin-biotin complex. Biochemistry 37, 76577663. (39) Virzi, F., Fritz, B., Rusckowski, M., Gionet, M., Misra, H., and Hnatowich, D. J. (1991) New indium-111 labeled biotin derivatives for improved immunotargeting. Int. J. Rad. Appl. Instrum. B 18, 719-726. (40) Jeong, J. M., Kinuya, S., Paik, C. H., Saga, T., Sood, V. K., Carrasquillo, J. A., Neumann, R. D., and Reynolds, J. C. (1994) Application of High Affinity Binding Concept to Radiolabel Avidin with Tc-99m Labeled Biotin and the Effect of pI on Biodistribution. Nucl. Med. Biol. 21, 935-940. (41) Hashmi, M., and Rosebrough, S. F. (1995) Synthesis, Pharmacokinetics, and Biodistribution of 67GA DeferoxamineacetylCysteinylbiotin. Drug Metab. Dispos. 23, 1362-1367. (42) Chang, F., Rusckowski, M., Qu, T., and Hnatowich, D. J. (1997) Early Results in the Irrational Design of New Bifunctional Chelators. Cancer (Suppl.) 80, 2347-2353. (43) Renn, O., Goodwin, D. A., Studer, M., Moran, J. K., Jacques, V., and Meares, C. F. (1996) New Approaches to Delivering Metal-Labeled Antibodies to Tumors: Synthesis and Characterization of New Biotinyl Chelate Conjugates for Pre-Targeted Diagnosis and Therapy. J. Controlled Release 39, 239-249. (44) Karacay, H., Sharkey, R. M., Govindan, S. V., McBride, W. J., Goldenberg, D. M., Hansen, H. J., and Griffiths, G. L. (1997) Development of a Streptavidin-Anti-Carcinoembryonic Antigen Antibody, Radiolabeled Biotin Pretargeting Method for Radioimmunotherapy of Colorectal Cancer. Reagent Development. Bioconjugate Chem. 8, 585-594. (45) Wilbur, D. S., Pathare, P. M., Hamlin, D. K., 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. (46) Wilbur, D. S., Pathare, P. M., Hamlin, D. K., Stayton, P. S., To, R., Klumb, L. A., Buhler, K. R., and Vessella, R. L. (1999) Development of new biotin/streptavidin reagents for pretargeting. Biomol. Eng. 16, 113-118. (47) Costello, S. H., Felix, R. T., and Giese, R. W. (1979) Enhancement of Immune Cellular Agglutination by Use of An Avidin-Biotin System. Clin. Chem. 25, 1572-1580. (48) Hofmann, K., Titus, G., Montibeller, J. A., and Finn, F. M. (1982) Avidin Binding of Carboxyl-Substituted Biotin and Analogues. Biochemistry 21, 978-984. (49) Shao, M. C., Chin, C. C., Caprioli, R. M., and Wold, F. (1987) The regulation of glycan processing in glycoproteins. The effect of avidin on individual steps in the processing of biotinylated glycan derivatives. J. Biol. Chem. 262, 29732979.

Relative Dissociation Rates of Biotin Derivatives (50) Bernstein, K. E., Welsh, S. L., and Inman, J. K. (1990) A Deeply Recessed Active Site in Angiotensin-Converting Enzyme is Indicated From The Binding Characteristics of BiotinSpacer-Inhibitor Reagents. Biochem. Biophys. Res. Commun. 167, 310-316. (51) Smith, J. S., Miller, B. T., Knock, S. L., and Kurosky, A. (1991) Biotinylated Peptides/Proteins. 1. Determination of Stoichiometry of Derivatization. Anal. Biochem. 197, 247253. (52) Polli, J. W., and Billingsley, M. L. (1991) Preparation, characterization and biological properties of biotinylated derivatives of calmodulin. Biochem. J. 275, 733-743. (53) Basak, A., Jean, F., Dugas, H., and Lazure, C. (1994) Biotinylation of an Enkephalin-Containing Heptapeptide via Various Spacer Arms. Synthesis, Comparative Binding Studies toward Avidin, and Application as Substrates in Enzymatic Reactions. Bioconjugate Chem. 5, 301-305. (54) Chauhan, J., Ebrahim, H., Bhullar, R. P., and Dakshinamurti, K. (1985) Human Serum Biotinidase. Ann. N. Y. Acad. Sci. 447, 386-388. (55) Wolf, B., Hymes, J., and Heard, G. S. (1990) Biotinidase. Methods Enzymol. 184, 103-111. (56) Hymes, J., and Wolf, B. (1996) Biotinidase and Its Roles in Biotin Metabolism. Clin. Chim. Acta 255, 1-11. (57) Axworthy, D. B., Theodore, L. J., Gustavson, L. M., and Reno, J. M. (1997) Biotinidase-Resistant Biotin-DOTA Conjugates. U.S. Patent 5,608,060. (58) Fujimori, K., Covell, D. G., Fletcher, J. E., and Weinstein, J. N. (1990) A Modeling Analysis of Monoclonal Antibody Percolation Through Tumors: A Binding-Site Barrier. J. Nucl. Med. 31, 1191-1198. (59) Piran, U., and Riordan, W. J. (1990) Dissociation Rate Constant of the Biotin-Streptavidin Complex. J. Immunol. Methods 133, 141-143. (60) Rosebrough, S. F. (1993) Plasma Stability and Pharmacokinetics of Radiolabeled Deferoxamine-Biotin Derivatives. J. Pharmacol. Exp. Ther. 265, 408-415. (61) Groman, E. V., Rothenberg, J. M., Bayer, E. A., and Wilchek, M. (1990) Enzymatic and Radioactive Assays for Biotin, Avidin, and Streptavidin. Methods Enzymol. 184, 208-217. (62) Livaniou, E., Evangelatos, G. P., and Ithakissios, D. S. (1987) Radioiodinated Biotin Derivatives for In Vitro Radioassays. J. Nucl. Med. 28, 1430-1434. (63) Livaniou, E., Evangelatos, G. P., and Ithakissios, D. S. (1987) Biotin radioligand assay with an 125I-labeled biotin derivative, avidin, and avidin double-antibody reagents. Clin. Chem. 33, 1983-1988. (64) Evangelatos, S. A., Kakabakos, S. E., Evangelatos, G. P., and Ithakissios, D. S. (1993) Determination of serum biotinidase activity with biotinyl derivatives of lodotyramines as substrates. J. Pharm. Sci. 82, 1228-1231. (65) Garlick, R. K., and Giese, R. W. (1988) Avidin binding of radiolabeled biotin derivatives. J. Biol. Chem. 263, 210-215. (66) Khawli, L. A., and Kassis, A. I. (1992) m-[125I]Iodoaniline: a Useful Reagent for Radiolabeling Biotin. Nucl. Med. Biol. 19, 297-301. (67) Najafi, A., and Peterson, A. (1993) Preparation and in vitro evaluation of “no-carrier-added” 18F-labeled biotin. Nucl. Med. Biol. 20, 401-405.

