Discovery of Nirmatrelvir Resistance Mutations in SARS-CoV-2 3CLpro: A Computational-Experimental Approach.

The COVID-19 pandemic has emphasized the urgency for effective antiviral therapies against SARS-CoV-2. Targeting the main protease (3CLpro) of the virus has emerged as a promising approach, and nirmatrelvir (PF-07321332), the active component of Pfizer's oral drug Paxlovid, has demonstrated remarkable clinical efficacy. However, the emergence of resistance mutations poses a challenge to its continued success. In this study, we employed alchemical free energy perturbation (FEP) alanine scanning to identify nirmatrelvir-resistance mutations within SARS-CoV-2 3CLpro. FEP identified several mutations, which were validated through in vitro IC50 experiments and found to result in 8- and 72-fold increases in nirmatrelvir IC50 values. Additionally, we constructed SARS-CoV-2 omicron replicons containing these mutations, and one of the mutants (S144A/E166A) displayed a 20-fold increase in EC50, confirming the role of FEP in identifying drug-resistance mutations. Our findings suggest that FEP can be a valuable tool in proactively monitoring the emergence of resistant strains and guiding the design of future inhibitors with reduced susceptibility to drug resistance. As nirmatrelvir is currently widely used for treating COVID-19, this research has important implications for surveillance efforts and antiviral development.

Photochemistry and Transmission Pump-Probe Spectroscopy of 2-Azidobiphenyls in Aqueous Nanocrystalline Suspensions: Simplified Kinetics in Crystalline Solids.

Aqueous nanocrystalline suspensions provide a simple and efficient medium for performing transmission spectroscopy measurements in the solid state. In this Letter we describe the use of laser flash photolysis methods to analyze the photochemistry of 2-azidobiphenyl and several aryl-substituted derivatives. We show that all the crystalline compounds analyzed in this study transform quantitatively into carbazole products via a crystal-to-crystal reconstructive phase transition. While the initial steps of the reaction cannot be followed within the time resolution of our instrument (ca. 8 ns), we detected the primary isocarbazole photoproducts and analyzed the kinetics of their formal 1,5-H shift reactions, which take place in time scales that range from a few nanoseconds to several microseconds. It is worth noting that the high reaction selectivity observed in the crystalline state translates into a clean and simple kinetic process compared to that in solution.

Rotational Dynamics of Diazabicyclo[2.2.2]octane in Isomorphous Halogen-Bonded Co-crystals: Entropic and Enthalpic Effects.

Based on rotational dynamics measurements carried out with isomorphic co-crystals formed by halogen-bonding (XB) between tritylacetylene halides (TrX) and diazabicyclo[2.2.2]octane (dabco), we were able to distinguish the sources of the enthalpic and entropic components in the rotational free energy barrier. We describe the formation of the 1:1 co-crystals (TrX···N(R)3N) obtained from 1 equiv of dabco and 1 equiv of either TrI or TrBr, respectively, to give 4a and 4b instead of the potential 2:1 complexes. The co-crystals were prepared by solvent evaporation and mechanochemical synthesis. No co-crystal with TrCl was obtained, reflecting the weaker nature of the TrCl···NR3 interaction. Single-crystal X-ray diffraction confirmed structures that resemble a spinning top on a tripod and revealed that the two XB co-crystals are isomorphous, with slightly different C-X···NR3 (X = I, Br) distances and packing interactions. Quadrupolar-echo 2H NMR experiments with 2H-labeled samples showed that fast rotation of dabco in these co-crystals follows a six-fold potential energy surface with three lowest energy minima. Variable-temperature 1H NMR spin-lattice relaxation (VT 1H T1) data revealed rotational dynamics with indistinguishable pre-exponential factors and small but distinguishable activation energies. The activation energy of 4b (Ea = 0.71 kcal mol-1) is the lowest reported in the field of amphidynamic crystals. Using the Eyring equation, we established that their activation entropy for rotation is small but negative (ΔS⧧ = -3.0 cal mol-1 K-1), while there is almost a 2-fold difference in activation enthalpies, with 4a having a higher barrier (ΔH⧧ = 0.95 kcal mol-1) than 4b (ΔH⧧ = 0.54 kcal mol-1). Analysis of the rotator cavity in the two co-crystals revealed subtle differences in steric interactions that account for their different activation energies.

