Synthesis of 11 alpha-hydroxyprogesterone haptens

C-4 and C-6 bridged haptens of 11 alpha-hydroxyprogesterone, an anti-androgen, were respectively synthesized via 4, 5 and 5 alpha, 6 alpha epoxide ring openings using 3-mercaptopropanoic acid. Substitution of 6-bromo derivative of progesterone using the same reagent unexpectedly afforded a C-4 substituted product instead of the normal C-6 substituted product previously reported in the literature.     

A novel immunization procedure involving pretreatment with cross-reacting steroid conjugated to a copolymer of D-glutamic acid and D-lysine for production of low cross-reactive antisera

Anti-testosterone and anti-5 alpha-dihydrotestosterone antisera have been produced by pretreatment of mice with a cross-reacting steroid coupled to a copolymer of D-glutamic acid and D-lysine to inhibit production of cross-reacting antibodies before immunization with the specific steroid conjugated to a protein. 15 beta-Carboxyethylmercapto derivatives were used as haptens. The cross-reactivities of the anti-testosterone and anti-5 alpha-dihydrotestosterone antisera with 5 alpha-dihydrotestosterone and with testosterone were 0.49 and 13.5%, respectively.     

Magnetic resonance studies of the binding site interactions between 19F-labeled nitrophenyl haptens and specific mouse myeloma immunoglobulin MOPC-315

The interactions between MOPC-315, a mouse myeloma protein with specificity for nitrophenyl haptens, and 19F-substituted haptens have been investigated using nuclear magnetic resonance (NMR) spectroscopy. The haptens studied are mono- or dinitrophenyl derivatives of gamma-aminobutyric acid, lysine, or glycine which have trifuoromethyl groups attached to the phenyl rings. Upon binding to immunoglobulin, the 19F nucleus experiences a downfield shift whose magnitude depends on the position of the trifluoromethyl group on the phenyl ring but is independent of other structural changes in the hapten such as the number of nitro groups attached to the phenyl ring. Further, the chemical shift of bound hapten is not influenced by the amount of the constant region attached to the binding site; we accordingly conclude that the presence of the distal, constant regions of the immunoglobulin molecule does not influence binding site interactions.     

Epitopic discrimination by monoclonal antibodies directed against the same alkylated nucleoside

Epitopic specificity of three monoclonal antibodies (mAb's) (coded as ER-6, ER-3, and EM-1) was examined through the utilization of haptenic structural analogs. The binding affinity expressed by the microscopic equilibrium constant (Ki) (Yuhasz, et al., Biochemistry 26, 2334-2342 (1987] of the immunizing hapten, O6-ethyl-2'-deoxy-guanosine (*G) and eight structural analogs, were analyzed by a nitrocellulose affinity filter assay (NAFA) and radioimmunoassay (RIA) for each mAb to determine the protein-hapten interaction between the epitope and the binding cavity. Several components of the *G hapten were determined to be critical for each mAb recognition, while all three mAb's were found to require the O6-ethyl moiety, conjugated guanine base ring, the glycosyl bond and the sugar ring C [1'] and C [2'] position. This investigation further probes and categorizes the binding specificity of the monoclonal antibodies after incorporation of the *G monomer into three short deoxyribooligomeric haptens: O6-ethyl-2'-deoxyguanylyl 3',5' deoxyadenosine (*GA), 2'-deoxyadenylyl 3',5' O6-ethyl-2'-deoxyguanylyl 3',5' 2'-deoxyadenosine (A*GA), and O6-ethyl-2'-deoxyguanylyl 3',5' 2'-deoxyadenylyl 3',5'-2'-deoxyadenylyl 3',5' 2'-deoxycytosine (*GAAC). Unlike the similar binding profiles for the monoclonal antibodies and the haptenic structural analogs, the binding profiles for the deoxyribooligomeric haptens were found to differ in their modes of recognition. These results will be compared to ascertain the key components of monomer and oligomer interaction of the binding cavity. It is important for investigations where monoclonal antibodies derived from small haptens are utilized in recognition of larger antigens containing those haptens.     

Crystal structure of a recombinant anti-estradiol Fab fragment in complex with 17beta -estradiol.

