Introduction

Cycloadditions are an important class of reactions for site‐specific labeling with applications in super‐resolution microscopy of cellular components.1–5 Key properties of these reactions are their bioorthogonality and high selectivity. By exploiting the concept of mutual orthogonality between several click‐type cycloaddition reactions, a combination of particularly slow and fast reactions enables the simultaneous labeling of multiple sites in a kinetically controlled fashion in vivo and in vitro.6–8 Recently, a set of orthogonal reactions has been used by Nikić et al.9 to label the insulin receptor and Influenza proteins on non‐canonical amino acids bearing strained 8‐membered rings in a time dependent manner. The click reactions utilized in this work are n‐propyl azide, 3‐benzyl tetrazine (H‐Tet) and 3‐benzyl‐6‐methyl tetrazine (Me‐Tet) ligations to three types of eight‐membered cyclic rings (Figure 1 a and Supporting Information Figure S1, IUPAC names in Supporting Information), namely i) racemic equatorial trans‐cyclooct‐2‐en‐1‐methylcarbamate (TCO*e) and racemic axial trans‐cyclooct‐2‐en‐1‐methylcarbamate (TCO*a), ii) two enantiomers of strained cyclooct‐2‐yn‐1‐methylcarbamate (SCO), and iii) endo‐ and exo‐bicyclonon‐5‐yn‐1‐methylcarbamate (BCNendo & BCNexo). The tetrazine ligation, a 4+2‐cycloaddition, is a strain‐promoted inverse electron demand Diels–Alder reaction (SPIEDAC),10–11 while the alkyne–azide ligation is a strain‐promoted Huisgen‐type 1,3‐dipolar 3+2‐cycloaddition (SPAAC).12–15 The large differences in reaction speed when employing supposedly similar ligation partners, such as H‐Tet versus Me‐Tet, or TCO* versus SCO, render dual color labeling of biological systems possible.14, 16

Figure 1 Open in figure viewer PowerPoint Kinetics of mutually orthogonal click reactions. a) Overview of the investigated reaction set between TCO*e/a isomers, BCNendo/exo isomers and SCO with azide, H‐Tet and Me‐Tet. Different isomers are marked as *. Differently sized arrows represent the reaction rates, where thicker means faster. Dashed lines show reactions that were too slow to be measured. Structures of all isomers are given in Figure S1. b) Correlation between experimental rate constants k and the calculated activation energies Eact for each pair of reactants. The solid line shows a linear regression. The SCO/BCN‐azide rates are taken from Borrmann et al.13 For the reactions too slow to be measured, we only obtained an upper limit for k as indicated by the error lines and omitted such points for the linear fit. Experimental error bars are smaller than the symbol size and are omitted for clarity.

Quantitatively predicting such differences and understanding their origin by quantum chemical calculations would increase the ability to further enhance the mutual orthogonality and augment the available set of reactions for preferential labeling. A large body of previous quantum‐chemical computational work has characterized and thereby significantly advanced our understanding of a number of highly related strain‐promoted click reactions.17–21 They mostly ascribed the differences in reactivity of these reactions, which have been shown to feature an inverse Diels–Alder electron demand, to contributions from orbital interactions, distortion and Pauli repulsion.22–23 The higher reactivity of tetrazines towards trans‐cyclooctene was attributed to their higher electrophilicity as compared to azides,19 while introducing nitrogens into the diene was proposed to decrease Pauli repulsion, rendering tetrazines the fastest reaction partner for cycloadditions with alkenes.23

Notwithstanding these advances, previous work has been focusing on highly simplified reactant scaffolds limiting the comparison of calculated activation energies to reaction rates determined experimentally for more complex scaffolds. However, attaching handles to the functional groups of azides, tetrazines and the different eight‐membered rings is the crucial requisite to label biomolecules, and may also critically affect the reactivity of the molecule, as experimental data increasingly certify that the speed of these reactions is highly susceptible to minor changes in substituents.9 Importantly, handles on cyclooctyne rings introduce a single stereocenter whereas handles on cyclooctene rings add an additional stereocenter. This results in two SCO enantiomers and two pairs of TCO* enantiomers (see Figure S1). The configuration of the stereocenters emerge as a crucial determinant for this class of click reactions.24–25 Considering the effect of the genuine handles used for functionalization is therefore indispensable for a direct validation of insights from quantum chemical calculations and, more importantly, for the application of these findings to the design of novel orthogonal reactions.

We addressed this fundamental challenge and herein present the first comprehensive set of measured rates and computed barriers for reactants with direct relevance for biological applications. We systematically computed the conformations and energies for the SPIEDAC and SPAAC reactions described above, using density functional theory (DFT). This resulted in a total of 48 reactions with configurationally distinct transition states and products, out of which 24 are chemically distinct enantiomers. The resulting system sizes required advanced sampling of the complex energy landscape, and the diminishing barriers of the high speed TCO* reactions rendered it necessary to incorporate van der Waals complexes into the reaction pathway. On this basis, unexpectedly, we find that SPIEDAC reactions involving SCO and tetrazines to follow normal electron demand, ascribed to the electrophilicity of the SCO. We thus term this class of reactions SPINEDAC (strain‐promoted inherently normal electron demand Diels–Alder cycloaddition). We observe isomeric configurations to critically fine‐tune the reactivity of the investigated click reactions. Our study aids the engineering of a currently widely used set of cycloadditions to further enhance their reactivity and mutual orthogonality for their applicability in biology.