m-[125I]iodoaniline: a useful reagent for radiolabeling biotin

Nucl. Med. Biol. Vol. 19, No. 3, pp. 297-301, 1992 Int. J. Radiat. Appl. In&urn. Parr B

0883-2897/92 $5.00 + 0.00

Pcrgamon Press plc

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m -[125I]Iodoaniline: a Useful Reagent Radiolabeling Biotin* LESLIE

A. KHAWLIT

and AMIN

for

1. KASSISt

Department of Radiology (Nuclear Medicine), Harvard Medical School, Shields Warren Radiation Laboratory, 50 Binney Street, Boston, MA 02115, U.S.A. (Received

2 August 1991)

Biotinyl-m-[‘*sI]iodoanilide (BIA) was synthesized by coupling biotin to m-[‘251]iodoanilinevia a mixed anhydride reaction. m-[‘2SI]Iodoaniline was produced from the tin precursor, which was prepared using a palladium catalyzed reaction of hexabutylditin with m-bromoaniline. The radioiodinated BIA derivative is characterized by a stable amide and/or intact ureido group on the biotin molecule; it may thus be a useful carrier for targeting radionuclides to avidin-conjugated antibodies previously localized on tumors.

Introduction has a very high affinity for avidin (Green, 1975) and streptavidin (Chaiet and Wolf, 1964); this interaction suggests that biotin may be a useful carrier for targeting radionuclides to avidin-conjugated antibodies previously localized on tumors (Hnatowich et al., 1987). One way to accomplish this would be to functionalize the biotin with a small molecule that could then be directly radiolabeled. This method is unsatisfactory, because biotin is sensitive to the oxidizing conditions needed for radiolabeling (Windholtz et al., 1983). A suitable approach would be to couple biotin to a pre-iodinated small molecule. Livaniou et al. (1987) have labeled biotin by connecting it through an amide linkage to radioiodinated tyramine, a molecule in which the iodine is substituted ortho to a hydroxyl group on an aromatic ring. Biotin thus radiolabeled suffers from varying degrees of in viva deiodination due to several factors, including the presence of a hydroxyl group ortho to the radiohalogen which decreases the carbon-iodine bond energy (Cottrell, 1958) and the structural similarity between the iodinated tyramine and thyroid hormones which makes the former susceptible to enzymatic processes (Dumas, 1973; Smallridge et al., 1981; Zalutsky and Narula, 1987). Biotin

*Presented in part at the Third International Conference on Monoclonai Antibody Immunoconjugates for C;?ncer, San Diexo, Calif.. 4-6 Februarv 1988 and at the 35th Annual Me&g of the Society of -Nuclear Medicine, San Francisco, Calif., 1417 June 1988. tPresent address: University of Southern California, School of Medicine, 204 Hoffman Medical Research Building, 2011 Zonal Avenue, Los Angeles, CA 90033, U.S.A. $Author for correspondence.

In this paper, we describe the synthesis of biotinylm-1 ‘25i’271]iodoanilide (4) (BIA) (Scheme 1) which is suitable for use in the biotin-avidin system. An iododemetallation reaction is used to label the small molecule 2 at an organotin bond (Coenen et al., 1983) in good radiochemical yield, and this radiolabeled molecule 3 is coupled to biotin by a mixed-anhydride method (Anderson et al., 1967). In order to evaluate the utility of the radioiodinated biotin amide (4) in the biotin-avidin system, we have studied its affinity for streptavidin in some detail.

Materials and Methods All chemicals were of reagent grade and were purchased from Aldrich Chemical Company (Milwaukee, Wis.). Tetrakis(triphenylphosphine)palladium was handled and stored under nitrogen. N,N-Dimethylformamide (DMF) was used immediately from a newly opened bottle. All solvents were of analytical grade and were used directly. Iodine- 125 as sodium iodide with a specific activity of 15.4 Ci/mg I was obtained from Amersham Corporation in sodium hydroxide solution (pH 7-l 1). Glassware was dried overnight at 150°C. Thin layer chromatography (TLC) was carried out on silica gel GHLF plates (Analtech No. 21521), developed as specified below, and visualized by U.V. and I, fumes; all tin products were also visualized using 5% phosphomolybdic acid in ethanol and subsequent heating. Flash chromatography was carried out according to the method of Still er al. (1978) using Kieselgel 60, 230-400 mesh (Merck No. 9385). Melting points were determined on a Fisher-Johns apparatus and are uncorrected. Proton (‘H) nuclear 291

