Synthesis of Cross-Chain Bridging Cryptands and Induction of Molecular Chirality.

In this study, we synthesized two cryptands featuring entangled tri- and tetra(ethylene glycol) linkers. The cryptand bearing short linkers was chiral without any asymmetric carbon atoms. After chiral high-performance liquid chromatography was used to separate the enantiomers, the absolute configuration of each cryptand was determined through single-crystal X-ray and circular dichroism analyses. The racemization of the cryptand possessing long linkers proceeded at room temperature.

The oxidative folding of nascent polypeptides provides electrons for reductive reactions in the ER.

The endoplasmic reticulum (ER) maintains an oxidative redox environment that is advantageous for the oxidative folding of nascent polypeptides entering the ER. Reductive reactions within the ER are also crucial for maintaining ER homeostasis. However, the mechanism by which electrons are supplied for the reductase activity within the ER remains unknown. Here, we identify ER oxidoreductin-1α (Ero1α) as an electron donor for ERdj5, an ER-resident disulfide reductase. During oxidative folding, Ero1α catalyzes disulfide formation in nascent polypeptides through protein disulfide isomerase (PDI) and then transfers the electrons to molecular oxygen via flavin adenine dinucleotide (FAD), ultimately yielding hydrogen peroxide (H2O2). Besides this canonical electron pathway, we reveal that ERdj5 accepts electrons from specific cysteine pairs in Ero1α, demonstrating that the oxidative folding of nascent polypeptides provides electrons for reductive reactions in the ER. Moreover, this electron transfer pathway also contributes to maintaining ER homeostasis by reducing H2O2 production in the ER.

Nonthermal acceleration of protein hydration by sub-terahertz irradiation.

The collective intermolecular dynamics of protein and water molecules, which overlap in the sub-terahertz (THz) frequency region, are relevant for expressing protein functions but remain largely unknown. This study used dielectric relaxation (DR) measurements to investigate how externally applied sub-THz electromagnetic fields perturb the rapid collective dynamics and influence the considerably slower chemical processes in protein-water systems. We analyzed an aqueous lysozyme solution, whose hydration is not thermally equilibrated. By detecting time-lapse differences in microwave DR, we demonstrated that sub-THz irradiation gradually decreases the dielectric permittivity of the lysozyme solution by reducing the orientational polarization of water molecules. Comprehensive analysis combining THz and nuclear magnetic resonance spectroscopies suggested that the gradual decrease in the dielectric permittivity is not induced by heating but is due to a slow shift toward the hydrophobic hydration structure in lysozyme. Our findings can be used to investigate hydration-mediated protein functions based on sub-THz irradiation.

Repetitive DNA symmetry elements negatively regulate gene expression in embryonic stem cells.

Transcription factor (TF) binding to genomic DNA elements constitutes one of the key mechanisms that regulates gene expression program in cells. Both consensus and nonconsensus DNA sequence elements influence the recognition specificity of TFs. Based on the analysis of experimentally determined c-Myc binding preferences to genomic DNA, here we statistically predict that certain repetitive, nonconsensus DNA symmetry elements can relatively reduce TF-DNA binding preferences. This is in contrast to a different set of repetitive, nonconsensus symmetry elements that can increase the strength of TF-DNA binding. Using c-Myc enhancer reporter system containing consensus motif flanked by nonconsensus sequences in embryonic stem cells, we directly demonstrate that the enrichment in such negatively regulating repetitive symmetry elements is sufficient to reduce the gene expression level compared with native genomic sequences. Negatively regulating repetitive symmetry elements around consensus c-Myc motif and DNA sequences containing consensus c-Myc motif flanked by entirely randomized sequences show similar expression baseline. A possible explanation for this observation is that rather than complete repression, negatively regulating repetitive symmetry elements play a regulatory role in fine-tuning the reduction of gene expression, most probably by binding TFs other than c-Myc.

Monitoring and Predicting the Size of Fine Particles Prepared in a Fluidized-Bed Granulator Using a Handheld-Type Raman Spectrometer.

