Introduction of aliphatic amino and hydroxy groups to keto steroids

Bioconlugete ctwm. 1002, 3, 182-180

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Introduction of Aliphatic Amino and Hydroxy Groups to Keto Steroids Using 0-Substituted Hydroxylamines+ Heikki Mikola' and Elina Hiinninen$ Wallac Chemical Laboratories, P.O. Box 10, SF-20101 Turku, Finland. Received January 7, 1992 (Aminooxy)butyl- and -hexylamines and alcohols were synthesized by the Ing-Manske modification of the Gabriel synthesis. The aminooxy group of these heterobifunctional spacer reagents is a far more powerful nucleophile than the amino or hydroxy group because of the oxygen atom adjacent to the amino group (a-effect). The aminooxy group reacts readily with keto groups while the amino or hydroxy (or other) group remains free for further reactions. These excellent heterobifunctional spacer reagents were used here to derivatize keto steroids in an alkaline alcoholicsolution. Using the described, general, and easy one-step synthesis, aliphatic amino or hydroxy groups with a spacer arm have been introduced to testosterone, 6-ketoestradiol, and cortisol.

INTRODUCTION

EXPERIMENTAL PROCEDURES

In many biochemical applications, haptens coupled covalently to other molecules are needed (1). For example in affinity chromatography and in immunoassays haptens immobilized on solid matrix are used. In antibody production the hapten needs to be coupled covalently to a carrier before immunization, and in competitive immunoassays the hapten is traditionally coupled to a label molecule to be used as a tracer. Usually before coupling, a spacer arm and a suitable reactive group have to be introduced to the hapten molecule, for example by using bifunctional spacer reagents. The coupling site, the chemical structure of the linkage, and the length of the spacer arm between the hapten and the label or the carrier have important roles in the recognition of the hapten by the antibody (1-5). 0-(Carboxymethyl)oximes, hemisuccinates, carboxyethyl ethers, and thioethers are frequently used as derivatives in steroid immunoassays (1). The spacer arm in all of these derivatives is quite short. a,w-Diaminohydrocarbons have been coupled to testosterone 3-10(carboxymethyl)oximel (6) to get longer spacer arms containing an aliphatic amino group. The yield of this reaction is quite low because of the large excess of diamino compound needed, which has to be removed from the reaction mixture. Amino sterols have also been used as starting materials for some bridged steroid derivatives (3, 7)instead of keto or hydroxy steroids. In the present study aliphatic (aminooxy)alkylamines and alcohols were synthesized. In a one-step synthesis these were used to incorporate a spacer arm with primary aliphatic amino or hydroxy group to the keto group of testosterone, 6-ketoestradiol, and cortisol. Similar (aminooxy)butyl compounds have so far been utilized as enzyme inhibitors or bacteriostatics (8-13) and to introduce aliphatic amino (14) or hydroxyl groups (15)to nucleotides. The use of these synthesized steroid derivatives in immunoassays will be described later.