LESLIE A. KIMWLI and AWN I. KASSIS

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Biotin

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magnetic resonance (NMR) spectra were measured on a Varian T-60 spectrometer. Chemical shifts (ppm) are reported downfield (6) relative to internal tetramethylsilane (TMS) standard. NMR concentrations were about 10% (w/v) in the indicated solvent. Elemental analyses were performed by Galbraith Laboratories, Knoxville, Tenn. Radioactive samples were measured using either an NaI autogamma counter (Packard Instruments) or an ion chamber dose calibrator (Nuclear Associates). Autoradiography was performed on Kodak XTL-2 film. m-Aminophenyltributylstannane

3-10 contained hexabutylditin, RI= 0.8, and fractions 32-40 contained the starting material 1, R,= 0.25. Fractions 18-30, which showed a single spot at Rf = 0.4, were combined and the solvent was evaporated to provide pure m-aminophenyltributylstannane (2) (2.3 g, 82%). ‘H-NMR (CDCl,, 6); 7.2 (triplet, lH, J = 7 Hz, aryl CS-H); 6.9 (doublet, lH, J = 7 Hz, aryl C4-H); 6.8 (singlet, lH, aryl C2-H); 6.6 (doublet, lH, J = 7 Hz, aryl C6-H); 3.6 (singlet, 2H, NH,); 0.9-1.7 (multiplets, 27H, 3 x n-C,H,). Anal. cald for C,H,,NSn: C, 56.57; H, 8.70; N, 3.66. Found: C, 56.56; H, 8.72; N, 3.72.

(2)

A mixture of m-bromoaniline (1) (1.26 g, 7.3 mmol), hexabutylditin (5.20 g, 8.9 mmol) and tetrakis( triphenylphosphine)palladium (79.0 mg, 0.07 mmol) in toluene (10 mL) was stirred and heated at 105°C for 12 h under nitrogen. The resulting black mixture was filtered, and the filtrate was evaporated to dryness under reduced pressure at 50°C. The residue obtained was dissolved in hexane, and the solution was applied to a flash chromatography column (30 x 200 mm) of Kieselgel 60. Elution was initiated with hexane (lOOmL, fractions l-10) followed by 2% ethyl acetate in hexane (100 mL, fractions 1l-20) and 4% ethyl acetate in hexane (200 mL, fractions 2140). All fractions were analyzed by TLC with EtOAc/hexane (4: 100) as solvent. Fractions

m Jodoaniline (3) To a stirred solution of sodium iodide (30 mg, 0.26mmol) in H,O (1OOpL) was added maminophenyltributylstannane (lOOmg, (2) 0.26 mmol) in methanol (1 mL) followed by a solution of N-chlorosuccinimide (NCS) (35 mg, 0.26 mmol) in methanol (1 mL). Reaction progress was followed by TLC with EtOAC/hexane, 2: 1, as solvent (starting material 2, 4 = 0.58). The reaction mixture was stirred at room temperature (RT) for 30 min. The mixture initially turned yellow from the formation of I+ and then rapidly became lighter until it was colorless. A minimum amount of 5% aqueous sodium bisulfite was added, the solution was evaporated, and the resulting mixture was chromatographed

Biotinyl-m-[‘251]iodoanilide on Kieselgel 60 using a 1 x 20 cm disposable column (Bio-Rad). Elution was initiated with hexane (9mL, fractions l-3) followed by 33% ethyl acetate in hexane (27mL, fractions 412). Fractions 6 and 7 were combined, and the solvent was removed in uacuo to give the desired product, m -iodoaniline (3) (54 mg, 94%): TLC (EtOAC/hexane, I :2) R,= 0.43. ‘HNMR (CDCI,, 6); 6.6-7.1 (multiplets, 4H, 4 aryl CH), 3.6 (singlet, 2H, NH,). m -[‘2sl]lodoaniline

(3)

