Bioconjugate Chem. 1994, 5, 167-171
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Synthesis of 3-Hydroxyestra-l,3,5(lO)-trien-17-oneand 3,17/3-Dihydroxyestra-1,3,5(10)-triene Gar-N-(eBiotinyl)caproamide, Tracer Substances for Developing Immunoassays for Estrone and Estradio1 Peter Luppa,*-+Christian Birkmayer,l and Hagen Hauptmannl Institute for Clinical Chemistry, Klinikum rechts der Isar, Technical University Munich, D-81675 Munich, Germany, and Institute for Organic Chemistry, University Regensburg, D-93053 Regensburg, Germany. Received October 5, 1993"
We describe the synthesis of 3-hydroxyestra-1,3,5(10)-trien-17-one Ga-N-(~-biotinyl)caproamide and 3,17p-dihydroxyestra-1,3,5( 10)-triene 6a-N-(~-biotinyl)caproamide from 3-hydroxyestra-1,3,5(10)-trien17-one and 3,17p-dihydroxyestra-l,3,5(10)-triene, via the 6-keto estrogenic derivatives. The reductive amination of these compounds is an effective step toward an epimeric mixture of the respective amines, which are easily biotinylated by use of N-(e-biotinylcaproy1)-N-hydroxysuccinimideester. The 6aepimers could be isolated from the alp-composition by application of isocratic HPLC, and overall yields were about 20% for the epimeric end products. The structures of the stereoisomers could clearly be assigned through 1H NMR studies. The ratios of the respective isomers obtained from the reductive amination were found to be 3(a):2@). The biotinylated estrogens can be used as tracers in a novel immunoassay concept for the determination of these analytes in human serum. Ring position 6 was selected for derivatization because of its distance from the functionalized positions 3 and 17and, therefore, of a negligible alteration of the tracer's structure in comparison to underivatized estrone or estradiol.
INTRODUCTION Numerous immunoassays based on nonradioactive labels for the determination of steroids in serum or other human body fluids were created in the last decade (1-4). Most of the competitive assays use either enzyme- or substratelabeled steroid tracers. These assays often lack analytical sensitivity and specificity due to an altered structure of the respective steroid tracers (5-10). This effect is observed in cases in which steroids have been derivatized a t the ring positions 3 or 17. By introducing the label in the same position as was used for the conjugation of the hapten steroid to a carrier protein for the purpose of antibody production, nearly identical recognition sites for the antisteroid antibody can be achieved. 3-Hydroxyestra1,3,5(10)-trien-17-one(estrone, E l ) and 3,17@-dihydroxyestra-l,3,5(1O)-triene (estradiol,E2) are usually conjugated to carrier proteins via their benzylic 6-position using the O-(carboxymethy1)oximation(CMO) method (11-1 7). But little is reported about structural derivatizations at that or the vicinal 7-position for the development of labeled steroidal tracers. Competitive nonradioactive immunoassays for E l as well as E2 can be set up by using biotinylated estrogen tracers, which easily bind to a streptavidin reporter enzyme conjugate (18). With appropriate substrates, the enzymemediated signal subsequently indicates the concentration of the endogenous steroids of interest. The biotin residue must be attached via a defined spacer group to the steroid. According to ref 19, this is due to the importance of *Address correspondence to this author at: Institut fur Klinische Chemie & Pathobiochemie,Klinikum rechts der Isar der TUMunchen,Ismaninger Str. 22, D-81675Munich, Germany.
Phone 0049 89 4140 4751; Fax 0049 89 41 805 175. Technical University Munich. t University Regensburg. e Abstract published in Advance ACS Abstracts, February f
15, 1994.