Materials. The reagents for syntheses were purchased either from Aldrich-Chemie, E. Merck, or Fluka and used without further purification. The solvents were p.a. grade either from E. Merck or Baker. TLC plates and silica for short-column chromatography were obtained from E. Merck. 'HNMR spectra were recorded on either JEOL GX 400 or Hitachi Perkin-Elmer R-600 spectrometers using tetramethylsilane (TMS) or 3-(trimethylsily1)propanesulfonic acid (DSS) as internal standard. UV spectra were obtained on a Shimadzu UV-2100 spectrophotometer. Mass spectra were recorded on a VG7070E mass spectrometer by using an electronic ionization energy of 70 or 15 eV (E1 70 eV and E1 15 eV) and chemical ionization with ammonia as reagent gas (CI). Melting points are uncorrected and were measured on a Gallenkamp capillary melting point apparatus. Syntheses of Aminooxy-Group-Containing Heterobifunctional Spacer Reagents. N-(CBromobuty1)phthalimide (I). A mixture of 55.6 g of potassium phthalimide, 95.5 mL of 1,4-dibromobutane,and 3.5 mL of dimethylformamide was stirred at 160 O C for about 17 h. The warm reaction mixture was filtered and the precipitate was washed with ethanol. The solvents were evaporated under reduced pressure, and the remainder was recrystallized twice from ethanol to produce N-(4bromobuty1)phthalimide (I). Yield 59.7 g (71%). Mp: 77-80 "C. N-(6-Bromohexy1)phthalimide(II). The title compound was synthesized by the method employed for N(4-bromobuty1)phthalimide(I) starting from 55.6 g of potassium phthalimide and 114.5 mL of 1,6-dibromohexane. Yield: 66.9 g (72%). Mp: 52-55 "C. N-[I-(Phthalimidooxy) butyllphthdimide (III). A mixture of 17.6 g of N-(4-bromobutyl)phthalimide(I), 11.0 g of N-hydroxyphthalimide, and 63 mL of dry dimethylformamide was heated to 60 OC. Triethylamine (9.5 mL) was added and the mixture was stirred a t room temperature for 30 h. Water (250 mL) was added and the precipitate was collected and washed with water. The crude product (111)was dried at room temperature. Yield: 18.4 g (81%). Mp: 159-161 OC. N-[6-(Phthalimidooxy)hexyllphthalimide (10. The title compound was synthesizedas N-[4-(phthalimidooxy)butyllphthalimide (111)from 17.6 g of N-(bbromohexyl)-

* Author to whom correspondence should be addressed. + Presented in part as a poster at the 8th International

IUPAC Conference on Organic Synthesis, Helsinki, Finland, July 23-27, 1990.

t Present address: Orion Corp., Farmos, P.O.Box 425, SF20101 Turku, Finland.

0 1992 American Chemical Society

Introductlon of Reactlve Spacer Arms to SteroMs

phthalimide (11) and 11.0 g of N-hydroxyphthalimide. Yield: 21.5 g (97%). Mp: 114-118 "C. 4-(Aminooxy)butylamineDihydrochloride (V). A mixture of 18.0 g of N- [4-(phthalimidooxy)butyl]phthalimide (III),9.5 mL of hydrazinehydrate, and 50 mL of ethanol was stirred for 2 h at 80 "C and then 2 days at room temperature. Concentrated hydrochloric acid (100 mL) was added and refluxed for 1h. Water (125 mL) was added and the ethanol was evaporated under reduced pressure. The precipitated phthalhydrazide was separated by filtration; the solution was concentrated,made alkaline, and evaporated to dryness. Ethanol (50 mL) was added to solubilize the product and the remaining solid was filtered. The ethanol solution was acidified using 6 M HC1, filtered, and evaporated to dryness. The solid product was crystallized from methanoVethy1acetate to give 6.8 g (97% ) of 4-(aminooxy)butylamine dihydrochloride (V). 