Synthesis and application of supramolecular catalysts in the oxidation of unactivated C(spᵌ)-H bonds

English Abstract Over recent decades, impressive progress was achieved in the selective functionalization of unactivated aliphatic C(sp3)-H bonds. Considering the inert character and ubiquity of these bonds, they are considered among the most challenging ones to selectively convert. However, at the same time, the selective reaction of C-H bonds, especially in the form of late-stage functionalizations, has great potential and is very attractive from an economic point of view. Synthetic chemists have established different approaches to achieve good activity and selectivity in C-H oxidations, including directed, undirected, and supramolecular strategies. To us, the supramolecular approach, in which binding between the catalyst and the substrate is achieved via multiple weak forces, was of great interest in particular. In the first part of this thesis, supramolecular catalysts consisting of a glycoluril-based molecular tweezer and well-established M(pdp) or M(mcp) catalysts were prepared. In total, five different versions were synthesized in ten steps each. In all cases, preferential oxidation of decylammonium at the intrinsically deactivated positions close to the ammonium salt was observed (C3/C4). However, slightly different results were obtained depending on the catalyst used. The best results regarding the selectivity for the deactivated positions C3/C4 were achieved with Fe(pdp)Twe. Other than decylammonium, a handful of other linear aliphatic ammonium salts, two ammonium substrates possessing a terpene substitution pattern, and two cyclohexane derivatives were investigated in the oxidation with Fe(pdp)Twe. Finally, we also studied the oxidation behavior of our catalyst in different solvents (MeCN, TFE, HFIP). In the second part, the non-heme Mn(mcp) catalyst was functionalized with two distinct cyclophane moieties, leading to Mn(mcp)CY3 and Mn(mcp)CY5. The catalysts were both made in a seven-step synthesis. We hypothesized that selective oxidations of aliphatic substrates lacking any functional handle might be possible in TFE and HFIP, due to a solvophobic effect. Unfortunately, we observed the same oxidation pattern with our supramolecular catalysts as with the unfunctionalized Mn(mcp) for all investigated substrates. However, it should be noted that the work on this concept is ongoing and that promising first results have been observed by another group member with different macrocycles

Inhibitor Synthesis and Biophysical Characterization of Protein–Ligand–Solvent Interactions An Analysis of the Thermodynamics and Kinetics of Ligand Binding to Thermolysin

