Accurate prediction of rate constants of Diels–Alder reactions and application to design of Diels–Alder ligation

Bioorthogonal reactions are useful tools to gain insights into the structure, dynamics, and function of biomolecules in the field of chemical biology. Recently, the Diels–Alder reaction has become a promising and attractive procedure for ligation in bioorthogonal chemistry because of its higher rate and selectivity in water. However, a drawback of the previous Diels–Alder ligation is that the widely used maleimide moiety as a typical Michael acceptor can readily undergo Michael addition with nucleophiles in living systems. Thus, it is important to develop a nucleophile-tolerant Diels–Alder system in order to extend the scope of the application of Diels–Alder ligation. To solve this problem, we found that the theoretical protocol M06-2X/6-31+G(d)//B3LYP/6-31G(d) can accurately predict the activation free energies of Diels–Alder reactions with a precision of 1.4 kcal mol−1 by benchmarking the calculations against the 72 available experimental data. Subsequently, the electronic effect and ring-strain effect on the Diels–Alder reaction were studied to guide the design of the new dienophiles. The criteria of the design is that the designed Diels–Alder reaction should have a lower barrier than the Michael addition, while at the same time it should show a similar (or even higher) reactivity as compared to the maleimide-involving Diels–Alder ligation. Among the designed dienophiles, three substituted cyclopropenes (i.e. 1,2-bis(trifluoromethyl)-, 1,2-bis(hydroxylmethyl)- and 1,2-bis(hydroxylmethyl)-3-carboxylcyclopropenes) meet our requirements. These substituted cyclopropene analogs could be synthesized and they are thermodynamically stable. As a result, we propose that 1,2-bis(trifluoromethyl)-, 1,2-bis(hydroxylmethyl)- and 1,2-bis(hydroxylmethyl)-3-carboxylcyclopropenes may be potential candidates for efficient and selective Diels–Alder ligation in living systems.