'H NMR 6 (D2O) 1.70 (4 H, m, -CH2CH2-), 3.03 (2 H, t, NHzCHz), 3.77 ppm (2 H, t, NH20CH2). MS: free base, m / z (relative intensity) (CI) 105 (37), 90 (31), 88 (30), 72 (100); [M + HI+ calcd for C4Hl2N20 + H, 105.1028, found 105.069; (E1 15 eV) 105 (5), 88 (ll),72 (loo), 55 (36); [M + HI+ found 105.10; (E1 70 eV) 88,72, 55. 6-(Aminooxy)hexylamineDihydrochloride (VI). The title compound was synthesized by the method employed for 4-(aminooxy)butylamine dihydrochloride (V). 'H NMR 6 (D20) 1.40 (4 H, m, -CHz-), 1.68 (4 H, m, -CHz-), 3.93 ppm (2 H, t, NH~OCHZ). 3.00 (2 H, t, NHzCH~), 4-Bromobutyl Acetate (VII). Acetyl bromide (10.0 g) was dropped into refluxing tetrahydrofuran (200 mL) and the product, 4-bromobutyl acetate (VII), was distilled at reduced pressure. Yield: 14 g (88%). Bp: 92 "C (12 mmHg). N-[(4-Acetylbutyl)oxylphthalimide (VIII). A mixture of 80 g of N-hydroxyphthalimide and 100 g of 4-bromobutyl acetate (VII) was added into 200 mL of dry dimethylformamide and 70 mL of dry triethylamine and stirred at room temperature for 16 h and at 100 "C for 30 min. The mixture was cooled to room temperature and poured into 2 L of water. The precipitate was collected, washed with water, dried in a vacuum desiccator, and used without further purification. 4-(Aminooxy)butanol (1x1.Crude N-[(4-acetylbutyl)oxylphthalimide (VIII) from the previous step was refluxed in a mixture of acetic acid (135 mL) and concentrated hydrochloric acid (75mL) for 45 min. After cooling, the precipitated phthalic acid was filtered off and the filtrate concentrated and coevaporated five times with 50 mL of water to dryness to remove the traces of acids. Water (100 mL) was added and the pH was adjusted to 13-14 using 30% sodium hydroxide. The product was extracted from water with ethyl acetate in a continuous extraction system for 8 h. Distillation at reduced pressure yielded 28 g (50%)of 4-(aminooxy)butanol (IX). Bp: 98 "C (1 mmHg). 'H NMR: 6 (DzO)1.62 (4 H, m, -CHz-), 3.63 (2 H, t, OHCHz), 3.76 ppm (2 H, t, NH20CH2). MS: free base, m/z (relative intensity) (CI) 106 (85), 88 (38), 73 (30),55 (100); [M + HI+calcd for C4HllN02 + H 106.0868, found 106.096; (E1 15 eV) 106 (3), 88 (4), 73 (72), 55 (100); [M + HI+ found 106.03; (E1 70 eV) 73,55. N-[(6-Bromohexyl)oxylphthalimide (X).A mixture of 8.16 g of N-hydroxyphthalimide and 30.5 g of 1,6-dibromohexane in 125 mL of dry dimethylformamide and 6.9 mL of triethylamine was stirred at 60 "C for 2 h and then at room temperature for 24 h. After filtration the reaction mixture was distilled at reduced pressure and the re-