In the pre-clinical development stages of most drug design campaigns, the equilibrium binding affinity of a prospective lead candidate, in the form of an IC50, Kd or ΔG° value, is the most commonly employed benchmark parameter for its effectiveness as a putative drug. Hydrogen bonding, van der Waals and electrostatic interactions, as well as hydrophobic effects are among the most prominent factors that contribute to binding. In structure based design approaches, these interactions can routinely be linked to a structural motif of a drug molecule, which can greatly assist in the construction of compounds with a desired set of properties. Equilibrium binding affinity can also be expressed in terms of kinetics, were the steady-state constant Kd is defined as the ratio of the rate constants of dissociation (kd) and association (ka). The thermodynamic expression ΔG° can be subdivided into an enthalpic (ΔH°) and an entropic (–TΔS°) term. In either case, the molecular mechanisms that define the kinetics of binding or the compensation of enthalpic and entropic contributions are not fully understood. The goal of this dissertation is the in-depth investigation of the molecular processes that drive protein–ligand interactions. A special focus is set on the partitioning of thermodynamic and kinetic parameters into their respective microscopic elements. For this, the metalloprotease thermolysin (TLN) is used as a model system. This protein is well characterized and represents a robust system with excellent crystallographic properties and a thoroughly documented inhibitor class. The first publication (Chapter 2) presents an improved strategy for the synthesis and purification of phosphonamidate peptides that are known as potent inhibitors of TLN. Due to the inherent instability of the phosphorous–nitrogen bond, the introduction of polar functional groups into the inhibitor scaffold is quite challenging. Here, a synthetic strategy is presented that minimizes the amount of hydrolysis during peptide coupling, deprotection and purification through the use of an allyl-based protection system and a solid-phase extraction (SPE) protocol for the final purification step. This allows the synthesis of highly pure TLN inhibitors incorporating a variety of functional groups for use in biophysical experiments. In the second publication (Chapter 3), a strategy for the design of inhibitors is highlighted, which relies on the targeted design of water networks that are formed around a protein–ligand complex. Based on information from a previous study, the shape of a hydrophobic portion of a TLN ligand is altered in a way that allows a beneficial stabilization of water molecules in the first solvation layer of the complex. Supported by molecular dynamics simulations, a series of diastereomeric inhibitors is synthesized and the binding process is characterized by X-ray crystallography, isothermal titration calorimetry (ITC) and surface plasmon resonance spectroscopy (SPR). The optimization of the hydrophobic P2’ moiety results in a 50-fold affinity enhancement compared to the original methyl substituted ligand. This improvement is mainly driven by a favorable enthalpic term that originates from the stabilization of water polygons in the solvation shell. In the follow-up study in Chapter 4, the binding signature of a series of inhibitors that place a charged and polar moiety in the solvent exposed S2’ pocket of TLN is investigated. Here, a partially hydrated ammonium group is gradually retracted deeper into the hydrophobic protein environment. From the crystal structures it is evident that the polar ligands do not recruit an increased amount of water molecules into their solvation layer when compared to related analogues that feature a purely aliphatic residue at the solvent interface. The penalty for the partial desolvation of the charged functional group, in combination with the lack of a strongly ordered water network, results in a severe affinity decrease that is driven by an unfavorable enthalpic term. The deep, hydrophobic S1’ pocket of TLN determines the substrate specificity of the protease and is commonly addressed by high affinity inhibitors. Experimental evidence from previous studies suggests, however, that this apolar crevice is only poorly solvated in the absence of an interaction partner. With the study in Chapter 5, an attempt for the experimental analysis of the hydration state of the S1’ pocket is presented. For this, a special inhibitor is designed that transforms the protein pocket into a cavity, while simultaneously providing enough empty space for the accommodation of several water molecules. A detailed analysis of an experimentally phased electron density map reveals that the cavity remains completely unsolvated and thus, vacuous. As an intriguing prospect for the exploitation of such poorly hydrated protein pockets in drug design, the placement of an iso-pentyl moiety in the ligand’s P1’ position results in a dramatic, enthalpically driven gain in affinity by a factor of 41 000. With a detailed structural analysis of a series of chemically diverse TLN inhibitors, the kinetics of the protein–ligand binding process are investigated in Chapter 6. From the SPR derived kinetic information, it becomes apparent that the nature of the functional group in the P2’ position of a thermolysin inhibitor has a significant impact on its dissociation kinetics. This property can be linked to the interaction between the respective functionality of a ligand and Asn112, a residue that lines the active site of the protease and is commonly believed to align a substrate for proteolytic cleavage. This residue undergoes a significant conformational change when the protein transitions from its closed state to its open form, from which a ligand is released. Interference with this retrograde induced-fit mechanism through strong hydrogen-bonding interactions to an inhibitor results in a pronounced deceleration of the dissociation process. The case of the known inhibitor ZFPLA demonstrates that a further restriction of the rotation of Asn112 by a steric barrier in the P1 position of a ligand, can reduce the rate constant of dissociation by a factor of 74 000. Fragment-based lead discovery has become a popular method for the generation of prospective drug molecules. The weak affinity of fragments and the necessity for high concentrations, however, can result in false-positive signals from the initial binding assays that routinely plague fragment-based screening. The pursuit of such a “red herring” can lead to a significant loss of time and resources. In Chapter 7, a molecule that emerged as one of the most potent binders from an elaborate fragment screen against the aspartic protease endothiapepsin is identified as a false-positive. Detailed crystallographic, HPLC and MS experiments reveal that the affinity detected in multiple assays can in fact be attributed to another compound. This entity is formed from the initially employed molecule in a reaction cascade that results in a major rearrangement of its heterocyclic core structure. Supported by quantum chemical calculations and NMR experiments, a mechanism for the formation of the elusive compound is proposed and its binding mode analyzed by X-ray crystallography

Computer simulations on oxidative stress-induced reactions in SARS-CoV-2 spike glycoprotein: a multi-scale approach