We prepared two kinds of fine particles by treating lactose monohydrate (Lac) with the same formulation in a fluidized-bed granulator, which differed in the spraying air pressure. Raman intensities of treated Lac during processing were measured using a handheld-type Raman spectrometer and plotted against particle size. As particle size increased, Raman intensity decreased in both operating conditions. A linear relationship between them was observed, and regression lines were obtained with good correlation coefficients as calibration curves in both operating conditions. We also prepared two other types of fine particles by treating Lac with the same formulation in a fluidized-bed granulator, which differed in the scale or processing mechanism. The particle size could be successfully predicted using the calibration curve obtained taking powder porosity into consideration. Based on this study, we present a new at-line approach in process analytical technology to monitor and predict particle size using a handheld-type Raman spectrometer.

Development of Low Molecular Weight Ligands for Integrin αvß3.

We designed and synthesized non-peptide organic molecular ligands for integrin αvß3. Candidate ligands featured amidino analog and carboxy groups as binding sites on either side of a spacer, which consisted of benzophenone or an analog, such as diphenyl sulfide, diphenyl sulfoxide, diphenyl sulfone, or diphenyl ether. Competitive binding assays to integrin αvß3 with respect to [125I]echistatin were used to determine inhibitory activity of the synthetic ligands. Ligands bearing 2-aminobenzimidazoyl and glycyl groups separated by a benzophenone spacer demonstrated more potent binding than did a linear Arg-Gly-Asp (RGD) tripeptide that represents the native integrin αvß3 binding motif. Ligands possessing 2-aminobenzimidazoyl and carboxy groups and diphenyl sulfoxide or diphenyl ether spacers inhibited binding of [125I]echistatin with IC50 values similar to that of the linear RGD tripeptide.

Four- and two-armed hetero porphyrin dimers: their specific recognition and self-sorting behaviours.

In this study we self-assembled the four-armed porphyrin hetero dimer capsule Cap4, stabilized through amidinium-carboxylate salt bridges, in CH2Cl2 and CHCl3. The dimer capsule Cap4 was kinetically and thermodynamically more stable than the corresponding two-armed dimer Cap2. The number of arms strongly influenced their recognition behaviour; guests possessing small aromatic faces (e.g., 1,3,5-trinitrobenzene) preferred residing in the cavity of the two-armed capsule Cap2, rather than in Cap4, both thermodynamically and kinetically; in contrast, large aromatic guests (e.g., 9,10-dibromoanthracene) were encapsulated predominantly by Cap4 because of favourable entropic effects. The number of arms enabled self-sorting behaviour of the dimer formation; complexation studies using an equimolar mixture of the four porphyrin constituents of the two capsules revealed the quantitative formation of the corresponding dimers Cap2 and Cap4. Furthermore, we examined the specific molecular recognition of Cap2 and Cap4; NMR experiments of mixtures of Cap2 and Cap4 in the presence of favourable guests for Cap2 and Cap4 revealed that these guest molecules were encapsulated selectively by their preferred hosts.

Synthesis of a Mechanically Planar Chiral and Axially Chiral [2]Rotaxane.

In this study, we synthesized a [2]rotaxane that was both mechanically planar chiral and axially chiral, comprising a symmetrical bis-crown ether featuring a biphenyl moiety (as the macrocyclic component) and a symmetrical bis-ammonium salt (as the dumbbell-shaped component).

Quantitative Difference in Solubility of Diastereomeric (2H/1H)-Isotopomers.

