Lubomír Rulíšek

Tyrosinase contains a coupled binuclear copper (CBC) active site, which in its bicuprous form (deoxy-Ty) binds O2 to form a side-on peroxide [Cu(II)2O2]2+ intermediate (oxy-Ty) that performs the regioselective monooxygenation of monophenols to catechols and their subsequent 2e− oxidation to quinones. Previously, we used spectroscopic, kinetic, and computational methods to elucidate the mechanism of the initial steps in the monooxygenation reaction. Herein, we investigated the final step in catecholate formation and provide experimental and computational results elucidating the catechol oxidation reaction, formally a proton-coupled 2e− process. We employed single-turnover stopped-flow absorption to observe the elusive catecholate-Cu(II)2OH intermediate via the anaerobic reaction of resting Cu(II)2OH Ty (met-Ty) with the catecholate substrate. This intermediate was cryo-trapped and characterized by electron paramagnetic resonance and X-ray absorption spectroscopies. These experimental results were correlated to quantum mechanics/molecular mechanics (QM/MM) and QM calculations to describe the complete catalytic cycle of tyrosinase, revealing that the 2e− oxidation of catechol to quinone involves two steps: (i) coupled 1H+/1e− transfer from the bound monoanionic catecholate to the bridged hydroxide of the met-Ty active site, followed by (ii) the second 1e− transfer. The latter is calculated to be the rate-limiting step in catechol oxidation, confirmed by experimental solvent kinetic isotope effect studies. Our computational data suggest that quinone release from the protein provides the necessary driving force for this reaction. These results establish a detailed description of this oxidase cycle of the tyrosinase mechanism and more broadly provide molecular-level insights into the diverse reactivity of CBC sites in biology.

Controlling selectivity in CO₂ reduction catalysis is essential for minimizing the formation of H₂ byproduct. We recently reported that the cobalt and iron complexes of the 1-([2,2′-bipyridin]-6-yl)-N-([2,2′-bipyridin]-6-ylmethyl)-N-(pyridin-2-ylmethyl) methanamine ligand (L, DBPy-PyA) are efficient catalysts to selectively generate H₂ and CO, respectively. Herein we demonstrate that selectivity can be controlled not only by exchanging the metal center but also adjusting the electronic properties of the ligand. Under electrochemical conditions in acetonitrile, with 2,2,2-trifluoroethanol as a proton source, the unsubstituted CoL and the electron-rich CoL^OMe predominantly produce H₂ (selectivity of 82% and 55%, respectively). In contrast, the electron-deficient CoL^CF3 favors CO formation with a selectivity up to 87%. DFT calculations show that formation of the metal-hydride is favored in the case of CoL and CoL^OMe, whereas it is substantially endergonic in the case of CoL^CF3. Concurrently, the binding of CO₂ and the evolution of the resulting intermediates in CoL^CF3 can benefit from ligand-assisted proton transfer, as confirmed by microkinetic modeling. Catalytic tests were then conducted under photochemical conditions, affording syngas with tunable CO/H₂ ratios that follow the electronic effects of the chosen catalyst. Overall, the results underscore how ligand tuning can be a powerful handle for controlling selectivity in molecular CO₂RR.

Is it possible to obtain a sufficiently accurate quantum mechanical (QM) energy of an arbitrary oligopeptide structure in an implicit solvent within a second? Herein, we explore the possibility of constructing potential energy surfaces of larger peptides from rigorous quantum chemical data acquired for hundreds of thousands of capped mono-, di-, and tripeptides. We demonstrate that modern machine-learning methods, in particular NequIP, when trained only on tripeptides, can already predict QM energies of random decapeptides with a root-mean-square error (RMSE) of –1. The models also perform well on other out-of-distribution tasks: conformer ranking of a 31-peptide (identifying the global minimum), geometry optimization (RMSD 0.009 Å), and prediction of side-chain interaction energies on PDB structures (with sub-kcal mol^–1 accuracy). We show that the success of the ML approach is critically dependent on two factors: (i) inclusion of off-equilibrium structures from hot MD sampling, which includes systematic sampling of dihedral angles, and (ii) training on energies of solvated structures, instead of gas-phase energies. Solvated systems are both easier to predict by ML and a more relevant model of typical biomolecular interactions. We make all datasets and models available at doi.org/10.5281/zenodo.15356387.

Structure-guided optimization of a peptidic chelating agent, combining high Pb( ii ) affinity, selectivity, and in silico modelling, enables efficient in vivo Pb detoxification, including brain Pb reduction and selective urinary excretion.

The central dogma of molecular biology describes how genetic information stored in nucleic acids guides the formation of structured proteins from a single molecular alphabet of twenty amino acids (C20). The extent to which C20 is uniquely capable of forming structural polymers remains a fundamental open question. Here we demonstrate that peptides built from other, "xeno" amino acid alphabets can adopt protein-like secondary structural motifs. Having designed two different xeno alphabets, with them we constructed both combinatorial random sequence libraries and specific, designed 25-mer sequences. We report sequence-dependent structural motifs that result according to circular dichroism, infrared spectroscopy and nuclear magnetic resonance interpreted by molecular dynamics simulations, supported by extensive quantum mechanical calculations. This evidence, including a solution-phase NMR-resolved helical motif, demonstrates that the potential for amino acids to form structure bearing sequences is not unique to life's alphabet, and thereby reveals a previously unexplored sequence-structure space. Results inform the search for extraterrestrial life, lay foundations for incorporating novel synthetic functional groups and heteroatoms into structure-bearing alphabets, and provide the first truly independent data with which to test and improve all that has been learned about protein folding from the study of life's 20 side chains.

