Viktorya Aviyente

Deamidation of Asparagine Residues : Direct Hydrolysis versus Succinimide-Mediated Deamidation Mechanisms

Quantum chemical calculations are reported to provide new insights on plausible mechanisms leading to the deamidation of asparagine residues in proteins and peptides. Direct hydrolysis to aspartic acid and several succinimide-mediated mechanisms have been described. The catalytic effect of water molecules has been explicitly analyzed. Calculations have been carried out at the density functional level (B3LYP/6-31+G**). Comparisons of free energy profiles show that the most favorable reaction mechanism goes through formation of a succinimide intermediate and involves tautomerization of the asparagine amide to the corresponding imidic acid as the initial reaction step. Another striking result is that direct water-assisted hydrolysis is competitive with the succinimide-mediated deamidation routes even in the absence of acid or base catalysis. The rate-determining step for the formation of the succinimide intermediate is cyclization, regardless of the mechanism. The rate-determining step for the complete deamidation is the hydrolysis of the succinimide intermediate. These results allow clarification of some well-known facts, such as the isolation of succinimide or the absence of iso-Asp among the reaction products observed in some experiments.

Solvent effects on glycine II. Water-assisted tautomerization

The water‐assisted tautomerization of glycine has been investigated at the B3LYP/6‐31+G** level using supermolecules containing up to six water molecules as well as considering a 1:1 glycine–water complex embedded in a continuum. The conformations of the tautomers in this mechanism do not display an intramolecular H bond, instead the functional groups are bridged by a water molecule. The replacement of the intramolecular H bond by the bridging water reduces the polarity of the NH bond in the zwitterion and increases that of the OH bond in the neutral, stabilizing the zwitterion. Both the charge transfer effects and electrostatic interactions stabilize the nonintramolecularly H‐bonded zwitterion conformer over the intramolecularly hydrogen bonded one. The nonintramolecularly H‐bonded neutral is favored only by charge transfer effects. Although there is no strong evidence whether the intramolecularly hydrogen bonded or non hydrogen bonded structures are favored in the bulk solution represented as a dielectric continuum, it is likely that the latter species are more stable. The free energy of activation of the water‐assisted mechanism is higher than the intramolecular proton transfer channel. However, when the presumably higher conformational energy of the zwitterion reacting in the intramolecular mechanism is taken into account, both mechanisms are observed to compete. The various conformers of the neutral glycine may form via multiple proton transfer reactions through several water molecules instead of a conformational rearrangement.

Solvent effects on glycine. I. A supermolecule modeling of tautomerization via intramolecular proton transfer

The relative stabilities of glycine tautomers involved in the intramolecular proton transfer are investigated computationally by considering glycine‐water complexes containing up to five water molecules. The supermolecule results are compared with continuum calculations. Specific solute‐solvent interactions and solvent induced changes in the solute wave function are considered using the natural bond orbitals (NBO) method. The stabilization of the zwitterion upon solvation is explained by the changes in the wave functions localized on the forming and breaking bonds as well as by the different interaction energies in the zwitterionic and neutral clusters. Only the neutral species exist in mono‐ and dihydrated clusters and in the gas phase. In the smaller clusters, zwitterions are mainly stabilized by conformational effects, whereas in larger clusters, in particular when glycine is solvated on both sides of its heavy atom backbone, polarization effects dominate the stability of a given tautomer. Generally, the strength of the solute‐solvent interactions is governed by the intermolecular charge transfer interactions. As the solvation progresses, the hypothetical gaseous zwitterion is better solvated than the gaseous neutral, making zwitterion to neutral tautomerization progressively less exothermic for clusters containing up to three water molecules, and endothermic for larger clusters. The neutral isomer does not exist for some solvent arrangements with five water molecules. Only solvent arrangements in which water molecules do not interact with the reactive proton are considered. Hence, the experimentally observed double well potential energy surface may be due to such an interaction or to a different reaction mechanism.

