Simulating Self-Assembly of Organosulfur Species on Gold Nanoparticles

This Thesis aims to establish an accurate but computationally effective method for simulating self-assembly of organosulfurs on gold nanoparticles (AuNPs), a process resulting in their functionalisation. A second gold rush is currently rekindling chemists’ interest in the synthesis of novel functionalised AuNPs: these can bear the most chemically diverse functional groups, making them employable in a wide variety of applications, from optoelectronics to catalysis. Some aspects of self-assembly remain experimentally unclear at the mechanistic and electronic levels: achieving its accurate reproduction in silico would indeed represent an important contribution in the synthesis of functionalised AuNPs. This task, however, has so far proven difficult to achieve. In this work, I set out and review four fundamental challenges facing the computational chemist aiming to simulate self-assembly, and describe the strategy chosen to overcome them, using thiols (RSH) as the reference organosulfur. These challenges involve proper reproduction of: I) gold’s relativistic effects and aurophilicity; II) the extensive surface reconstruction occurring upon self-assembly, with formation of RS–Au–SR staples and hydrogen loss; III) the large scale ligands involved in the process and their interactions; and IV) the fluctuating solvent environment in which it occurs. Confined to the AuNP core and RSH headgroups, challenges I and II involve complex electronic properties and entail electronic change, with bonds being cleaved (S–H) and reformed (S–Au, possibly H–H): overcoming them requires explicit simulation of electrons with a QM method (DFT). Challenges III and IV involve the entire RSH-AuNP system, including RSH tails of typically ∼10^2 atoms: QM methods become impracticable at these system sizes, and a less costly classical forcefield treatment (MM) is necessary in this case, at least in part. The work presented here then proceeds towards the stated aim by attempting to resolve each of these challenges I–IV. The eventually devised solution proposes a combination of classical molecular dynamics (MD), followed by the hybrid QM/MM method ONIOM, which allows to combine the ‘best of the QM and MM worlds’ and is well established for other systems. To overcome challenge I, various effective core potentials (ECPs); basis sets; and density functionals are evaluated based on their ability to predict properties and geometries of several pristine AuNPs. These properties and geometries are either derived experimentally, or from high-level ab initio calculations. The chosen QM method PBE/LANL2DZ is then further tested on various systems, assessing its ability (challenge II) to reproduce hydrogen loss and staple formation. Upon proposing to tackle challenge III using ONIOM (with the OPLS-AA forcefield for the MM part), the method’s performance is first compared to that of full QM (PBE/LANL2DZ) in terms of accuracy and efficiency, and in a variety of contexts, including on AuNPs featuring a 38-atom gold core. Once these calculations confirm the considerable time gains afforded by the introduction of ONIOM, I then demonstrate its full applicability in the optimisation of a large, experimentally plausible functionalised AuNP. Finally, I propose to tackle challenge IV by introducing a classical MD simulation stage to precede QM/MM optimisation. As a test, MD is used to generate statistically significant sets of 8-atom AuNPs coated with alkylthiols of different chain lengths, which are then optimised, thereby successfully reproducing the early stages of reconstruction. I then conclude by successfully testing this MD + ONIOM approach on two much larger functionalised AuNPs, having 20-atom gold cores and sixteen or seventeen 64-atom ligands. My Thesis highlights both the strengths and limitations of the ONIOM approach in simulating such a complex process as organosulfur self-assembly on AuNPs. Nonetheless, the chosen MD + ONIOM strategy can indeed reproduce key aspects of self-assembly with increased CPU-efficiency, and, importantly, makes electronically plausible predictions: it therefore represents a viable route for the in silico investigation of this process, and an encouraging fulfilment of my initial aim.Open Acces

Unpicking the Cause of Stereoselectivity in Actinorhodin Ketoreductase Variants with Atomistic Simulations

