Driving Forces Controlling Host-Guest Recognition in Supercritical Carbon Dioxide Solvent.

The formation of supramolecular host-guest complexes is a very useful and widely employed tool in chemistry. However, supramolecular chemistry in non-conventional solvents such as supercritical carbon dioxide (scCO2 ), one of the most promising sustainable solvents, is still in its infancy. In this work, we explored a successful route to the development of green processes in supercritical CO2 by combining a theoretical approach with experiments. We were able to synthesize and characterize an inclusion complex between a polar aromatic molecule (benzoic acid) and peracetylated-ß-cyclodextrin, which is soluble in the supercritical medium. This finding opens the way to wide, environmental friendly, applications of scCO2 in many areas of chemistry, including supramolecular synthesis, reactivity and catalysis, micro and nano-particle formation, molecular recognition, as well as enhanced extraction processes with increased selectivity.

Taste for chiral guests: investigating the stereoselective binding of peptides to ß-cyclodextrins.

Obtaining compounds of diastereomeric purity is extremely important in the field of biological and pharmaceutical industry, where amino acids and peptides are widely employed. In this work, we theoretically investigate the possibility of chiral separation of peptides by ß-cyclodextrins (ß-CDs), providing a description of the associated interaction mechanisms by means of molecular dynamics (MD) simulations. The formation of host/guest complexes by including a model peptide in the macrocycle cavity is analyzed and discussed. We consider the terminally blocked phenylalanine dipeptide (Ace-Phe-Nme), in the L- and D-configurations, to be involved in the host/guest recognition process. The CD-peptide free energies of binding for the two enantiomers are evaluated through a combined approach that assumes: (1) extracting a set of independent molecular structures from the MD simulation, (2) evaluating the interaction energies for the host/guest complexes by hybrid quantum mechanics/molecular mechanics (QM/MM) calculations carried out on each structure, for which we also compute, (3) the solvation energies through the Poisson-Boltzmann surface area method. We find that chiral discrimination by the CD macrocycle is of the order of 1 kcal/mol, which is comparable to experimental data for similar systems. According to our results, the Ace-(D)Phe-Nme isomer leads to a more stable complex with a ß-CD compared to the Ace-(L)Phe-Nme isomer. Nevertheless, we show that the chiral selectivity of ß-CDs may strongly depend on the secondary structure of larger peptides. Although the free energy differences are relatively small, the predicted selectivities can be rationalized in terms of host/guest hydrogen bonds and hydration effects. Indeed, the two enantiomers display different interaction modes with the cyclodextrin macrocavity and different mobility within the cavity. This finding suggests a new interpretation for the interactions that play a key role in chiral recognition, which may be exploited to design more efficient and selective chiral separations of peptides.

A new glimpse into the CO(2)-philicity of carbonyl compounds.

We report a theoretical study on non-conventional structures of 1:1 complexes between carbon dioxide and carbonyl compounds. These structures have never been reported before but are relevant for understanding the solubility of carbonyl compounds in supercritical CO(2). The work is based on the results of ab initio calculations at the MP2 and CCSD(T) levels using aug-cc-pVDZ and aug-cc-pVTZ basis sets. Investigated systems include aldehydes, ketones and esters, together with some fluorinated derivatives. The results are interpreted in terms of natural bond orbital analyses. Harmonic vibrational frequency calculations have also been done in order to compare them with available experimental data. We show for the first time that complexes where CO(2) behaves globally as a Lewis base are stable in the case of ketones and esters, but not in the case of aldehydes, and their stability is similar to that of traditional complexes in which CO(2) behaves as a Lewis acid. This finding considerably modifies the concept of CO(2)-philicity and may have important ramifications in the development of green reactions in supercritical CO(2).

Cavity closure dynamics of peracetylated ß-cyclodextrins in supercritical carbon dioxide.

Structural properties of peracetylated ß-cyclodextrin in supercritical carbon dioxide were investigated by means of molecular dynamics simulations. The study indicated a strong reduction of the cavity accessibility to guest molecules, compared to native ß-cyclodextrin in water. Indeed, the cavity is self-closed during the largest part of the simulation, which agrees well with suggestions made on the basis on high-pressure NMR experiments. Self-closure happens because one glucose unit undergoes a main conformational change (from chair to skew) that brings one of the acetyl groups in the wide rim of the cyclodextrin to the cavity interior. This arrangement turns out to be quite favorable, persisting for several nanoseconds. In addition to the wide rim self-closure, a narrow rim self-closure may also occur, though it is less likely and exhibits short duration (<1 ns). Therefore, the number of solvent molecules reaching the cavity interior is much smaller than that found in the case of native ß-cyclodextrin in water after correction to account for different molar densities. These findings support the weak tendency of the macromolecule to form host-guest complexes in this nonconventional medium, as reported by some experiments. Finally, Lewis acid/base interactions between the acetyl carbonyl groups and the solvent CO(2) molecules were analyzed through ab initio calculations that revealed the existence of a quite favorable four-member ring structure not yet reported. The ensemble of these results can contribute to establish general thermodynamic principles controlling the formation of inclusion complexes in supercritical CO(2), where the hydrophilicity/hydrophobicity balance is not applicable.