Bloconjugate Chem., Vol. 3,No. 2, 1992 183

mainder was crystallized from ethanol to produce N-[(6bromohexyl)oxylphthalimide(X). Yield: 11g (68%).Mp: 57-62 "C. N-[(6-Acetylhexyl)oxylphthalimide(XI).A mixture of 0.98 g of N-[(6-bromohexyl)oxylphthalimide (X) and 2.46 g of sodium acetate in 50 mL of acetic acid was refluxed for 36 h and evaporated to dryness. The product, N-[(6-acetylhexyl)oxylphthalimide(XI),wasusedwithout further purification. 6-(Aminooxy)hexanolHydrochloride (XII). A mixture of 1.83g of crude N- [(6-acetylhexy1)oxylphthalimide(XI) and 1.17 mL of hydrazine hydrate in 5 mL of ethanol was stirred overnight at 62 "C. Concentrated hydrochloric acid (10 mL) was added and refluxed for 2.5 h. Water (20 mL) was added and the ethanol was evaporated. The precipitated phthalhydrazide was separated by filtration and the solution was concentrated. The concentrate was purified with a short silica column using first 30% methanol in chloroform and then 50% methanol in chloroform, which eluted the product, 6-(aminooxy)hexan01 hydrochloride (XII). 'H NMR: 6 (DzO) 1.68 (4 H, m, -CHz-), 1.42 (4 H, m, -CH2-), 3.60 (2 H, t, OHCHz), 3.73 ppm (2 H, t, NHzOCH2). General Procedures for the Synthesis of Steroid O-Alkyloximes. Testosterone 3-[O-(6-Aminohexy1)oximel. A solution of 100 mg of testosterone and 360 mg of 6-(aminooxy)hexylaminedihydrochloride (VI) in 3 mL of 90% ethanol and 150 mg of sodium acetate was stirred at room temperature for 1h and then refluxed for 4 h. The reaction mixture was filtered and evaporated to dryness. The product,testosterone 3-[0-(6-aminohexyl)oximel,was purified with a short silica column using gradient elution from 0 to 20% of methanol in chloroform. Yield: 120 mg (85%). 'H NMR 6 (CDCl3 + CD30D) 0.77 (3 H, ~ , 1 8 CHd, 1.07 (3 H, s,19-CH3),1.42 (4 H, m, -CHzCHz-), 1.74 (4 H, m, -CH&Hz-), 2.93 (2 H, d, CHZNHZ),3.61 (1H, d, J = 8.5 Hz, 17a-H),4.02 (2 H, t, NOCHz), 5.74 and 6.36 ppm (1H, 2 s, 4-H anti- and syn-isomer, respectively). MS: (E1 70 eV) m / z (relative intensity) 402 (81, 372 (51, 314 (31, 286 (381, 116 (100); M+ calcd for C ~ ~ H ~ Z N Z O Z 402.3236, found 402.288. 6-Ketoestradiol6-[0-(6-Hydroxyhexyl)oxime].A solution of 290 mg of 6-ketoestradiol and 400 mg of 6-(aminooxy)hexanol hydrochloride (XII) in 20 mL of 90% ethanol and 650 mg of sodium acetate was treated as above. The product, estradiol 6-[O-(6-hydroxyhexyl)oximelwas purified by thin-layer chromatography using 20 % methanol in chloroformto develop the plates. Yield: 130 mg (30%). 'H NMR 6 (CDCl3 + CD30D) 0.78 (3 H, ~ , 1 8 CH3), 1.48 (m, -CHzCH2-), 1.78 (m, -CHzCH2-), 3.00 (m, CHzOH), 3.73 (1H, m, 17a-H),4.18 (2 H, t, NOCHz), 6.89 (1H, dd, J = 2.8 and 8.5,2-H), 7.20 (1H, d, J = 8.5,1-H), 7.43 ppm (1 H, d, J = 2.8, 4-H). MS: (E1 70 eV) m / z (relative intensity) 401 (13), 371 (51,313 (31,284 (loo),226 (5), 172 (191,117 (50);M+ calcd for C24H35N04 401.2557, found 401.225. Cortisol 3- [0-(6-Aminohexyl)oximel.A solution of 1 g of cortisol and 0.7 mL of pyrrolidine in 15mL of methanol was stirred for 15 min. Pyrrolidine (0.7 mL) and 566 mg of 6-(aminooxy)hexylamine dihydrochloride (VI) were added, and the reaction mixture was heated to 60 "C for 5 min and then stirred at room temperature for 2 h. The mixture was evaporated to dryness and purified with a short silica column using 20 % methanol in chloroform as eluent. Yield: 300 mg (26%). 'H NMR 6 (CDC13+ CD3OD) 0.87 (3 H, s, l8-CH3), 1.32-1.47 (7 H, m, -CH~CHZand 19-C&), 1.78 (4 H, m, -CHzCHz-), 2.96 (2 H, d, CH2NH2),4.02 (2 H, t, NOCHz),4.39 (1H, m, lla-H), 5.66and

-

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Bloconlugate Chem., Vol. 3, No. 2, 1992

6.25 ppm (1H, 2 s, 4-H anti- and syn-isomer,respectively). MS: (E1 15 eV) m / z (relative intensity) 446 (5), 368 (8), 301 (371, 256 (18), 207 (40), 135 (100); (CI) (relative intensity) 477 (4),447 (€9,433 (ll),419 (57),391 (1001,363 (28), 279 (47), 173 (64); [M + HI+ calcd for C27H~N205 + H 477.3328, found 477.25.