The crystal structure of a Fab fragment of an anti-17beta-estradiol antibody 57-2 was determined in the absence and presence of the steroid ligand, 17beta-estradiol (E2), at 2.5 and 2.15-A resolutions, respectively. The antibody binds the steroid in a deep hydrophobic pocket formed at the interface between the variable domains. No major structural rearrangements take place upon ligand binding; however, a large part of the heavy chain variable domain near the binding pocket is unusually flexible and is partly stabilized when the steroid is bound. The nonpolar steroid skeleton of E2 is recognized by a number of hydrophobic interactions, whereas the two hydroxyl groups of E2 are hydrogen-bonded to the protein. Especially, the 17-hydroxyl group of E2 is recognized by an intricate hydrogen bonding network in which the 17-hydroxyl itself forms a rare four-center hydrogen bond with three polar amino acids; this hydrogen bonding arrangement accounts for the low cross-reactivity of the antibody with other estrogens such as estrone. The CDRH3 loop plays a prominent role in ligand binding. All the complementarity-determining regions of the light chain make direct contacts with the steroid, even CDRL2, which is rarely directly involved in the binding of haptens.     

Biology and mode of action of pure antioestrogens

The properties of a series of 7 alpha-alkyl analogues of oestradiol are described. Studies of chemical structure and activity in the immature rat uterotrophic/antiuterotrophic assay revealed that molecules containing a terminal functional group (acid, alcohol, amine, amide) linked to the steroid by a decamethylene bridge possess both oestradiol agonist and antagonist activity. However, certain amides, exemplified by the compound ICI 164,384 [N-n-butyl-11-(3,17 beta-dihydroxyoestra-1,3,5(10)-trien-7 alpha-yl)-N-methylundecanamide], were devoid of oestrogenic activity but possessed potent antioestrogenic activity. Comparison of receptor binding and biological potency of steroid 7 alpha- and 7 beta-isomers showed that activity is confined largely to the 7 alpha-isomer. Comparison of the effects of tamoxifen and ICI 164,384 on progesterone receptor (PR) concentration in the rat uterus showed that, unlike tamoxifen, ICI 164,384 did not induce PR and blocked induction of PR by oestradiol. Chronic treatment of mature female rats with ICI 164,384 led to an ovariectomy-like regression of the uterus without affecting LH secretion or the rate of growth. ICI 164,384 was also an effective antitumour agent in rats bearing carcinogen-induced mammary tumours.     

An investigation of antibody acyl hydrolysis catalysis using a large set of related haptens

An aspect of catalytic antibody research that receives little attention in the literature involves hapten systems that fail to elicit antibody catalysts despite a high affinity immune response and hapten designs that resemble those known to elicit catalysts. We have investigated a series of 12 phosphate and phosphonate haptens in a total of three animal systems. Dramatic and reproducible differences were observed in the catalytic activities of polyclonal antibodies elicited by the different haptens. A phosphate hapten with a phenyl ring on the side of the hapten opposite the linker elicited reproducibly high levels of polyclonal antibody catalytic activity. The other 11 haptens, most with benzyl groups on the side of the hapten opposite the linker, elicited immune responses in which catalytic activity was significantly weaker in terms of the level of observed catalytic activity, as well as frequency of elicited catalysts. Our results indicate that subtle features of transition state analogue hapten structure can have a dramatic and reproducible influence over the catalytic activity of elicited antibodies in related haptens. Whatever the explanation, subtle changes in mechanistic features due to altered leaving group ability/location or overall hapten flexibility, the comprehensive data presented here indicate that phenyl or 4-nitrophenyl leaving groups located opposite the hapten linker are to be preferred in order to elicit highly active antibody catalysts for acyl hydrolysis reactions.     

Coupling of biologically active steroids to conjugating arms through ether linkages for use in immunochemistry.

Conjugation of haptens through ether linkages avoids leakage problems in immunoassays, but this procedure is not easily applied to most steroids that bear low reacting hydroxyls. A new technique allowing the ether coupling of biologically active steroids with conjugating arms in mild conditions compatible with thermosensitive protecting groups is presented. In the first step, the solvent (an aromatic hydrocarbon) was dehydrated by azeotropic distillation in a soxhlet apparatus using a cartridge filled with 0.3 nm and 0.4 nm molecular sieves. In this protected medium, a thallium steroid alkoxide was completely formed by reaction of the steroid with thallium ethoxide and by the continuous elimination of ethanol. The halogenated chain was then introduced into the same medium and reacted in the absence of moisture to give the ether. 17beta-Hydroxy and 11alpha-hydroxy derivatives were involved in this reaction. The coupling was effective for all of the compounds tested after 2-36 h of reaction time and at temperatures between 80 and 140 degrees C. The conjugates were at least 95% pure, and yields ranged from 15 to 95%.     