1043-1802/94/2905-0167$04.50/0
diminishing sterical hindrances between the biotin moiety and the antiestrogen antibody. Additionally, the chemical structure of the spacer has a relevant impact on the antibody recognition: the structure must be similar to the O-(carboxymethy1)oximelinkage which is used in the immunization procedure. 6-Aminoestrogens were previously described by Hamacher and Christ (20, 21) and Chesne et al. (22) as intermediates for the synthesis of antineoplastics against the estrogen receptor. 6-Biotinylation of E2 was first reported by Tiefenauer and Bodmer, who described E2 tracers with different spacer groups between the E2 6-position and the biotin moiety and their potential use in enzyme immunoassays (19,23-25). The aim of our work was to develop efficient syntheses of 3-hydroxyestra-1,3,5(lO)-trien-17-one 6a-N-(e-biotinyl)caproamide(Bio-El) and 3,17P-dihydroxyestra-l,3,5(10)-triene 6-N-(~-biotinyl)caproamide (Bio-EB), including defined isolations and characterizations of the 6a-epimers, for establishing new immunoassays for E l and E2. EXPERIMENTAL PROCEDURES General Methods. Melting points (uncorrected) were determined on a Reichert Thermovar. 'H NMR spectra were recorded on Beckman E 360 (60 MHz), Bruker AC 250 F (250 MHz), and Bruker ARX 400 (400 MHz). MedSi was used as the reference signal (6 0.00 ppm). IR spectra were obtained from KBr on a Beckman spectrophotometer 24. UV spectra were measured in MeOH on a Hitachi spectrophotometer U 2000; dioxane was used for 11. MS analyses were performed for 70 eV E1 with a Varian MAT 112sand for FAB/high-resolution MS with a Finigan MAT 90. For column chromatography silica gel (grain 0.0630.200) was used, and TLC was performed using silica gel 60 F254 (0.2 mm); both silica materials were obtained from E. Merck (Darmstadt,Germany). The preparative HPLC system consisted of an HPLC pump Knauer 64, a Vertex column (250 mm, 16-mmdiameter), filled with LiChrosorb 0 1994 American Chemical Society
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100 (5pm), both from Knauer (Bad Homburg, Germany), and a UV detector ERC 7210 (ERC, Alteglofsheim, Germany). Chemicals were obtained from Merck, except for E l and E2, which were purchased from Sigma (Deisenhofen, Germany), and N-(e-biotinylcaproy1)-Nhydroxysuccinimide ester from Boehringer Mannheim (Mannheim, Germany). Preparation of 3-Hydroxyestra-1,3,5(lO)-trien-l7one 6-N-(e-Biotinyl)caproamide(6). 3-Hydroxyestra-1,3,5(lO)-trien-l7-one17-(cyclic 1,2ethanediyl acetal) (2) was prepared from 1 according to 180-182 (lit. 26 mp ref 26 with a yield of 75%. Mp ("0: ("C) 182-185). 3-Hydroxyestra-1,3,5(lO)-trien-l7-one3-acetate 17(cyclic 1,2-ethanediylacetal) (3) was prepared according to ref 27 with a yield of 70%. Mp ("C): 103-104 (lit. (27) mp ("C) 105-105.5). 