Covalent binding to proteins to form neoantigens is thought to be central to the pathogenesis of penicillin hypersensitivity reactions. We have undertaken detailed mass spectrometric studies to define the mechanism and protein chemistry of hapten formation from benzylpenicillin (BP) and its rearrangement product, benzylpenicillenic acid (PA). Mass spectrometric analysis of human serum albumin exposed to BP and PA in vitro revealed that at low concentrations (drug protein molar ratio 0.001:1) and during short time incubations BP and PA selectively target different residues, Lys199 and Lys525, respectively. Molecular modeling showed that the selectivity was a function of noncovalent interaction before covalent modification. With increased exposure to higher concentrations of BP and PA, multiple epitopes were detected on albumin, demonstrating that the multiplicity of hapten formation is a function of time and concentration. More importantly, we have demonstrated direct evidence that PA is a hapten accounting for the diastereoisomeric BP antigen formation in albumin isolated from the blood of patients receiving penicillin. Furthermore, PA was found to be more potent than BP with respect to stimulation of T cells from patients with penicillin hypersensitivity, illustrating the functional relevance of diastereoisomeric hapten formation.

which after mutarotation at C(1) may transfer the acyl group back to the 1R-OH. This has been conclusively demonstrated by NMR spectroscopy. 14,30 Other rearranged forms of AGs are also known. When the Mitsunobu synthesis 32 (see Synthesis) is used, lactones of type 5 are byproducts, sometimes in appreciable yields. The mechanism of formation is not clear but presumably involves cyclization of the first-formed intermediate ester. If there were an in vivo route to such lactones via free carboxyl acyl glucuronides, they would doubtless be potent acylating agents, transferring in this case the sugar along with the acyl residue.Concerning 1β-AGs of benzoic acids, the chemical stability/ reactivity may be predicted with some confidence by a Hammett correlation, 33 as discussed in detail later (NMR section). Some preliminary studies have been carried out on the computational chemistry modeling of the relative stabilities of the isomers of the AGs of 2-, 3-, and 4-trifluoromethylbenzoic acids. 34 For nonarylcarboxylic acids, the structure-reactivity is more complex. Certainly branching at the R-carbon is a factor leading to increased stability; thus the AGs of gemfibrozil 6, 35 clofibric acid 7 36 and valproic acid 8, 37 all doubly R-substituted, display long half-lives (from about 6-70 h in pH 7.4 buffer 37 ).AGs derived from NSAIDs such as naproxen 9 and ibuprofen 10 generally have half-lives from 1 to 4 h under the same conditions. 38 In such compounds, an additional complication is the different rates of rearrangement of the (R)-and (S)-AG diastereoisomers. In the case of 2-phenylpropanoic acid itself 11, 38 this rate was found to be about twice as fast for the (2R)as for the (2S)-AG epimer, and this ratio is typical of the 2-aryl propanoate class. Scheme 2. Possible Fates of AGs Scheme 3. Reactivity of Acyl Glucuronides a a Clearly the rearrangement (pathway 2) requires migration of the acyl group at least as far as O(3).

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