Monoclonal antibodies that recognize minimal differences in the three-dimensional structures of metal-chelate complexes

Immunization of BALB/c mice with a cadmium-chelate-protein conjugate resulted in the isolation of two hybridoma cell lines (A4 and E5) that synthesized antibodies with different variable regions, but similar metal-chelate affinity. The ability of these two monoclonal antibodies to interact with 12 different metal-chelate complexes was studied using the KinExA 3000 immunoassay instrument. The two antibodies showed the highest affinity for cadmium and mercury complexes of ethylenediamine N,N,N',N'-tetraacetic acid (EDTA). The E5 antibody bound to EDTA complexes of cadmium and mercury with equilibrium dissociation constants (K(d)) of 1.62 x 10(-)(9) M and 3.64 x 10(-)(9) M, respectively. The corresponding values for the A4 antibody were 14.7 x 10(-)(9) M and 3.56 x 10(-)(9) M. Addition of a cyclohexyl ring to the EDTA backbone increased the affinity of E5 for the metal-chelate haptens, while decreasing the binding of A4 to the same haptens. Based on available crystal structures, molecular models were constructed for five different divalent metal-chelate complexes. The models were compared to determine structural features of the haptens that may influence antibody recognition. Difference distance matrixes were used to identify areas of the metal-chelate haptens that differed in three-dimensional space. Antibody affinity correlated well with the extent of total structural difference for these metal-EDTA complexes.     

Effects of hapten epitope structure and hapten-self conjugation pattern on T cell specificity and Ir gene control in hapten-self cytotoxic and helper T cell responses

Mouse strains of H-2b haplotype exhibit much weaker cytotoxic T lymphocyte (CTL) responses to haptens reactive with amino groups of cell surface (NH2-reactive haptens) compared with H-2k strains. However, H-2b strains can generate high CTL responses to haptens reactive with sulfhydryl groups of cell surface (SH-reactive haptens). The present study investigates the role of haptenic structure and hapten-cell surface reaction patterns in influencing the generation of the T cell specificity as well as the H-2-linked genetic control. CTL and helper T cell responses were generated against two structurally related haptens, N-iodoacetyl-N'-(5-sulfonic-1-naphthyl) ethylene-diamine (SH-reactive AEDANS; AED-SH) and 5-sulfo-1-naphthoxy acetic acid N-hydroxysuccinimide ester (NH2-reactive form of AEDANS; AED-NH2) by immunizing C57BL/6N (H-2b) mice with these hapten-modified syngeneic spleen cells. Spleen cells from primed C57BL/6N mice generated strong CTL and helper T cell activities upon in vitro restimulation with the respective hapten-modified self. The generation of potent anti-AED-NH2 CTL and helper T cell responses in C57BL/6N mice sharply contrasted with the failure of NH2-reactive haptens studied thus far to generate strong anti-hapten cytotoxic responses in H-2b mice. Antibodies induced against the above two haptens exhibited extensive cross-reactivity detected by hemagglutination, whereas CTL and helper T cells clearly discriminated the structural difference between AED-NH2 and AED-SH haptens. The hapten specificity in T cell recognition was also observed between AED-NH2 and trinitrophenyl (TNP) haptens, which were demonstrated to functionally modify similar cell surface sites. These results indicate that hapten epitope structure and hapten-cell membrane conjugation patterns influence the generation of H-2-linked genetic control and T cell specificity in anti-hapten self cytotoxic as well as helper T cell responses.     

NK Cells Respond to Haptens by the Activation of Calcium Permeable Plasma Membrane Channels

Natural Killer (NK) cells mediate innate immunity to infected and transformed cells. Yet, NK cells can also mount hapten-specific recall responses thereby contributing to contact hypersensitivity (CHS). However, since NK cells lack antigen receptors that are used by the adaptive immune system to recognize haptens, it is not clear if NK cells respond directly to haptens and, if so, what mediates these responses. Here we show that among four haptens the two that are known to induce NK cell-dependent CHS trigger the rapid influx of extracellular Ca²⁺ into NK cells and lymphocyte cell lines. Thus lymphocytes can respond to haptens independent of antigen presentation and antigen receptors. We identify the Ca²⁺-permeable cation channel TRPC3 as a component of the lymphocyte response to one of these haptens. These data suggest that the response to the second hapten is based on a distinct mechanism, consistent with the capacity of NK cells to discriminate haptens. These findings raise the possibility that antigen-receptor independent activation of immune cells contributes to CHS.     

