Indentation and self-healing mechanisms of a self-assembled monolayer:a combined experimental and modeling study

A combination of in situ vibrational sum-frequency generation (SFG) spectroscopy and molecular-dynamics (MD) simulations has allowed us to study the effects of indentation of self-assembled octadecylphosphonic acid (ODPA) monolayers on α-Al2O3(0001). Stress-induced changes in the vibrational signatures of C–H stretching vibrations in SFG spectra and the results of MD simulations provide clear evidence for an increase in gauche-defect density in the monolayer as a response to indentation. A stress-dependent analysis indicates that the defect density reaches saturation at approximately 155 MPa. After stress is released, the MD simulations show an almost instantaneous healing of pressure-induced defects in good agreement with experimental results. The lateral extent of the contact areas was studied with colocalized SFG spectroscopy and compared to theoretical predictions for pressure gradients from Hertzian contact theory. SFG experiments reveal a gradual increase in gauche-defect density with pressure before saturation close to the contact center. Furthermore, our MD simulations show a spatial anisotropy of pressure-induced effects within ODPA domains: molecules tilted in the direction of the pressure gradient increase in tilt angle while those on the opposite side form gauche-defects

If It Is Hard, It Is Worth Doing: Engineering Radical Enzymes from Anaerobes

With a pressing need for sustainable chemistries, radical enzymes from anaerobes offer a shortcut for many chemical transformations and deliver highly sought-after functionalizations such as late-stage C-H functionalization, C-C bond formation, and carbon-skeleton rearrangements, among others. The challenges in handling these oxygen-sensitive enzymes are reflected in their limited industrial exploitation, despite what they may deliver. With an influx of structures and mechanistic understanding, the scope for designed radical enzymes to deliver wanted processes becomes ever closer. Combined with new advances in computational methods and workflows for these complex systems, the outlook for an increased use of radical enzymes in future processes is exciting

Ammonium Complexes of Orthoester Cryptands Are Inherently Dynamic and Adaptive

© 2019 American Chemical Society. Fluxional chemical species such as bullvalene have been a valuable source of inspiration and fundamental insight into the nature of chemical bonds. A supramolecular analogue of bullvalene, i.e., a "fluxional host-guest system", in which the ensemble of a well-defined host and guest is engaged in continuous, degenerate constitutional rearrangements, is still elusive, however. Here, we report experimental and computational evidence for guest-induced dynamic covalent rearrangements in the ammonium complexes of self-assembled orthoester cryptands. This unique behavior is made possible by the ammonium guest playing a dual role: it is sufficiently acidic to initiate dynamic covalent exchange reactions at the orthoester bridgeheads, and as a hydrogen bond donor it acts as a supramolecular template, governing the outcome of a multitude of possible intra- and intermolecular rearrangement reactions. One particularly striking example of inherent dynamic behavior was observed in host-guest complex [NH4+o-Me2-2.1.1], which spontaneously rearranged into the larger and thermodynamically more stable complex [NH4+o-Me2-2.2.1], even though this process led to the formation of poor host o-Me2-1.1.1 as a consequence of the excess of one subcomponent (diethylene glycol; "1" in our nomenclature). These inherently adaptive host-guest networks represent a unique platform for exploring the interrelationship between kinetic and thermodynamic stability. For instance, as a result of optimal NH4+ binding, complex [NH4+o-Me2-2.2.1] was found to be thermodynamically stable (negligible intermolecular rearrangements over weeks), whereas computational studies indicate that the compound is far from kinetically stable (intramolecular rearrangements)

Revealing solvent-dependent folding behaviour of mycolic acids from Mycobacterium tuberculosis by advanced simulation analysis