Tale of Twisted Molecules. Atropselective Photoreactions: Taming Light Induced Asymmetric Transformations through Non-biaryl Atropisomers.

Photochemical transformations are a powerful tool in organic synthesis to access structurally complex and diverse synthetic building blocks. However, this great potential remains untapped in the mainstream synthetic community due to the challenges associated with stereocontrol originating from excited state(s). The finite lifetime of an excited state and nearly barrierless subsequent processes present significant challenges in manipulating the stereochemical outcome of a photochemical reaction. Several methodologies were developed to address this bottleneck including photoreactions in confined media and preorganization through noncovalent interactions resulting in stereoenhancement. Yet, stereocontrol in photochemical reactions that happen in solution in the absence of organized assemblies remained largely unaddressed. In an effort to develop a general and reliable methodology, our lab has been exploring non-biaryl atropisomers as an avenue to perform asymmetric phototransformations. Atropisomers are chiral molecules that arise due to the restricted rotation around a single bond (chiral axis) whose energy barrier to rotation is determined by nonbonding interactions (most often by steric hindrance) with appropriate substituents. Thus, atropisomeric substrates are chirally preorganized during the photochemical transformation and translate their chiral information to the expected photoproducts. This strategy, where "axial to point chirality transfer" occurs during the photochemical reaction, is a hybrid of the successful Curran's prochiral auxiliary approach involving atropisomers in thermal reactions and the Havinga's NEER principle (nonequilibrating excited-state rotamers) for photochemical transformations. We have investigated this strategy in order to probe various aspects such as regio-, enantio-, diastereo-, and chemoselectivity in several synthetically useful phototransformations including 6π-photocyclization, 4π-ring closure, Norrish-Yang photoreactions, Paternò-Büchi reaction, and [2 + 2]- and [5 + 2]-photocycloaddition. The investigations detailed in this Account clearly signify the scope of our strategy in accessing chirally enriched products during phototransformations. Simple design modifications such as tailoring the steric handle in atropisomers to hold reactive units resulted in permanently locked/traceless axial chirality in addition to incorporating multiple stereocenters in already complex scaffolds obtained from phototransformation. Further improvements allowed us to employ low energy visible light rather than high energy UV light without compromising the stereoenrichment in the photoproducts. Continued investigations on atropisomeric scaffolds have unraveled new design features, with outcomes that are unique and unprecedented for excited state reactivity. For example, we have established that reactive spin states (singlet or triplet excited state) profoundly influence the stereochemical outcome of an atropselective phototransformation. In general, the photochemistry and photophysics of atropisomeric substrates differ significantly from their achiral counterparts irrespective of having the same chromophore initiating the excited state reactivity. The ability of axially chiral chromophores to impart stereoenrichment in the intramolecular photoreactions appears to be promising. A challenging endeavor for the "axial to point chirality transfer" strategy is to enhance stereoenrichment or alter chemical reactivity in intermolecular photoreactions. Insights gained from our investigations will serve as a platform to venture into more complicated yet fruitful research in terms of broad synthetic utility.

Photoreactions with a Twist: Atropisomerism-Driven Divergent Reactivity of Enones with UV and Visible Light.

Light-induced transformation of atropisomeric and achiral enones displays divergent reactivity. Photocyclization leading to 3,4-dihydroquinolin-2-one was observed with achiral enone carboxamide, whereas the atropisomeric enone carboxamides underwent hydrogen abstraction leading to spiro-ß-lactams. Detailed photochemical, photophysical, and theoretical investigations have provided insight into this divergent reactivity and selectivity.

Structure-Kinetics Correlations in Isostructural Crystals of α-(ortho-Tolyl)-acetophenones: Pinning Down Electronic Effects Using Laser-Flash Photolysis in the Solid State.

Aqueous suspensions of nanocrystals in the 200-500 nm size range of isostructural α-(ortho-tolyl)-acetophenone (1a) and α-(ortho-tolyl)-para-methylacetophenone (1b) displayed good absorption characteristics for flash photolysis experiments in a flow system, with transient spectra and decay kinetics with a quality that is similar to that recorded in solution. In contrast to solution measurements, reactions in the solid state were characterized by a rate limiting hydrogen transfer reaction from the triplet excited state and a very short-lived biradical intermediate, which does not accumulate. Notably, the rate for δ-hydrogen atom transfer of 1a (2.7 × 10(7) s(-1)) in the crystalline phase is 18-fold larger than that of 1b (1.5 × 10(6) s(-1)). With nearly identical molecular and crystal structures, this decrease in the rate of δ-hydrogen abstraction can be assigned unambiguously to an electronic effect by the para-methyl group in 1b, which increases the contribution of the (3)π,π* configuration relative to the reactive (3)n,π* configuration in the lowest triplet excited state. These results highlight the potential of relating single crystal X-ray structural data with absolute kinetics from laser flash photolysis.

