Mechanism of inhibition of SARS-CoV-2 Mpro by N3 peptidyl Michael acceptor explained by QM/MM simulations and design of new derivatives with tunable chemical reactivity

The SARS-CoV-2 main protease (M^pro) is essential for replication of the virus responsible for the COVID-19 pandemic, and one of the main targets for drug design. Here, we simulate the inhibition process of SARS-CoV-2 M^pro with a known Michael acceptor (peptidyl) inhibitor, N3. The free energy landscape for the mechanism of the formation of the covalent enzyme-inhibitor product is computed with QM/MM molecular dynamics methods. The simulations show a two-step mechanism, and give structures and calculated barriers in good agreement with experiment. Using these results and information from our previous investigation on the proteolysis reaction of SARS-CoV-2 M^pro, we design two new, synthetically accessible N3-analogues as potential inhibitors, in which the recognition and warhead motifs are modified. QM/MM modelling of the mechanism of inhibition of M^pro by these novel compounds indicates that both may be promising candidates as drug leads against COVID-19, one as an irreversible inhibitor and one as a potential reversible inhibitor.

QM/MM simulations identify the mechanism of reaction of N3, a covalent peptidyl inhibitor of SARS-CoV-2 main protease. Modelling of two novel proposed compounds, B1 and B2, suggests that reversibility of covalent inhibition could be tailored.     

‘Sacrificial’ supramolecular assembly and pressure-induced polymerization: toward sequence-defined functionalized nanothreads

Limited supramolecular strategies have been utilized to synthesize sequence-defined polymers, despite the prominence of noncovalent interactions in materials design. Herein, we illustrate the utility of ‘sacrificial’ aryl-perfluoroaryl supramolecular synthons to synthesize sp³-hybridized nanothreads from sp²-enriched reactants. Our strategy features A–B reactant pairs in the form of a phenol:pentafluorophenol co-crystal that is preorganized for an electronically-biased and sequence-defined polymerization. The polymerization, initiated at 12 GPa, affords an alternating copolymer featuring exogenous –OH functionalities. The external substitution is confirmed through IR spectroscopy. Importantly, the inclusion of the functional unit provides the first experimental glimpse at reaction mechanism: keto–enol tautomerization that can only occur during cycloaddition is observed through IR spectroscopy. Our approach realizes the first example of a functionalized nanothread and attains sequence definition through sacrificial supramolecular preorganization and presents a further approach for de novo design of complex nanothreads.

Supramolecular synthons are exploited to synthesize –OH functionalized sp³-rich sequence-defined nanothreads using pressure-induced polymerization of a phenol:pentafluorophenol co-crystal.      

Developing potent BTKC481S PROTACs for ibrutinib-resistant malignant lymphoma

Ibrutinib is a first-line treatment drug for B-cell malignancies. However, resistance to ibrutinib has been reported due to BTK^C481S mutation. Although PROTAC strategy is expected to overcome this clinical resistance, it has limitations such as large molecular weight and moderate bioactivity, which restrict its potential clinical application. Herein, we report a new type of potent BTK^C481S-targeting PROTAC degrader. Through design, computer-assisted optimization and SAR studies, we have developed a representative BTK^C481S degrader L6 with a much smaller molecular weight and improved solubility. Notably, L6 demonstrates better BTK degrading activity and lower IC₅₀ value in ibrutinib-resistant cell line than the first-generation BTK degrader P13I. Optimization strategy of L6 provides a general approach in the development of PROTACs targeting BTK and other proteins for future study.     