Oxidative stress, which occurs when an organism is exposed to an adverse stimulus that results in a misbalance of antioxidant and pro-oxidants species, is the common denominator of diseases considered as a risk factor for SARS-CoV-2 lethality. Indeed, reactive oxygen species caused by oxidative stress have been related to many virus pathogenicity. In this work, simulations have been performed on the receptor binding domain of SARS-CoV-2 spike glycoprotein to study what residues are more susceptible to be attacked by ·OH, which is one of the most reactive radicals associated to oxidative stress. The results indicate that isoleucine (ILE) probably plays a crucial role in modification processes driven by radicals. Accordingly, QM/MM-MD simulations have been conducted to study both the ·OH-mediated hydrogen abstraction of ILE residues and the induced modification of the resulting ILE radical through hydroxylation or nitrosylation reactions. All in all, in silico studies show the importance of the chemical environment triggered by oxidative stress on the modifications of the virus, which is expected to help for foreseeing the identification or development of antioxidants as therapeutic drugs. Graphic abstrac

Design of Novel Anticancer Drugs Utilizing Busulfan for Optimizing Pharmacological Properties and Pattern Recognition Techniques for Elucidation of Clinical Efficacy

Chronic myelogenous leukemia (CML) is a disorder in which an excessive number of blood stem cells develop into the white blood cell group called granulocytes. The anticancer drug Busulfan is a cell cycle non-specific alkylating agent which is utilized to maintain white blood cell counts below 15000 cells/microliter. The side effects induced by busulfan are significant and affirms the intimation for new drug constructs. Fifteen analogous compounds were generated from the molecular structure of busulfan . These compounds retain the double methanesulfonate functional groups descriptive of this class of alkylating anticancer drugs. However, the carbon chain substituent separating the methanesulfonate is highly modified in order to allow significant changes in drug properties that may produce favorable characteristics that benefit clinical application. Important properties such as Log P, polar surface area, formula weight, molecular volume, Log BB, and violations of the Rule of 5 were determined to ascertain similarities and differences. All fifteen analog compounds retained zero violations of the Rule of 5, which suggests favorable properties for useful drug availability. Values of Log BB and BB remained the same throughout at -0.841 and 0.144, respectively. In addition, values of polar surface area and number of oxygens and nitrogens remained the same throughout at 86.752 A3 and 6, respectively. However, formula weight, number of atoms, number of rotatable bonds varied significantly with Log P varying across a broad range (-0.428 to 2.734). The variance in Log P within this group of methane sulfonate compounds allows new and potentially highly beneficial pharmacological properties for clinical application. Pattern recognition techniques such as cluster analysis, non-metric multidimensional scaling, discriminant analysis, and K-means cluster analysis discerned underlying relationships within this group of anticancer drugs and to the parent busulfan. This work shows that pattern recognition methods combined with molecular modeling can discover and elucidate novel drug designs for the clinical treatment of CML

Analyses of All Possible Point Mutations within a Protein Reveals Relationships between Function and Experimental Fitness: A Dissertation

The primary amino acid sequence of a protein governs its specific cellular functions. Since the cracking of the genetic code in the late 1950’s, it has been possible to predict the amino acid sequence of a given protein from the DNA sequence of a gene. Nevertheless, the ability to predict a protein’s function from its primary sequence remains a great challenge in biology. In order to address this problem, we combined recent advances in next generation sequencing technologies with systematic mutagenesis strategies to assess the function of thousands of protein variants in a single experiment. Using this strategy, my dissertation describes the effects of most possible single point mutants in the multifunctional Ubiquitin protein in yeast. The effects of these mutants on the essential activation of ubiquitin by the ubiquitin activating protein (E1, Uba1p) as well as their effects on overall yeast growth were measured. Ubiquitin mutants defective for E1 activation were found to correlate with growth defects, although in a non-linear fashion. Further examination of select point mutants indicated that E1 activation deficiencies predict downstream defects in Ubiquitin function, resulting in the observed growth phenotypes. These results indicate that there may be selective pressure for the activity of the E1enzyme to selectively activate ubiquitin protein variants that do not result in functional downstream defects. Additionally, I will describe the use of similar techniques to discover drug resistant mutants of the oncogenic protein BRAFV600E in human melanoma cell lines as an example of the widespread applicability of our strategy for addressing the relationship between protein function and biological fitness

Theoretical and Experimental Investigation of Non-Covalent Interactions in S-Nitrosothiols and Thio-Carboxylic Acids