Mikola and Hanninen

Scheme I. Synthesis Dihydroc hlorides

(Aminooxy)alkylamine

of

0

n

RESULTS AND DISCUSSION

0-(Carboxymethy1)oximes have been used extensively while conjugating steroids to other molecules. During the synthesis of such steroid derivatives (aminooxy)acetic acid has been used as a heterobifunctional spacer reagent. In spite of this the same method of incorporating other spacer arms and aliphatic functional groups to steroids has remained almost unnoticed. Here the method is used to couple some aminooxy-group-containingbifunctional reagents to keto steroids, Methods of synthesizing (aminooxy)alkylamines and alcoholsare described as examples of how to synthesize these bifunctional spacer reagents, which are quite stable and useful, as hydrochlorides.While using other aminooxy compounds, like (aminooxy)alkyl carboxylic acids or thiols, other reactive groups can easily be introduced to keto compounds by the same method. There are quite a number of methods of synthesizing aminooxy compounds (0-substituted hydroxylamines) (13, 16). Here the Ing-Manske modification of the Gabriel synthesis (the phthalimido method) (17) was used because of our experience with phthalimido derivatives of dibromoalkanes during the synthesis of chemiluminescent derivatives of steroids (18). Other suitable methods for synthesizing these aminooxy compounds are, for example, the use of benzohydroxamic acid (12)or ethyl N-hydroxyacetimidate (19). In both cases the conditions during the reaction and hydrolysis of the protecting groups are relatively mild. The mass spectrometric characterization of the synthesized aminooxy compounds seems to indicate that these molecules can act as ionization reactants. Under normal electronic ionization conditions (70 eV), no molecular ions were detectable, and with a very high concentration of the aminooxy compound and a lower ionization energy (15 eV), the molecular ions could be detected as [M + HI+. When normal chemical ionization with ammonia was applied, the molecular ions were clearly obtainable as [M + HI+. In all the spectra, characteristic fragmentation was observed and the major ions detected originated from loss of ONH2, and NH2 or OH, or both. Synthesis of (Aminooxy)alkylamineDihydrochlorides. (Aminooxy)alkylamine dihydrochlorides (V, VI) have been synthesized according to the method of Adarichev and co-workers (14) as shown in the Scheme I. Synthesis of Aminooxy Alcohols. 4-(Aminooxy)butanol (1x1 was synthesized using the method of Petrenko and co-workers (15) as presented in the Scheme 11. 4-(Aminooxy)butanol (IX) was easily extracted from an alkaline water solution by a continuous extraction system and purified by distillation. 6-(Aminooxy)hexanol hydrochloride ( X I I ) was synthesized as presented in the Scheme 111. The protecting phthalimido and acetyl groups were then cleaved by hydrazine hydrate and concentrated hydrochloric acid treatment, although the same method as with 4-(aminooxy)butanol could be also used. 6-(Aminooxy)hexanol hydrochloride was purified by using short-column chromatography. Introduction of the Spacer Arm and Reactive Group to Keto Steroids (Scheme IV). The aminooxy group reacts in refluxing alkaline (NaOH or CH300Na)

0

0

I

;

;?hydrate

H2N(CH2).O.NH2 2HCI

-

V VI

(nd) (n=6)

Scheme 11. Synthesis of (Aminooxy)butanol

+

CHlCOBr

CH$OO(CHi)dBr

VI1

/ J

N-hydroxyphthdmidc EtqN. DMF

0

I

1. acetic acid. HCI

1.NaOH

IX

HO(CH2)PNH,

Scheme 111. Hydrochloride

of

Synthesis

(Aminooxy)hexanol

sodium acetate acetic acid

n

I

I hydrazme hydrate

2 HCI

HO(CH&ONH,

HCI

XI1

alcoholic solution readily with keto groups of steroids to produce alkyl oximes (20). The aminooxy group is far more reactive than the amino or hydroxy group because of the oxygen atom adjacent to the amino group (the CYeffect), which greatly increases the nucleophility of the amino group (21,22). For an example of the use of these

Introduction of Reactive Spacer Arms to Steroids

Bloconjugate Chem., Vol. 3, No. 2, 1992

Scheme IV. Synthesis of Testosterone 0-Alkyloximes

1

E ~ O Hsodium , acetate

185

separated for example on sulfoethyl Sephadex LH-20 (22) or to some extent on silica TLC plates using 20 % methanol in chloroform to develop the plates. The yield, which varied from 26 to 85 5% ,is greatly influenced by the molar ratio of the reagents (steroid/aminooxy compound),which varied in this study from 1/1.6to 1/73during the synthesis of testosterone and cortisol, respectively. The amino, hydroxy, or other similarly prepared derivatives of bridged steroids can be coupled to other molecules and used, for example, in steroid immunoassays (25). LITERATURE CITED (1) Pratt, J. J. (1978) Steroid immunoassay in clinical chemistry. Clin. Chem. 24, 1869-1890.