Selective covalent targeting of SARS-CoV-2 main protease by enantiopure chlorofluoroacetamide

The coronavirus disease 2019 (COVID-19) pandemic has necessitated the development of antiviral agents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The main protease (M^pro) is a promising target for COVID-19 treatment. Here, we report an irreversible SARS-CoV-2 M^pro inhibitor possessing chlorofluoroacetamide (CFA) as a warhead for the covalent modification of M^pro. Ugi multicomponent reaction using chlorofluoroacetic acid enabled the rapid synthesis of dipeptidic CFA derivatives that identified 18 as a potent inhibitor of SARS-CoV-2 M^pro. Among the four stereoisomers, (R,R)-18 exhibited a markedly higher inhibitory activity against M^pro than the other isomers. Reaction kinetics and computational docking studies suggest that the R configuration of the CFA warhead is crucial for the rapid covalent inhibition of M^pro. Our findings highlight the prominent influence of the CFA chirality on the covalent modification of proteinous cysteines and provide the basis for improving the potency and selectivity of CFA-based covalent inhibitors.

Chlorofluoroacetamide (CFA) was used as the warhead for covalent targeting of SARS-CoV-2 M^pro. The chirality at CFA showed marked influence on inhibitory activity, suggesting stereospecific activation of CFA for cysteine modification in the protein.     

Revealing the molecular mechanisms of proteolysis of SARS-CoV-2 Mpro by QM/MM computational methods

SARS-CoV-2 M^pro is one of the enzymes essential for the replication process of the virus responsible for the COVID-19 pandemic. This work is focused on exploring its proteolysis reaction by means of QM/MM methods. The resulting free energy landscape of the process provides valuable information on the species appearing along the reaction path and suggests that the mechanism of action of this enzyme, taking place in four steps, slightly differs from that of other cysteine proteases. Our predictions, which are in agreement with some recently published experimental data, can be used to guide the design of COVID-19 antiviral compounds with clinical potential.

The molecular mechanism of the proteolysis reaction catalyzed by SARS-CoV-2 M^pro, one of the enzymes essential for the replication process of the virus responsible for the COVID-19 pandemic, is described using computational QM/MM methods.     

Visible-light-driven palladium-catalyzed Dowd–Beckwith ring expansion/C–C bond formation cascade

A visible-light-induced palladium-catalyzed Dowd–Beckwith ring expansion/C–C bond formation cascade is described. A range of six to nine-membered β-alkenylated cyclic ketones possessing a quaternary carbon center were accessed under mild conditions. Besides styrenes, the electron-rich alkenes such as silyl enol ethers and enamides were also compatible, providing the desired β-alkylated cyclic ketones in moderate to good yields.

An intermolecular Dowd–Beckwith ring expansion/C–C bond formation is achieved through light-induced palladium catalysis. Not only styrenes but also the electron-rich alkenes such as silyl enol ethers and enamides were also compatible in this reaction.     

Nickel-catalyzed C–O/N–H,C–S/N–H,and C–CN/N–H annulation of aromatic amides with alkynes: C–O,C–S,and C–CN activation

The Ni-catalyzed reaction of ortho-phenoxy-substituted aromatic amides with alkynes in the presence of LiO^tBu as a base results in C–O/N–H annulation with the formation of 1(2H)-isoquinolinones. The use of a base is essential for the reaction to proceed. The reaction proceeds, even in the absence of a ligand, and under mild reaction conditions (40 °C). An electron-donating group on the aromatic ring facilitates the reaction. The reaction was also applicable to carbamate (C–O bond activation), methylthio (C–S bond activation), and cyano (C–CN bond activation) groups as leaving groups.

The Ni-catalyzed reaction of ortho-phenoxy-substituted aromatic amides with alkynes in the presence of LiO^tBu as a base results in C–O/N–H annulation with the formation of 1(2H)-isoquinolinones.     

