Epitope analysis: N⁴‐Formylation of the D‐benzylpenicilloyl antigenic structure

Abstract

Benzylpenicilloic acidAcetic anhydride treatment of 6-(benzylpenicilloylamido)-meric mixtures. The inhibitory capacity of these haptens was hexanoic acid bis(benzy1ammonium) salt (1 a) in formic acid assessed in a precipitating system involving anti-D-a-benzylgave the N4-formylated derivatives 2a and 2 b as diastereo-penicilloyl antibody and D-a-benzylpenicilloyl antigen.Recently developed epitope mapping techniques give new insights into problems of immunochemical specificity. It thus becomes apparent that protein epitopes are considerably larger than the penta-or hexapeptide moieties previously considered. Furthermore, within antibody paratopes, some locations seem to be very sensitive to structural changes of the interacting epitope, whereas others are not 2-4). With regard to epitopic, protein-linked small molecules, it now appears that three types of cross-reactivities can be distinguished ' I. These are a) broad cross-reactivity or low specificity exemplified by antibody interactions involving alcuronium-type compounds; b) intermediate crossreactivity suggested by most of the experience gained in the penicillin allergy field; c) strict specificity shown e.g. by derivatives of the pyrazolinone and pyrazolidinedione series.It is well-established that penicilloyl haptens containing various side chains show good cross-reactivity when interacting with polyclonal anti-benzylpenicilloyl antibodies from the rabbit6,7J. It is also known that the 5-membered thiazolidine can be replaced by a 6-membered dihydrothiazine ring present in cephalosporins without marked loss of binding capacity ' ). A possible interpretation could be that in polyclonal antipenicilloyl responses a proportion of the antibody population is regularly directed against the side chain and another one against the thiazolidine moiety. Whether this is correct, awaits confirmation.In order to provide a basis for further studies, we have applied formylation in order to alter the thiazolidine structure in an immunochemically recognizable way. The procedure, which can be carried out with a variety of penicillins, involves formylation of penicilloylamides, in the first place that of 6-(penicilloylamido)hexanoic acids with formic acid in the presence of acetic anhydride. It then becomes possible to study the N4-formylated and the unformylated forms of penicilloyl structures by interacting them with selected antipenicilloyl antibodies.A problem is the possible formation of four diastereomeric forms, D-a, D-0, D-y and D-6, involving the centers C-5 and C-6 of the penicilloyl structure (Scheme 3). It is known that D-a-penicilloylamides show mutarotation in alkaline as well as in acid ~o l u t i o n~~'~) , and cupric ions and presumably several other heavy metal ions catalyze mutarotation even in neutral aqueous solution 'IJ.