pH-dependent reaction triggering in PmHMGR crystals for time-resolved crystallography.

Serial crystallography and time-resolved data collection can readily be employed to investigate the catalytic mechanism of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl (HMG)-coenzyme-A (CoA) reductase (PmHMGR) by changing the environmental conditions in the crystal and so manipulating the reaction rate. This enzyme uses a complex mechanism to convert mevalonate to HMG-CoA using the co-substrate CoA and cofactor NAD+. The multi-step reaction mechanism involves an exchange of bound NAD+ and large conformational changes by a 50-residue subdomain. The enzymatic reaction can be run in both forward and reverse directions in solution and is catalytically active in the crystal for multiple reaction steps. Initially, the enzyme was found to be inactive in the crystal starting with bound mevalonate, CoA, and NAD+. To observe the reaction from this direction, we examined the effects of crystallization buffer constituents and pH on enzyme turnover, discovering a strong inhibition in the crystallization buffer and a controllable increase in enzyme turnover as a function of pH. The inhibition is dependent on ionic concentration of the crystallization precipitant ammonium sulfate but independent of its ionic composition. Crystallographic studies show that the observed inhibition only affects the oxidation of mevalonate but not the subsequent reactions of the intermediate mevaldehyde. Calculations of the pKa values for the enzyme active site residues suggest that the effect of pH on turnover is due to the changing protonation state of His381. We have now exploited the changes in ionic inhibition in combination with the pH-dependent increase in turnover as a novel approach for triggering the PmHMGR reaction in crystals and capturing information about its intermediate states along the reaction pathway.

Data-Efficient, Chemistry-Aware Machine Learning Predictions of Diels-Alder Reaction Outcomes.

The application of machine learning models to the prediction of reaction outcomes currently needs large and/or highly featurized data sets. We show that a chemistry-aware model, NERF, which mimics the bonding changes that occur during reactions, allows for highly accurate predictions of the outcomes of Diels-Alder reactions using a relatively small training set, with no pretraining and no additional features. We establish a diverse data set of 9537 intramolecular, hetero-, aromatic, and inverse electron demand Diels-Alder reactions. This data set is used to train a NERF model, and the performance is compared against state-of-the-art classification and generative machine learning models across low- and high-data regimes, with and without pretraining. The predictive accuracy (regio- and site selectivity in the major product) achieved by NERF exceeds 90% when as little as 40% of the data set is used for training. Another high-performing model, Chemformer, requires a larger training data set (>45%) and pretraining to reach 90% Top-1 accuracy. Accurate predictions of less-represented reaction subclasses, such as those involving heteroatomic or aromatic substrates, require higher percentages of training data. We also show how NERF can use small amounts of additional training data to quickly learn new systems and improve its overall understanding of reactivity. Synthetic chemists stand to benefit as this model can be rapidly expanded and tailored to areas of chemistry corresponding to the low-data regime.

Finding Relevant Retrosynthetic Disconnections for Stereocontrolled Reactions.

Machine learning-driven computer-aided synthesis planning (CASP) tools have become important tools for idea generation in the design of complex molecule synthesis but do not adequately address the stereochemical features of the target compounds. A novel approach to automated extraction of templates used in CASP that includes stereochemical information included in the US Patent and Trademark Office (USPTO) and an internal AstraZeneca database containing reactions from Reaxys, Pistachio, and AstraZeneca electronic lab notebooks is implemented in the freely available AiZynthFinder software. Three hundred sixty-seven templates covering reagent- and substrate-controlled as well as stereospecific reactions were extracted from the USPTO, while 20,724 templates were from the AstraZeneca database. The performance of these templates in multistep CASP is evaluated for 936 targets from the ChEMBL database and an in-house selection of 791 AZ designs. The potential and limitations are discussed for four case studies from ChEMBL and examples of FDA-approved drugs.

A Quantum-Guided Molecular Mechanics Force Field for the Ferrocene Scaffold.