Inclusion Complexes of β-Cyclodextrin with Keto/Enol Tautomers of 2-Acetyl-1-tetralone. A Comparative Study

The keto–enol interconversion of 2-acetyl-1-tetralone (ATLO) and of 2-acetyl-cyclohexanone (ACHE) occurs at measurable rates in aqueous acid or neutral medium. This finding allowed us to determine the keto–enol equilibrium constants, K _E, by following two distinct methods. Both methodologies afford results in complete agreement. The first one is a test of the Beer-Lambert law under two different experimental conditions that contain the substrate only in the enol form or in a mixture of both tautomers in equilibrium. The second method analyses the UV-absorption spectrum of each substrate under keto–enol equilibrium in aqueous β-cyclodextrin (β-CD) solutions of variable concentration: the presence of β-CD increases the percentage of the enol due to the formation of 1:1 inclusion complexes between this tautomer and β-CD. Rates of keto–enol tautomerization, in neutral and acid medium, and of nitrosation in acid medium under non equilibrium conditions have also been measured. Throughout the study, the presentation of the results is done by comparing the different behaviour observed between ATLO and ACHE. While the enol of ACHE included into the β-CD cavity shows to be unreactive either in tautomerization or in nitrosation, in the case of ATLO it is observed tautomerization through the complexed enol. In addition, with ACHE only the enol tautomer forms inclusion complexes with β-CD, whereas with ATLO the keto tautomer entries also to the β-CD cavity, however the stability constant with the enol is near 3-fold that of the keto isomer. These main differences can be rationalized on the basis of the molecular structure of these diketones.This revised version was published online in July 2005 with a corrected issue number.     

Symmetry of three-center,four-electron bonds

Three-center, four-electron bonds provide unusually strong interactions; however, their nature remains ununderstood. Investigations of the strength, symmetry and the covalent versus electrostatic character of three-center hydrogen bonds have vastly contributed to the understanding of chemical bonding, whereas the assessments of the analogous three-center halogen, chalcogen, tetrel and metallic -type long bonding are still lagging behind. Herein, we disclose the X-ray crystallographic, NMR spectroscopic and computational investigation of three-center, four-electron [D–X–D]⁺ bonding for a variety of cations (X⁺ = H⁺, Li⁺, Na⁺, F⁺, Cl⁺, Br⁺, I⁺, Ag⁺ and Au⁺) using a benchmark bidentate model system. Formation of a three-center bond, [D–X–D]⁺ is accompanied by an at least 30% shortening of the D–X bonds. We introduce a numerical index that correlates symmetry to the ionic size and the electron affinity of the central cation, X⁺. Providing an improved understanding of the fundamental factors determining bond symmetry on a comprehensive level is expected to facilitate future developments and applications of secondary bonding and hypervalent chemistry.

The factors determining the symmetry and the fundamental nature of the three-center, four-electron bonds are assessed.     

The role of the active site tyrosine in the mechanism of lytic polysaccharide monooxygenase

Catalytic breakdown of polysaccharides can be achieved more efficiently by means of the enzymes lytic polysaccharide monooxygenases (LPMOs). However, the LPMO mechanism has remained controversial, preventing full exploitation of their potential. One of the controversies has centered around an active site tyrosine, present in most LPMO classes. Recent investigations have for the first time obtained direct (spectroscopic) evidence for the possibility of chemical modification of this tyrosine. However, the spectroscopic features obtained in the different investigations are remarkably different, with absorption maximum at 420 and 490 nm, respectively. In this paper we use density functional theory (DFT) in a QM/MM formulation to reconcile these (apparently) conflicting results. By modeling the spectroscopy as well as the underlying reaction mechanism we can show how formation of two isomers (both involving deprotonation of tyrosine) explains the difference in the observed spectroscopic features. Both isomers have a [TyrO–Cu–OH]⁺ moiety with the OH in either the cis- or trans-position to a deprotonated tyrosine. Although the cis-[TyrO–Cu–OH]⁺ moiety is well positioned for oxidation of the substrate, preliminary calculations with the substrate reveal that the reactivity is at best moderate, making a protective role of tyrosine more likely.

With QM/MM, we investigate the mechanism of tyrosine deprotonation in lytic polysaccharide monooxygenases. Our results support deprotonation and our calculated UV-vis spectra show that two isomers must be formed to match recent experiments.     

