Intersystem Crossing Enables 4‑Thiothymidine to Act as a Photosensitizer in Photodynamic Therapy: An Ab Initio QM/MM Study

Motivated by its potential use as a photosensitizer in photodynamic therapy, we report the first ab initio quantum mechanics/molecular mechanics (QM/MM) study of 4-thiothymidine in aqueous solution. The core chromophore 4-thiothymine was described using the multiconfigurational CASSCF and CASPT2 QM methods, while the ribose and the solvent water molecules were treated at the MM level (CHARMM and TIP3P, respectively). The minima of the five lowest electronic states (S₀, S₁, S₂, T₁, and T₂) and six minimum-energy intersections were fully optimized at the QM­(CASSCF)/MM level, and their energies were further refined by single-point QM­(CASPT2)/MM and CASPT2 calculations. The relevant spin–orbit couplings were also computed. We find that (1) there are three efficient photophysical pathways that account for the experimentally observed ultrafast formation of the lowest triplet state with a quantum yield of nearly unity, (2) the striking qualitative differences in the photophysical behavior of 4-thiothymine and thymine originate from the different electronic structure of their S₁ states, and (3) environmental effects play an important role. The present QM/MM calculations provide mechanistic insight that may guide the design of improved photosensitizers for photodynamic therapy

Computational Insights into an Enzyme-Catalyzed [4+2] Cycloaddition

The enzyme SpnF, involved in the biosynthesis of spinosyn A, catalyzes a formal [4+2] cycloaddition of a 22-membered macrolactone, which may proceed as a concerted [4+2] Diels–Alder reaction or a stepwise [6+4] cycloaddition followed by a Cope rearrangement. Quantum mechanics/molecular mechanics (QM/MM) calculations combined with free energy simulations show that the Diels–Alder pathway is favored in the enzyme environment. OM2/CHARMM free energy simulations for the SpnF-catalyzed reaction predict a free energy barrier of 22 kcal/mol for the concerted Diels–Alder process and provide no evidence of a competitive stepwise pathway. Compared with the gas phase, the enzyme lowers the Diels–Alder barrier significantly, consistent with experimental observations. Inspection of the optimized geometries indicates that the enzyme may prearrange the substrate within the active site to accelerate the [4+2] cycloaddition and impede the [6+4] cycloaddition through interactions with active-site residues. Judging from partial charge analysis, we find that the hydrogen bond between the Thr196 residue of SpnF and the substrate C15 carbonyl group contributes to the enhancement of the rate of the Diels–Alder reaction. QM/MM simulations show that the substrate can easily adopt a reactive conformation in the active site of SpnF because interconversion between the C5–C6 s-<i>trans</i> and s-<i>cis</i> conformers is facile. Our QM/MM study suggests that the enzyme SpnF does behave as a Diels-Alderase

Role of Two Alternate Water Networks in Compound I Formation in P450eryF

The P450eryF enzyme (CYP107A1) hydroxylates 6-deoxyerythronolide B to erythronolide B during erythromycin synthesis by <i>Saccharopolyspora erythraea</i>. In many P450 enzymes, a conserved “acid-alcohol pair” is believed to participate in the proton shuttling pathway for O₂ activation that generates the reactive oxidant (Compound I, Cpd I). In CYP107A1, the alcohol-containing amino acid is replaced with alanine. The crystal structure of DEB bound to CYP107A1 indicates that one of the substrate hydroxyl groups (5-OH) may facilitate proton transfer during O₂ activation. We applied molecular dynamics (MD) and hybrid quantum mechanics/molecular mechanics (QM/MM) techniques to investigate substrate-mediated O₂ activation in CYP107A1. In the QM/MM calculations, the QM region was treated by density functional theory, and the MM region was represented by the CHARMM force field. The MD simulations suggest the existence of two water networks around the active site, the one found in the crystal structure involving E360 and an alternative one involving E244. According to the QM/MM calculations, the first proton transfer that converts the peroxo to the hydroperoxo intermediate (Compound 0, Cpd 0) proceeds via the E244 water network with direct involvement of the 5-OH group of the substrate. For the second proton transfer from Cpd 0 to Cpd I, the computed barriers for the rate-limiting homolytic O–O cleavage are similar for the E360 and E244 pathways, and hence both glutamate residues may serve as proton source in this step

Quantum Mechanics/Molecular Mechanics Study of Oxygen Binding in Hemocyanin

We report a combined quantum mechanics/molecular mechanics (QM/MM) study on the mechanism of reversible dioxygen binding in the active site of hemocyanin (Hc). The QM region is treated by broken-symmetry density functional theory (DFT) with spin projection corrections. The X-ray structures of deoxygenated (deoxyHc) and oxygenated (oxyHc) hemocyanin are well reproduced by QM/MM geometry optimizations. The computed relative energies strongly depend on the chosen density functional. They are consistent with the available thermodynamic data for oxygen binding in hemocyanin and in synthetic model complexes when the BH&HLYP hybrid functional with 50% Hartree–Fock exchange is used. According to the QM­(BH&HLYP)/MM results, the reaction proceeds stepwise with two sequential electron transfer (ET) processes in the triplet state followed by an intersystem crossing to the singlet product. The first ET step leads to a nonbridged superoxo Cu_B^II–O₂^•– intermediate via a low-barrier transition state. The second ET step is even more facile and yields a side-on oxyHc complex with the characteristic Cu₂O₂ butterfly core, accompanied by triplet-singlet intersystem crossing. The computed barriers are very small so that the two ET processes are expected to very rapid and nearly simultaneous

Exploring the Triplet Excited State Potential Energy Surfaces of a Cyclometalated Pt(II) Complex: Is There Non-Kasha Emissive Behavior?

