Electronic spectra of the isomeric 4-benzylidene-2-phenyl-Δ2-oxazolin-5-ones and the products of their reaction with nucleophiles including α-chymotrypsin : Kinetics of the hydrolysis of the isomeric enzyme derivatives

The 4-cis- and trans-benzylidene-2-phenyl-Δ²oxazolin-5-ones have been prepared and evaluated as chromophoric reagents for the investigation of the active site of α-chymotrypsin. The UV spectra of the isomeric oxazolinones are discussed and compared with those of similar compounds, notably the 1,4-diphenyl-1,3-butadienes, A novel spectroscopic consequence of geometrical isomerism in conjugated chromophores is reported. The facile isomerization of the cis-oxazolinone has been investigated by spectrophotometry and product isolation. Both oxazolinones react rapidly with the enzyme to provide products whose UV spectra are consistent with their assignment as α-benzamido- cinnamoyl-enzymes. The uncertainties in these assignments resulting from the presence in the oxazolinones of multiple electrophilic centres are discussed. The pseudo first-order rate constants for the hydrolysis of the products of interaction of α-chymotrypsin with the isomeric oxazolinones were determined at 25·0°, I = 00·1 in the pH range 7–10–5. The pH-rate profile for the hydrolysis of the product formed by reaction of the trans-oxazolinone is consistent with this reaction being deacylation of α-benzamido-trans-cinnamoyl-α-chymotrypsin catalysed by the enzyme's electron relay system (pKa 7·8, ks= 0·159s^?1). The pH rate profile for the hydrolysis of the product formed by reaction of the cis-oxazolinone is more complex. The profile could include a component catalysed by the relay system (pKa approx 8, $\text{k}$, = 10^?3 s^?1) but the predominating reaction appears to be an unusually rapid reaction of the derivatized enzyme with hydroxide ion (k = 233 ± 10 M^?1s^?1). Possible interpretations of these pH-rate profiles are discussed.