Molecular control over vitrimer-like mechanics – tuneable dynamic motifs based on the Hammett equation in polyimine materials

In this work, we demonstrate that fine-grained, quantitative control over macroscopic dynamic material properties can be achieved using the Hammett equation in tuneable dynamic covalent polyimine materials. Via this established physical-organic principle, operating on the molecular level, one can fine-tune and control the dynamic material properties on the macroscopic level, by systematic variation of dynamic covalent bond dynamics through selection of the appropriate substituent of the aromatic imine building blocks. Five tuneable, crosslinked polyimine network materials, derived from dianiline monomers with varying Hammett parameter (σ) were studied by rheology, revealing a distinct correlation between the σ value and a range of corresponding dynamic material properties. Firstly, the linear correlation of the kinetic activation energy (E_a) for the imine exchange to the σ value, enabled us to tune the E_a from 16 to 85 kJ mol⁻¹. Furthermore, the creep behaviour (γ), glass transition (T_g) and the topology freezing transition temperature (T_v), all showed a strong, often linear, dependence on the σ value of the dianiline monomer. These combined results demonstrate for the first time how dynamic material properties can be directly tuned and designed in a quantitative – and therefore predictable – manner through correlations based on the Hammett equation. Moreover, the polyimine materials were found to be strong elastic rubbers (G′ > 1 MPa at room temperature) that were stable up to 300 °C, as confirmed by TGA. Lastly, the dynamic nature of the imine bond enabled not only recycling, but also intrinsic self-healing of the materials over multiple cycles without the need for solvent, catalysts or addition of external chemicals.

Controlling macroscopic material properties of dynamic covalent polyimines via the electronic effect of dianiline monomers based on the Hammett equation.     

Glycoside hydrolase stabilization of transition state charge: new directions for inhibitor design

Carbasugars are structural mimics of naturally occurring carbohydrates that can interact with and inhibit enzymes involved in carbohydrate processing. In particular, carbasugars have attracted attention as inhibitors of glycoside hydrolases (GHs) and as therapeutic leads in several disease areas. However, it is unclear how the carbasugars are recognized and processed by GHs. Here, we report the synthesis of three carbasugar isotopologues and provide a detailed transition state (TS) analysis for the formation of the initial GH-carbasugar covalent intermediate, as well as for hydrolysis of this intermediate, using a combination of experimentally measured kinetic isotope effects and hybrid QM/MM calculations. We find that the α-galactosidase from Thermotoga maritima effectively stabilizes TS charge development on a remote C5-allylic center acting in concert with the reacting carbasugar, and catalysis proceeds via an exploded, or loose, S_N2 transition state with no discrete enzyme-bound cationic intermediate. We conclude that, in complement to what we know about the TS structures of enzyme-natural substrate complexes, knowledge of the TS structures of enzymes reacting with non-natural carbasugar substrates shows that GHs can stabilize a wider range of positively charged TS structures than previously thought. Furthermore, this enhanced understanding will enable the design of new carbasugar GH transition state analogues to be used as, for example, chemical biology tools and pharmaceutical lead compounds.

Positive charge stabilized on remote C5-allylic center with catalysis occurring via a loose S_N2 transition state.     

Palladium-catalyzed direct asymmetric C–H bond functionalization enabled by the directing group strategy

In the past decade, selective C–C and C-heteroatom bond construction through palladium-catalyzed direct C–H bond functionalization has been extensively studied by employing a variety of directing groups. Within this category, direct asymmetric C(sp²)–H and C(sp³)–H activation for the construction of highly enantiomerically enriched skeletons still progressed at a slow pace. This minireview briefly introduces the major advances in the field for palladium-catalyzed direct asymmetric C–H bond functionalization via the directing group strategy.

This minireview introduces Pd-catalyzed direct asymmetric C–H functionalization reactions using a directing group strategy.     