Mycobacterium tuberculosis remains a persistent pathogen, partly due to its lipid rich cell wall, of which mycolic acids (MAs) are a major component. The fluidity and conformational flexibilities of different MAs in the bacterial cell wall significantly influence its properties, function, and observed pathogenicity; thus, a proper conformational description of different MAs in different environments (e.g., in vacuum, in solution, in monolayers) can inform about their potential role in the complex setup of the bacterial cell wall. Previously, we have shown that molecular dynamics (MD) simulations of MA folding in vacuo can be used to characterize MA conformers in seven groupings relating to bending at the functional groups (W, U and Z-conformations). Providing a new OPLS-based forcefield parameterization for the critical cyclopropyl group of MAs and extensive simulations in explicit solvents (TIP4P water, hexane), we now present a more complete picture of MA folding properties together with improved simulation analysis techniques. We show that the ‘WUZ’ distance-based analysis can be used to pinpoint conformers with hairpin bends at the functional groups, with these conformers constituting only a fraction of accessible conformations. Applying principle component analysis (PCA) and refinement using free energy landscapes (FELs), we are able to discriminate a complete and unique set of conformational preferences for representative alpha-, methoxy- and keto-MAs, with overall preference for folded conformations. A control backbone-MA without any mero-chain functional groups showed significantly less folding in the mero-chain, confirming the role of functionalization in directing folding. Keto-MA showed the highest percentage of WUZ-type conformations and, in particular, a tendency to fold at its alpha-methyl trans-cyclopropane group, in agreement with results from Villeneuve et al. MAs demonstrate similar folding in vacuum and water, with a majority of folded conformations around the W-conformation, although the molecules are more flexible in vacuum than in water. Exchange between conformations, with a disperse distribution that includes unfolded conformers, is common in hexane for all MAs, although with more organization for Keto-MA. Globular, folded conformations are newly defined and may be specifically relevant in biofilms

Activation of Glycyl Radical Enzymes─Multiscale Modeling Insights into Catalysis and Radical Control in a Pyruvate Formate-Lyase-Activating Enzyme

Pyruvate formate-lyase (PFL) is a glycyl radical enzyme (GRE) playing a pivotal role in the metabolism of strict and facultative anaerobes. Its activation is carried out by a PFL-activating enzyme, a member of the radical S-adenosylmethionine (rSAM) superfamily of metalloenzymes, which introduces a glycyl radical into the Gly radical domain of PFL. The activation mechanism is still not fully understood and is structurally based on a complex with a short model peptide of PFL. Here, we present extensive molecular dynamics simulations in combination with quantum mechanics/molecular mechanics (QM/MM)-based kinetic and thermodynamic reaction evaluations of a more complete activation model comprising the 49 amino acid long C-terminus region of PFL. We reveal the benefits and pitfalls of the current activation model, providing evidence that the bound peptide conformation does not resemble the bound protein-protein complex conformation with PFL, with implications for the activation process. Substitution of the central glycine with (S)- and (R)-alanine showed excellent binding of (R)-alanine over unstable binding of (S)-alanine. Radical stabilization calculations indicate that a higher radical stability of the glycyl radical might not be the sole origin of the evolutionary development of GREs. QM/MM-derived radical formation kinetics further demonstrate feasible activation barriers for both peptide and C-terminus activation, demonstrating why the crystalized model peptide system is an excellent inhibitory system for natural activation. This new evidence supports the theory that GREs converged on glycyl radical formation due to the better conformational accessibility of the glycine radical loop, rather than the highest radical stability of the formed peptide radicals

Computationally driven design of an artificial metalloenzyme using supramolecular anchoring strategies of iridium complexes to alcohol dehydrogenase

Artificial metalloenzymes (ArMs) confer non-biological reactivities to biomolecules, whilst taking advantage of the biomolecular architecture in terms of their selectivity and renewable origin. In particular, the design of ArMs by the supramolecular anchoring of metal catalysts to protein hosts provides flexible and easy to optimise systems. The use of cofactor dependent enzymes as hosts gives the advantage of both a (hydrophobic) binding site for the substrate and a cofactor pocket to accommodate the catalyst. Here, we present a computationally driven design approach of ArMs for the transfer hydrogenation reaction of cyclic imines, starting from the NADP+-dependent alcohol dehydrogenase from Thermoanaerobacter brockii (TbADH). We tested and developed a molecular docking workflow to define and optimize iridium catalysts with high affinity for the cofactor binding site of TbADH. The workflow uses high throughput docking of compound libraries to identify key structural motifs for high affinity, followed by higher accuracy docking methods on smaller, focused ligand and catalyst libraries. Iridium sulfonamide catalysts were selected and synthesised, containing either a triol, a furane, or a carboxylic acid to provide the interaction with the cofactor binding pocket. IC50 values of the resulting complexes during TbADH-catalysed alcohol oxidation were determined by competition experiments and were between 4.410 mM and 0.052 mM, demonstrating the affinity of the iridium complexes for either the substrate or the cofactor binding pocket of TbADH. The catalytic activity of the free iridium complexes in solution showed a maximal turnover number (TON) of 90 for the reduction of salsolidine by the triol-functionalised iridium catalyst, whilst in the presence of TbADH, only the iridium catalyst with the triol anchoring functionality showed activity for the same reaction (TON of 36 after 24 h). The observation that the artificial metalloenzymes developed here lacked stereoselectivity demonstrates the need for the further investigation and optimisation of the ArM. Our results serve as a starting point for the design of robust artificial metalloenzymes, exploiting supramolecular anchoring to natural NAD(P)H binding pockets