3-Hydroxyestra-1,3,5(lO)-triene-6,17-dione3-Acetate 17-(Cyclic 1,2-ethanediyl acetal) (4). According to a method given by Garza and Rao (28), a stirred suspension of 2.8 g (28 mmol) of Cr03/50 mL of CH2Cl2 was cooled t o 4 0 "C under N2. A 2.58-g (28mmol) portion of 3,5-dimethylpyrazole was added. After 15 min, 1.00 g (2.81 mmol) of 3 in 10 mL of CHzClz was added. The solution was stirred for 6 h a t -25 "C. The residue was chromatographed on silica gel/petroleum ether using a petroleum ether/ethyl acetate eluent (4:l v/v) (Rf = 0.2). A total of 518 mg (1.4 mmol, 50%)of the crude ketone was obtained. After recrystallization from MeOH, 310 mg (840 pmol, 30%)of colorless ketone 4 was obtained. Mp (OC): 161-163. IR (cm-l): 2945,2880 (CH); 1755(aliphaticester C=O); 1675 (ketone); 1190 (cyclic ether (dioxolane)). MS (70eV EI, m/z):370 (100,M+). lH NMR (60 MHz, CDCl3, ppm): 7.6 (d, l H , J = 8 Hz); 7.5-7.0 (m, 2H, 2,4 CH); 3.8 (m, 4H, dioxolane CH); 2.2 (s, 3H, 17-OCOCH3);0.83 (s, 3H, 18-CH). UV: 245 nm, log e = 3.84; 297 nm, log t = 2.74. Anal. Calcd: C, 71.33; H, 7.08; 0, 21.59. Found: C, 71.41; H, 6.84; 0, 21.08. 6-Amino-3-hydroxyestra-1,3,5(lO)-trien-l7-one 17(Cyclic 1,2-ethanediyl acetal) ( 5 ) . A 370-mg (1mmol) portion of the ketone 4 and 390 mg (6 mmol) of NaCNBH3 were dissolved under N2 in 4.5 mL of buffer (40.5 g of dried NH40Ac in 150 mL of dry MeOH (24))and refluxed at 65 "C for 48 h. The clear reaction solution was worked up by removing the solvent and treating the residue with saturated aqueous NaHC03 and diethyl ether. The combined aqueous phases were again extracted with diethyl ether. After the pooled ether phases were dried, the solvent was removed at room temperature (rt). A total of 250 mg of a slightly yellow amorphous product was obtained, corresponding to a 75 % yield of the amine 5. IR (cm-I): 3340, 3320, 3260 (amine NH). MS (70 eV EI, m/z):329 (13, M+);312 (M+- NH3). 'H NMR (250 MHz, CDCl3, ppm): 7.14-6.65 (m, 4H, 1-CH, 2-CH, 3-COH, 4-CH); 3.97-3.44 (m, 5H, 6-CH, 17-C-dioxolane);0.86,0.85 (s, 3H, 18-CH) (a/@-ratio:k1.5; epimeric excess: 20%). UV: 282 nm, log 6 = 3.32; 218 nm, log t = 3.88. 3-Hydroxyestra-1,3,5(lO)-trien-l7-one 6-N-(e-Biotiny1)caproamide (6). A 53-mg (160 pmol) portion of the amine 5, 87 mg (191 pmol) of N-(6-biotinylcaproy1)-Nhydroxysuccinimide ester, 32 mg (44 pL, d = 0.73, 320 pmol) of triethylamine, and 1 mL of DMF were stirred under N2 for 24 h at rt prior to evaporation of the solvent. The residue was treated with 2 mL of dry saturated methanolic hydrochloric acid (pH = 2) for 24 h at rt. All reaction compounds except 6 were removed by high vacuum. The substance was purified before HPLC on a
Luppa et al.
silica gel/petroleum ether column using a MeOH/CH2C12 (k4.5) (viv) eluent (Rf = 0.5). Isolation of the 6a-Epimer 6a from the 6alBEpimeric Mixture 6. Isocratic HPLC: eluent, 7% MeOH/93% CH2C12; flow rate, 10 mL/min. 6a: 13-14 min. 6b: 14-20 min. 6a. MS: HR-MS (PI-LISIMS) (glycerin/MeOH): calcd for CaH49N405S (MH+)625.3424, found 625.3420; difference, 0.6 ppm. IH NMR (400 MHz, CDC13, ppm): 9.70 (s, 1H);7.26-6.69 (m, 3H, 1-CH,2-CH, 4-CH); 6.37 (d, l H , JNH-6 = 9 Hz, 6a-CNH);5.24 (ddd, l H , JNH= - ~9 Hz, J6-7 = 11.7 and 6.4 Hz, 60-CH); 0.89 (s,3H, 18-CH). UV: 279 nm, log t = 3.54; 220 nm, log t = 4.26. Preparation of 3,17B-Dihydroxyestra-1,3,5(10)triene 6-N-(e-Biotinyl)caproamide(12). 3,178-Dihydroxyestra-l,3,5(10)-triene 3,178-Diacetate (8) was prepared from 7 according to the method of ref 29. The yield was 90%. Mp ("C): 121-123 (lit. (21) mp ("C) 123-125). 3,17B-Dihydroxyestra-1,3,5(lO)-trien-6-one3,17-Diacetate (9). Preparation was performed analogous to that of 4 from 8 with similar yields. Mp ("C): 173 (lit. (21) mp ("C) 173). 