In this Article, we address the complexity of the emissive processes of a square-planar heteroleptic Pt­(II) complex bearing 2-phenylpyridine (ppy) as cyclometalated ligand and an acetylacetonate derivative (dbm) as ancillary ligand. The origins of emission were identified with the help of density functional theory (DFT) and quadratic response (QR) time-dependent (TD)-DFT calculations including spin–orbit coupling (SOC). To unveil the photodeactivation mechanisms, we explored the triplet potential energy surfaces and computed the SOCs and the radiative decay rates (<i>k</i>_r) from possible emissive states. We find that emission likely originates from a higher-lying ³MLCT/³LLCT state and not from the Kasha-like ³MLCT/³LC_dbm state. The temperature-dependent nonradiative deactivation mechanisms were also elucidated. The active role of metal-centered (³MC) triplet excited states is confirmed for these deactivation pathways

Analytical Gradients for Density Functional Calculations with Approximate Spin Projection

We have derived and implemented analytical gradients for broken-symmetry unrestricted density functional calculations (BS-UDFT) with removal of spin contamination by Yamaguchi’s approximate spin projection method. Geometry optimizations with these analytical gradients (AGAP-opt) yield results consistent with those obtained with the previously available numerical gradients (NAP-opt). The AGAP-opt approach is found to be more precise, efficient, and robust than NAP-opt. It allows full geometry optimizations for large open-shell systems. We report results for three types of organic diradicals and for a binuclear vanadium­(II) complex to demonstrate the merits of removing the spin contamination effects during geometry optimization (AGAP-opt vs BS-UDFT) and to illustrate the superior performance of the analytical gradients (AGAP-opt vs NAP-opt). The results for the vanadium­(II) complex indicate that the AGAP-opt method is capable of handling pronounced spin contamination effects in large binuclear transition metal complexes with two magnetic centers

On the Effect of Varying Constraints in the Quantum Mechanics Only Modeling of Enzymatic Reactions: The Case of Acetylene Hydratase

Quantum mechanics only (QM-only) studies of enzymatic reactions employ a coordinate-locking scheme, in which certain key atoms at the periphery of the chosen cluster model are fixed to their crystal structure positions. We report a case study on acetylene hydratase to assess the uncertainties introduced by this scheme. Random displacements of 0.1, 0.15, and 0.2 Å were applied at the ten terminal atoms fixed in the chosen 116-atom cluster model to generate sets of ten distorted structures for each given displacement. The relevant stationary points were reoptimized under these modified constraints to determine the variations of the computed energies and geometries induced by the displacements of the fixed atoms. Displacements of 0.1 Å cause a relatively minor perturbation that can be accommodated during geometry optimization, resulting in rather small changes in key bond distances and relative energies (typically of the order of 0.01 Å and 1 kcal/mol), whereas displacements of 0.2 Å lead to larger fluctuations (typically twice as high) and may sometimes even cause convergence to different local minima during geometry optimization. A literature survey indicates that protein crystal structures with a resolution higher than 2.0 Å are normally associated with a coordinate error of less than 0.1 Å for the backbone atoms. Judging from the present results for acetylene hydratase, such uncertainties seem tolerable in the design of QM-only models with more than 100 atoms, which are flexible enough to adapt during geometry optimization and thus keep the associate uncertainties in the computed energies and bond distances at tolerable levels (around 1 kcal/mol and 0.01 Å, respectively). On the other hand, crystal structures with significantly lower resolution should be used with great caution when setting up QM-only models because the resulting uncertainties in the computational results may become larger than acceptable. The present conclusions are mostly based on systematic DFT­(B3LYP) calculations with a medium-size basis set. Test calculations on selected structures confirm that similar results are obtained for larger basis sets, different functionals (ωB97X, BMK, M06), and upon including solvation and zero-point corrections, even though the fluctuations in the computed relative energies become somewhat larger in some cases

Why Is the Oxidation State of Iron Crucial for the Activity of Heme-Dependent Aldoxime Dehydratase? A QM/MM Study