Ketoreductase enzymes (KRs) with a high degree of regio- and stereoselectivity are useful biocatalysts for the production of small, specific chiral alcohols from achiral ketones. Actinorhodin KR (actKR), part of a type II polyketide synthase involved in the biosynthesis of the antibiotic actinorhodin, can also turn over small ketones. In vitro studies assessing stereocontrol in actKR have found that, in the “reverse” direction, the wild-type (WT) enzyme’s mild preference for S-α-tetralol is enhanced by certain mutations (e.g., P94L) and entirely reversed by others (e.g., V151L) in favor of R-α-tetralol. Here, we employ computationally cost-effective atomistic simulations to rationalize these trends in WT, P94L, and V151L actKR using trans-1-decalone (1) as the model substrate. Three potential factors (FI–FIII) are investigated: frequency of pro-R vs pro-S reactive poses (FI) is assessed with classical molecular dynamics (MD), binding affinity of pro-R vs pro-S orientations (FII) is compared using the binding free energy method MM/PBSA, and differences in reaction barriers toward trans-1-decalol (FIII) are assessed by hybrid semiempirical quantum/classical (QM/MM) MD simulations with umbrella sampling, benchmarked with density functional theory. No single factor is found to dominate stereocontrol: FI largely determines the selectivity of V151L actKR, whereas FIII is more dominant in the case of P94L. It is also found that formation of S-trans-1-decalol or R-trans-1-decalol mainly arises from the reduction of the trans-1-decalone enantiomers (4aS,8aR)-1 or (4aR,8aS)-1, respectively. Our work highlights the complexity of enzyme stereoselectivity as well as the usefulness of atomistic simulations to aid the design of stereoselective biocatalysts

Path to Actinorhodin:Regio- and Stereoselective Ketone Reduction by a Type II Polyketide Ketoreductase Revealed in Atomistic Detail

In type II polyketide synthases (PKSs), which typically biosynthesize several antibiotic and antitumor compounds, the substrate is a growing polyketide chain, shuttled between individual PKS enzymes, while covalently tethered to an acyl carrier protein (ACP): this requires the ACP interacting with a series of different enzymes in succession. During biosynthesis of the antibiotic actinorhodin, produced by Streptomyces coelicolor, one such key binding event is between an ACP carrying a 16-carbon octaketide chain (actACP) and a ketoreductase (actKR). Once the octaketide is bound inside actKR, it is likely cyclized between C7 and C12 and regioselective reduction of the ketone at C9 occurs: how these elegant chemical and conformational changes are controlled is not yet known. Here, we perform protein-protein docking, protein NMR, and extensive molecular dynamics simulations to reveal a probable mode of association between actACP and actKR; we obtain and analyze a detailed model of the C7-C12-cyclized octaketide within the actKR active site; and we confirm this model through multiscale (QM/MM) reaction simulations of the key ketoreduction step. Molecular dynamics simulations show that the most thermodynamically stable cyclized octaketide isomer (7R,12R) also gives rise to the most reaction competent conformations for ketoreduction. Subsequent reaction simulations show that ketoreduction is stereoselective as well as regioselective, resulting in an S-alcohol. Our simulations further indicate several conserved residues that may be involved in selectivity of C7-12 cyclization and C9 ketoreduction. Detailed insights obtained on ACP-based substrate presentation in type II PKSs can help design ACP-ketoreductase systems with altered regio- or stereoselectivity

A Fe2+-dependent self-inhibited state influences the druggability of human collagen lysyl hydroxylase (LH/PLOD) enzymes