3,17p-Dihydroxyestra-1,3,5( lO)-trien-6-one(10) was prepared from 9 according to ref 21 with a quantitative yield. Mp ("C): 276-279 (lit. (21) mp ("C) 282). 6-Amino-3,17@-dihydroxyestra-1,3,5( 10)-triene (1 1) was prepared in a manner similar to the synthesis of 5 from 10 with a yield of 85 7% of the 40-mixture. IR (cm-l): 3420 (free OH); 3345, 3280 (amine NH); 2940 (CH aliphatic); 1600 (NHdef)(24). MS (70 eV EI, mlz): 287 (7, M+),270 (100, M+ -NH3) (24). 'H NMR (250MH2, CD3OD, ppm): 7.15-6.6 (m,3H, 1,2,4-CH);4.00 (m, l H , 6-CH); 3.65 (m, l H , 17-CH); 0.81, 0.75 (s, lH, 18-CH) (24) ( a / 0-ratio: 1:2.3;epimeric excess, 40%). UV (dioxane): 280 nm, log t = 3.38; 222 nm, log t = 4.03 (24). 3,178-Dihydroxyestra-l,3,5(10)-trieneGa-N-(e-Biotiny1)caproamide (12). A 22-mg (76.7 pmol) portion of the amine 11,42 mg (92.5 Fmol) of N-(6-biotinylcaproy1)N-hydroxysuccinimide ester, 18.25 mg (25 pL, d = 0.73) of triethylamine, and 1mL of DMF were stirred under N2 for 24 h at rt prior to evaporation of the solvent. The residue was taken up with 1.5 mL of MeOH and purified on a chromatographic column filled with 7 g of silica gel/ petroleum ether, using a benzene/MeOH eluent (4:l v/v) (Rf = 0.24). Isolation of the 6a-Epimer 12a from the 648Epimeric Mixture 12. Isocratic HPLC: eluent, 9% MeOH/91% CH2C12; flow rate, 9.5 mL/min. 12a: 1717.75 min, 1.4 mg (2.23 pmol). 12b: 18.75-20 min, 1.7 mg 12a: MS: HR-MS (PI-LISIMS) (2.7 pmol). (glycerin/MeOH): calcd for CaH51N405S (MH+)627.3581, found 627.3621; difference, 6.3 ppm; calcd for C34H50N405SNa (MNa+)649.3399,found 649.3408;difference, 1.3ppm (23). lH NMR (400 MHz, CDC13, ppm): 9.43 (s, 1H); =9 7.52-6.76 (m, aromatic CH, OH); 6.26 (d, l H , JNH-B Hz, 6a-NH); 6.01 (t, lH, J = 6 Hz); 5.22 (ddd, l H , J N H - ~ ~ = 9 Hz, J6-7 = 10 and 6 Hz, 60-CH); 0.77 (s, 3H, 18-CH). UV: 280 nm, log t = 3.13 (23). 6-Amino-3,17B-dihydroxyestra-1,3,5( 10)-triene 178Acetate (13) was prepared in a manner analogous to that of 5 from 9 with a yield of 70 7%. Preparation of 3-Hydroxyestra-1,3,5(lO)-trien-l7one 6-Benzamide (14) and 3,178-Dihydroxyestra-1,3,5(10)-triene178-Acetate 6-Benzamide 3-Benzoate (15). 3-Hydroxyestra-1,3,5(10)-trien-17-one6-benzamide (14). A 22-mg (67 pmol) portion of amine 5 and 40 mg (177 pmol) of benzoic anhydride were stirred in 1 mL of pyridine for 24 h at rt. All reaction components except the steroid were removed by vacuum. The protecting
Bioconjugate Chem., Vol. 5, No. 2, 1994
Technical Notes
Scheme 1. 6-Position
189
Biotinylation of 3-Hydroxyestra-1,3,5(lO)-trien-l7-one and 3,17&Dihydroxyestra-1,3,5(10)-triene in the
Ho
& & Ho
1
I
2
8
I
Am
& 3
I
I
Am
JJpcp :@ 0
Ho
R-C-0
HN-C-Ph
4
10
HO
HO
HN-C-Ph
14
15
& t
5
NHI
11
HO HN-C-(WcBwo
12
groups were then removed successively by a dry methanolic hydrochloride solution (pH = 2) and a NaOH/MeOH solution (3 mL of saturated aqueous NaOH and 1 mL of MeOH). Isolation of the Ga-Epimer 14a from the Sa/8Epimeric Mixture 14. Isocratic HPLC conditions: eluent, 1%MeOH/CH2C12; flow rate, 10 mL/min. 14a: 12.0-13.3 min, 1.196 mg. 14b: 14.5-16.5 min, 0.45 mg. 14a: MS (70 eV EI, m/z): 389 (3, M+); 312 (3, M+ - Ph); 286 (100, M+ - PhCONH2). UV: 278 nm, log t = 2.45; 219 nm, log t = 3.41. lH NMR (400 MHz, CDC13, ppm): 7.77-