ann

isomer (-60 %)

syn isomer (-40

9%)

bifunctional aminooxy compounds, testosterone 3-, 6-ketoestradiol 6-, and cortisol 3-(O-alkyloximes) were synthesized. Testosterone and estradiol alkyloximes were synthesized from aminooxy compound and testosterone and 6-ketoestradiol, respectively, in refluxing alcoholic solution. When preparing cortisol 3-derivatives, the 3keto group has to be activated, for example, by using pyrrolidine (23) because of the other reactive keto group at 21-position. The same method can be used while derivatizing other 3-keto steroids and has to be used while derivatizing 3-keto steroids which have several keto groups, e.g. progesterone. The chemical structure linking the bridge to the steroid is the same as in the 0-(carboxymethyl)oximes, but the bridge itself is three or five carbon atoms longer. The mass spectra of the synthesized steroid derivatives clearly showed the cleavage of the introduced side chain. With electronic ionization of 15eV, no molecular ion could be detected in the spectrum of the cortisol derivative because of the obvious fragmentation of the parent cortisol structure. While using chemical ionization the [M HI+ of the cortisol derivative was detectable. In the UV spectra of the synthesized cortisol and testosterone derivativesa characteristic shift of the absorption maximum from about 240 nm for 3-keto steroids to 250 nm for the corresponding oxime derivatives has been detected. In estradiol derivatives a similar shift of the maxima from 256 and 327 nm for 6-ketoestradiol to 260 and 311nm for the oxime derivative has also been observed. Since the formed C=N double bond restricts free rotation, the oximes of steroids exist as syn- and antiisomers. In estradiol oximes these isomers could not be detected from the lH NMR spectra, where the chemical shift of the aromatic proton at the C-4 carbon of 6-alkyloximes and underivatized estradiol were 7.43 and 6.56 ppm, respectively. In oxime derivatives of 4-en-3-one steroids, such as cortisol and testosterone, these isomers can be detected from the lNMR spectra. The most striking difference between the 'H NMR spectra of these two isomers is the chemical shift of the olefinic proton at the C-4 carbon. The chemical shifts were 5.66 and 5.74 ppm for the anti-isomer and 6.25 and 6.36 ppm for the synisomer of cortisol and testosterone 3-(alkyloximes), respectively,while these shifts for underivatized cortisol and testosterone were 5.65 and 5.73 ppm, respectively. The ratio (syn/anti) of these two isomers in these reaction conditions varied from U1.2 for cortisol to U1.5 for testosterone. The ratio is influenced by the reaction conditions and solvent used ( 2 4 ) . The isomers can be

