Photochemical Reaction of Ketoprofen with Proteinogenic Amino Acids.

Ketoprofen (KP) is one of the most popular nonsteroidal anti-inflammatory drugs; however, drug-induced photosensitivity of KP has been reported as a serious adverse effect. KP incorporated into a protein can produce an allergen under UV irradiation, which causes drug-induced photosensitivity. The photochemistry of KP with 20 kinds of proteinogenic amino acids in phosphate buffer solutions at pH 7.4 was studied by transient absorption spectroscopy. The KP carboxylate anion (KP-) gave rise to a carbanion via a decarboxylation within a laser pulse, and the carbanion yielded 3-ethylbenzophenone ketyl biradical (3-EBPH) through a proton transfer reaction. Twelve kinds of proteinogenic amino acids obviously accelerated the reaction. Structural information on the complexes of KP docked in the binding sites of human serum albumin (HSA) was obtained by molecular mechanics (MM) and molecular dynamics (MD) calculations. The photochemical reaction of KP- with amino acid residues in HSA was discussed on the basis of the experimental and calculational results. The information on the reactivity of KP with the amino acids and the stable structures of the KP-HSA complexes should be essential for understanding of the initial step for drug-induced photosensitivity.

De novo design of a nanopore for single-molecule detection that incorporates a ß-hairpin peptide.

The amino-acid sequence of a protein encodes information on its three-dimensional structure and specific functionality. De novo design has emerged as a method to manipulate the primary structure for the development of artificial proteins and peptides with desired functionality. This paper describes the de novo design of a pore-forming peptide, named SV28, that has a ß-hairpin structure and assembles to form a stable nanopore in a bilayer lipid membrane. This large synthetic nanopore is an entirely artificial device for practical applications. The peptide forms multidispersely sized nanopore structures ranging from 1.7 to 6.3 nm in diameter and can detect DNAs. To form a monodispersely sized nanopore, we redesigned the SV28 by introducing a glycine-kink mutation. The resulting redesigned peptide forms a monodisperse pore with a diameter of 1.7 nm leading to detection of a single polypeptide chain. Such de novo design of a ß-hairpin peptide has the potential to create artificial nanopores, which can be size adjusted to a target molecule.

Thermal and Nonthermal Microwave Effects of Ethanol and Hexane-Mixed Solution as Revealed by In Situ Microwave Irradiation Nuclear Magnetic Resonance Spectroscopy and Molecular Dynamics Simulation.

Microwave heating is widely used to accelerate the organic synthesis reaction. However, the role of the nonthermal microwave effect in the chemical reaction has not yet been well characterized. The microwave heating processes of an ethanol-hexane mixed solution were investigated using in situ microwave irradiation nuclear magnetic resonance spectroscopy and molecular dynamics (MD) simulation. The temperature of the solution under microwave irradiation was estimated from the temperature dependence of the 1H chemical shifts (chemical shift calibrated (CSC)-temperature). The CSC-temperature increased to 58 °C for CH2 and CH3 protons, while it increased to 42 °C for OH protons during microwave irradiation. The CSC-temperature of CH2 and CH3 protons reflects the bulk temperature of solution by the thermal microwave effect. The lower CSC-temperature of the OH proton can be attributed to a nonthermal microwave effect. MD simulation revealed that electron dipole moments of OH groups ordered along the oscillated electric field decreased the entropy by absorbing microwave energy and simultaneously increased the entropy by dissipating energy to the solution as the thermal and nonthermal microwave effect. Ordered polar molecules interact to increase hydrogen bonds between OH groups as the nonthermal microwave effect, which explains the lower CSC-temperature of the OH protons. The nonthermal microwave effects contribute to the intrinsic acceleration of the organic reaction.

Separation of D-amino acid-containing peptide phenylseptin using 3,3'-phenyl-1,1'-binaphthyl-18-crown-6-ether columns.

Several D-amino acid-containing peptides (DAACPs) with antimicrobial, cardio-excitatory, or neuronal activities have been found in several species. Here, we demonstrated the chiral separation of the antimicrobial peptide diastereomers, D-phenylseptin and L-phenylseptin using (S) and (R) 3,3'-phenyl-1,1'-binaphthyl-18-crown-6-ether columns (CR-I (+) and CR-I (-), respectively) and also investigated the underlying mechanism. First, using D-amino acid-containing tripeptide Phe-Phe-Phe-OH, we found that CR-I (+) could be used to recognize diastereomeric tripeptides containing an L-amino acid as the first residue. On the contrary, CR-I (-) enabled separation of a series of diastereomers with D-amino acid as the first residue. Therefore, we achieved separation of the stereoisomers using the chiral columns depending on the position of the D- amino acid in the peptide and demonstrated the orthogonality of separations of the chiral columns. Then, using CR-I (+), we separated amphibian antimicrobial peptide diastereomers, L- and D-phenylseptin, which have the sequences, L-Phe-L-Phe-L-Phe and L-Phe-D-Phe-L-Phe at their N-termini, respectively. In order to understand the host-guest interactions, we performed molecular dynamics simulations for L-Phe-L-Phe-L-Phe tripeptide-CR-I molecule complex systems. Three hydrogen bonds between the N-terminal amine group -NH3+ and the crown ether oxygens were the dominant interactions. The hydrophobic interactions between phenyl-rings in the chiral selector unit of CR-I (+) and the side chains of 2nd and 3rd residues of the peptide also contributed to the affinity. Our results show that the CR-I (+)-column can be applied for the separation of endogenous DAACPs generated by the post-translational modification.

The role of d-allo-isoleucine in the deposition of the anti-Leishmania peptide bombinin H4 as revealed by 31P solid-state NMR, VCD spectroscopy, and MD simulation.

Bombinin H4 is an antimicrobial peptide that was isolated from the toad Bombina variegata. Bombinin H family peptides are active against gram-positive, gram-negative bacteria, and fungi as well as the parasite Leishmania. Among them, bombinin H4 (H4), which contains d-allo-isoleucine (d-allo-Ile) as the second residue in its sequence, is the most active, and its l-isomer is bombinin H2 (H2). H4 has a significantly lower LC50 than H2 against Leishmania. However, the atomic-level mechanism of the membrane interaction and higher activity of H4 has not been clarified. In this work, we investigated the behavior of the conformations and interactions of H2 and H4 with the Leishmania membrane using 31P solid-state nuclear magnetic resonance (NMR), vibrational circular dichroism (VCD) spectroscopy, and molecular dynamics (MD) simulations. The generation of isotropic 31P NMR signals depending on the peptide concentration indicated the abilities of H2 and H4 to exert antimicrobial activity via membrane disruption. The VCD experiment and density functional theory calculation confirmed the different stability and conformations of the N-termini of H2 and H4. MD simulations revealed that the N-terminus of H4 is more stable than that of H2 in the membrane, in line with the VCD experiment data. VCD and MD analyses demonstrated that the first l-Ile and second d-allo-Ile of H4 tend to take a cis conformation. These residues function as an anchor and facilitate the easy winding of the helical conformation of H4 in the membrane. It may assist to quickly reach to the threshold concentration of H4 on the Leishmania membrane. This article is part of a Special Issue entitled: d-Amino acids: biology in the mirror, edited by Dr. Loredano Pollegioni, Dr. Jean-Pierre Mothet and Dr. Molla Gianluca.