Role of Conformational Dynamics in the Evolution of Retro-Aldolase Activity

Enzymes exist as ensembles of conformations that are important for function. Tuning these populations of conformational states through mutation enables evolution toward additional activities. Here we computationally evaluate the population shifts induced by distal and active site mutations in a family of computationally designed and experimentally optimized retro-aldolases. The conformational landscape of these enzymes was significantly altered during evolution, as pre-existing catalytically active conformational substates became major states in the most evolved variants. We further demonstrate that key residues responsible for these substate conversions can be predicted computationally. Significantly, the identified residues coincide with those positions mutated in the laboratory evolution experiments. This study establishes that distal mutations that affect enzyme catalytic activity can be predicted computationally and thus provides the enzyme (re)­design field with a rational strategy to determine promising sites for enhancing activity through mutation

Endohedral Metal-Induced Regioselective Formation of Bis-Prato Adduct of Y₃N@<i>I</i>_<i>h</i>‑C₈₀ and Gd₃N@<i>I</i>_<i>h</i>‑C₈₀

Regioselective bisaddition of M₃N@<i>I</i>_<i>h</i>-C₈₀ (M = Y, Gd) was observed for the first time in the Prato reaction with <i>N</i>-ethylglycine and formaldehyde. The main kinetic bisadduct of Y₃N@C₈₀ was determined to be a [6,6],[6,6] adduct by ¹H and ¹³C NMR and vis/NIR spectroscopy, and it converted to a mixture of regioisomers upon heating via a sigmatropic rearrangement. The main kinetic bisadduct of Gd₃N@C₈₀ (the [6,6],[6,6] adduct on the basis of vis/NIR data) existed stably under thermal conditions without isomerization. The likely position of the second addition of the Gd₃N@C₈₀ bisadduct was predicted by DFT calculations

(4 + 2) and (2 + 2) Cycloadditions of Benzyne to C₆₀ and Zig-Zag Single-Walled Carbon Nanotubes: The Effect of the Curvature

Addition of benzyne to carbon nanostructures can proceed via (4 + 2) (1,4-addition) or (2 + 2) (1,2-addition) cycloadditions depending on the species under consideration. In this work, we analyze by means of density functional theory (DFT) calculations the reaction mechanisms for the (4 + 2) and (2 + 2) cycloadditions of benzyne to nanostructures of different curvature, namely, C₆₀ and a series of zigzag single-walled carbon nanotubes. Our DFT calculations reveal that, except for the concerted (4 + 2) cycloaddition of benzyne to zigzag single-walled carbon nanotubes, all cycloadditions studied are stepwise processes with the initial formation of a biradical singly bonded intermediate. From this intermediate, the rotation of the benzyne moiety determines the course of the reaction. The Gibbs energy profiles lead to the following conclusions: (i) except for the 1,4-addition of benzyne to a six-membered ring of C₆₀, all 1,2- and 1,4-additions studied are exothermic processes; (ii) for C₆₀ the (2 + 2) benzyne cycloaddition is the most favored reaction pathway; (iii) for zigzag single-walled carbon nanotubes, the (4 + 2) benzyne cycloaddition is preferred over the (2 + 2) reaction pathway; and (iv) there is a gradual decrease in the exothermicity of the reaction and an increase of energy barriers as the diameter of the nanostructure of carbon is increased. By making use of the activation strain model, it is found that the deformation of the initial reactants in the rate-determining transition state is the key factor determining the chemoselectivity of the cycloadditions with benzyne

Diels–Alder Reactions of Graphene: Computational Predictions of Products and Sites of Reaction

The cycloaddition reactions and noncovalent π interactions of 2,3-dimethoxybutadiene (DMBD), 9-methylanthracene (MeA), tetracyanoethylene (TCNE), and maleic anhydride (MA) with graphene models have been investigated using density functional theory (DFT) calculations. Reaction enthalpies have been obtained to assess the reactivity and selectivity of covalent and noncovalent functionalization. Results indicate that graphene edges may be functionalized by the four reagents through cycloaddition reactions, while the interior regions cannot react. Noncovalent complexation is much more favorable than cycloaddition reactions on interior bonds of graphene. The relative reactivities of different sites in graphene are related to loss of aromaticity and can be predicted using Hückel molecular orbital (HMO) localization energy calculations

The Frozen Cage Model: A Computationally Low-Cost Tool for Predicting the Exohedral Regioselectivity of Cycloaddition Reactions Involving Endohedral Metallofullerenes

Functionalization of endohedral metallofullerenes (EMFs) is an active line of research that is important for obtaining nanomaterials with unique properties that might be used in a variety of fields, ranging from molecular electronics to biomedical applications. Such functionalization is commonly achieved by means of cycloaddition reactions. The scarcity of both experimental and theoretical studies analyzing the exohedral regioselectivity of cycloaddition reactions involving EMFs translates into a poor understanding of the EMF reactivity. From a theoretical point of view, the main obstacle is the high computational cost associated with this kind of studies. To alleviate the situation, we propose an approach named the frozen cage model (FCM) based on single point energy calculations at the optimized geometries of the empty cage products. The FCM represents a fast and computationally inexpensive way to perform accurate qualitative predictions of the exohedral regioselectivity of cycloaddition reactions in EMFs. Analysis of the Dimroth approximation, the activation strain or distortion/interaction model, and the noncluster energies in the Diels–Alder cycloaddition of <i>s-cis</i>-1,3-butadiene to X@<i>D</i>_3<i>h</i>-C₇₈ (X = Ti₂C₂, Sc₃N, and Y₃N) EMFs provides a justification of the method