Bioconjugate Chem., Vol. 11, No. 4, 2000 583 (68) Shoup, T. M., Fischman, A. J., Jaywook, S., Babich, J. W., Strauss, H. W., and Elmaleh, D. R. (1994) Synthesis of Fluorine-18-Labeled Biotin Derivatives: Biodistribution and Infection Localization. J. Nucl. Med. 35, 1685-1690. (69) Kortylewicz, Z. P., Baranowska-Kortylewicz, J., Adelstein, S. J., Carmel, A. D., and Kassis, A. I. (1994) Radiolabeled Biotin Amides from Triazenyl Precursors: Synthesis, Binding, and In-Vivo Properties. J. Labelled Compd. Radiopharm. 34, 1129-1146. (70) Foulon, C. F., Adelstein, S. J., and Kassis, A. I. (1996) Onestep synthesis of radioiodinated biotin derivatives. Bioorg. Med. Chem. Lett. 6, 779-784. (71) Foulon, C. F., Schoultz, B. W., and Zalutsky, M. R. (1997) Preparation and Biological Evaluation of an Astatine-211 Labeled Biotin Conjugate: Biotinyl-3-[211At]astatoanilide. Nucl. Med. Biol. 24, 135-143. (72) Foulon, C. F., Alston, K. L., and Zalutsky, M. R. (1997) Synthesis and Preliminary Biological Evaluation of (3-Iodobenzoyl)norbiotinamide and ((5-Iodo-3-pyridinyl)carbonyl)norbiotinamide: Two Radioiodinated Biotin Conjugates with Improved Stability. Bioconjugate Chem. 8, 179-186. (73) Rowlinson-Busza, G., Hnatowich, D. J., Rusckowski, M., Snook, D., and Epenetos, A. A. (1993) Xenograft Localization Using Pretargeted Streptavidin-conjugated Monoclonal Antibody and 111In-Labeled Biotin. Antibody, Immunoconjugates, Radiopharm. 6, 97-109. (74) Modorati, G., Brancato, R., Paganelli, G., Magnani, P., Pavoni, R., and Fazio, F. (1994) Immunoscintigraphy with three step monoclonal pretargeting technique in diagnosis of uveal melanoma: preliminary results. Br. J. Ophthalmol. 78, 19-23. (75) Alvarez-Diez, T. M., Polihronis, J., and Reilly, R. M. (1996) Pretargeted Tumour Imaging with Streptavidin Immunoconjugates of Monoclonal Antibody CC49 and 111In-DTPA-Biocytin. Nucl. Med. Biol. 23, 459-466. (76) Breitz, H. B., Weiden, P. L., Beaumier, P. A., Axworthy, D. B., Seiler, C., Su, F.-M., Graves, S., Bryan, K., and Reno, J. M. (2000) Clinical Optimization of Pretargeted Radioimmunotherapy with Antibody-Streptavidin Conjugate and 90Y-DOTA-Biotin. J. Nucl. Med. 41, 131-140. (77) Axworthy, D. B., Reno, J. M., Hylarides, M. D., Mallett, R. W., Theodore, L. J., Gustavson, L. M., Su, F., Hobson, L. J., Beaumier, P. L., and Fritzberg, A. R. (2000) Cure of human carcinoma xenografts by a single dose of pretargeted yttrium90 with negligible toxicity. Proc. Natl. Acad. Sci. U.S.A. 97, 1802-1807. (78) 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. (79) Wilbur, D. S., Hamlin, D. K., Chyan, M.-K., Pathare, P. M., and Kegley, B. B. (2000) Biotin Reagents in Antibody Pretargeting. 5. Additional Studies of Biotin Conjugate Design to Provide Biotinidase Stability. Bioconjugate Chem. (manuscript in preparation).

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Biotin Reagents for Antibody Pretargeting. 4. Selection of Biotin

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