Peptide binding to ß-cyclodextrins: structure, dynamics, energetics, and electronic effects.

Peptide-cyclodextrin and protein-cyclodextrin host-guest complexes are becoming more and more important for industrial applications, in particular in the fields of pharmaceutical and food chemistry. They have already deserved many experimental investigations although the effect of complex formation in terms of peptide (or protein) structure is not well-known yet. Theoretical calculations represent a unique tool to analyze such effects, and with this aim we have carried out in the present investigation molecular dynamics simulations and combined quantum mechanics-molecular mechanics calculations. We have studied complexes formed between the model Ace-Phe-Nme peptide and the ß-cyclodextrin (ß-CD) macromolecule, and our analysis focuses on the following points: (1) how is the peptide structure modified in going from bulk water to CD environment (backbone torsion angles), (2) which are the main peptide-CD interactions, in particular in terms of hydrogen bonds, (3) which relative peptide-CD orientation is preferred and which are the structural and energetic differences between them, and (4) how the electronic properties of the peptide changes under complex formation. Overall, our calculations show that in the most stable configuration, the backbone chain lies in the narrow rim of the CD. Strong hydrogen bonds form between the H atoms of the peptidic NH groups and oxygen atoms of the secondary OH groups in the CD. These and other (weaker) hydrogen bonds formed by the carbonyl groups reduce considerably the flexibility of the peptide structure, compared to bulk water, and produce a marked increase of the local dipole moment by favoring configurations in which the two C═O bonds point toward the same direction. This effect might have important consequences in terms of the peptide secondary structure, although this hypothesis needs to be tested using larger peptide models.

X-ray, ESR, and quantum mechanics studies unravel a spin well in the cofactor-less urate oxidase.

Urate oxidase (EC 1.7.3.3 or UOX) catalyzes the conversion of uric acid using gaseous molecular oxygen to 5-hydroxyisourate and hydrogen peroxide in absence of any cofactor or transition metal. The catalytic mechanism was investigated using X-ray diffraction, electron spin resonance spectroscopy (ESR), and quantum mechanics calculations. The X-ray structure of the anaerobic enzyme-substrate complex gives credit to substrate activation before the dioxygen fixation in the peroxo hole, where incoming and outgoing reagents (dioxygen, water, and hydrogen peroxide molecules) are handled. ESR spectroscopy establishes the initial monoelectron activation of the substrate without the participation of dioxygen. In addition, both X-ray structure and quantum mechanic calculations promote a conserved base oxidative system as the main structural features in UOX that protonates/deprotonates and activate the substrate into the doublet state now able to satisfy the Wigner's spin selection rule for reaction with molecular oxygen in its triplet ground state.

Coupling and uncoupling mechanisms in the methoxythreonine mutant of cytochrome P450cam: a quantum mechanical/molecular mechanical study.

The Thr252 residue plays a vital role in the catalytic cycle of cytochrome P450cam during the formation of the active species (Compound I) from its precursor (Compound 0). We investigate the effect of replacing Thr252 by methoxythreonine (MeO-Thr) on this protonation reaction (coupling) and on the competing formation of the ferric resting state and H2O2 (uncoupling) by combined quantum mechanical/molecular mechanical (QM/MM) methods. For each reaction, two possible mechanisms are studied, and for each of these the residues Asp251 and Glu366 are considered as proton sources. The computed QM/MM barriers indicate that uncoupling is unfavorable in the case of the Thr252MeO-Thr mutant, whereas there are two energetically feasible proton transfer pathways for coupling. The corresponding rate-limiting barriers for the formation of Compound I are higher in the mutant than in the wild-type enzyme. These findings are consistent with the experimental observations that the Thr252MeO-Thr mutant forms the alcohol product exclusively (via Compound I), but at lower reaction rates compared with the wild-type enzyme.

Intrinsic reactivity of uric acid with dioxygen: Towards the elucidation of the catalytic mechanism of urate oxidase.

Urate oxidase catalyzes the transformation of uric acid in 5-hydroxyisourate, an unstable compound which is latter decomposed into allantoïn. Crystallographic data have shown that urate oxidase binds a dianion urate species deprotonated in N3 and N7, while kinetics experiments have highlighted the existence of several intermediates during catalysis. We have employed a quantum mechanical approach to analyze why urate oxidase is selective for one particular dianion and to explore all possible reaction pathways for the oxidation of one uric acid species by molecular dioxygen in presence of water. Our results indicate the urate dianion deprotonated in N3 and N7 is among all urate species that can coexist in solution it is the compound which will lose the most easiestly one electron in presence of molecular dioxygen. In addition, the transformation of this dianion in 5-hydroxyisourate is thermodynamically the most favorable reaction. Finally, several reaction pathways can be drawn, each starting with the spontaneous transfer of one electron from the urate dianion to molecular dioxygen. During that period, the existence of a 5-hydroperoxyisourate intermediate, which has been proposed elsewhere, does not seem mandatory.

