Computational chemistry and biochemistry began with Isaac Newton's classical mechanics in the 17th century and the establishment of quantum mechanics in the 1920s. Enabled by extraordinary advances in computers, in the last half century, this field has become a robust partner with experiment. The challenges facing computational chemists and biochemists, the Holy Grails of the field, are described. These include the development of a highly accurate density functional, ideally one that has universal chemical accuracy, and accurate polarizable force fields, as well as methods to handle efficiently the massive number of computations that must be performed for molecular dynamics and for the computation of flexible systems such as proteins. We estimate when the breakthroughs that will make computation a powerful engine for chemical discovery and design will be achieved. The Holy Grails of this field involve methods to enable the accurate and efficient prediction of structures and properties of complex biological systems and materials. The principal Holy Grail is a routine computational method for the prediction and design of multicomponent, often heterogeneous, functional systems and devices.

The Diels−Alder reactions of seven 1,2,4,5-tetrazines with unstrained and strained alkenes and alkynes were studied with quantum mechanical calculations (M06-2X density functional theory) and analyzed with the distortion/interaction model. The higher reactivities of alkenes compared to alkynes in the Diels−Alder reactions with tetrazines arise from the differences in both interaction and distortion energies. Alkenes have HOMO energies higher than those of alkynes and therefore stronger interaction energies in inverseelectron-demand Diels−Alder reactions with tetrazines. We have also found that the energies to distort alkenes into the Diels−Alder transition-state geometries are smaller than for alkynes in these reactions. The strained dienophiles, trans-cyclooctene and cyclooctyne, are much more reactive than unstrained trans-2-butene and 2-butyne, because they are predistorted toward the Diels−Alder transition structures. The reactivities of substituted tetrazines correlate with the electron-withdrawing abilities of the substituents. Electron-withdrawing groups lower the LUMO+1 of tetrazines, resulting in stronger interactions with the HOMO of dienophiles. Moreover, electron-withdrawing substituents destabilize the tetrazines, and this leads to smaller distortion energies in the Diels−Alder transition states.

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A new class of bioorthogonal reagents, 1,2,4-triazines, is described. These scaffolds are stable in biological media and capable of robust reactivity with trans-cyclooctene (TCO). The enhanced stability of the triazine scaffold enabled its direct use in recombinant protein production. The triazine-TCO reaction can also be used in tandem with other bioorthogonal cycloaddition reactions. These features fill current voids in the bioorthogonal toolkit.

The azide−dibenzocyclooctyne and transcyclooctene−tetrazine cycloadditions are both bioorthogonal and mutually orthogonal: trans-cyclooctene derivatives greatly prefer to react with tetrazines rather than azides, while dibenzocyclooctyne derivatives react with azides but not with tetrazines under physiological conditions. DFT calculations used to identify the origins of this extraordinary selectivity are reported, and design principles to guide discovery of new orthogonal cycloadditions are proposed. Two new bioorthogonal reagents, methylcyclopropene and 3,3,6,6-tetramethylthiacycloheptyne, are predicted to be mutually orthogonal in azide and tetrazine cycloadditions.A zide and tetrazine cycloadditions have become central reactions in the rapidly developing field of cellular component labeling with bioorthogonal reactions. 1−3 Bertozzi and co-workers have developed strain-promoted (3 + 2) cycloaddition reactions between azides and cyclooctynes since 2004 (Scheme 1a). 4 These reactions proceed at a rate that is sufficient for in vivo labeling without the toxic copper(I) catalysts traditionally employed in "click chemistry" involving azide cycloadditions. Several groups have developed structurally varied cyclooctyne derivatives with different chemical reactivities and physical properties. 5 Another breakthrough in this area came in 2008 with the application of inverse-electrondemand Diels−Alder reactions of 1,2,4,5-tetrazines and strained alkenes (Scheme 1b). 6 In particular, the trans-cyclooctene− tetrazine (4 + 2) cycloaddition, which has an extremely high bimolecular rate constant (k 2 = 10 2 −10 4 M −1 s −1 ), 7 is much faster than the azide−cyclooctyne (3 + 2) cycloaddition (k 2 = 10 −3 −1 M −1 s −1 ). 1c Recently, Hilderbrand and co-workers demonstrated that two bioorthogonal cycloaddition pairs are mutually orthogonal. 8 That is, as shown in Scheme 2a, transcyclooctene derivatives greatly prefer to react with tetrazines rather than azides, while dibenzocyclooctyne derivatives react with azides but not with tetrazines under physiological conditions (Scheme 2b). On the basis of this discovery, Hilderbrand and co-workers successfully realized the simultaneous labeling and imaging of two different cancer cell types in biological environments. 8 At almost the same time, Schultz, Lemke, and co-workers found that trans-cyclooctenes show extremely high selectivity toward tetrazines rather than azides in protein labeling experiments. 9 However, the cyclooctynemodified proteins couple with both tetrazine-functionalized and azide-functionalized dyes. 9 The similar reactivities of cyclooctynes with azides and tetrazines was also demonstrated in separate kinetic studies by the Bertozzi and Wang groups: tetrazines react with cyclooctynes only 1−2 orders of magnitude faster than azides do (Scheme 2c). 10 transCyclooctene, cyclooctyne, and dibenzocyclooctyne are all highly strained molecules; why do their selectivities toward azides and tetrazines under bioorthogonal cycloadditions differ