Effect of N-o-nitrobenzylation on conformation and membrane permeability of linear peptides.

In this study, we explored the potential of the photoremovable o-nitrobenzyl (oNB) group as a tool to manipulate the membrane permeability and regulate the conformation of linear peptides by means of experimental and computational studies. We found that the introduction of one or more oNB groups markedly increased the permeability and altered the conformation, as compared to the corresponding unmodified peptides. We thoroughly investigated the impact of peptide length, number of oNB group, oNB insertion position, and introduction of N- and C-terminal protecting groups on the passive membrane permeability by means of parallel artificial membrane permeability assay (PAMPA). Photoreaction of peptides containing one or two oNB groups proceeded cleanly in moderate to high yields, releasing the unprotected parent linear peptide. The oNB-modified peptides showed a cis/trans conformational equilibrium, while after photolysis, the unprotected linear peptides showed only the trans-amide conformation. Furthermore, a comprehensive comparison of oNB-modified peptides and N-methylated peptides was conducted, encompassing conformational analysis and physicochemical properties. N-Substituted peptides favored a folded-like structure, which may contribute to the improvement in permeability.

Stabilization of sp-Hybridized Nitrogen Cation by Lewis Acid-Base Complex Formation with Intramolecular Iodine.

Here we show that the sp-hybridized nitrogen cation is strongly stabilized by a peri-iodine substituent in the tetralone system. The cation is captured by anionic species such as CF3 CO2 - , affording hypervalent iodine(III) compounds with a short nitrogen-iodine (N-I) bond, in which the cation serves as a Lewis acid. Notably, the O-I bond of the O-trifluoroacetate or O-acetate is intrinsically weaker than the N-I bond due to its more ionic character and is further weakened by protonation in trifluoroacetic acid. As a result, the oxygen ligand can dissociate in the presence of a Brønsted acid, affording a I+ cation intermediate that retains the N-I bond. We isolated the cation as the tetrafluoroborate, and characterized it experimentally by 1 H NMR spectroscopy and X-ray structure analysis, and theoretically by means of DFT calculation. The results suggest that the N-I bonded cation is intrinsically stable, and is weakly coordinated with water and the BF4 counter anion or trifluoroacetate anion. This cation can be employed as a reagent for α-oxidation of ketones.

Isosteric Replacement of Ester Linkage of Lysophospholipids with Heteroaromatic Rings Retains Potency and Subtype Selectivity.

Our group has reported various derivatives of lysophosphatidylserine (LysoPS) as potent and subtype-selective agonists for G-protein-coupled receptors (GPCRs). However, the ester linkage between the glycerol moiety and fatty acid or fatty acid surrogate is present in all of them. In order to develop these LysoPS analogs as drug candidates, appropriate pharmacokinetic consideration is essential. Here, we found that the ester bond of LysoPS is highly susceptible to metabolic degradation in mouse blood. Accordingly, we examined isosteric replacement of the ester linkage with heteroaromatic rings. The resulting compounds showed excellent retention of potency and receptor subtype selectivity, as well as increased metabolic stability in vitro.

Isolation and Reactions of Imidoyl Fluorides Generated from Oxime Using the Diethylaminosulfur Trifluoride/Tetrahydrofuran (DAST-THF) System.

Fluorination of oximes with the relatively mild diethylaminosulfur trifluoride/tetrahydrofuran (DAST-THF) system affords imidoyl fluorides. These compounds were isolated, and their structures were confirmed by X-ray single-crystal structure analysis. Reaction of imidoyl fluorides with various nucleophiles efficiently afforded amides, amidines, thioamides, and amine derivatives in high yields. Furthermore, one-pot reaction of in situ generated imidoyl fluorides from oximes was also applicable to efficient synthesis of these products. The oxime stereochemistry and acid-labile protecting group remained intact in this system.

Exploration of LPS2 agonist binding modes using the combination of a new hydrophobic scaffold and homology modeling.

Lysophosphatidylserine (LysoPS) is an endogenous pan-agonist of three G-protein coupled receptors (GPCRs): LPS1/GPR34, LPS2/P2Y10, and LPS3/GPR174, and we previously reported a series of LysoPS-based agonists of these receptors. Interestingly, we found that LPS1 agonist activity was very sensitive to structural change at the hydrophobic fatty acid moiety, whereas LPS2 agonist activity was not. Here, to probe the molecular basis of LPS2 agonist binding, we developed a new class of hydrophobic fatty acid surrogates having a biphenyl-ether scaffold. The LPS2 agonist activity of these compounds proved sensitive to molecular modification of the hydrophobic skeleton. Thus, we next constructed an LPS2 model by homology modeling and docking/molecular dynamics (MD) simulation, and validated it by means of SAR studies together with point mutations of selected receptor amino-acid residues. The putative ligand-binding site of LPS2 is Γ-shaped, with a hydrophilic site horizontally embedded in the receptor transmembrane helix bundles and a perpendicular hydrophobic groove adjoining transmembrane domains 4 and 5 that is open to the membrane bilayer. The binding poses of LPS2 agonists to this site are consistent with easy incorporation of various kinds of fatty acid surrogates. Structural development based on this model afforded a series of potent and selective LPS2 full agonists, which showed enhanced in vitro actin stress fiber formation effect.