Covalently Patterned Graphene Surfaces by a Force-Accelerated Diels–Alder Reaction

Cyclopentadienes (CPs) with Raman and electrochemically active tags were patterned covalently onto graphene surfaces using force-accelerated Diels–Alder (DA) reactions that were induced by an array of elastomeric tips mounted onto the piezoelectric actuators of an atomic force microscope. These force-accelerated cycloadditions are a feasible route to locally alter the chemical composition of graphene defects and edge sites under ambient atmosphere and temperature over large areas (∼1 cm²)

Acceleration of an Aromatic Claisen Rearrangement via a Designed Spiroligozyme Catalyst that Mimics the Ketosteroid Isomerase Catalytic Dyad

A series of hydrogen-bonding catalysts have been designed for the aromatic Claisen rearrangement of a 1,1-dimethylallyl coumarin. These catalysts were designed as mimics of the two-point hydrogen-bonding interaction present in ketosteroid isomerase that has been proposed to stabilize a developing negative charge on the ether oxygen in the migration of the double bond. Two hydrogen bond donating groups, a phenol alcohol and a carboxylic acid, were grafted onto a conformationally restrained spirocyclic scaffold, and together they enhance the rate of the Claisen rearrangement by a factor of 58 over the background reaction. Theoretical calculations correctly predict the most active catalyst and suggest that both preorganization and favorable interactions with the transition state of the reaction are responsible for the observed rate enhancement

Why Bistetracenes Are Much Less Reactive Than Pentacenes in Diels–Alder Reactions with Fullerenes

The Diels–Alder (DA) reactions of pentacene (PT), 6,13-bis­(2-trimethylsilylethynyl)­pentacene (TMS-PT), bistetracene (BT), and 8,17-bis­(2-trimethylsilylethynyl)­bistetracene (TMS-BT) with the [6,6] double bond of [60]­fullerene have been investigated by density functional theory calculations. Reaction barriers and free energies have been obtained to assess the effects of frameworks and substituent groups on the DA reactivity and product stability. Calculations indicate that TMS-BT is about 5 orders of magnitude less reactive than TMS-PT in the reactions with [60]­fullerene. This accounts for the observed much higher stability of TIPS-BT than TIPS-PT when mixed with PCBM. Surprisingly, calculations predict that the bulky silylethynyl substituents of TMS-PT and TMS-BT have only a small influence on reaction barriers. However, the silylethynyl substituents significantly destabilize the corresponding products due to steric repulsions in the adducts. This is confirmed by experimental results here. Architectures of the polycyclic aromatic hydrocarbons (PAHs) play a crucial role in determining both the DA barrier and the adduct stability. The reactivities of different sites in various PAHs are related to the loss of aromaticity, which can be predicted using the simple Hückel molecular orbital localization energy calculations

Bis-1,3-dipolar Cycloadditions on Endohedral Fullerenes M₃N@<i>I</i>_<i>h</i>‑C₈₀ (M = Sc, Lu): Remarkable Endohedral-Cluster Regiochemical Control

In this work, we briefly report some attempts to control regioisomeric bisadditions on Sc₃N@<i>I</i>_<i>h</i>-C₈₀ and Lu₃N@<i>I</i>_<i>h</i>-C₈₀ using the tether-controlled multifunctionalization method. We then describe the use of independent (nontethered) bis-1,3-dipolar cycloaddition reactions and the characterization of 5 new bisadducts, 3 for Sc₃N@C₈₀ and 2 for Lu₃N@C₈₀, which have never been reported before. Unexpectedly and remarkably, 4 of these compounds exhibit relatively high symmetry and 2 of these bisadducts are the first examples of intrinsically chiral endohedral compounds, due to the addition pattern, not to the presence of chiral centers on the addends. Since an analysis of the statistically possible number of bisadduct isomers on an <i>I</i>_<i>h</i>-C₈₀ cage has not been reported, we present it here

Acceleration of an Aromatic Claisen Rearrangement via a Designed Spiroligozyme Catalyst that Mimics the Ketosteroid Isomerase Catalytic Dyad

A series of hydrogen-bonding catalysts have been designed for the aromatic Claisen rearrangement of a 1,1-dimethylallyl coumarin. These catalysts were designed as mimics of the two-point hydrogen-bonding interaction present in ketosteroid isomerase that has been proposed to stabilize a developing negative charge on the ether oxygen in the migration of the double bond. Two hydrogen bond donating groups, a phenol alcohol and a carboxylic acid, were grafted onto a conformationally restrained spirocyclic scaffold, and together they enhance the rate of the Claisen rearrangement by a factor of 58 over the background reaction. Theoretical calculations correctly predict the most active catalyst and suggest that both preorganization and favorable interactions with the transition state of the reaction are responsible for the observed rate enhancement