Electron-deficient dendralenes, bearing enone substructures and possessing an unfavorable disposition of like charges at the neighboring carbons, undergo nucleophilic 1,4-addition (Michael) or 1,6-addition (anti-Michael). Diverse products are obtained, including those of simple addition as well as cyclic and ortho-fused systems arising via multistep sequences, depending on the structure of the substrate and the nature of the nucleophile. Attack of a hydride at an enone fragment triggers the formation of multisubstituted pyranones and furans; furan formation was also initiated by thiolates. A notable exception is the derivative with a five-membered cyclic enone, which prefers simple additions followed by the reshuffling of the double bonds for both H^- and RS^- nucleophiles. By contrast, the latter enone is the only one that can react with stabilized C-nucleophiles, yielding bicyclic compounds. Domino cyclizations can also be induced by the enolization of the enone with DBU, giving mostly polysubstituted furans. However, the dendralene with a five-membered cyclic enone and its analogue with a six-membered ring behave differently: The former gives a mixture, while the latter prefers the formation of an isocoumarin derivative, which is driven by aromatization. DFT calculations have shown that the additions of thiolates are mostly governed by the thermodynamic stability of possible products arising from complex equilibrium processes.

Recent experiments highlighted the significance of Au(I)···H interactions in the structures of Au(I)-containing materials as well as in catalysis. However, they also raised the important question of whether similar interactions could be observed for Ag(I) and Cu(I) analogues of Au(I). Herein, we present experimental and computational evidence for the formation of hydrogen bonds between the Ag(I) center and the dimethylammonium group of protonated bis(1-adamantyl)(2-(dimethylamino)phenyl)phosphine ligand (1). We support this in the gas phase by employing infrared (IR) photodissociation spectroscopy correlated with quantum chemical (QC) computations. For the Ag(I) complex (1AgCl₂H) in solution, we show by correlating the nuclear magnetic resonance data and QC calculations that the formation of the Ag(I)···H bond competes with the formation of the Cl···H bond depending on the polarity of the environment. We evaluated interaction energies between the dimethylammonium group and the central metal across the coinage metal series (Au/Ag/Cu) using the relativistic CCSD(T) method. Furthermore, employing wave function analysis, we gained qualitative insight into Au/Ag/Cu···H bonding at the electronic level. The definition of Ag(I)···H interactions thus extends horizons in silver(I) chemistry.

Skeletal editing of 5-membered carbocycles to 6-membered heteroaromatic compounds represents an attractive concept that would enable late-stage heteroarene construction. However, the current state of the art does not offer many synthetic strategies on how to achieve such a goal. One of those is based on a reaction of aromatic tertiary alcohols with sodium azide under acidic conditions, i.e., the Schmidt reaction. Herein, we present a hitherto unexplored double Schmidt reaction toward aromatic compounds possessing two pyridine rings. It is the first example of a conversion of 5,8-disubstituted indeno­[2,1-c]­fluorene-5,8-diols to m,n-diaza­[5]­helicenes in high yields. We also demonstrate that the regioisomeric ratio can be controlled, to a certain extent, by tuning conditions. Mechanistic investigation encompassing experimental as well as DFT calculations sheds light on the course of the reaction and provides a rationale for the observed regioselectivity and its control. In addition, structures of several diaza[5]­helicenes and intermediates were unequivocally confirmed by single crystal X-ray diffraction analyses. The double Schmidt rearrangement strategy was also applied for the transformation of enantioenriched [7]­helical indeno­[2,1-c]­fluorene-5,8-diols into the corresponding enantioenriched m,n-diaza­[7]­helicenes without significant loss of enantiopurity. Their structures were also unequivocally confirmed by single crystal X-ray diffraction analyses.

We designed a minimalistic zinc(II)‐binding peptide featuring the Cys ₂ His ₂ zinc‐finger motif. To this aim, several tens of thousands of (His/Cys)‐X _n ‐(His/Cys) protein fragments ( n =2–20) were first extracted from the 3D protein structures deposited in Protein Data Bank (PDB). Based on geometrical constraints positioning two Cys (C) and two His (H) side chains at the vertices of a tetrahedron, approximately 22 000 sequences of the (H/C)‐X _i ‐(H/C)‐X _j ‐(H/C)‐X _k ‐(H/C) type, satisfying N _{metal–binding H} = N _{metal‐binding C} =2, were processed. Several other criteria, such as the secondary structure content and predicted fold stability, were then used to select the best candidates. To prove the viability of the computational design experimentally, three peptides were synthesized and subjected to isothermal calorimetry (ITC) measurements to determine the binding constants with Zn ²⁺ , including the entropy and enthalpy terms. For the strongest Zn ²⁺ ions binding peptide, P1 , the dissociation constant was shown to be in the nanomolar range ( K _D =~220 nM; corresponding to Δ G _bind =−9.1 kcal mol ⁻¹ ). In addition, ITC showed that the [ P1 : Zn ²⁺ ] complex forms in 1 : 1 stoichiometry and two protons are released upon binding, which suggests that the zinc coordination involves both cysteines. NMR experiments also indicated that the structure of the [ P1 : Zn ²⁺ ] complex might be quite similar to the computationally predicted one. In summary, our proof‐of‐principle study highlights the usefulness of our computational protocol for designing novel metal‐binding peptides.