Self-Assembly, Adaptive Response, and in,out-Stereoisomerism of Large Orthoformate Cryptands

© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. We report on triethylene glycol-based orthoformate cryptands, which adapt their bridgehead configurations in response to metal templates and intramolecular hydrogen bonding in a complex manner. In contrast to smaller 1.1.1-orthoformate cryptands, the inversion from out,out-2.2.2 to in,in-2.2.2 occurs spontaneously by thermal homeomorphic isomerization, i. e., without bond breakage. The global thermodynamic minimum of the entire network, which includes an unprecedented third isomer (in,out-2.2.2), could only be reached under conditions that facilitate dynamic covalent exchange. Both inversion processes were studied in detail, including DFT calculations and MD simulations, which were particularly helpful for explaining differences between equilibrium compositions in solvents chloroform and acetonitrile. Unexpectedly, the system could be driven to the in,out-2.2.2 state by using a metal template with a size mismatch with respect to the out,out-2.2.2 cage

Radical Stabilization Energies for Enzyme Engineering: Tackling the Substrate Scope of the Radical Enzyme QueE

© 2019 American Chemical Society. Experimental assessment of catalytic reaction mechanisms and profiles of radical enzymes can be severely challenging due to the reactive nature of the intermediates and sensitivity of cofactors such as iron-sulfur clusters. Here, we present an enzyme-directed computational methodology for the assessment of thermodynamic reaction profiles and screening for radical stabilization energies (RSEs) for the assessment of catalytic turnovers in radical enzymes. We have applied this new screening method to the radical S-adenosylmethione enzyme 7-carboxy-7-deazaguanine synthase (QueE), following a detailed molecular dynamics (MD) analysis that clarifies the role of both specific enzyme residues and bound Mg2+, Ca2+, or Na+. The MD simulations provided the basis for a statistical approach to sample different conformational outcomes. RSE calculation at the M06-2X/6-31+G∗ level of theory provided the most computationally cost-effective assessment of enzyme-based energies, facilitated by an initial triage using semiempirical methods. The impact of intermolecular interactions on RSE was clearly established, and application to the assessment of potential alternative substrates (focusing on radical clock type rearrangements) proposes a selection of carbon-substituted analogues that would react to afford cyclopropylcarbinyl radical intermediates as candidates for catalytic turnover by QueE

Effect of Oriented Electric Fields on Biologically Relevant Iron–Sulfur Clusters: Tuning Redox Reactivity for Catalysis

Enzyme-based iron-sulfur clusters, exemplified in families such as hydrogenases, nitrogenases, and radical S-Adenosylmethionine enzymes, feature in many essential biological processes. The functionality of biological iron-sulfur clusters extends beyond simple electron transfer, relying primarily on the redox activity of the clusters, with a remarkable diversity for different enzymes. The active-site structure and the electrostatic environment in which the cluster resides direct this redox reactivity. Oriented electric fields in enzymatic active sites can be significantly strong, and understanding the extent of their effect on iron-sulfur cluster reactivity can inform first steps toward rationally engineering their reactivity. An extensive systematic density functional theory-based screening approach using OPBE/TZP has afforded a simple electric field-effect representation. The results demonstrate that the orientation of an external electric field of strength 28.8 MV cm-1 at the center of the cluster can have a significant effect on its relative stability in the order of 35 kJ mol-1. This shows clear implications for the reactivity of iron-sulfur clusters in enzymes. The results also demonstrate that the orientation of the electric field can alter the most stable broken-symmetry state, which further has implications on the directionality of initiated electron-Transfer reactions. These insights open the path for manipulating the enzymatic redox reactivity of iron-sulfur cluster-containing enzymes by rationally engineering oriented electric fields within the enzymes

Dynamic Covalent Self‐Assembly of Chloride‐ and Ion‐Pair‐Templated Cryptates

While supramolecular hosts capable of binding and transporting anions and ion pairs are now widely available, self-assembled architectures are still rare, even though they offer an inherent mechanism for the release of the guest ion(s). In this work, we report the dynamic covalent self-assembly of tripodal, urea-based anion cryptates that are held together by two orthoester bridgeheads. These hosts exhibit affinity for anions such as Cl−, Br− or I− in the moderate range that is typically advantageous for applications in membrane transport. In unprecedented experiments, we were able to dissociate the Cs⋅Cl ion pair by simultaneously assembling suitably sized orthoester hosts around the Cs+ and the Cl− ion