Multifunctional human collagen lysyl hydroxylase (LH/PLOD) enzymes catalyze post-translational hydroxylation and subsequent glycosylation of collagens, enabling their maturation and supramolecular organization in the extracellular matrix (ECM). Recently, the overexpression of LH/PLODs in the tumor microenvironment results in abnormal accumulation of these collagen post-translational modifications, which has been correlated with increased metastatic progression of a wide variety of solid tumors. These observations make LH/PLODs excellent candidates for prospective treatment of aggressive cancers. The recent years have witnessed significant research efforts to facilitate drug discovery on LH/PLODs, including molecular structure characterizations and development of reliable high-throughput enzymatic assays. Using a combination of biochemistry and in silico studies, we characterized the dual role of Fe2+ as simultaneous cofactor and inhibitor of lysyl hydroxylase activity and studied the effect of a promiscuous Fe2+ chelating agent, 2,2'-bipyridil, broadly considered a lysyl hydroxylase inhibitor. We found that at low concentrations, 2,2'-bipyridil unexpectedly enhances the LH enzymatic activity by reducing the inhibitory effect of excess Fe2+. Together, our results show a fine balance between Fe2+-dependent enzymatic activity and Fe2+-induced self-inhibited states, highlighting exquisite differences between LH/PLODs and related Fe2+, 2-oxoglutarate dioxygenases and suggesting that conventional structure-based approaches may not be suited for successful inhibitor development. These insights address outstanding questions regarding druggability of LH/PLOD lysyl hydroxylase catalytic site and provide a solid ground for upcoming drug discovery and screening campaigns

Electronic and relativistic contributions to ion-pairing in polyoxometalate model systems

Ion pairs and solubility related to ion-pairing in water influence many processes in nature and in synthesis including efficient drug delivery, contaminant transport in the environment, and self-assembly of materials in water. Ion pairs are difficult to observe spectroscopically because they generally do not persist unless extreme solution conditions are applied. Here we demonstrate two advanced techniques coupled with computational studies that quantify the persistence of ion pairs in simple solutions and offer explanations for observed solubility trends. The system of study, ([(CH3)4N]+,Cs)8[M6O19] (M = Nb,Ta), is a set of unique polyoxometalate salts whose water solubility increases with increasing ion-pairing, contrary to most ionic salts. The techniques employed to characterize Cs+ association with [M6O19]8− and related clusters in simple aqueous media are 133Cs NMR (nuclear magnetic resonance) quadrupolar relaxation rate and PDF (pair distribution function) from X-ray scattering. The NMR measurements consistently showed more extensive ion-pairing of Cs+ with the Ta-analogue than the Nb-analogue, although the electrostatics of the ions should be identical. Computational studies also ascertained more persistent Cs+–[Ta6O19] ion pairs than Cs+–[Nb6O19] ion pairs, and bond energy decomposition analyses determined relativistic effects to be the differentiating factor between the two. These distinctions are likely responsible for many of the unexplained differences between aqueous Nb and Ta chemistry, while they are so similar in the solid state. The X-ray scattering studies show atomic level detail of this ion association that has not been prior observed, enabling confidence in our structures for calculations of Cs-cluster association energies. Moreover, detailed NMR studies allow quantification of the number of Cs+ associated with a single [Nb6O19]8− or [Ta6O19]8− anion which agrees with the PDF analyses

Chemical perturbation of oncogenic protein folding: from the prediction of locally unstable structures to the design of disruptors of Hsp90-Client interactions

Protein folding quality control in cells requires the activity of a class of proteins known as molecular chaperones. Heat shock protein‐90 (Hsp90), a multidomain ATP driven molecular machine, is a prime representative of this family of proteins. Interactions between Hsp90, its co‐chaperones, and client proteins have been shown to be important in facilitating the correct folding and activation of clients. Hsp90 levels and functions are elevated in tumor cells. Here, we computationally predict the regions on the native structures of clients c‐Abl, c‐Src, Cdk4, B‐Raf and Glucocorticoid Receptor, that have the highest probability of undergoing local unfolding, despite being ordered in their native structures. Such regions represent potential ideal interaction points with the Hsp90‐system. We synthesize mimics spanning these regions and confirm their interaction with partners of the Hsp90 complex (Hsp90, Cdc37 and Aha1) by Nuclear Magnetic Resonance (NMR). Designed mimics selectively disrupt the association of their respective clients with the Hsp90 machinery, leaving unrelated clients unperturbed and causing apoptosis in cancer cells. Overall, selective targeting of Hsp90 protein–protein interactions is achieved without causing indiscriminate degradation of all clients, setting the stage for the development of therapeutics based on specific chaperone:client perturbation

Designing molecular spanners to throw in the protein networks.