Elimination of water from the carboxyl group of GlyGlyH

The elimination of water from the carboxyl group of protonated diglycine has been investigated by density functional theory calculations. The resulting structure is identical to the b2 ion formed in the mass spectrometric fragmentation of protonated peptides (therefore named “b2” in this study). The most stable geometry of the fragment ion (“b2”) is an O-protonated diketopiperazine. However, its formation is kinetically disfavored as it requires a free energy of 58.2 kcal/mol. The experimentally observed N-protonated oxazolone is 3.0 kcal/mol less stable. The lowest energy pathway for the formation of the “b2” ion requires a free energy of 37.5 kcal/mol and involves the proton transfer from the amide oxygen of protonated diglycine to the hydroxyl oxygen. Fragmentation initiated by proton transfer from the terminal nitrogen has also a comparable free energy of activation (39.4 kcal/mol). Proton transfer initiating the fragmentation, from the highly basic terminal nitrogen or amide oxygen to the less basic hydroxyl oxygen is feasible at energies reached in usual mass spectrometric experiments. Amide N-protonated diglycine structures are precursors of mainly y1 ions rather than “b2” ions. In the lowest energy fragmentation channels, proton transfer to the hydroxylic oxygen, bond breaking and formation of an oxazolone ring occur concertedly but asynchronously. Proton transfer to hydroxyl oxygen and cleavage of the corresponding C-O bond take place at the early stages of the fragmentation step, while ring closure to form an oxazolone geometry occurs at the later stages of the transition. The experimentally observed low kinetic energy release is expected to be due to the existence of a strongly hydrogen bonded protonated oxazolone-water complex in the exit channel. Whereas the threshold energy for “b2” ion formation (37.1 kcal/mol) is lower than for the y1 ion (38.4 kcal/mol), the former requires a tight transition state with an activation entropy, ΔS‡ = −1.2 cal/mol.K and the latter has a loose transition state with ΔS‡ = +8.8 cal/mol.K. This leads to y1 being the major fragment ion over a wide energy range.

Effect of Lewis Acid Catalysts on Diels−Alder and Hetero-Diels−Alder Cycloadditions Sharing a Common Transition State

The thermal and Lewis acid catalyzed cycloadditions of beta,gamma-unsaturated alpha-ketophosphonates and nitroalkenes with cyclopentadiene have been explored by using density functional theory (DFT) methods. In both cases, only a single highly asynchronous bis-pericyclic transition state yielding both Diels-Alder and hetero-Diels-Alder cycloadducts could be located. Stepwise pathways were found to be higher in energy. On the potential energy surface, the bis-pericyclic cycloaddition transition state is followed by the Claisen rearrangement transition state. No intermediates were located between these transition states. Claisen rearrangement transition states are also highly asynchronous, but bond lengths are skewed in the opposite direction compared to the bis-pericyclic transition states. The relative positions of the bis-pericyclic and Claisen rearrangement transition states may control periselectivity due to the shape of the potential energy surface and corresponding dynamical influences. Inspection of the thermal potential energy surface (PES) indicates that a majority of downhill paths after the bis-pericyclic transition state lead to the Diels-Alder cycloadducts, whereas a smaller number of downhill paths reach the hetero-Diels-Alder products with no intervening energy barrier. Lewis acid catalysts alter the shape of the surface by shifting the cycloaddition and the Claisen rearrangement transition states in opposite directions. This topographical change qualitatively affects the branching ratio after the bis-pericyclic transition state and ultimately reverses the periselectivity of the cycloaddition giving a preference for hetero-Diels-Alder cycloadducts.

Structures and reactivity of gaseous glycine and its derivatives

Abstract B3LYP/6-31++G∗∗ has been used to model the conformers of glycine, protonated glycine, the unimolecular fragmentation, the proton transfer and the bimolecular proton exchange reactions with NH 3 . B3LYP/6-31++G∗∗ has located all the conformers—except one—located previously with electron correlation methods. The results show that the performance of B3LYP/6-31++G∗∗ is significantly better than that of the HF method and in most cases as good as the ab initio theories such as MP2, CCSD, and CISD. We have thus used B3LYP/6-31++G∗∗ in order to understand the unimolecular fragmentation and bimolecular reactions of glycine with NH 3 . The proton has been found to be mobile over all basic sites when a threshold of ∼33 kcal/mol is reached. A barrier of ∼50 kcal/mol exists for the fragmentation reaction, H 2 O and CO being sequentially eliminated. The amino protons are exchanged with an onium mechanism and the carboxylic proton is exchanged via a salt bridge complex.

Computational study on nonenzymatic peptide bond cleavage at asparagine and aspartic acid.

