Research Article Received 24 June 2010, Revised 20 October 2010, Accepted 24 November 2010 Published online 17 February 2011 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/jlcr.1864 Synthesis and characterization of radiolabeled 17b-estradiol conjugates Suman L. Shrestha,a Xuelian Bai,b David J. Smith,c Heldur Hakk,cà Francis X. M. Casey,b Gerald L. Larsen,c and G. Padmanabhana The use of radioactive tracers for environmental fate and transport studies of emerging contaminants, especially for those that are labile, offers convenience in tracking study compounds and their metabolites, and in calculating mass balances. The aim of this study was to synthesize radiolabeled glucuronide and sulfate conjugates of 17b-estradiol (17b-E2). The conjugates 17b-[4-14C]estradiol-3-glucuronide ([14C]17b-E2-3-G) and 17b-[4-14C]estradiol-17-sulfate ([14C]17b-E2-17-S) were synthesized utilizing immobilized enzyme and chemical syntheses, respectively. Microsomal proteins from the liver of a phenobarbital induced pig (Sus scrofa domestica) were harvested and used to glucuronidate [14C]17b-E2. Synthesis of [14C]17b-E2-17-S consisted of a three-step chemical process – introducing a blocking group at the C-3 position of [14C]17b- E2, sulfation at C-17 position, and subsequent deblocking to yield the desired synthetic product. Successful syntheses of [14C]17b-E2-3-G and [14C]17b-E2-17-S were achieved as verified by liquid chromatography, radiochemical analyses, quadrupole-time-of-flight (QTOF) mass spectrometry, and 1H and 13C nuclear magnetic resonance spectroscopy. Radiochemical yields of 84 and 44% were achieved for 17b-E2-3-G and 17b-E2-17-S, respectively. Synthetic products were purified using high-performance liquid chromatography and radiochemical purities of 98% or greater were obtained. Keywords: 17b-estradiol; immobilized enzyme; conjugation; glucuronide; sulfate; steroid hormone Introduction function in the detections of ‘free’ steroidal estrogens in the environment. Although, estrogen conjugates are biologically Medical research has used radiolabeled estrogenic compounds inactive,24 they can potentially be cleaved by microbial enzymes 1 2 to study breast and uterine cancers, estrogenic receptors, and to form the more potent parent compound.24 3 as imaging agents in breast tumors. More recently though, Conjugation reactions are a common vertebrate mechanism 14 the radiolabeled hormonal compounds (e.g. [ C]estradiol, in which hormones, drugs, toxicants, and non-nutritive organic 14 14 4–8 3 6 [ C]estrone, and [ C] testosterone and 6,7- H-estradiol ) molecules are eliminated.25 During conjugation a charged, polar have been used to study the fate and transport of steroids in the moiety is attached to a hydrophobic compound (e.g. estrogen), environment. Exposure to exogenous reproductive hormones which increases its water solubility and excretion in urine or bile. 9–11 has been associated with adverse effects in certain aquatic Estrogens are typically conjugated with glucuronic or sulfuric 12–14 and terrestrial species. Human waste treatment and animal acid at the C-3 and/or the C-17 positions24 (Schemes 1 and 2). feeding operations (AFOs) are sources of estradiol (E2), estrone Glucuronidation of estrogen is catalyzed by uridine 50-diphospho- (E1), and estriol (E3) to the environment. Estradiol is the most glucuronosyltransferase (UGT) enzymes in the endoplasmic 15–18 potent of these natural estrogens. reticulum and sulfation is catalyzed by cytosolic sulfotransferases Laboratory studies suggest estrogens should have little to no (SULTs).26 mobility and should not persist in the environment because they The environmental fate of estradiol conjugates has not been 4,8,19 bind rapidly and strongly to soil and degrade within hours. extensively studied, possibly because radiolabeled conjugates Field studies, however, have indicated that estrogens are present are not commercially available. The availability of radiolabeled in the environment at frequencies and concentrations that imply conjugated hormones would enable studies to be conducted 20,21 they are moderately mobile and persistent. Estrogen that would improve the understanding of the fate and transport conjugates, which have different water solubilities, sorption of these labile compounds in the environment. The objective of coefficients, and degradation rates relative to their ‘free’ estrogen counterparts, may offer insights into why steroidal estrogens are frequently detected in the environment.20 Swine (sus scrofa aDepartment of Civil Engineering, North Dakota State University (NDSU), Fargo, domesticus), poultry (gallus domesticus), and cattle (bos taurus) ND 58105, USA excrete 96, 69, and 42%, respectively, of the estrogens as bDepartment of Soil Science, NDSU, Fargo, ND 58105, USA conjugates.22 In fact, appreciable amounts of 17b-estradiol (17b-E2) in conjugated forms have been measured in swine cBiosciences Research Laboratory, USDA-ARS, Fargo, ND 58102, USA manure slurry (liquid urine and feces) from AFO manure storage 23 *Correspondence to: Heldur Hakk, USDA ARS Biosciences Research Laboratory, lagoons. Conjugates form a major portion of total environ- 1605 Albrecht Blvd, Fargo, ND 58102-2765, USA. mental estrogen loading from AFOs and might play a significant E-mail: [email protected] 267 J. Label Compd. Radiopharm 2011, 54 267–271 Copyright r 2011 John Wiley & Sons, Ltd. S. L. Shrestha et al. Scheme 1. Glucuronidation of the hydroxyl group at C-3 of 17-b-estradiol by uridine 50-diphospho-glucuronosyltransferase (UGT). Scheme 2. Chemical synthesis of [14C]17b-estradiol-17-sulfate conjugate from [14C]17b-estradiol. this paper is to provide a method to synthesize carbon-14- Use Committee guidelines. The hog was intramuscularly (2 d) labeled 17b-E2-3-G and 17b-E2-17-S. then intraperitoneally dosed (2 d) with approximately 20 mg/kg phenobarbital for 4 consecutive days, after which, the hog Experimental was euthanized. The liver was homogenized and microsomes were isolated via differential centrifugation. Proteins were Materials solubilized and immobilized onto Sepharose beads27 and were [14C]17b-E2 (55 mCi/mmol) (1) was purchased from American stored in a 1:1 suspension with 0.1 M Tris buffer (pH 7.4) at 41C Radiolabeled Chemicals (St Louis, MO). Unlabeled 1,UDP until use. glucuronic acid, magnesium chloride, ethanol, potassium phos- phate monobasic, potassium phosphate dibasic, potassium hydro- Liquid scintillation counting xide, hydrochloric acid, ethyl acetate, pyridine, sodium hydroxide, chlorosulfonic acid, and acetic acid were obtained from Sigma- Radioactivity was quantitated with a Packard 1900 CA scintilla- tion analyzer (Downers Grove, IL), and samples were dissolved in Aldrich. Triethylamine (Fluka); benzoyl chloride (Bayer); trisodium TM phosphate (Mallinkrodt, Paris, KY) were obtained from other EcoLite scintillation cocktail. sources. Acetonitrile (ACN) was obtained from EMD Chemicals (Gibbstown, NJ). Scintillation fluid EcoLiteTM was obtained from MP High-performance liquid chromatography Biomedicals (Santa Ana, CA). SPE cartridges Bond ElutTM C18 (6 g, s Analytical high-performance liquid chromatography (HPLC) for 2 20 mL) and Sep-Pak Vac C18 were obtained from Varian (Harbor was performed using a Waters 600E System Controller and City, CA) and Waters (Milford, MA), respectively. pump (Milford, MA), equipped with a Jasco FP 920 fluorescence Uridine 50-diphospho-glucuronosyltransferase (UGT) detector (Jasco, Easton, MD) with the following conditions: Phenomenex-C18, 4.6  250 mm, 5 mm; A: 10% ACN in 50 mM A castrated, cross-bred hog weighing 24.4 kg was used as the ammonium acetate (pH 4.5), B: 90% ACN in 50 mM ammonium 268 source of the UGT enzymes, following USDA Animal Care and acetate (pH 4.5); gradient: 20–100% B, 29 min, 100% B, 3 min www.jlcr.org Copyright r 2011 John Wiley & Sons, Ltd. J. Label Compd. Radiopharm 2011, 54 267–271 S. L. Shrestha et al. hold, 1.0 mL/min, excitation and emission wavelengths of 280 Synthesis of 17b-[4-14C]estradiol-17-sulfate (5) and 312 nm, respectively. Prep-HPLC was performed on Jones Chromatography-C18, 10  250 mm, 5 mm; A: 5% ACN in 50 mM 14 b ammonium acetate (pH 4.5), B: 90% ACN in water; isocratic 85% [ C]17 -estradiol-3-benzoate (3) solvent A, 15% solvent B; 4.7 mL/min. Radiolabeled 1 (259.5 mg, 0.95 mmol, 47.7 mCi) was mixed with For 5, analytical HPLC was performed on a Gilson System unlabeled 1 (11.43 mg, 42 mmol) in ethanol and the solvent was 45NC Gradient Analytical instrument (Gilson Medical Electronics, evaporated.28 The residue was re-dissolved in 2 mL of acetoni- Middleton, WI) equipped with a variable wavelength UV trile, and 13 mL triethylamine and 11 mL benzoyl chloride detector with the following conditions: Radial-Pak-C18, (13.3 mg, 94.7 mmol) were added; the reaction mixture was 8  100 mm (Waters Associates, Milford, MA); A: 10:90 metha- stirred at room temperature for 2 h and subsequently dried nol/water, B: 90:10 methanol/water; gradient: 20% B to 100% B, under a stream of nitrogen. To the white residue, 4 mL of 0.1 M 28 min., 4 min hold; 1.0 mL/min 220 nm. HPLC for 3 was trisodium phosphate solution was added and the mixture was conducted using following conditions: Radial-Pak-C18, sonicated for 30 min resulting in a light yellow suspension. The 8  100 mm; A: 10:90 methanol/water, B: 90:10 methanol/water; suspension was extracted with ethyl acetate (3 mL  3), and gradient: 20% B to 100% B, 30 min, 15 min hold; 1.0 mL/min, UV the organic solvent was evaporated under a stream of nitrogen. 220 nm. The residue (3) was dissolved in ethyl acetate (3 mL) and water (1 mL) for further purification using HPLC. The yield of 3 was 13 Mass spectral analysis 59.3% and radiochemical purity was 98%. C NMR (MeOH-d4)d: Negative ion LC/MS was performed with a Waters Alliance 2695 165.94, 150.13, 139.49, 139.38, 134.86, 130.99, 130.99, 129.83, 129.83, 127.47, 122.63, 119.79, 82.45, 51.33, 45.55, 44.34, 40.14, HPLC (Symmetry-C18, 2.1  100 mm; A: 40% ACN in water, 1 B: 60% ACN in water; gradient: 40–100% B, 10 min, 5 min hold, 37.99, 30.69, 30.56, 28.27, 27.48, 24.04, 11.68.