Ferrocene derivatives have a wide range of applications, including as ligands in asymmetric catalysis, due to their chemical stability, rigid backbone, steric bulk, and ability to encode stereochemical information via planar chirality. Unfortunately, few of the available molecular mechanics force fields incorporate parameters for the accurate study of this important building block. Here, we present a MM3* force field for ferrocenyl ligands, which was generated using the quantum-guided molecular mechanics (Q2MM) method. Detailed validation by comparison to DFT calculations and crystal structures demonstrates the accuracy of the parameters and uncovers the physical origin of deviations through excess energy analysis. Combining the ferrocene force field with a force field for Pd-allyl complexes and comparing the crystal structures shows the compatibility with previously developed MM3* force fields. Finally, the ferrocene force field was combined with a previously published transition-state force field to predict the stereochemical outcomes of the aminations of Pd-allyl complexes with different amines and different chiral ferrocenyl ligands, with an R2 of ∼0.91 over 10 examples.

Strategic elements in computer-assisted retrosynthesis: A case study of the pupukeanane natural products.

Computer-assisted synthesis planning represents a growing area of research, especially for complex molecule synthesis. Here, we present a case study involving the pupukeanane natural products, which are complex, marine-derived, natural products with unique tricyclic scaffolds. Proposed routes to members of each skeletal class informed by pathways generated using the program Synthia™ are compared to previous syntheses of these molecules. In addition, novel synthesis routes are proposed to pupukeanane congeners that have not been prepared previously.

HSP90 inhibitors reduce cholesterol storage in Niemann-Pick type C1 mutant fibroblasts.

Niemann-Pick type C1 (NPC1) disease is a lysosomal lipid storage disorder caused by mutations of the NPC1 gene. More than 300 disease-associated mutations are reported in patients, resulting in abnormal accumulation of unesterified cholesterol, glycosphingolipids, and other lipids in late endosomes and lysosomes (LE/Ly) of many cell types. Previously, we showed that treatment of many different NPC1 mutant fibroblasts with histone deacetylase inhibitors resulted in reduction of cholesterol storage, and we found that this was associated with enhanced exit of the NPC1 protein from the endoplasmic reticulum and delivery to LE/Ly. This suggested that histone deacetylase inhibitors may work through changes in protein chaperones to enhance the folding of NPC1 mutants, allowing them to be delivered to LE/Ly. In this study, we evaluated the effect of several HSP90 inhibitors on NPC1I1061T skin fibroblasts. We found that HSP90 inhibition resulted in clearance of cholesterol from LE/Ly, and this was associated with enhanced delivery of the mutant NPC1I1061T protein to LE/Ly. We also observed that inhibition of HSP90 increased the expression of HSP70, and overexpression of HSP70 also reduced cholesterol storage in NPC1I1061T fibroblasts. However, we did not see correction of cholesterol storage by arimoclomol, a drug that is reported to increase HSP70 expression, at doses up to 0.5 mM. The increase in other chaperones as a consequence of HSP90 improves folding of NPC1 protein and relieves cholesterol accumulation in NPC1 mutant fibroblasts.

Stereoselectivity Predictions for the Pd-Catalyzed 1,4-Conjugate Addition Using Quantum-Guided Molecular Mechanics.

The conjugate addition of aryl boronic acids to enones is a powerful synthetic tool to introduce quaternary chiral centers, but the experimentally observed stereoselectivities vary widely, and the identification of suitable substrate-ligand combinations requires significant effort. We describe the development and application of a transition-state force field (TSFF) by the quantum-guided molecular mechanics (Q2MM) method that is validated using an automated screen of 9 ligands, 38 aryl boronic acids, and 22 enones, leading to a MUE of 1.8 kJ/mol and a R2 value of 0.877 over 82 examples. A detailed error analysis identified the structural origin for the deviations in the small group of outliers. The TSFF was then used to predict the stereoselectivity for 27 ligands and 59 enones. The vast majority of the virtual screening results are in line with the expected results. Selected results for 6-substituted pyrox ligands, which were not part of the training set, were followed up by density functional theory and experimental studies.

Design, Synthesis, and Evaluation of a Luminescent Cholesterol Mimic.