6.75 (m,9H, 1-CH,2-CH, benzoxy-aromate CH, OH); 6.35 (d, 1H, J N H=- ~ 9.5 Hz, NH); 5.55 (ddd, 1H, J ~ - N=H9.5 Hz, Jg7 = 6.11 Hz, 6p-CH);0.92 (9,3H, 18-CH). 'H NMR (400 MHz, CDsOD, ppm): 7.95-6.63 (m, 8H, 1-CH,2-CH, = 6.1 and 4-CH, benzoxy-aromate CHI; 5.43 (dd, l H , 5 ~ 7 11.2 Hz, GP-CH); 0.96 (s, 3H, 18-CH). 3,178-Dihydroxyestra-1,3,5( 10)-triene 17B-Acetate 6-Benzamide 3-Benzoate (15). A 53-mg (161 pmol) portion of 13 and 75 mg (332 pmol) of benzoic anhydride were stirred for 20 h at rt in 1 mL of pyridine. After the solvent was removed, the crude product was purified on
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Bioconjugate Chem., Vol. 5, No. 2, 1994
a silica gel/petroleum ether column using petroleum ether/ ethyl acetate (1:l v/v), yielding 80 mg (150 pmol, 92%)of purified 15. IR (cm-l): 3065 (CH aromatic); 2920 (CH aliphatic); 1780(3-benzoxyC=O); 1725 (17-acetoxyC=O); 1685 (amide I); 1660 (amide 11). Isolation of the 6a-Epimer 15a from the 6a/pMixture 15. Eluent: 1.25% MeOH/CH2C12. Flow rate: 10 mL/min. 15a: 8.0-9.5 min, 6.59 mg. 15b: 9.5-10.3 min, 4.47 mg. 15a: MS (70 eV EI, 4 2 ) : 537 (0.02,M+(I)); 433 (2, M+(II));416 (2, M+ -PhCONH2); 312 (100, M+(II) - PhCONH2). 'H NMR (400 MHz, CDCl3, ppm): 8.136.74 (m, 13H, 1-CH, 2-CH, 4-CH, 3-benzoxy-H, 6-benza= 9 Hz, NH); 5.52 (m, mide aromate H); 6.32 (d, l H , JNH-B 1H); 4.69 (t, l H , J = 8 Hz, 17-C-H); 2.06 (s, 3H, 17-OCOCH3);0.84 (s, 3H, 18-CH). 'H NMR (400 MHz, CD30D, ppm): 7.98-6.62 (m,8H, 1-CH, 2-CH, 4-CH, 6-benzamide aromate H); 5.39 (dd, l H , J6-7= = 11 Hz, Jc7eq= 7 Hz, 6P-CH); 4.68 (t, l H , J = 8 Hz, 17-CH); 2.04 (9, 3H, 17-OCOCH3);0.90 (s, 3H, 18-CH). UV: 277 nm, log e = 2.64; 233 nm, log t = 3.33. RESULTS AND DISCUSSION
This paper describes the preparation of immunochemical tracer conjugates of estrogens by attaching biotin residues via a -NHCO(CH2)5NH- spacer arm to E l and E2 at C-6. The five-step syntheses of the tracer substances 6 and 12 from the estrogens 1 and 7 are given in Scheme 1. The overall yields are 15% for 6 and 20% for 12. On the basis of syntheses of Garza (28) and Tiefenauer (24), our synthetic approaches for 6 and 12 exhibit the following features: the extractive procedure for the isolation of the epimeric amino compounds 5 and 11, obtained by reductive amination of the respective 6-keto precursors, is convenient and simple. The biotinylation reaction was modified by employing approximately equimolar ratios of substrate and biotinylating agent with nearly quantitative yields. The separation of the respective biotinylated epimers was achieved by HPLC using an isocratic eluent system on a LiChrosorb 100 column. To identify the exact stereochemistry at the benzylic ring position 6, HPLC-separable 6-benzamide derivatives 14 and 15 were synthesized. In the course of NMR investigations of the amines 5, 11, and 13, we conclude that-in contrast to Tiefenauer et al. (24)-the reductive amination step is not characterized by a-stereospecificity. Epimeric excesses for the a-compounds of 5 and 11 were 20% and 40% as determined from their C-18 signals. To prove our assumption and to confirm the configuration of the tracers 6 and 12, we set out to characterize the isolated 6a-epimers by NMR spectroscopy. This approach is based on the work of Wintersteiner et