Discovery of SARS-CoV-2 Mpro peptide inhibitors from modelling substrate and ligand binding

The main protease (M^pro) of SARS-CoV-2 is central to viral maturation and is a promising drug target, but little is known about structural aspects of how it binds to its 11 natural cleavage sites. We used biophysical and crystallographic data and an array of biomolecular simulation techniques, including automated docking, molecular dynamics (MD) and interactive MD in virtual reality, QM/MM, and linear-scaling DFT, to investigate the molecular features underlying recognition of the natural M^pro substrates. We extensively analysed the subsite interactions of modelled 11-residue cleavage site peptides, crystallographic ligands, and docked COVID Moonshot-designed covalent inhibitors. Our modelling studies reveal remarkable consistency in the hydrogen bonding patterns of the natural M^pro substrates, particularly on the N-terminal side of the scissile bond. They highlight the critical role of interactions beyond the immediate active site in recognition and catalysis, in particular plasticity at the S2 site. Building on our initial M^pro-substrate models, we used predictive saturation variation scanning (PreSaVS) to design peptides with improved affinity. Non-denaturing mass spectrometry and other biophysical analyses confirm these new and effective ‘peptibitors’ inhibit M^pro competitively. Our combined results provide new insights and highlight opportunities for the development of M^pro inhibitors as anti-COVID-19 drugs.

The main protease (M^pro) of SARS-CoV-2 is central to viral maturation and is a promising drug target. In silico methods reveal structural aspects of how it binds to its 11 natural cleavage sites, the design of novel peptide inhibitors, and insights into drug design.     

A quantum algorithm for spin chemistry: a Bayesian exchange coupling parameter calculator with broken-symmetry wave functions

The Heisenberg exchange coupling parameter J (H = −2JS_i · S_j) characterises the isotropic magnetic interaction between unpaired electrons, and it is one of the most important spin Hamiltonian parameters of multi-spin open shell systems. The J value is related to the energy difference between high-spin and low-spin states, and thus computing the energies of individual spin states are necessary to obtain the J values from quantum chemical calculations. Here, we propose a quantum algorithm, B̲ayesian ex̲change coupling parameter calculator with b̲roken-symmetry wave functions (BxB), which is capable of computing the J value directly, without calculating the energies of individual spin states. The BxB algorithm is composed of the quantum simulations of the time evolution of a broken-symmetry wave function under the Hamiltonian with an additional term jS², the wave function overlap estimation with the SWAP test, and Bayesian optimisation of the parameter j. Numerical quantum circuit simulations for H₂ under a covalent bond dissociation, C, O, Si, NH, OH⁺, CH₂, NF, O₂, and triple bond dissociated N₂ molecule revealed that the BxB can compute the J value within 1 kcal mol⁻¹ of errors with less computational costs than conventional quantum phase estimation-based approaches.

A quantum algorithm “Bayesian exchange coupling parameter calculator with broken-symmetry wave function (BxB)” enables us to calculate Heisenberg exchange coupling parameter J without inspecting total energies of individual spin states, within 1 kcal mol⁻¹ of energy tolerance.     

Diverse ring-opening reactions of rhodium η4-azaborete complexes

Sequential treatment of [Rh(COE)₂Cl]₂ (COE = cyclooctene) with PⁱPr₃, alkyne derivatives and ^tBuN BMes (Mes = 2,4,6-trimethylphenyl) provided functionalized rhodium η⁴-1,2-azaborete complexes of the form (η⁴-azaborete)RhCl(PⁱPr₃). The scope of this reaction was expanded to encompass alkynes with hydrogen, alkyl, aryl, ferrocenyl, alkynyl, azaborinyl and boronate ester substituents. Treatment of these complexes with PMe₃ led to insertion of the rhodium atom into the B–C bond of the BNC₂ ring, forming 1-rhoda-3,2-azaboroles. Addition of N-heterocyclic carbenes to azaborete complexes led to highly unusual rearrangements to rhodium η²,κ¹-allenylborylamino complexes via deprotonation and C–N bond cleavage. Heating and photolysis of an azaborete complex also led to rupture of the C–N bond followed by subsequent rearrangements, yielding an η⁴-aminoborylallene complex and two isomeric η⁴-butadiene complexes.