Enantiospecific photochemical 6π-ring closure of α-substituted atropisomeric acrylanilides--role of alkali metal ions.

Direct irradiation of atropisomeric α-substituted acrylanilides in the presence of alkali metal ions gave high ee values in the 3,4-dihydro-2-quinolin-2-one photoproduct, while in the absence of alkali metal ions, racemic photoproduct was observed. The heavy atom effect leading to enhanced triplet yields alters the reactive pathway leading to the observed enantioselectivity in the photoproduct.

Enantiospecific photochemical transformations under elevated pressure.

Enantiospecific axial-to-point chiral transfer in light-induced transformations was efficient under elevated pressure at high temperatures. Model photoreactions with atropisomeric compounds showed higher enantioselectivity in the photoproducts under elevated pressure. The ee values in the photoproducts were rationalized based on the increased stability of optically pure atropisomeric compounds at elevated pressure, even at high temperatures.

Fun with photons: selective light induced reactions in solution and in water soluble nano-containers.

Two distinct strategies for controlling selectivity, in particular stereoselectivity in photochemical reactions are reviewed. In the first strategy, supramolecular approach using cucurbituril nano-containers in catalytic amounts is employed to control selectivity during photochemical transformations. In the second approach, a generalized methodology for carrying out light-induced transformations in solution at ambient conditions is detailed where axially chiral motifs are employed to enantiospecifically transfer the axial chirality in the reactant to point chirality in the photoproduct(s).

Enantiospecific 6π-photocyclization of atropisomeric α-substituted acrylanilides in the solid-state: role of crystalline confinement on enantiospecificity.

Direct irradiation of optically-pure axially-chiral α-substituted acrylanilides in the crystalline state leads to 3,4-dihydro-2-quinolin-2-one photoproduct with an enantiomeric ratio of 85 : 15 while a racemic mixture of photoproduct is observed in solution.

Reactive spin state dependent enantiospecific photocyclization of axially chiral α-substituted acrylanilides.

The enantiomeric ratio (e.r.) in the 3,4-dihydroquinolin-2-one photoproduct during 6π-photocyclization of α-substituted axially chiral ortho-tert-butyl-acrylanilides depends on the nature of the reactive spin state (singlet or triplet), where the singlet-spin state reactivity gives a racemic mixture and the triplet reactivity gives an e.r. value >95 : 5.

Enantiospecific photochemical Norrish/Yang type II reaction of nonbiaryl atropchiral alpha-oxoamides in solution--axial to point chirality transfer.

Alpha-oxoamides 1 with o-tert-butyl substitution on the N-phenyl moiety were found to be stable axially chiral atropisomers. These axially chiral alpha-oxoamides undergo enantiospecific photochemical gamma-Hydrogen abstraction in CHCl(3) to yield beta-lactams with high enantioselectivity (e.r. approximately 90:10) in solution. The extent of enantioselectivity was found to be dependent on the reaction temperature.

6pi-Photocyclization of O-tert-butylacrylanilides. N-substitution dictates the regiochemistry of cyclization.

O-tert-Butylacrylanilides with N-H substitution undergo 6pi-photocyclization at the unsubstituted ortho carbon, whereas the corresponding N-methyl derivatives cyclize at the ortho carbon bearing the tert-butyl with the eventual loss of 2-methylpropene.

Light-induced transfer of molecular chirality in solution: enantiospecific photocyclization of molecularly chiral acrylanilides.

Molecularly chiral o-tert-butylacrylanilides undergo enantiospecific 6pi-photocyclization to yield 3,4-dihydroquinolin-2-ones with very high enantioselectivity (>90%) in solution. The photocyclization results in the removal of the ortho tert-butyl substituent, presumably via a zwitterionic intermediate. Beta-substitution in the alkene is found to be critical for the transfer of molecular chirality (axial chirality) in the reactant to point chirality in the photoproduct(s).