The development of new chemical tools with improved properties is essential to chemical and cell biology. Of particular interest is the development of mimics of small molecules with important cellular function that allow the direct observation of their trafficking in a cell. To this end, a novel 15-azasterol has been designed and synthesized as a luminescent cholesterol mimic for the monitoring of cholesterol trafficking. The brightness of this probe, which is ∼32-times greater than the widely used dehydroergosterol probe, is combined with resistance to photobleaching in solution and in human fibroblasts and an exceptionally large Stokes-like shift of ∼150-200 nm. The photophysical properties of the probe have been studied experimentally and computationally, suggesting an intersystem crossing to the triplet excited state with subsequent phosphorescent decay. Molecular dynamics simulations show a similar binding mode of cholesterol and the azasterol probe to NPC proteins, demonstrating the structural similarity of the probe to cholesterol.

A molecular mechanism of artemisinin resistance in Plasmodium falciparum malaria.

Artemisinins are the cornerstone of anti-malarial drugs. Emergence and spread of resistance to them raises risk of wiping out recent gains achieved in reducing worldwide malaria burden and threatens future malaria control and elimination on a global level. Genome-wide association studies (GWAS) have revealed parasite genetic loci associated with artemisinin resistance. However, there is no consensus on biochemical targets of artemisinin. Whether and how these targets interact with genes identified by GWAS, remains unknown. Here we provide biochemical and cellular evidence that artemisinins are potent inhibitors of Plasmodium falciparum phosphatidylinositol-3-kinase (PfPI3K), revealing an unexpected mechanism of action. In resistant clinical strains, increased PfPI3K was associated with the C580Y mutation in P. falciparum Kelch13 (PfKelch13), a primary marker of artemisinin resistance. Polyubiquitination of PfPI3K and its binding to PfKelch13 were reduced by the PfKelch13 mutation, which limited proteolysis of PfPI3K and thus increased levels of the kinase, as well as its lipid product phosphatidylinositol-3-phosphate (PI3P). We find PI3P levels to be predictive of artemisinin resistance in both clinical and engineered laboratory parasites as well as across non-isogenic strains. Elevated PI3P induced artemisinin resistance in absence of PfKelch13 mutations, but remained responsive to regulation by PfKelch13. Evidence is presented for PI3P-dependent signalling in which transgenic expression of an additional kinase confers resistance. Together these data present PI3P as the key mediator of artemisinin resistance and the sole PfPI3K as an important target for malaria elimination.

Transition State Force Field for the Asymmetric Redox-Relay Heck Reaction.

A transition state force field (TSFF) was developed using the quantum-guided molecular mechanics (Q2MM) method to describe the stereodetermining migratory insertion step of the enantioselective redox-relay Heck reaction for a range of multisubstituted alkenes. We show that the TSFF is highly predictive through an external validation of the TSFF against 151 experimentally determined stereoselectivities resulting in an R2 of 0.89 and MUE of 1.8 kJ/mol. In addition, limitations in the underlying force field were identified by comparison of the TSFF results to DFT level calculations. A novel application of the TSFF was demonstrated for 31 cases where the enantiomer predicted by the TSFF differed from the originally published values. Experimental determination of the absolute configuration demonstrated that the computational predictions were accurate, suggesting that TSFFs can be used for the rapid prediction of the absolute stereochemistry for a class of reactions. Finally, a virtual ligand screen was conducted utilizing both the TSFF and a simple molecular correlation method. Both methods were similarly predictive, but the TSFF was able to show greater utility through transferability, speed, and interpretability.

Regioselective Alkylation of Pyridinium Riboses.

The nucleophilic addition of alkyl, benzyl, and allyl organozinc reagents to protected pyridinium riboses proceeds under mild conditions and with yields of >90% in several cases and complete regioselectivity for the 4-position.

Discovery of selective small-molecule HDAC6 inhibitor for overcoming proteasome inhibitor resistance in multiple myeloma.

