Figure 7 Barbiturate structures.
In the case of both warfarin and pentobarbital, the molecule has a single asymmetric center in a nonrigid position of the molecule. When one considers that, in the case of pentobarbital, an exchange of positions between a hydrogen atom and methyl group reverses the stereochemistry, the selectivity of the antibodies for such substances is impressive. If the single asymmetric center were part of a more rigid system, such as a ring, one might anticipate even greater possibilities for enantioselectivity. This was shown to be the case with hexobarbital (Fig. 7 [14a]) in which the asymmetric center is the 5 carbon atom of the ring.
Synthesis of enantiomeric analogs of this drug made use of stereoselective reactions beginning with the enantiomers of 2,2,2-trifluoro-l(9-anthryl)-ethanol. Esterification of each enantiomer with 2-cyano-2-cyclo~ hexylidene acetic acid yielded an optically active compound, which was methylated to create an additional asymmetric center. The methylation was stereoselective, and one diastereoisomer could be crystallized out in high optical purity. Reaction of the optically pure esters with methylurea led to optically pure enantiomers of hexobarbital, which by bromination/ reduction procedures could be converted to tritium-labeled hexobarbital (Fig. 7 [14b]). If 4,4-dimethoxybutylurea was used in place of methylurea, eventually an optically active analog having the nitrogen of the hexobarbital linked to a butyraldehyde moiety resulted. This could then be conjugated to the amino groups of bovine serum albumin to give an immunogen (Fig. 7 [14c]). Possibly because of the greater rigidity of the asymmetric center, the rf-hexobarbital cross-reacted with the antiserum to the I immunogen to the extent of only 0.005%; the cross-reaction of /-hexobarbital with the d antiserum was only 0,0005% (53).
To develop assays for d- and /-methadone, the hemisuccinates of a-l-methadol (Fig. 4 [4a]) were conjugated to bovine thyroglobulin (54). The resulting immunogens (Fig. 4 [4b]) caused formation of antisera that were quite selective when used with enantiomerically pure tritium-labeled d- or /-methadone. The cross-reaction of the / isomer with the d antiserum was less than 1%, and cross-reaction of /-methadone with the d antiserum was about 3%. The racemate exhibited cross-reactions of 56-57% (55).
Ephedrine is a diastereoisomer of pseudoephedrine (Fig. 4 [7a]) having the erythro configuration. The enantiomeric immunogens of this compound were prepared (57) in a manner similar to that described for ¿/-pseudoephedrine (40). Tritium-labeled d,/-ephedrine was used as the radioligand. Cross-reactions of antisera with the opposite enantiomer were less than 2%. When plasma from a subject given d,/-ephedrine was analyzed by R1A, the sum of the two enantiomer concentrations agreed closely with the total ephedrine concentration determined by GLC-electron capture detection (57).
With the advent of hybridoma technology the question arises as to whether, given a racemic immunogen, one would be able to generate hybridomas producing monoclonal antibodies to both isomers with high enantioselectivity. This is supported by the observed enantioselectivity of polyclonal antisera to racemic immunogens discussed earlier and the results obtained with nicotine (67), The selectivity of such antibodies was less clear cut with a diastereoisomeric compound. Monoclonal antibodies were developed to the nerve agent soman (Fig. 8 [15a]) by immunizing mice with a phenyldiazonium analog of soman conjugated to keyhold limpet hemocyanin or bovine serum albumin (Fig, 8 [15b]). Soman has two asymmetric centers; one on carbon [C(±)] and one on phosphorus [P(±)]. One antibody-producing clone was obtained from each protein. A competitive inhibition enzyme assay was used to test binding affinity. The first monoclonal antibody had the following order of affinity: C(+)P(+) S C(-)P(+) < C(-)P(-) < C(+)P(-). The second antibody also exhibited a preference for the more toxic P(—) diastereoisomers. Unfortunately, the observed affinity constants were very low (covering a range from 5 x 103 L/mol to < 103 L/mol for the first antibody and from 5.9 X 105 to 5.2 x 10* for the second antibody). Other selectivity observations indicated that only two loci on the soman molecule (the P=0 oxygen and f-butyl group)
b. X = — O —N = N — (Protein),M * ■«- Chiral center
provided the major source of interaction with the antibodies. Because three loci are needed for enantioselectivity, the rather poor range of enantioseiectivities may be explained. The immunogen used for this coupling contains a highly immunodominant azophenyl residue, and it is not surprising that the resulting antibodies have low affinity for the actual soman molecule itself (56).
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