QM/MM study of the second proton transfer in the catalytic cycle of the D251N mutant of cytochrome P450cam.

Protonation of Compound 0 in the catalytic cycle of cytochrome P450cam may lead to the formation of either the reactive Compound I (coupling) or the ferric resting state (uncoupling). In this work, we investigate the effect of the D251N mutation on the coupling and uncoupling reaction by combined quantum mechanics/molecular mechanics (QM/MM) calculations. The mutated Asn251 residue has two possible orientations, i.e. directed toward the active site (no flip) or away from the active site (flip), with the latter one being preferred in classical molecular dynamics (MD) simulations. The possible proton transfer mechanisms in the coupling and uncoupling reaction were studied for three models of the D251N mutant, i.e. no flip (model I), flip (model II), and flip with an extra water (model III). According to the QM/MM calculations, the uncoupling reaction is always less favorable than the coupling reaction. The coupling reaction in the D251N mutant follows the same mechanism as in the wild-type enzyme, with initial O-O cleavage followed by proton transfer. The barrier for the initial step is similar in all D251N models, but the proton transfer is most facile in model III. The hydroxide anion formed in model III is not reprotonated easily by neighboring residues, while proton delivery from bulk solvent seems possible via a water network that remains intact during 2 ns classical MD simulation. The computational results are consistent with the experimental findings that the coupling reaction dominates the consumption of dioxygen in the D251N mutant, but with lower activity than in the wild-type enzyme.

How is the reactivity of cytochrome P450cam affected by Thr252X mutation? A QM/MM study for X = serine, valine, alanine, glycine.

Proton transfer reactions play a vital role in the catalytic cycle of cytochrome P450cam and are responsible for the formation of the iron-oxo species called Compound I (Cpd I) that is supposed to be the active oxidant. Depending on the course of the proton transfer, protonation of the last observable intermediate (ferric hydroperoxo complex, Cpd 0) can lead to either the formation of Cpd I (coupling reaction) or the ferric resting state (uncoupling reaction). The ratio of these two processes is drastically affected by mutation of the Thr252 residue. In this work, we study the effect of Thr252X (X = serine, valine, alanine, glycine) mutations on the formation of Cpd I by means of hybrid quantum mechanical/molecular mechanical (QM/MM) calculations and classical simulations. In the wild-type enzyme, the coupling reaction is favored since its rate-limiting barrier is 13 kcal/mol lower than that for uncoupling. This difference is reduced to 7 kcal/mol in the serine mutant. In the case of valine, alanine, and glycine mutants, an additional water molecule enters the active site and lowers the activation energy of the uncoupling reaction significantly. With the additional water molecule, coupling and uncoupling have similar barriers in the valine mutant, and the uncoupling reaction becomes favored in the alanine and glycine mutants. These findings agree very well with experimental results and thus confirm the assumption that uncontrolled proton delivery by solvent water networks is responsible for the uncoupling reaction. The present study provides a detailed mechanistic understanding of the role of the Thr252 residue.

Oxygen pressurized X-ray crystallography: probing the dioxygen binding site in cofactorless urate oxidase and implications for its catalytic mechanism.

The localization of dioxygen sites in oxygen-binding proteins is a nontrivial experimental task and is often suggested through indirect methods such as using xenon or halide anions as oxygen probes. In this study, a straightforward method based on x-ray crystallography under high pressure of pure oxygen has been developed. An application is given on urate oxidase (UOX), a cofactorless enzyme that catalyzes the oxidation of uric acid to 5-hydroxyisourate in the presence of dioxygen. UOX crystals in complex with a competitive inhibitor of its natural substrate are submitted to an increasing pressure of 1.0, 2.5, or 4.0 MPa of gaseous oxygen. The results clearly show that dioxygen binds within the active site at a location where a water molecule is usually observed but does not bind in the already characterized specific hydrophobic pocket of xenon. Moreover, crystallizing UOX in the presence of a large excess of chloride (NaCl) shows that one chloride ion goes at the same location as the oxygen. The dioxygen hydrophilic environment (an asparagine, a histidine, and a threonine residues), its absence within the xenon binding site, and its location identical to a water molecule or a chloride ion suggest that the dioxygen site is mainly polar. The implication of the dioxygen location on the mechanism is discussed with respect to the experimentally suggested transient intermediates during the reaction cascade.