Proteins govern most aspects of cellular life and, through specific interfaces, are typically involved in intricate protein–protein interaction (PPI) networks and signaling pathways. Subtle up- or downregulation of key protein functions and PPIs results in disease; still, the preferred option to contrast the role of a protein in disease and healthy conditions alike remains its outright shutdown through orthosteric ligands that block its active site. Here, we explore subtler alternatives to modulate proteins and PPIs. Driven by a view of proteins as dynamic entities, we discuss ways to identify allosteric binding sites, which, when targeted by tailored ligands, can induce significant changes in the active site of a protein, and lead to agonistic or antagonistic effects. We also summarize the selective regulation of specific PPIs—either direct or allosteric—and show that effects can be stabilizing as well as destabilizing, depending on how the conformational equilibrium of a protein is shifted

Unpicking the Cause of Stereoselectivity in Actinorhodin Ketoreductase Variants with Atomistic Simulations

Ketoreductase enzymes (KRs) with a high degree of regio- and stereoselectivity are useful biocatalysts for the production of small, specific chiral alcohols from achiral ketones. Actinorhodin KR (actKR), part of a type II polyketide synthase involved in the biosynthesis of the antibiotic actinorhodin, can also turn over small ketones. In vitro studies assessing stereocontrol in actKR have found that, in the “reverse” direction, the wild-type (WT) enzyme’s mild preference for S-α-tetralol is enhanced in certain mutants (e.g. P94L); and entirely reversed in others (e.g. V151L) in favor of R-α-tetralol. Here, we employ efficient atomistic simulations to rationalize these trends in WT, P94L, and V151L actKR, using trans-1-decalone (1) as the model substrate. Three potential factors (FI-FIII) are investigated: frequency of pro-R vs. pro-S reactive poses (FI) is assessed with classical molecular dynamics (MD); binding affinity of pro-R vs. pro-S orientations (FII) is compared using the binding free energy method MM/PBSA; and differences in reaction barriers towards trans-1-decalol (FIII) are assessed by hybrid semiempirical quantum / classical (QM/MM) MD simulations with umbrella sampling, benchmarked with density functional theory. No single factor is found to dominate stereocontrol: FI largely determines the selectivity of V151L actKR, whereas FIII is more dominant in the case of P94L. It is also found that formation of S-trans-1-decalol or R-trans-1-decalol mainly arises from the reduction of the trans-1-decalone enantiomers (4aS,8aR)-1 or (4aR,8aS)-1, respectively. Our work highlights the complexity of enzyme stereoselectivity as well as the usefulness of atomistic simulations to aid the design of stereoselective biocatalysts

Simulating the Favorable Aggregation of Monolacunary Keggin Anions

We here present a series of classical molecular dynamics simulations (MD) on aqueous solutions of the salts Li₅AlW₁₂O₄₀ and Li₉AlW₁₁O₃₉, providing us with valuable insight on their aggregative behavior. Analysis of relative dipole moment orientation in pairs of aggregated [AlW₁₁O₃₉]^9– excludes that their large dipole moment is behind their greater propensity to aggregate. On the other hand, MD simulations of the aqueous Li⁺ salt of the fictitious [AlW₁₂O₄₀]^9–as high in charge as [AlW₁₁O₃₉]^9–, but lacking dipole moment and tetrahedral in shape like [AlW₁₂O₄₀]^5–reveal that it is in fact the higher negative charge itself that promotes aggregation, by allowing to recruit a higher number of Li⁺ countercations, which then act as an electrostatic glue. The lower charge on [AlW₁₂O₄₀]^5–, on the other hand, is not able to muster enough Li⁺ countercations for it to aggregate favorably