Multiple myeloma (MM) has proven clinically susceptible to modulation of pathways of protein homeostasis. Blockade of proteasomal degradation of polyubiquitinated misfolded proteins by the proteasome inhibitor bortezomib (BTZ) achieves responses and prolongs survival in MM, but long-term treatment with BTZ leads to drug-resistant relapse in most patients. In a proof-of-concept study, we previously demonstrated that blocking aggresomal breakdown of polyubiquitinated misfolded proteins with the histone deacetylase 6 (HDAC6) inhibitor tubacin enhances BTZ-induced cytotoxicity in MM cells in vitro. However, these foundational studies were limited by the pharmacologic liabilities of tubacin as a chemical probe with only in vitro utility. Emerging from a focused library synthesis, a potent, selective, and bioavailable HDAC6 inhibitor, WT161, was created to study the mechanism of action of HDAC6 inhibition in MM alone and in combination with BTZ. WT161 in combination with BTZ triggers significant accumulation of polyubiquitinated proteins and cell stress, followed by caspase activation and apoptosis. More importantly, this combination treatment was effective in BTZ-resistant cells and in the presence of bone marrow stromal cells, which have been shown to mediate MM cell drug resistance. The activity of WT161 was confirmed in our human MM cell xenograft mouse model and established the framework for clinical trials of the combination treatment to improve patient outcomes in MM.

Understanding Rate Acceleration and Stereoinduction of an Asymmetric Giese Reaction Mediated by a Chiral Rhodium Catalyst.

The surprising acceleration of the addition of electron-rich radicals to α,ß-unsaturated 2-acyl imidazoles by a chiral-at-metal rhodium catalyst is investigated. M06/Lanl2DZ (Rh),6-31G(d) calculations reproduce the observed rate acceleration and shed light on a catalyst design where a rigid chiral pocket with a steric interaction >5 Å from the chiral metal center leads to the observed high stereoinduction. Analysis of the molecular orbitals of two key addition transition states emphasize the role of the catalyst as a Lewis acid without significant charge transfer.

Visible-Light-Activated Asymmetric ß-C-H Functionalization of Acceptor-Substituted Ketones with 1,2-Dicarbonyl Compounds.

We report a visible-light-activated asymmetric ß-C(sp3)-H functionalization of 2-acyl imidazoles and 2-acylpyridines with 1,2-dicarbonyl compounds (typically α-ketoesters) catalyzed by a tailored stereogenic-at-rhodium Lewis acid catalyst. The C-C bond formation products are obtained in high yields (up to 99%) and with excellent stereoselectivities (up to >20:1 dr and up to >99% ee). Experimental and computational studies support a mechanism in which a photoactivated Rh-enolate transfers a single electron to the 1,2-dicarbonyl compound followed by proton transfer and a subsequent stereocontrolled radical-radical recombination.

Direct Visible-Light-Excited Asymmetric Lewis Acid Catalysis of Intermolecular [2+2] Photocycloadditions.

A reaction design is reported in which a substrate-bound chiral Lewis acid complex absorbs visible light and generates an excited state that directly reacts with a cosubstrate in a highly stereocontrolled fashion. Specifically, a chiral rhodium complex catalyzes visible-light-activated intermolecular [2+2] cycloadditions, providing a wide range of cyclobutanes with up to >99% ee and up to >20:1 d.r. Noteworthy is the ability to create vicinal all-carbon-quaternary stereocenters including spiro centers in an intermolecular fashion.

Prediction of Stereochemistry using Q2MM.

