Alpha-pyrones have been used for applications ranging from total synthesis to antibiotics. However, their application as dienes in bioorthogonal reactions has not been extensively explored. In previous work, we demonstrated the promising application of ester-functionalized pyrones in bioorthogonal protein labeling. Here, we constructed a library of substituted pyrones to evaluate their potential in bioorthogonal reactions by exploring the relationships among structure, reactivity, and bioorthogonality. We found that most pyrone derivatives with electron-withdrawing groups exhibited reactivity toward