Many achiral organic compounds become chiral by an isotopic substitution of one of the enantiotopic moieties in their structures. Although spectroscopic methods can recognize the molecular chirality due to an isotopic substitution, the effects of isotopically chiral compounds in enantioselective reactions have remained unsolved because the small chirality arises only from the difference between the number of neutrons in the atomic nuclei. The difference between the diastereomeric isotopomers of reactive sources should be the key to these effects. However, the energy difference between them is difficult to calculate, even using present computational methods, and differences in physical properties have not yet been reported. Here, we demonstrate that the small energy difference between the diastereomeric isotopomers at the molecular level can be enhanced to appear as a solubility difference between the diastereomeric (2H/1H) isotopomers of α-aminonitriles, synthesized from an isotopically chiral amine, achiral aldehyde, and HCN. This small, but measurable, difference induces the chiral (d/l) imbalance in the suspended α-aminonitrile; therefore, a second enhancement in the solid-state chirality proceeds to afford a highly stereoimproved aminonitrile (>99% selectivity) whose handedness arises completely from the excess enantiomer of isotopically chiral amine, even in a low enantiomeric excess and low deuterium-labeling ratio. Because α-aminonitriles can be hydrolyzed to chiral α-amino acids with the removal of an isotope-labeling moiety, the current sequence of reactions represents a highly enantioselective Strecker amino acid synthesis induced by the chiral hydrogen (2H/1H) isotopomer. Thus, hydrogen isotopic chirality links directly with the homochirality of α-amino acids via a double enhancement of α-aminonitrile, the chiral intermediate of a proposed prebiotic mechanism.

Function-Related Dynamics in Multi-Spanning Helical Membrane Proteins Revealed by Solution NMR.

A primary biological function of multi-spanning membrane proteins is to transfer information and/or materials through a membrane by changing their conformations. Therefore, particular dynamics of the membrane proteins are tightly associated with their function. The semi-atomic resolution dynamics information revealed by NMR is able to discriminate function-related dynamics from random fluctuations. This review will discuss several studies in which quantitative dynamics information by solution NMR has contributed to revealing the structural basis of the function of multi-spanning membrane proteins, such as ion channels, GPCRs, and transporters.

Potential pharmacokinetic interaction between orally administered drug and osmotically active excipients in pediatric polypharmacy.

Poorly absorbable sugar alcohols (e.g., mannitol, sorbitol, and maltitol) are the excipients frequently contained in pediatric dosage forms. Due to their osmotically active properties, certain amount of sugar alcohols reportedly reduces oral bioavailability of concomitant drugs. This fact implies the possible pharmacokinetic interaction between orally administered drug and sugar alcohols which are present in other concomitant medications. The purpose of this study was to identify the possibility and likeliness of the osmotically active excipient-induced pharmacokinetic interaction in pediatric polypharmacy. Previously developed in silico model that captured the osmotic effect of sugar alcohols in adults was expanded to pediatric population. This mathematical model successfully explained the impaired bioavailability of lamivudine by the co-administered sorbitol in other dosage forms. In the meantime, sugar alcohol contents in marketed pediatric dosage forms were investigated by reverse engineering technology. Considering the critical administration dose of sugar alcohols estimated by in silico model, it was revealed that 25 out of 153 pediatric dosage forms were identified as possible perpetrators even under the approved administration and dosage in Japan. This study shed light on the potential pharmacokinetic interaction that cannot be dismissed throughout the pediatric pharmaceutical dosage form design and development.

Nonthermal excitation effects mediated by sub-terahertz radiation on hydrogen exchange in ubiquitin.

Water dynamics in the hydration layers of biomolecules play crucial roles in a wide range of biological functions. A hydrated protein contains multiple components of diffusional and vibrational dynamics of water and protein, which may be coupled at ∼0.1-THz frequency (10-ps timescale) at room temperature. However, the microscopic description of biomolecular functions based on various modes of protein-water-coupled motions remains elusive. A novel approach for perturbing the hydration dynamics in the subterahertz frequency range and probing them at the atomic level is therefore warranted. In this study, we investigated the effect of klystron-based, intense 0.1-THz excitation on the slow dynamics of ubiquitin using NMR-based measurements of hydrogen-deuterium exchange. We demonstrated that the subterahertz irradiation accelerated the hydrogen-deuterium exchange of the amides located in the interior of the protein and hydrophobic surfaces while decelerating this exchange in the amides located in the surface loop and short 310 helix regions. This subterahertz-radiation-induced effect was qualitatively contradictory to the increased-temperature-induced effect. Our results suggest that the heterogeneous water dynamics occurring at the protein-water interface include components that are nonthermally excited by the subterahertz radiation. Such subterahertz-excited components may be linked to the slow function-related dynamics of the protein.