To a 5-mL test tube containing sodium [‘251]iodide (10.5 PCi) was added 20 p L of a 330-mM solution of m-aminophenyltributylstannane (2) in methanol and 50 p L of a 72-mM solution of NCS in methanol. The reaction was stirred vigorously for 30 min at RT and quenched with 5% aqueous sodium bisulfite. The resulting solution was evaporated under a stream of nitrogen, and the mixture was chromatographed on Kieselgel 60 using a 8 x IOOmm disposable column (Bio-Rad). Elution was initiated with hexane (5 mL, fractions l-5) followed by 35% ethyl acetate in hexane (15 mL, fractions 620). Fractions 10-13, which showed one spot (R, 0.43) on TLC (EtOAC/hexane, 1: 2) autoradiography that comigrated with m-iodoaniline were combined. Their radioactive content was determined to be 9.4 PCi of m-[‘2SI]iodoaniline (3) (90% radiochemical yield). Fractions 5-8 constituted 5% of the radioactivity and contained two compounds with R, values of 0.60 and 0.74. The identity of the side products was not determined. Biotinyl-m

-iodoanilide (4)

A solution of biotin (220 mg, 0.90 mmol) in 7 mL anhydrous DMF was stirred and cooled in a salt-ice bath. Triethylamine (0.14 mL, 1.05 mmol) was added followed by isobutyl chloroformate (0.13 mL, 1.05 mmol), and a white precipitate was formed. After 10 min, m-iodoaniline (200 mg, 0.90 mmol) dissolved in 0.5 mL DMF was added and stirring was continued for 5 min at salt-ice-bath temperature and for 15 min at RT. The solution was evaporated under reduced pressure at 7O”C, and 0.3 mL of 5% sodium bicarbonate was added. The residue obtained was dissolved in chloroform and applied to a flash chromatography column (30 x 200 mm) of Kieselgel 60 equilibrated with CHCl,/EtOAc, 3 : 1. Elution was initiated with the same solvent system (80mL, fractions l-8) followed by CHCl,/EtOAc/MeOH, 3: 1: 1 (120 mL, fractions 9-20). All fractions were analyzed by TLC with CHCl,/EtOAc/MeOH, 3: 1: 1. Fractions 3-6 contained a side product identified as isobutyl-N-m-iodophenylcarbamate (5), R, = 0.89, while fractions 7-l 1 contained the starting material, m -iodoaniline (3), R, = 0.8 1. Fractions 13-l 7, which showed a single spot at R, = 0.5 1, were combined and the solvent was evaporated to give pure biotinyl-miodoanilide (4) (285 mg, 71%): m.p. 143-145°C. ‘HNMR (Me,SO-d,, 6); 1.5 (multiplets, 8H); 2.3

299

(multiplets, 2H); 2.8 (multiplets, 1H); 4.3 (multiplets, 2H); 6.4 (broad singlet, 2H); 6.9-8.1 (multiplets, 4H, 4 aryl CH); 9.8 (singlet, lH, NH). Anal. cald for &,H2,N,02SI: C, 43.15; H, 4.52; N, 9.43. Found: C, 42.66; H, 4.61; N, 9.62. Biotinyl-m-[‘251]iodoanilide

(4)

A solution of 10 mg biotin in 1 mL of anhydrous DMF was cooled in a salt-ice bath. Triethylamine (6 FL) was added followed by isobutyl chloroformate (6 pL). After IOmin, m-[‘251]iodoaniline (3) (4.1 PCi) dissolved in 50 PL of DMF was added, and the mixture was stirred vigorously for 15 min at RT before the reaction was quenched with 50 ,uL of 5% aqueous sodium bicarbonate. The resulting mixture was chromatographed on Kieselgel 60 using an 8 x IOOmm disposable column (Bio-Rad). Elution was initiated with CHCl,/EtOAc, 3: 1 (10 mL, fractions l-5) followed by CHCl,/EtOAc/MeOH, 3: 1: 1 (12 mL, fractions 6-16). Fractions 8-10 showed one spot, Rf= 0.51, on TLC with CHCl,/EtOAc/MeOH, 3 : 1: 1, and autoradiography indicated that the radioactivity comigrated with biotinyl-m-iodoanilide. The desired fractions were combined to give 3.32pCi of biotinyl-m-[‘251]iodoanilide (4), (80% radiochemical yield). Fractions 4 and 5 constituted 6% of the radioactivity accounted for by the side product 5 with an R, value of 0.89. Binding capacity bio tin amide

qf streptavidin

to radioiodinated

Radioiodinated biotin amide (4) (200 ,uL, 36 nCi) was added to 5-mL test tubes containing increasing volumes (lo-100 pL) of streptavidin solution (10 pg/mL) in 0.01 M phosphate buffered saline, pH 7.2 (PBS). The volume in each tube was adjusted to 3OOpL with PBS. The tubes were mixed and incubated for 30 min at RT. Aliquots were analyzed using an instant thin layer chromatography system consisting of silica gel impregnated glass fibers (Gelman Sciences, No. 61886). Strips (2 x 20cm) were spotted with 1 PL of sample, air dried and immediately eluted with MeOH/H,O, 80:20, for approx. 12cm, again air dried, cut in half and counted to determine streptavidin bound radioactivity. The radiolabeled biotin-streptavidin complex stayed at the origin, while the radioiodinated biotin amide moved with the solvent front.