al. (30), who found for the 60-proton of the 3,6a,l7P-estratriol triacetate epimer the couplingpattern of an ABX system with coupling constants of 5.5 and 8.5 Hz, while the AB part of the 6a-proton's signal of the 6P-isomer gave the coupling constant of 3 Hz. The chromatographic separation of the amines was not feasible; therefore, we derivatized 5 and 13 with benzoic anhydride to obtain the stable benzamides 14 and 15. The respective NMR-pure a-epimers could be isolated by HPLC. We found that the amide proton also interacts with the 6-proton, thereby giving rise to complex signals. We removed the amide's interference by H/D exchange with CD30D. A first-order evaluation of the four-line pattern found for the 6P-proton of 14a and Ea, according to Wintersteiner, resulted in coupling constants of 6.1, 11.3 Hz, and 6.6,11.4 Hz, respectively. Analogous values were found for the 6-biotinylated estrogens: 11.7, 6.4 Hz for 6a, as well as 6.0, 10.0 Hz for 12a.
Luppa et al.
In summary, we propose effective synthetic pathways of both 6a-biotinylated E l and E2; the respective E l derivative has not been described in literature. 6-Biotinylated E2 compounds, however, were already reported by Bodmer and Tiefenauer. But in contrast to data for the synthesis of these tracers compounds given in 24, and for the respective HPLC purifications, described in 19, 23, our synthetic pathway combined with the appropriate isocratic HPLC separation technique offers improved accessibility and yields stereochemical pure products. Concerning the ability of the new tracers for E l and E2 immunoassays, it must be considered that due to the mode of conjugation at C-6, the structural determinants of BioE l and Bio-E2 are nearly unchanged in comparison to unsubstituted E l and E2, since the position 6 in the B-ring is most distant from the prominent positions 3 and 17. Thus, the antiestrogen antibodies are unable to distinguish between the tracers and the endogenous steroids E l or E2. By use of these compounds, a novel competitive immunoassay concept for the determination of E l and E2 in serum could be realized. Results will be presented elsewhere. The E l enzyme immunoassay is run with the following components: Bio-El tracer, polyclonal anti-El antibody, and reporter enzyme linked to streptavidin. In pilot experiments we checked the competitive character of the Bio-El tracer in competition to ring A-tritiated El. It could be demonstrated that Bio-El adequately displaces tritiated E l from the antibody in the expected competitive way (data not shown). As shown in ref 19, the -NHCO(CH&NH- spacer arm, linking biotin to the estrogen, satisfactorily fulfillsthe structural requirements of optimal antigen-antibody interaction. It is worth mentioning that biotinylated steroids, as stable immunochemicalprobes, in connection with streptavidin-conjugated reporter enzymes, should allow the development of a series of competitive immunoassays for the determination of the respective steroids in human serum as well as their potential application in steroid receptor studies. ACKNOWLEDGMENT
This article is dedicated to Prof. G. M h k l on the occasion of his 65th birthday. The authors thank Mrs. A. Rossner for technical assistance, Prof. E. Kuss for reading the manuscript, and the Fond der Deutschen Chemischen Industrie for financial support. LITERATURE CITED (1) Pal, S. B., Ed. (1986)Immunoassay technology,Vol. 2, Walter