+

(2) Lavastre, I., Besancon, J., Brossier, P., and Moise, C. (1990) The synthesis of metallocene-labelled drugs for biological assays. Appl. Organomet. Chem. 4 , 9-17. (3) Tiefenauer, L. X., and Andres, R. Y. (1990) Biotinyl-estradiol derivatives in enzyme immunoassays: Structural requirements for optimal antibody binding. J. Steroid Biochem. 35, 633-639. (4) Bermudez, J. A., Coronado, V., Mijares, A., Leon, C., Velazquez, A., Noble, P., and Mateos, J. L. (1975) Stereochemical approach to increase the specificity of steroid antibodies. J . Steroid Biochem. 6 , 283-290. (5) Mikola, H., and Miettinen, P. (1991) Preparation of europium labeled derivatives of cortisol for time-resolved fluoroimmunoassays. Steroids 56, 17-21. (6) Evrain, Ch., Rajkowski, K. M., Cittanova, N., and Jayle, M. F. (1980) The preparation of three fluorescence-labelled derivatives of testosterone. Steroids 35, 611-619. (7) Cuilleron, C. Y., Mappus, E., Forest, M. G., and Bertrand, J. (1981) Synthesis and stereochemistry of 78- and 7a-amino-, acetamido-, hemisuccinamido- and terephthalamido derivatives of testosterone. Steroids 38, 607-632. (8) McHale, D., Green, J., and Mamalis, P. (1960) Amino-oxyderivatives. Part I. Some a-amino-oxy-acids and a-aminooxy-hydrazides. J. Chem. SOC.225-229. (9) Mamalis, P., Green, J., and McHale, D. (1960) Amino-oxyderivatives. Part 11. Some derivatives of N-hydroxydiguanide. J. Chem. SOC.229-238. (10) Schulmann, E. L., Paquette, L. A., Heinzelman, R. V., Wallach, D. P., DaVanzo, J. P., and Greig, M. E. (1962) The synthesis and y-aminobutyric acid transaminase inhibition of aminooxy acids and related compounds. J. Med. Chem. 5 , 464-477. (11) Satshenko,L. P., Severin,E. S.,and Khomutov,R. M. (1968) On the inhibition of decarboxylase of L-glutamic acid by derivatives of hydroxylamine and relative compounds. Biokhimiya (Moscow) 33, 142-147. (12) Pankaskie, M. C., and Scholtz, S. A. (1989) An improved synthetic route to aminoxypropylamine (APA) and related homologs. Synth. Commun. 19, 339-344. (13) McKay, A. F., Garmaise, D. L., Paris, G. Y., and Gelblum, S. (1960) Bacteriostats. 111. Oxyamines and their derivatives. Can. J. Chem. 38, 343-358. (14) Adarichev,V. A., Dymshits, G. M., Kalachikov, S. M., Pozdnyakov, P. I., and Salganik, R. I. (1987)Introduction of aliphatic amino groups into DNA and their labelling with fluorochromes in preparation of molecular hibridisation probes. Bioorg. Chem. 13, 1066-1069. (15) Petrenko, V. A., and Pozdnyakov, P. I. (1983) Synthesis of triester analogs of di(deoxynuc1eoside)phosphates containing hydroxylamine residue. Bioorg. Chem. 9, 832-837. (16) Ilvespaa, A. O., and Marxer, A. (1964) 0-Substituted hydroxylamines and their derivatives. Chimia 18, 1-36. (17) Rougny, A., and Daudon, M. (1976) Use of N-hydroxylimides for the synthesis of primary alkoxylamines. Bull. SOC. Chim. Fr. 5-6, 833-838. (18) Lindstrom, L., Meurling, L., and Lovgren, T. (1982) The measurement of serum cortisol by a solid-phase chemiluminescence immunoassay. J. Steroid Biochem. 16, 577-580.

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(19) Khomutov, R. M. (1961) Hydroxylamine derivatives I. Synthesis of 0-substituted hydroxylamines. Zh. Obshch. Khim. SSSR 31, 1992-1995. (20) Erlanger, B. F., Borek, F., Beiser, S. M., and Lieberman, S. (1957) Steroid-protein conjugates I. Preparation and characterization of conjugates of bovine serum albumin with testosterone and with cortisone. J. Biol. Chem. 228, 713-727. (21) Klopman, G., Tsuda, K., Louis, J. B., and Davis, R. E. (1970) SupernucleophilesI. The alpha effect. Tetrahedron 26,45494554. (22) Grekov, A. P., and Vaselov, V. Y. (1978) a-effect in the chemistry of organic compounds. Usp.Khim. 47,1200-1230.

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(23) Janoski, A. H., Shulman, F. C., and Wright, G. E. (1974) Selective 3-(O-carboxymethyl)oxime formation in steroidal 3,20-diones for hapten immunospecificity. Steroids 23, 4964. (24) Axelson,M.,Sjovall,J.,Drakenberg,T.,andForsen,S.(1978) Separation and configuration of syn and anti isomers of testosterone oxime. Anal. Lett. B l l , 229-237. (25) Mikola, H., Hiinninen, E., and HGglund, A-C. (1990) Preparation of europium labeled steroid derivatives for timeresolved fluoroimmunoassays(Abstract). J.Steroid Biochem. 36 Suppl. 115s.