Nonenzymatic peptide bond cleavage at asparagine (Asn) and glutamine (Gln) residues has been observed during peptide deamidation experiments; cleavage has also been reported at aspartic acid (Asp) and glutamic acid (Glu) residues. Although peptide backbone cleavage at Asn is known to be slower than deamidation, fragmentation products are often observed during peptide deamidation experiments. In this study, mechanisms leading to the cleavage of the carboxyl-side peptide bond of Asn and Asp residues were investigated using computational methods (B3LYP/6-31+G**). Single-point solvent calculations at the B3LYP/6-31++G** level were carried out in water, utilizing the integral equation formalism-polarizable continuum (IEF-PCM) model. Mechanism and energetics of peptide fragmentation at Asn were comparatively analyzed with previous calculations on deamidation of Asn. When deamidation proceeds through direct hydrolysis of the Asn side chain or through cyclic imide formationvia a tautomerization routeit exhibits lower activation barriers than peptide bond cleavage at Asn. The fundamental distinction between the mechanisms leading to deamidationvia a succinimideand backbone cleavage was found to be the difference in nucleophilic entities involved in the cyclization process (backbone versus side-chain amide nitrogen). If deamidation is prevented by protein three-dimensional structure, cleavage may become a competing pathway. Fragmentation of the peptide backbone at Asp was also computationally studied to understand the likelihood of Asn deamidation preceding backbone cleavage. The activation barrier for backbone cleavage at Asp residues is much lower (approximately 10 kcal/mol) than that at Asn. This suggests that peptide bond cleavage at Asn residues is more likely to take place after it has deamidated into Asp.

Conformational properties of amphotericin B amide derivatives – impact on selective toxicity

Even though it is highly toxic, Amphotericin B (AmB), an amphipathic polyene macrolide antibiotic, is used in the treatment of severe systemic fungal infections as a life-saving drug. To examine the influence of conformational factors on selective toxicity of these compounds, we have investigated the conformational properties of five AmB amide derivatives. It was found that the extended conformation with torsional angles (φ,ψ)=(290°,180° ) is a common minimum of the potential energy surfaces (PES) of unsubstituted AmB and its amide derivatives. The extended conformation of the studied compounds allows for the formation of an intermolecular hydrogen bond network between adjacent antibiotic molecules in the open channel configuration. Therefore, the extended conformation is expected to be the dominant conformer in an open AmB (or its amide derivatives) membrane channel. The derivative compounds for calculations were chosen according to their selective toxicity compared to AmB and they had a wide range of selective toxicity. Except for two AmB derivatives, the PES maps of the derivatives reveal that the molecules can coexist in more than one conformer. Taking into account the cumulative conclusions drawn from the earlier MD simulation studies of AmB membrane channel, the results of the potential energy surface maps, and the physical considerations of the molecular structures, we hypothesize a new model of structure-selective toxicity of AmB derivatives. In this proposed model the presence of the extended conformation as the only well defined global conformer for AmB derivatives is taken as the indicator of their higher selective toxicity. This model successfully explains our results. To further test our model, we also investigated an AmB derivative whose selective toxicity has not been experimentally measured before. Our prediction for the selective toxicity of this compound can be tested in experiments to validate or invalidate the proposed model.

Computational study of factors controlling the boat and chair transition states of Ireland-Claisen rearrangements.

The origins of the boat transition state preference in the Ireland-Claisen rearrangements studied experimentally by Kishi and co-workers have been explored computationally with Density Functional Theory. Steric interactions in the chair transition states were identified as the principal reason for the boat transition state preference.

Cyclopolymerization Reactions of Diallyl Monomers: Exploring Electronic and Steric Effects Using DFT Reactivity Indices

The regioselectivity in the cyclopolymerization of diallyl monomers is investigated using DFT-based reactivity indices. In the first part, the experimentally observed mode of cyclization (exo versus endo) of 11 selected radicals involved in this process is reproduced by the computation of activation energies, entropies, enthalpies, and Gibbs free energies for the 5- and 6-membered cyclization reactions. The application of a recently proposed energy partitioning of the activation barriers shows that the regioselectivity cannot be explained by the steric effect alone. Next, a number of relevant DFT-based reactivity indices, such as non-spin-polarized and spin-polarized Fukui functions, spin densities, and dual descriptors, were applied to probe the role of the polar and stereoelectronic effects in this reaction. The dual descriptor has been found to reproduce best the experimental trends, confirming the important role of the stereoelectronic effects.