Rhodium η⁴-azaborete complexes can be transformed into a variety of species with ring-opened, BN-containing ligands by treatment with Lewis bases.     

Preparation and characterization of the enol of acetamide: 1-aminoethenol,a high-energy prebiotic molecule

Amide tautomers, which constitute the higher-energy amide bond linkage, not only are key for a variety of biological but also prebiotic processes. In this work, we present the gas-phase synthesis of 1-aminoethenol, the higher-energy tautomer of acetamide, that has not been spectroscopically identified to date. The title compound was prepared by flash vacuum pyrolysis of malonamic acid and was characterized employing matrix isolation infrared as well as ultraviolet/visible spectroscopy. Coupled-cluster computations at the AE-CCSD(T)/cc-pVTZ level of theory support the spectroscopic assignments. Upon photolysis at λ > 270 nm, the enol rearranges to acetamide as well as ketene and ammonia. As the latter two are even higher in energy, they constitute viable starting materials for formation of the title compound.

Amide tautomers, which constitute the higher-energy amide bond linkage, not only are key for a variety of biological but also prebiotic processes.     

Catalyst control of selectivity in the C–O bond alumination of biomass derived furans

Non-catalysed and catalysed reactions of aluminium reagents with furans, dihydrofurans and dihydropyrans were investigated and lead to ring-expanded products due to the insertion of the aluminium reagent into a C–O bond of the heterocycle. Specifically, the reaction of [{(ArNCMe)₂CH}Al] (Ar = 2,6-di-iso-propylphenyl, 1) with furans proceeded between 25 and 80 °C leading to dearomatised products due to the net transformation of a sp² C–O bond into a sp² C–Al bond. The kinetics of the reaction of 1 with furan were found to be 1st order with respect to 1 with activation parameters ΔH^‡ = +19.7 (±2.7) kcal mol⁻¹, ΔS^‡ = −18.8 (±7.8) cal K⁻¹ mol⁻¹ and ΔG^‡_{298 K} = +25.3 (±0.5) kcal mol⁻¹ and a KIE of 1.0 ± 0.1. DFT calculations support a stepwise mechanism involving an initial (4 + 1) cycloaddition of 1 with furan to form a bicyclic intermediate that rearranges by an α-migration. The selectivity of ring-expansion is influenced by factors that weaken the sp² C–O bond through population of the σ*-orbital. Inclusion of [Pd(PCy₃)₂] as a catalyst in these reactions results in expansion of the substrate scope to include 2,3-dihydrofurans and 3,4-dihydropyrans and improves selectivity. Under catalysed conditions, the C–O bond that breaks is that adjacent to the sp²C–H bond. The aluminium(iii) dihydride reagent [{(MesNCMe)₂CH}AlH₂] (Mes = 2,4,6-trimethylphenyl, 2) can also be used under catalytic conditions to effect a dehydrogenative ring-expansion of furans. Further mechanistic analysis shows that C–O bond functionalisation occurs via an initial C–H bond alumination. Kinetic products can be isolated that are derived from installation of the aluminium reagent at the 2-position of the heterocycle. C–H alumination occurs with a KIE of 4.8 ± 0.3 consistent with a turnover limiting step involving oxidative addition of the C–H bond to the palladium catalyst. Isomerisation of the kinetic C–H aluminated product to the thermodynamic C–O ring expansion product is an intramolecular process that is again catalysed by [Pd(PCy₃)₂]. DFT calculations suggest that the key C–O bond breaking step involves attack of an aluminium based metalloligand on the 2-palladated heterocycle. The new methodology has been applied to important platform chemicals from biomass.

Non-catalysed and catalysed reactions of aluminium reagents with furans, dihydrofurans and dihydropyrans were investigated and lead to ring-expanded products due to the insertion of the aluminium reagent into a C–O bond of the heterocycle.