Aldoxime dehydratase is a heme-containing enzyme that utilizes the ferrous rather than the ferric ion to catalyze the synthesis of nitriles by dehydration of the substrate. We report a theoretical study of this enzyme aimed at elucidating its catalytic mechanism and understanding this oxidation state preference (Fe²⁺ versus Fe³⁺). The uncatalyzed dehydration reaction was modeled by including three and four water molecules to assist in the proton transfer, but the computed barriers were very high at both the DFT (B3LYP) and coupled cluster CCSD­(T) levels. The enzymatic dehydration of <i>Z</i>-acetaldoxime was explored through QM/MM calculation using two different QM regions and covering all three possible spin states. The reaction starts by substrate coordination to Fe²⁺ via its nitrogen atom to form a six-coordinated singlet reactant complex. The ferrous heme catalyzes the N–O bond cleavage by transferring one electron to the antibond in the singlet state, while His320 functions as a general acid to deliver a proton to the leaving hydroxide, thus facilitating its departure. The key intermediate is identified as an Fe^III(CH₃CHN^•) species (triplet or open-shell singlet), with the closed-shell singlet Fe^II(CH₃CHN⁺) being about 6 kcal/mol higher. Subsequently, the same His320 residue abstracts the α-proton, coupled with electron transfer back to the iron center. Both steps are calculated to have feasible barriers (14–15 kcal/mol), in agreement with experimental kinetic studies. For the same mode of substrate coordination, the ferric heme does not catalyze the N–O bond cleavage, because the reaction is endothermic by about 40 kcal/mol, mainly due to the energetic penalty for oxidizing the ferric heme. The alternative binding option, in which the anionic aldoxime coordinates to the ferric ion via its oxyanion, also results in a high barrier (around 30 kcal/mol), mainly because of the large endothermicity associated with the generation of a suitable base (neutral His320) for proton abstraction

Origin of Inversion versus Retention in the Oxidative Addition of 3‑Chloro-cyclopentene to Pd(0)L_<i>n</i>

The preference for <i>syn</i> versus <i>anti</i> oxidative addition of 3-chloro-cyclopentene to Pd(0)­L_<i>n</i> was investigated using density functional theory (L = PH₃, PMe₃, PF₃, ethylene, maleic anhydride, pyridine, imidazol-2-ylidene). Both mono- and bis-ligation modes were studied (<i>n</i> = 1 and 2). The pathways were analyzed at the B2PLYP-D3/def2-TZVPP//TPSS-D3/def2-TZVP level, and an interaction/distortion analysis was performed at the ZORA-TPSS-D3/TZ2P level for elucidating the origin of the selectivity preferences. Mechanistically, the <i>anti</i> addition follows an S_N2 type mechanism, whereas the <i>syn</i> addition has partial S_N1 and S_N2′ character. Contrary to the traditional rationale that orbital interactions are dominant in the <i>anti</i> pathway, analysis of the variation of the interaction components along the intrinsic reaction coordinate shows that the <i>syn</i> pathway exhibits stronger overall orbital interactions. This orbital preference for the <i>syn</i> pathway diminishes with increasing donor capacity of the ligand. It is caused by the donation of the isolated p orbitals on the migrating chlorine atom to the PdL_<i>n</i> fragment, which is lacking in the <i>anti</i> pathway, whereas the HOMO–LUMO overlap between the fragments is greater for the <i>anti</i> pathway. Electrostatically, the <i>syn</i> pathway is preferred for weakly donating and withdrawing ligands, whereas the <i>anti</i> pathway is favored with strongly donating ligands

Determinants of Regioselectivity and Chemoselectivity in Fosfomycin Resistance Protein FosA from QM/MM Calculations

FosA is a manganese-dependent enzyme that utilizes a Mn²⁺ ion to catalyze the inactivation of the fosfomycin antibiotic by glutathione (GSH) addition. We report a theoretical study on the catalytic mechanism and the factors governing the regioselectivity and chemoselectivity of FosA. Density functional theory (DFT) calculations on the uncatalyzed reaction give high barriers and almost no regioselectivity even when adding two water molecules to assist the proton transfer. According to quantum mechanics/molecular mechanics (QM/MM) calculations on the full solvated protein, the enzyme-catalyzed glutathione addition reaction involves two major chemical steps that both proceed in the sextet state: proton transfer from the GSH thiol group to the Tyr39 anion and nucleophilic attack by the GSH thiolate leading to epoxide ring-opening. The second step is rate-limiting and is facilitated by the presence of the high-spin Mn²⁺ ion that functions as a Lewis acid and stabilizes the leaving oxyanion through direct coordination. The barrier for C1 attack is computed to be 8.9 kcal/mol lower than that for C2 attack, in agreement with the experimentally observed regioselectivity of the enzyme. Further QM/MM calculations on the alternative water attack predict a concerted mechanism for this reaction, where the deprotonation of water, nucleophilic attack, and epoxide ring-opening take place via the same transition state. The calculated barrier is 8.3 kcal/mol higher than that for GSH attack, in line with the observed chemoselectivity of the enzyme, which manages to catalyze the addition of GSH in the presence of water molecules around its active site. The catalytic efficiency, regioselectivity, and chemoselectivity of FosA are rationalized in terms of the influence of the active-site protein environment and the different stabilization of the distorted substrates in the relevant transition states