A Chiral [3]Rotaxane Comprising Achiral Bis-macrocyclic and Dumbbell-Shaped Components.

In this study, we synthesized a molecularly chiral [3]rotaxane comprising a calix-bis-crown ether (as the macrocyclic component) and two unsymmetrical dialkylammonium salts (as dumbbell-shaped components) without any chirality in any of the individual components. Chiral high-performance liquid chromatography was used to separate the enantiomers, which were characterized by circular dichroism spectroscopy. Density functional theory calculations gave an insight into the absolute configuration of each [3]rotaxane.

Conformational Plasticity of Cyclic Ras-Inhibitor Peptides Defines Cell Permeabilization Activity.

Cyclorasins 9A5 and 9A54 are 11-mer cyclic peptides that inhibit the Ras-Raf protein interaction. The peptides share a cell-penetrating peptide (CPP)-like motif; however, only cyclorasin 9A5 can permeabilize cells to exhibit strong cell-based activity. To unveil the structural origin underlying their distinct cellular permeabilization activities, we compared the three-dimensional structures of cyclorasins 9A5 and 9A54 in water and in the less polar solvent dimethyl sulfoxide (DMSO) by solution NMR. We found that cyclorasin 9A5 changes its extended conformation in water to a compact amphipathic structure with converged aromatic residues surrounded by Arg residues in DMSO, which might contribute to its cell permeabilization activity. However, cyclorasin 9A54 cannot adopt this amphipathic structure, due to the steric hindrance between two neighboring bulky amino-acid sidechains, Tle-2 and dVal-3. We also found that the bulkiness of the sidechains at positions 2 and 3 negatively affects the cell permeabilization activities, indicating that the conformational plasticity that allows the peptides to form the amphipathic structure is important for their cell permeabilization activities.

Base-induced multi-state fluorescence of a trefoil-shaped salicylaldehyde azine derivative.

A trefoil-shaped salicylaldehyde azine derivative bearing multiple acidic protons displays base-induced multi-state luminescence. The azine was prepared through the reaction of 1,3,5-triformylphloroglucinol with 4-methoxysalicylaldehyde hydrazone. 1H NMR spectroscopy revealed that the azine existed in solution at room temperature as an equilibrium mixture of two geometric isomers. The three-step deprotonation (four-state change) of the azine in solution was confirmed using 1H NMR, UV-vis absorption, and emission spectroscopy.

The Provisional No-Effect Threshold of Sugar Alcohols on Oral Drug Absorption Estimated by Physiologically Based Biopharmaceutics Model.

Sugar alcohols reduce oral drug bioavailability by osmotic effects, but the magnitude of these effects differs among different drugs. This study aimed to identify the drug-related critical attributes of osmotic effects and estimate the impact of a "practical" sugar alcohol dose on the pharmacokinetics of various molecules using modeling and simulation approaches. We developed a physiologically based biopharmaceutics model that considers the dose-dependent effects of sugar alcohols on the gastrointestinal physiology. The developed model captured the effects of sugar alcohols on ranitidine hydrochloride, metoprolol tartrate, theophylline, cimetidine, and lamivudine. Sensitivity analysis provided quantitative insights into the effects of sugar alcohols dependent on different drug permeability. In addition, our developed model indicated for the first time that a high systemic elimination rate is crucial for the reduction in maximum plasma concentration even for highly permeable drugs. Nonetheless, mannitol/sorbitol level of less than 400 mg had minor effects on the pharmacokinetics of the most sensitive drugs, indicating a provisional no-effect threshold dose. This mechanistic approach provides comprehensive estimation of osmotic effects on variety of drugs. Subsequently, these findings may invoke scientific discussion on the criteria for excipient changes in the context of biowaiver guidelines (e.g. biopharmaceutics classification system-based biowaiver).

Role of NMR in High Ordered Structure Characterization of Monoclonal Antibodies.