Total synthesis of diazasteroids (1).

Contrary to previous reports, the condensation product of 8-aminoquinoline (1) and 2-carbethoxycyclopentanone (2) undergoes ring closure with polyphosphoric acid to give the 1, 11-diazasteroid 5. Catalytic hydrogenation reduced the A ring and the double bond to produce 6. 8-Aminotetrahydroquinoline (7) and 3-carbethoxy-2-piperidone condense to a tricyclic intermediate (11), which could not be cyclized to a steroid, however.     

A sensitive enzyme-linked immunosorbent assay (ELISA) for testosterone: use of a novel heterologous hapten conjugated to penicillinase.

A microplate enzyme-linked immunosorbent assay (ELISA) has been developed for the measurement of testosterone in plasma. The assay uses a heterologous system consisting of a novel hapten 4-(17 beta-hydroxy-3-oxoestra-4,9-dien-11 beta-yl)butanoic acid (1) conjugated to penicillinase (beta-lactamase). The key reaction in the synthesis of the hapten was the cuprate-mediated 1,4-conjugate addition on 3,3,17,17-bis-ethylenedioxy-5 alpha,10 alpha-oxido-estr-9(11)-ene by the Grignard reagent derived from trimethyl 4-bromoorthobutyrate; this regiospecifically introduces the 11 beta-butanoate function. The hapten-penicillinase conjugate was used in the assay in conjunction with the immunoglobulin G (IgG) fraction derived from a previously characterized, highly specific, antitestosterone serum raised against a testosterone-19-O-carboxymethyl ether-bovine serum albumin (T-19-O-CME-BSA) conjugate. This unique system represents one incorporating three elements of structural heterology: bridge, site, and ring heterology between the antigen hapten and enzyme-linked hapten. The limit of detection was 10 pg of testosterone with a sensitivity range between 15 and 1,000 pg. A low level of cross-reactivity with 5 alpha-dihydrotestosterone (6.17%) and 11 beta-hydroxytestosterone (1.03%) was noted. No interference was noted with other common androgens, estradiol, or progesterone. The sensitivity and selectivity observed in the assay may be attributable to the selection of penicillinase as the enzyme marker and the elements of conformational heterology between the antigen-linked and enzyme-conjugated steroid haptens.     

Analysis of anabolic steroids in the horse: development of a generic ELISA for the screening of 17alpha-alkyl anabolic steroid metabolites

Due to the potential for misuse of a wide range of anabolic steroids in horse racing, a screening test to detect multiple compounds, via a common class of metabolites, would be a valuable forensic tool. An enzyme-linked immunosorbent assay (ELISA) has been developed to detect 17alpha-alkyl anabolic steroid metabolites in equine urine. 16beta-Hydroxymestanolone (16beta,17beta-dihydroxy-17alpha-methyl-5alpha-androstan-3-one) was synthesised in six steps from commercially available epiandrosterone (3beta-hydroxy-5alpha-androstan-17-one). Polyclonal antibodies were raised in sheep, employing mestanolone (17beta-hydroxy-17alpha-methyl-5alpha-androstan-3-one) or 16beta-hydroxymestanolone conjugated to human serum albumin, via a 3-carboxymethyloxime linker, as antigens. Antibody cross-reactivities were determined by assessing the ability of a library of 54 representative steroids to competitively bind the antibodies. Antibodies raised against 16beta-hydroxymestanolone showed excellent cross-reactivities for all of the 16beta,17beta-dihydroxy-17alpha-methyl steroids analysed and an ELISA has been developed to detect these steroid metabolites. Using this 16beta-hydroxymestanolone assay, urine samples from horses administered with stanozolol (17alpha-methyl-pyrazolo[4',3':2,3]-5alpha-androstan-17beta-ol), were analysed raw, following beta-glucuronidase hydrolysis, and following solid-phase extraction (SPE) procedures. The suppressed absorbances observed were consistent with detection of the metabolite 16beta-hydroxystanozolol. Positive screening results were confirmed by comparison with standard LCMS analyses. Antibodies raised against mestanolone were also used to develop an ELISA and this was used to detect metabolites retaining the parent D-ring structure following methandriol (17alpha-methylandrost-5-ene-3beta,17beta-diol) administration. The ELISA methods developed have application as primary screening tools for detection of new and known anabolic steroid metabolites.     

