Atropine Stereochemistry

*Chiral center FIGURE 4 Structures of drugs and immunogens.

tiation as one goes further from the link to protein. This antiserum had good selectivity vs. the isomeric methadols, but exhibited 12% cross-reaction with the N-desmethyl metabolite norLAMM. When norLAMM levels were twice those of LAMM, a 25% positive error was introduced. Such ratios can occur as early as 11 hr after LAMM administration (unpublished results) and may be more likely on repeated dosage, due to the longer half-life of the metabolite. This highlights the fact that enantioselec-tivity alone is not enough. Selectivity vs. metabolites and other closely related compounds must also be taken into consideration.

3. Atropine

S-atropine (Fig. 4 [5a]) is markedly more potent than the R isomer in a series of tests (31) and binds much more strongly to the muscarinic receptor (32). Indeed, given the propensity of this particular compound for racemiza-tion (33) and therefore its lack of complete optical purity, it would appear that essentially all the biological activity resides in the S enantiomer.

Various preparations of antisera to atropine have been reported. A racemic hemisuccinate ester was prepared and conjugated to bovine serum albumin by the carbodiimide technique. Antisera formed to the original immunogen selectively bound the R isomer (34), but a later antiserum prepared by this approach was reported to bind both R and S forms with "equal efficiency" (35). R, S-atropine was treated with diazotized p-amino-benzoic acid, and the resulting compound (which was not further characterized) was used for conjugation to bovine serum albumin by means of a carbodiimide-mediated reaction. Antisera resulting from use of this material were quite selective for the R isomer, with a cross-reaction of only about 2% for the S isomer (36). Virtanen et al. followed this procedure with S-atropine. Their antiserum bound equally to S- and R,S-atropine, as measured by displacement of tritium-labeled R,S-atropine (37). In another study (31), both racemic atropine and the S isomer were coupled to human serum albumin by the technique of Wurtzburger et al. (36). Antisera were obtained that were selective for both the R and S isomers (33).

The work with atropine presents a confusing picture. Use of racemic radioligand in these studies may contribute to the observed cross-reactivity picture. The rather facile enolization and hence racemization of atropine-type structures is another problem. Finally, the structure of the product of atropine and diazotized p-aminobenzoic acid has never been established conclusively. For these reasons, the radioreceptor assay for this compound (see below) is generally preferable.

4. propranolol

The importance of propranolol (Fig. 4 [6a]) has led to the development of a number of immunoassays for it. Most of the workers dealt with the racemic compound. However, Kawashima et al.; in an early demonstration of the utility of enantioselective assays, developed a procedure for analysis of the I isomer (38). These workers made a hemisuccinyl derivative of /-propranolol and coupled it to bovine serum albumin by a mixed anhydride technique. Antisera were generated in rabbits, and the radioligand was racemic tritiated propranolol. With this antiserum, this group measured plasma concentrations of the I isomer in rat biood (38), as well as in mice (39), Because they also had generated an antiserum against the racemic material that had little capability for distinguishing d-, /-, or d,i-propranolol, they also determined total propranolol and by difference could obtain an estimate of the d isomer as well. In the rat they found a higher initial concentration of d than / isomer, but much higher amounts of the / isomer in the heart. This was attributed to selective uptake of the / isomer by receptors. Because of the relatively high (7%) cross-reaction of the d isomer with the / antiserum, concentrations of the / isomer were corrected. The actual concentration of / was equal to [(measured concentration of / isomer) - 0.07(measured concentration of (¿,/-propranolol)]/0.93.

5. Pseudoephedrine

Findlay and co-workers developed an RIA for rf-pseudoephedrine (Fig, 4 [7a]) (40). The compound was allowed to add to methyl acrylate. After hydrolysis, the resulting carboxylic acid was conjugated to bovine serum albumin by means of a carbodiimide procedure to form an immunogen (Fig, 4 [7b]). The radioligand for the binding studies was either tritium-labeled d,/-pseudoephedrine (5 Ci/mmol) or a conjugate of the d-pseudoephedrine-methyl acrylate adduct (analogous to [7b]) with tyrosine methyl ester labeled with 125I. Charcoal or polyethylene glycol was used to separate free and bound ligand. With the tritium-labeled radioligand, serum could be analyzed directly, but plasma had to be extracted if the !25I radioligand was used. The plasma extract was treated with methyl acrylate to convert the ¿-pseudoephedrine to a compound more closely resembling the original immunogen. By this procedure it was possible to achieve sensitivities down to 0.2 ng/mL. When the iodinated radioligand was used, the cross-reaction with /-pseudoephedrine was less than 0.003%. With the tritium-labeled d,l radioligand, the cross-reaction with the / isomer was 0,01-0.05%. /-Ephedrine, a diastereoisomer, cross-reacted to the extent of 0.13-0.32%.

6. S-Bioallethrin

S-Bioallethrin (Fig. 5 [9a]) is a synthetic pyrethroid insecticide with three asymmetric centers. The 5 designation refers to the 1-R, 3-R, 4'-S isomer and is not a true designation of stereochemistry. Conversion of the terminal allylic group to —CH2CH2CH2OH, formation of hemisuccinate,

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