Friedel-Crafts Acylation of Aminocarboxylic Acids in Strong Brønsted Acid Promoted by Lewis Base P4O10.

The amino group in aminocarboxylic acids is sufficiently basic to be protonated in strong acids, and consequently, ionization of the carboxylic acid to an acylium ion is blocked due to charge-charge repulsion. Thus, acylation of aromatic compounds is significantly retarded in Friedel-Craft type reactions. We found that Friedel-Crafts acylation with aminocarboxylic acids can proceed smoothly even in a strong Brønsted acid (triflic acid, TfOH) if the Lewis base P4O10 is added. Here we describe the Friedel-Crafts acylation reactions of anthranilic acid and α- to δ-aminocarboxylic acids with benzene derivatives in the presence of P4O10. Non-amino-containing carboxylic acids as well as N-containing heteroaromatic carboxylic acids are available, and α-amino acids can be directly utilized without any protective group. Most substrates afford acylation products in high yields, although some epimerization/racemization may occur. Density functional theory (DFT) calculations suggested that P4O10 neutralizes the protonated amine, converting the N-H covalent bond to a N-hydrogen bond and allowing the carboxylic acid OH functionality to serve as a good leaving group.

Neighboring Group Participation of Nitrogen Cation to Form an Unusual Six-Membered Ring Intermediate During Oxime Rearrangement.

Neighboring group participation involving a 6-membered ring structure is rare, despite the privilege of 6-membered ring transition structures in organic chemistry. We examined the putative role of a 6-membered cyclic intermediate with neighboring group participation of nitrogen cation in syn-migration of peri-ester indanone oximes. Direct observation of a peri-methyl ester-iminylium intermediate in solution by means of 1H NMR supported the existence of the 6-membered cation intermediate. Density functional theory (DFT) calculations also supported the intervention of this intermediate in the rearrangement and indicated that it has a planar structure stabilized by electron delocalization.

Contribution of Solvents to Geometrical Preference in the Z/E Equilibrium of N-Phenylthioacetamide.

We studied the Z/E preference of N-phenylthioacetamide (thioacetanilide) derivatives in various solvents by means of 1H NMR spectroscopy, as well as molecular dynamics (MD) and other computational analyses. Our experimental results indicate that the Z/E isomer preference of secondary (NH)thioamides of N-phenylthioacetamides shows substantial solvent dependency, whereas the corresponding amides do not show solvent dependency of the Z/E isomer ratios. Detailed study of the solvent effects based on molecular dynamics simulations revealed that there are two main modes of hydrogen (H)-bond formation between solvent and (NH)thioacetamide, which influence the Z/E isomer preference of (NH)thioamides. DFT calculations of NH-thioamide in the presence of one or two explicit solvent molecules in the continuum solvent model can effectively mimic the solvation by multiple solvent molecules surrounding the thioamide in MD simulations and shed light on the precise nature of the interactions between thioamide and solvent. Orbital interaction analysis showed that, counterintuitively, the Z/E preference of NH-thioacetamides is mainly determined by steric repulsion, while that of sterically congested N-methylthioacetamides is mainly determined by thioamide conjugation.

Switching Lysophosphatidylserine G Protein-Coupled Receptor Agonists to Antagonists by Acylation of the Hydrophilic Serine Amine.

Three human G protein-coupled receptors (GPCRs)-GPR34/LPS1, P2Y10/LPS2, and GPR174/LPS3-are activated specifically by lysophosphatidylserine (LysoPS), an endogenous hydrolysis product of a cell membrane component, phosphatidylserine (PS). LysoPS consists of l-serine, glycerol, and fatty acid moieties connected by phosphodiester and ester linkages. We previously generated potent and selective GPCR agonists by modification of the three modules and the ester linkage. Here, we show that a novel modification of the hydrophilic serine moiety, that is, N-acylations of the serine amine, converted a GPR174 agonist to potent GPR174 antagonists. Structural exploration of the amide functionality provided access to a range of activities from agonist to partial agonist to antagonist. The present study would provide a new strategy for the development of lysophospholipid receptor antagonists.

Unexpectedly rigid short peptide foldamers in which NH-π and CH-π interactions are preserved in solution.

NH-π and CH-π interactions, due to their weak character, are not easily identified in solution. We report a group of isolable short peptides with stable folds, in which NH-π and CH-π main chain-side chain interactions can be detected in solution by means of NMR and ATR-IR spectroscopy.

Elaboration of Non-naturally Occurring Helical Tripeptides as p53-MDM2/MDMX Interaction Inhibitors.