Antibody Detection of Marihuana

A reliable chemical means of assaying small quantities of marihuana or tetrahydrocannabinol (THC) isomers in body fluids and cell preparations has long been sought. A possible immunological approach was suggested by work with oestrogen haptens. It was established earlier that diazobenzoic acid under appropriate conditions enters the A ring by virtue of the phenolic function of C-3, yielding oestrogen azobenzoic acid derivatives^{1, 2}. These compounds are coupled readily through the benzoyl carboxyl to proteins and solid matrices containing accessible NH₂ groups, retaining the important antigenic specificities contributed by the reactive ?OH and = O groups as well as those associated with the ringed backbone^{1, 3}. Other biologically important molecules which lend themselves to similar manipulation are the principal psychotomimetics. In our search for useful haptens and active antibodies to be used in a reliable model assay system, we have chosen to report the investigation of THC. Similar results have been achieved with the phenanthrene alkaloids (S. J. G., unpublished) preserving all the determinant functional groups ^* unlike the reagent for morphine assay recently reported⁴.     

6-hydroxy to 6'-amino tethered ring-to-ring macrocyclic aminoglycosides as probes for APH(3')-IIIa kinase

Based on molecular modeling and available X-ray structure data on aminoglycosides complexed with a bacterial ribosomal surrogate or with a kinase, two analogues of paromomycin were prepared by tethering the 6-OH and the 6'-NH(2) group with a five-carbon bridge. Only one of two possible hydroxyl groups was phosphorylated by the kinase. The application of ring closure metathesis is presented for the first time to construct bridged macrocyclic analogues in the aminoglycoside series.     

Guaiane dimers from Xylopia vielana

From the leaves of Xylopia vielana (Annonaceae) the three dimeric guaianes vielanin A-C were isolated and structurally elucidated by mass and NMR spectroscopy as 1-3. The structure of 1 contains a bridged ring system formed probably via a Diels-Alder reaction of two different guaiane monomers. Compounds 2 and 3 represent symmetric cyclobutanes formally generated from two equal guaiane moieties by [2 + 2] cycloaddition.     

Recent advances in the development of steroid sulphatase inhibitors

Inhibition of steroid sulphatase is now an important target for the development of new drugs for the treatment of women with endocrine-dependent breast tumours. The first potent sulphatase inhibitor identified, oestrone-3-O-sulphamate (EMATE) proved, unexpectedly, to be oestrogenic. A number of strategies have therefore been adopted to design and synthesize a non-oestrogenic inhibitor. For this, a number of modifications have been made to the A and D rings of the oestrone nucleus. 2 Methoxyoestrone-3-O-sulphamate, while having similar in vitro and in vivo sulphatase inhibitory potency to that of EMATE, was devoid of oestrogenic activity when tested at 2 mg/kg in an ovariectomised rat uterine weight gain assay. 17-Deoxyoestrone-3-O-sulphamate was also a potent steroid sulphatase inhibitor and while it was devoid of oestrogenic activity when tested at 0.1 mg/kg, did stimulate uterine growth at 1.0 mg/kg. As an alternative approach to the use of steroid-based inhibitors a number of single ring, bicyclic non-fused ring, and two fused ring sulphamate analogues were designed, synthesized and tested for their ability to inhibit steroid sulphatase activity. In general, although the single ring and bicyclic non-fused ring sulphamate analogues could inhibit sulphatase activity, they were considerably less potent than EMATE. The mono- and bis-sulphamate derivatives of 5,7-dihydroxyisoflavone were relatively potent, inhibiting in vivo steroid sulphatase activity by 62 and 81% respectively at a single oral dose of 10 mg/kg. A study of the structure-activity relationship of a series of coumarin-based sulphamates has led to the development of a number of potent non-steroidal inhibitors, one of which has a similar potency to that of EMATE. The identification of potent steroid- and non-steroid-based sulphatase inhibitors will enable the therapeutic value of this therapy to be examined in the near future.     

Synthesis of aminophosphonate haptens for an aminoacylation reaction between methyl glucoside and a beta-alanyl ester

Two 2-aminophosphonate haptens derived from methyl alpha-D-glucopyranoside were synthesized to mimic the transition-state of a transesterification reaction between methyl alpha-D-glucopyranoside and 4-nitrophenylester of tert-BOC-beta-alanine. Two sets of monoclonal antibodies were generated against these haptens.