Obtaining high ordered structure (HOS) information is of importance to guarantee the efficacy and safety of monoclonal antibodies (mAbs) in clinical application. Assessment of HOS should ideally be performed in a non-invasive manner under their formulated storage conditions, as any perturbation can introduce unexpected detritions. However, most of the currently available techniques only indirectly report HOS of mAbs and/or require a certain condition to conduct the analyses. Besides, the flexible multidomain architecture of mAbs has hampered atomic-resolution structural analyses using X-ray crystallography and cryo-electron microscopy. In contrast, the ability of nuclear magnetic resonance (NMR) spectroscopy to structurally analyze biomolecules in various conditions in a non-invasive and quantitative manner is suitable to meet the needs. However, the application of NMR to mAbs is not straightforward due to the high molecular weight of the system. In this review, we will discuss how NMR techniques have been applied to HOS analysis of mAbs, along with the recent advances of the novel 15N direct detection NMR strategy that allows for obtaining the structural fingerprint of mAbs at lower temperatures under multiple formulation conditions. The potential application of these NMR strategies will benefit next-generation mAbs, such as antibody-drug conjugates and bispecific antibodies.

Locking the Dynamic Axial Chirality of Biphenyl Crown Ethers through Threading.

This paper describes the syntheses of [2]rotaxanes comprising 23- and 26-membered biphenyl crown ethers as the macrocyclic components and secondary ammonium ions as the dumbbell-shaped components, and the locking of the dynamic axial chirality of the biphenyl moieties in these structures. Chiral high-performance liquid chromatography (HPLC) revealed that our [2]rotaxane featuring the 26-membered crown ether racemized at room temperature, but the racemization of the [2]rotaxane featuring the 23-membered crown ether did not proceed at room temperature over a period of three days. After separation of the enantiomers of the [2]rotaxane incorporating the 23-membered crown ether through chiral HPLC, we studied its racemization at elevated temperature. The rate of stereoinversion in dimethylsulfoxide (a polar solvent) was faster than that in o-dichlorobenzene (a nonpolar solvent), and herein we discuss these kinetic parameters.

Targeting the cryptic sites: NMR-based strategy to improve protein druggability by controlling the conformational equilibrium.

Cryptic ligand binding sites, which are not evident in the unligated structures, are beneficial in tackling with difficult but attractive drug targets, such as protein-protein interactions (PPIs). However, cryptic sites have thus far not been rationally pursued in the early stages of drug development. Here, we demonstrated by nuclear magnetic resonance that the cryptic site in Bcl-xL exists in a conformational equilibrium between the open and closed conformations under the unligated condition. While the fraction of the open conformation in the unligated wild-type Bcl-xL is estimated to be low, F143W mutation that is distal from the ligand binding site can substantially elevate the population. The F143W mutant showed a higher hit rate in a phage-display peptide screening, and the hit peptide bound to the cryptic site of the wild-type Bcl-xL. Therefore, by controlling the conformational equilibrium in the cryptic site, the opportunity to identify a PPI inhibitor could be improved.

Control of Transcription Initiation by Biased Thermal Fluctuations on Repetitive Genomic Sequences.

In the process of transcription initiation by RNA polymerase, promoter DNA sequences affect multiple reaction pathways determining the productivity of transcription. However, the question of how the molecular mechanism of transcription initiation depends on the sequence properties of promoter DNA remains poorly understood. Here, combining the statistical mechanical approach with high-throughput sequencing results, we characterize abortive transcription and pausing during transcription initiation by Escherichia coli RNA polymerase at a genome-wide level. Our results suggest that initially transcribed sequences, when enriched with thymine bases, contain the signal for inducing abortive transcription, whereas certain repetitive sequence elements embedded in promoter regions constitute the signal for inducing pausing. Both signals decrease the productivity of transcription initiation. Based on solution NMR and in vitro transcription measurements, we suggest that repetitive sequence elements within the promoter DNA modulate the nonlocal base pair stability of its double-stranded form. This stability profoundly influences the reaction coordinates of the productive initiation via pausing.