de Gruyter, Berlin. (2) Chan, D. W., and Perlstein, M. T., Eds. (1987) Immunoassay. A practical guide, Academic press, New York. (3) Albertson, B. D., and Haseltine, F. P., Eds. (1988) Nonradiometric assays. Technology and application in polypeptide and steroid hormone detection, Alan R. Liss, New York. (4) Gosling, J. P. (1990)A decade of development in immunoassay methodology. Clin. Chem. 36, 1408-1427. (5) Folan, J., Gosling, J. P., and Fottrell, P. F. (1988) Solidphase enzymoimmunoassay of estrone in serum. Clin. Chem. 34, 1843-1846. (6) Folan, J.,Gosling, J. P., Finn, M. F., and Fottrell, P. F. (1989) Solid-phase enzymoimmunoassay of estrone in saliva. Clin. Chem. 35, 569-572. (7) Elder, P. A,, Manley, L., and Lewis, J. G. (1990) Use of a monoclonal antibody to estrone-3-glucuronide in an enzymelinked immunosorbent assay (ELISA). J. Steroid Biochem. 36, 439-443. (8) Barnard, G., Kohen, F., Mikola, H., and Lovgren, T. (1989) Measurement of estrone-3-glucuronide in urine by rapid,
Technical Notes
homogeneous time-resolved fluoroimmunoassay. Clin. Chem. 35, 555-559. (9) Kim, J. B.,Barnard, G. J., Collins, W. P., Kohen,F., Lindner, H. R., and Eshhar, Z. (1982) Measurement of plasmaestradiol17p by solid-phase chemiluminescence immunoassay. Clin. Chem. 28, 1120-1124. 0 ) De Boever, J., Kohen, F., Usanachitt, C., Vandekerckhove, D., Leyseele, D., and Vandewalle, L. (1986) Direct chemiluminescence immunoassay for estradiol in serum. Clin. Chem. 32, 1895-1900. 1) Kuss, E., Goebel, R., and Enderle, H. (1973) Influence of oxo-, and/or hydroxy-groups a t C-16/C-17 of estrogens on affinity to anti-estrone, anti-estradiol-l7a- and anti-estradiol176-antisera. Hoppe-Seyler's Z. Physiol. Chem. 354, 347364. (12) Doerr, P., Goebel, R., and Kuss, E. (1973) Specific radioimmunologic determination of plasma oestradiol in males without chromatography. Acta Endocrinol. (Copenh.) 72, -~ Suppl. 173, 108. (13) Goebel, R., and Kuss, E. (1973) Radioimmunological determination of plasma estriol in pregnancy with anti-estriolC6 conjugate-antiserum. Acta Endocrinol. (Copenh.) 72, Suppl. 173, 109. (14) Exley, D., Johnson,M. W., andDean,P.D. G. (1971)Antisera highly specific for 17P-oestradiol. Steroids 18, 605-620. (15) Exley, D. (1972) Specificities of antibodies to oestrogens. J . Steroid Biochem. 3, 497-501. (16) Jeffcoate, S. L., and Searle, J. E. (1972) Preparation of a specific antiserum to estradiol-17p coupled to protein through the B-ring. Steroids 19, 181-188. (17) Lindner, H. R., Perel, E., Friedlander, A., and Zeitlin, A. (1972) Specificity of antibodies to ovarian hormones in relation to the site of attachment of the steroid hapten to the peptide carrier. Steroids 19, 357-375. (18) Diamandis, E. P., and Christopoulos, T. K. (1991)The biotin(strept)avidin system: principles and applications in biotechnology. Clin. Chem. 37, 625-636. (19) Tiefenauer, L. X., and Andres, R. Y. (1990)Biotinyl-estradiol derivatives in enzyme immunoassays: structural requirements for optimal antibody binding. J . Steroid Biochem. 35, 633639.
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