S-Nitrosothiols (RSNOs) are ubiquitous biomolecules whose chemistry is tightly controlled in unknown. In this work, we demonstrate, using high-level ab initio and DFT calculations, the ability of RSNOs to participate in intermolecular interactions with electron pair donors/Lewis bases (LBs) via a σ-hole, a region of positive electrostatic potential on the molecular surface at the extension of the N–S bond. Analysis of the nature of the intermolecular interactions in σ-hole bound RSNO-LB complexes shows the dominant role of electrostatic and dispersion interactions. Importantly, σ-hole binding is able to modulate the properties of RSNOs by changing the balance between two chemically opposite (antagonistic) resonance components, R–S+=N–O– (D) and R–S–/NO+ (I), which are, in addition to the main resonance structure R–S–N=O, necessary to describe the unusual electronic structure of RSNOs. σ-Hole binding at the sulfur atom of RSNO promotes the resonance structure D and reduces the resonance structure I, thereby stabilizing the weak N–S bond and making the sulfur atom more electrophilic. On the other hand, increasing the D-character of RSNO by other means (e.g. via N- or O-coordination of a Lewis acid) enhances the σ-hole bonding. Our calculations suggest that in the protein environment a combination of σ-hole bonding of a negatively charged amino acid sidechain at the sulfur atom and N- or O-coordination of a positively charged amino acid sidechain is expected to have a profound effect on the RSNO electronic structure and reactivity.Additionally, protein functionalities are highly dependent on the pKa value of their amino acids. The sequence of deprotonation in thiol containing amino acid side chains determine their nucleophilicity and reactivity. Cysteine as a sulfur containing amino acid is actively involved in the oxidases, reductases and disulfide isomerases through thiol-disulfide exchange reactions. In this study, we investigated the sequence of deprotonation between thiol and carboxylic acid as two active and determining groups in protein structures. This study have been performed in two different types of molecules. First, thiosalicylic acid was considered as an aromatic geminal bifunctional model molecule. The sequence of ionization was analyzed both computationally through DFT calculations and experimentally through UV-vis, NMR and X-ray diffraction measurements. Our experimental analysis were in agreement with our computational analysis confirming the fact that the sequence of deprotonation in bifunctional aromatic thiol-carboxylic acid is not following the classic rules of ionization. Second, we extended our experiment in to aliphatic molecules with vicinal and geminal thiol-carboxylic acid groups. In this part computational studies illustrate untraditional fashion of deprotonation which was incompatible with experimental X-ray diffraction measurements

Natural Organic Matter : From Contaminant Interactions to Structural Aspects

Natural organic matter (NOM) is one of the most abundant forms of organic matter on the earth\u27s surface. Because of its agricultural and environmental implications, it is essential to have a basic understanding of the structure and composition of NOM. This dissertation is an attempt to look how the studies of soil / sediment interaction with hydrophobic organic contaminants contribute to understanding the structure of NOM. The potential of polyaromatic hydrocarbon (PAH) and its metabolite to form bound residues was quantified. The formation of bound residues by naphthalene and its metabolite, cis-na phthalene-1,2-dihydrodiol, in a sediment, a silty loam soil and a peat under both sterile and nonsterile conditions was examined. The results showed that bound residue formation is low for naphthalene and between 5 and 20 times higher for the metabolite. The formation of naphthalene bound residues is probably due mainly to noncovalent interactions between the contaminant and natural organic matter. The formation of the diol bound residue is primarily the result of the covalent binding of the contaminant molecules to parts of organic matter. Noncovalent interactions between NOM and PAHs were also investigated. This investigation proceeded through thermodynamical analysis of the sorption of PAHs on whole and lipid-extracted geosorbents. Removal of the lipids was found to increase the sorption capacity of the samples as well as the exothermicity of the process. The entropy changes were small and positive for the whole geosorbent samples, but smaller and more negative when the lipids were removed. This indicated that the interaction of PAHs with soils and sediments in the absence of extractable lipids is stronger and the mechanisms involved may be different, changing from a partitioning-like mechanism to specific adsorption. Because of the competition between lipids and PAHs for the same sorption sites, the lipids can be viewed as an implicit sorbate . This conclusion points to an alternative explanation for sorption behavior of NOM that does not make any specific assumptions about the structure of NOM. The final part of the dissertation deals with the glass transition of NOM, considered proof of a polymeric nature, that have been inferred from sorption studies. Both thermal analysis and variable temperature solid-state NMR were unable to give a definitive answer concerning the existence of a geosorbent glass transition. However, the data do show the presence of a thermal event in geosorbent. This was identified as being due to the melting of the crystallites present in the geosorbents and not to a glass transition. Based on the findings of this research, this dissertation has shown that NOM models derived exclusively from sorption studies should be very cautiously applied until supported by direct experimental verification