Bioorthogonal chemical reactions are a thriving area of chemical research in recent years as an unprecedented technique to dissect native biological processes through chemistry-enabled strategies. However, current concepts of bioorthogonal chemistry have largely centered on “bond formation” reactions between two mutually reactive bioorthogonal handles.

Recently, in a reverse strategy, a collection of “bond cleavage” reactions has emerged with excellent biocompatibility. These reactions have expanded our bioorthogonal chemistry repertoire, enabling an array of exciting new biological applications that range from the chemically controlled spatial and temporal activation of intracellular proteins and small-molecule drugs to the direct manipulation of intact cells under physiological conditions.

The advancement of bioorthogonal cleavage reactions (BCRs) has expanded the scope of the bioorthogonal chemistry toolkit, leading to a diverse array of innovative biological applications. These include but are not limited to precise spatial and temporal activation of intracellular probes, prodrugs, proteins, glycans, and nucleic acids.

Based on our engineering modifications to Escherichia coli Nissle 1917 (EcN), we aim to design it as a solid tumor-localized drug delivery platform. The specific design involves site-specific incorporation of the unnatural amino acid Tet-v2.0 into the CsgA protein of ECN through genetic engineering. Subsequently, the engineered bacteria are localized at the solid tumor site, followed by systemic administration of the trans-cyclooctenes(TCO)-caged prodrug. Only the prodrug molecules distributed at the target site can undergo decaging through a tetrazine side-chain reaction with Tet-v2.0, restoring them to activate drug molecules with cytotoxicity.

The Tet-v2.0 we selected is a non-natural amino acid with a tetrazine side chain developed by the research group of Ryan A. Mehl at Oregon State University, derived structurally from Tyrosine.

When the tetrazine containing amino acid, Tet-v2.0, is incorporated into proteins with genetic code expansion, clean in vivo reactions can be achieved with rates approaching 103 M-1 s-1 and 105 M-1 s-1 for TCO and sTCO labels respectively.

While there is a wealth of characterization on the parameters and properties of the BCR reaction currently, there is a relative scarcity of studies focusing on reaction analysis in specific contexts, especially those employing quantum chemical calculations as a central analytical tool. In this regard, we aim to conduct an in-depth investigation into the reaction details between the Tet-v2.0 residue and TCO prodrug molecules using quantum chemical computation as a tool.

Here, the computational software employed is Gaussian, which operates by solving the Schrödinger equation using various approximation methods of different accuracies. Specifically, in ab initio calculations, the process begins by simplifying the system using the Born-Oppenheimer approximation, solving the Hartree-Fock self-consistent field equations, introducing multi-level perturbation electronic correlation energy corrections, and ultimately obtaining an approximate solution to the Schrödinger equation: On the other hand, in density functional theory, the solution to the exchange-correlation potential term in the Kohn-Sham equation is approached through methods like local density approximation, generalized gradient approximation, and hybrid functionals. This process yields an approximate solution to the K-S equation, which is equivalent to an approximate solution to the Schrödinger equation.

Regarding the specifics of our study, Tet-v2.0 is integrated into the protein scaffold, restricting its movement to within a certain radius where it can rotate but is not freely mobile. Simultaneously, the TCO prodrug molecule is freely distributed, and there are no constraints on the relative movement between the two entities. Therefore, to simplify calculations and reduce computational load, we neglect the protein scaffold portion of Tet-v2.0 and instead model it in the form of a small molecule with fixed coordinates.

Modeling of Tet-v2.0 and TCO molecules is carried out using Gauss View. Calculations were carried out at the ωB97X-D/6- 311G(d,p) level of theory for geometry optimizations and ωB97X-D18/def2-TZVP level of theory for energy calculations.

Subsequently, guided by the principles of chemical transition state theory, further arrangements and combinations of the two ground-state molecules are made using Gauss View to identify potential approximate transition state conformations. At the ωB97X-D/6- 311G(d,p) level of theory, set the solvent as water, and utilize opt=(calcfc,ts,noeigen) as the key phrase for high-precision transition state optimization, in which calcfc denotes the precise calculation of the initial structure's Hessian matrix and noeigen signifies that the number of eigenvalues of the Hessian matrix is not checked at each step during the optimization process. Finally, after optimization, an excellent transition state conformation is achieved. Subsequently, perform transition state frequency and energy calculations under the ωB97X-D18/def2-TZVP functional and orbital accuracy. Consistent with theoretical analysis, the transition state exhibits only one imaginary frequency, indicating that it indeed represents the energy saddle point, aligning with the definition of a transition state.

After obtaining the corrected transition state energy, compare it with the energies of the two ground-state molecules. According to chemical transition state theory, the difference between the ground state and the energy maximum point (transition state) represents the activation energy of the chemical reaction. Hence, the activation energy of the BCR can be determined.

This value falls within the moderate to high range for IEDDA reactions, corroborating reaction rates in the range of 10³ M^-1s^-1 to 10⁵ M^-1s^-1.