Results and Discussion Biotiny1-m-[‘25”271]iodoanilide (BIA) (4) can be used in many applications (Buckland, 1986; Livaniou et al., 1987) including binding to avidin-conjugated antibodies previously localized on tumors. The synthesis of the radiolabeled biotin was accomplished in three steps (Scheme 1). First m-aminophenyltributylstannane (2) was obtained by the reaction of hexabutylditin with m-bromoaniline using a palladium catalyst, and then the radioiodinated derivative 3 was

300

LESLIE A. KHAWLIand AMINI. K~ssrs

obtained via an iodination-destannylation reaction. Finally, the mixed anhydride of biotin was generated in situ and readily converted to BIA (4) by reaction with compound 3. Wursthorn and Kuiviia (1977) had reported the preparation of p-aminophenyistannane. In our hands, however, this reaction was not satisfactory. ’ H-NMR analysis of the reaction mixture after purification revealed the presence of tin-containing starting material in addition to aniline. The para-substituted aryi stannanes containing an electron-releasing group such as NH, have a high rate of cleavage of the aryi-tin bond (Eaborn et al., 1967). We have found in marked contrast that the me&-substituted aryi stannanes have a low rate of cleavage of aryl-tin bonds. The stannyiation of m-bromoaniline (1) was performed by modifying the method of Kosugi et al. (1981). By adjusting the amount of hexabutylditin to m-bromoaniiine in the reaction mixture and maintaining the temperature near iOYC, thus minimizing unwanted side products, we were able to increase the isolated yield of 2 from 32 to 82%. This stannane intermediate can be made in good yield, purified and stored in the refrigerator for at least 3 months, and the radiolabeling step can be completed at a later time before coupling to biotin. The chemistry of the coupling of the biotincarboxylic portion to m-[‘251]iodoaniline (3) is summarized in Scheme 1. The mixed anhydride of biotin was generated in situ before conversion to stable biotinyi-m-[‘251]iodoanilide (4). We were able to improve the radiochemical yield of 4 to 80% and to minimize the unwanted side product identified as N-m -[‘251’27 Iliodophenyi carbamate (5) by adjusting the concentration of reactants and moderating the temperature. The radioiodinated biotin amide (4) is easily separated from the side product 5 by flash chromatography using silica gel (Fig. 1). Many different biotinyi derivatives have been synthesized (Becker et al., 1971; Jasiewicz et al., 1976; Bayer and Wiichek, 1980; Roffman et al., 1986) in which the presence of exogenous groups attached to the biotin-carboxyiic portion does not interfere with the formation of the biotin-avidin complex

Fig. 2. Percentage of binding of radioiodinated biotin derivative as function of streptavidin concentration.

(Achuta Murthy and Mistry, 1977). The approach described in this paper, in which iodinated aniline (3) is attached to the carboxyiic site of the biotin, also produces a haiogenated biotin complex characterized by an intact ureido group which is an important requirement for the formation of the biotin-avidin complex (Bonjour, 1984). In fact, the binding of the radioiodinated biotin to increasing amounts of streptavidin was found to be linear (Fig. 2). When the mixture was refrigerated in solution, the binding level remained almost stable for several weeks. In conclusion, we have described a procedure for radiolabeling biotin that is particularly attractive for the study of systems in which biotin labeled with a variety of radiohalides (e.g. alpha emitters such as astatine-211 or beta emitters such as iodine-131) can be targeted to avidin-conjugated antibodies previously localized on tumors. Acknowledgemenrs-This work was supported in part by DOE DE-FG02-86ER60460 and by NC1 5 R01 CA 15523. Dr Khawli was a research fellow under NIH ST32 CA 09536-02.

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0

1

3

5 FRACTKW

Fig. 1. Radiochromatogram

7

9

11

h’UW8ER

of radioiodinated biotin reaction mixture.

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L

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