The standard method of screening ligands for selectivity in asymmetric, transition metal-catalyzed reactions requires experimental testing of hundreds of ligands from ligand libraries. This "trial and error" process is costly in terms of time as well as resources and, in general, is scientifically and intellectually unsatisfying as it reveals little about the underlying mechanism behind the selectivity. The accurate computational prediction of stereoselectivity in enantioselective catalysis requires adequate conformational sampling of the selectivity-determining transition state but has to be fast enough to compete with experimental screening techniques to be useful for the synthetic chemist. Although electronic structure calculations are accurate and general, they are too slow to allow for sampling or fast screening of ligand libraries. The combined requirements can be fulfilled by using appropriately fitted transition state force fields (TSFFs) that represent the transition state as a minimum and allow fast conformational sampling using Monte Carlo. Quantum-guided molecular mechanics (Q2MM) is an automated force field parametrization method that generates accurate, reaction-specific TSFFs by fitting the functional form of an arbitrary force field using only electronic structure calculations by minimization of an objective function. A key feature that distinguishes the Q2MM method from many other automated parametrization procedures is the use of the Hessian matrix in addition to geometric parameters and relative energies. This alleviates the known problems of overfitting of TSFFs. After validation of the TSFF by comparison to electronic structure results for a test set and available experimental data, the stereoselectivity of a reaction can be calculated by summation over the Boltzman-averaged relative energies of the conformations leading to the different stereoisomers. The Q2MM method has been applied successfully to perform virtual ligand screens on a range of transition metal-catalyzed reactions that are important from both an industrial and an academic perspective. In this Account, we provide an overview of the continued improvement of the prediction of stereochemistry using Q2MM-derived TSFFs using four examples from different stages of development: (i) Pd-catalyzed allylation, (ii) OsO4-catalyzed asymmetric dihydroxylation (AD) of alkenes, (iii) Rh-catalyzed hydrogenation of enamides, and (iv) Ru-catalyzed hydrogenation of ketones. In the current form, correlation coefficients of 0.8-0.9 between calculated and experimental ee values are typical for a wide range of substrate-ligand combinations, and suitable ligands can be predicted for a given substrate with ∼80% accuracy. Although the generation of a TSFF requires an initial effort and will therefore be most useful for widely used reactions that require frequent screening campaigns, the method allows for a rapid virtual screen of large ligand libraries to focus experimental efforts on the most promising substrate-ligand combinations.

Diastereoselective Synthesis of Highly Substituted Tetrahydrofurans by Pd-Catalyzed Tandem Oxidative Cyclization-Redox Relay Reactions Controlled by Intramolecular Hydrogen Bonding.

Palladium-catalyzed oxidative cyclization of alkenols provides a convenient entry into cyclic ethers but typically proceeds with little or no diastereoselectivity for cyclization of trisubstituted olefins to form tetrahydrofurans due to the similar energies of competing 5-membered transition-state conformations. Herein, a new variant of this reaction has been developed in which a PdCl2/1,4-benzoquinone catalyst system coupled with introduction of a hydrogen-bond acceptor in the substrate enhances both diastereoselectivity and reactivity. Cyclization occurs with 5-exo Markovnikov regioselectivity. Mechanistic and computational studies support an anti-oxypalladation pathway in which intramolecular hydrogen bonding increases the nucleophilicity of the alcohol and enforces conformational constraints that enhance diastereoselectivity. The cyclization is followed by a tandem redox-relay process that provides versatile side-chain functionalities for further derivatization.

Synthesis and Biochemical Evaluation of Biotinylated Conjugates of Largazole Analogues: Selective Class I Histone Deacetylase Inhibitors.

The synthesis of biotinylated conjugates of synthetic analogues of the potent and selective histone deacetylase (HDAC) inhibitor largazole is reported. The thiazole moiety of the parent compound's cap group was derivatized to allow the chemical conjugation to biotin. The derivatized largazole analogues were assayed across a panel of HDACs 1-9 and retained potent and selective inhibitory activity towards the class I HDAC isoforms. The biotinylated conjugate was further shown to pull down HDACs 1, 2, and 3.

Metal-Templated Design: Enantioselective Hydrogen-Bond-Driven Catalysis Requiring Only Parts-per-Million Catalyst Loading.

Based on a metal-templated approach using a rigid and globular structural scaffold in the form of a bis-cyclometalated octahedral iridium complex, an exceptionally active hydrogen-bond-mediated asymmetric catalyst was developed and its mode of action investigated by crystallography, NMR, computation, kinetic experiments, comparison with a rhodium congener, and reactions in the presence of competing H-bond donors and acceptors. Relying exclusively on weak forces, the enantioselective conjugate reduction of nitroalkenes can be executed at catalyst loadings as low as 0.004 mol% (40 ppm), representing turnover numbers of up to 20 250. A rate acceleration by the catalyst of 2.5 × 10(5) was determined. The origin of the catalysis is traced to an effective stabilization of developing charges in the transition state by carefully orchestrated hydrogen-bonding and van der Waals interactions between catalyst and substrates. This study demonstrates that the proficiency of asymmetric catalysis merely driven by hydrogen-bonding and van der Waals interactions can rival traditional activation through direct transition metal coordination of the substrate.