Protein-protein interactions (PPIs) are often mediated by helical, strand and/or coil secondary structures at the interface regions. We previously showed that non-naturally occurring, stable helical trimers of bicyclic ß-amino acids (Abh) with all-trans amide bonds can block the p53-MDM2/MDMX α-helix-helix interaction, which plays a role in regulating p53 function. Here, we conducted docking and molecular dynamics calculations to guide the structural optimization of our reported compounds, focusing on modifications of the C-terminal/N-terminal residues. We confirmed that the modified peptides directly bind to MDM2 by means of thermal shift assay, isothermal titration calorimetry, and enzyme-linked immunosorbent assay (ELISA) experiments. Biological activity assay in human osteosarcoma cell line SJSA-1, which has wild-type p53 and amplification of the Mdm2 gene, indicated that these peptides are membrane-permeable p53-MDM2/MDMX interaction antagonists that can rescue p53 function in the cells.

Capsanthin Production in Escherichia coli by Overexpression of Capsanthin/Capsorubin Synthase from Capsicum annuum.

Capsanthin, a characteristic red carotenoid found in the fruits of red pepper (Capsicum annuum), is widely consumed as a food and a functional coloring additive. An enzyme catalyzing capsanthin synthesis was identified as capsanthin/capsorubin synthase (CCS) in the 1990s, but no microbial production of capsanthin has been reported. We report here the first successful attempt to biosynthesize capsanthin in Escherichia coli by carotenoid-pathway engineering. Our initial attempt to coexpress eight enzyme genes required for capsanthin biosynthesis did not detect the desired product. The dual activity of CCS as a lycopene ß-cyclase as well as a capsanthin/capsorubin synthase likely complicated the task. We demonstrated that a particularly high expression level of the CCS gene and the minimization of byproducts by regulating the seven upstream carotenogenic genes were crucial for capsanthin formation in E. coli. Our results provide a platform for further study of CCS activity and capsanthin production in microorganisms.

Non-naturally Occurring Regio Isomer of Lysophosphatidylserine Exhibits Potent Agonistic Activity toward G Protein-Coupled Receptors.

Lysophosphatidylserine (LysoPS), an endogenous ligand of G protein-coupled receptors, consists of l-serine, glycerol, and fatty acid moieties connected by phosphodiester and ester linkages, respectively. An ester linkage of phosphatidylserine can be hydrolyzed at the 1-position or at the 2-position to give 2-acyl lysophospholipid or 1-acyl lysophospholipid, respectively. 2-Acyl lysophospholipid is in nonenzymatic equilibrium with 1-acyl lysophospholipid in vivo. On the other hand, 3-acyl lysophospholipid is not found, at least in mammals, raising the question of whether the reason for this might be that the 3-acyl isomer lacks the biological activities of the other isomers. Here, to test this idea, we designed and synthesized a series of new 3-acyl lysophospholipids. Structure-activity relationship studies of more than 100 "glycol surrogate" derivatives led to the identification of potent and selective agonists for LysoPS receptors GPR34 and P2Y10. Thus, the non-natural 3-acyl compounds are indeed active and appear to be biologically orthogonal with respect to the physiologically relevant 1- and 2-acyl lysophospholipids.

Conformational preference of bicyclic ß-amino acid dipeptides.

Bridged bicyclic amino acids have high potential applicability as self-organized, conformationally constrained synthetic building blocks that do not require assistance from hydrogen bond formation. We systematically investigated the intrinsic conformational propensities of dipeptides of bridged bicyclic ß-amino acids by means of accelerated molecular dynamics simulation and density functional theory (DFT) calculations in methanol, chloroform, and water. While the main-chain conformation, represented by φ and θ values, is fixed by the nature of the bicyclic ring structure, rotation of the C-terminal carbonyl group (ψ) is also restricted, converging to one or two minima. In endo-type dipeptides, in which the two N- and C-terminal amides are spatially close to each other, the C-terminal amide plane is placed horizontally. In exo-type dipeptides, in which the two amides are on opposite sides of the ring plane, the C-terminal carbonyl group can take two types of positions: either parallel/antiparallel with the N-terminal carbonyl or beneath the bicyclic ring, forcing the amide NHMe moiety to lie outside of the ring. We also examined the cis-trans preference of model bicyclic amides. Although the parent amides exhibit cis-trans equilibrium without any preference, addition of a methyl group on one of the bridgehead positions tips the equilibrium towards trans.

Membrane Phospholipid Analogues as Molecular Rulers to Probe the Position of the Hydrophobic Contact Point of Lysophospholipid Ligands on the Surface of G-Protein-Coupled Receptor during Membrane Approach.

When lipid mediators bind to G-protein-coupled receptors (GPCRs), the ligand first enters the lipid bilayer, then diffuses laterally in the cell membrane to make hydrophobic contact with the receptor protein, and finally enters the receptor's binding pocket. In this process, the location of the hydrophobic contact point on the surface of the receptor has been little discussed even in cases in which the crystal structure has been determined, because the ligand binding pocket is buried inside the transmembrane (TM) domains. Here, we coupled an activator ligand to a series of membrane phospholipid surrogates, which constrain the depth of entry of the ligand into the lipid bilayer. Consequently, via measurement of the receptor-activating activity as a function of the depth of entry into the membrane, these surrogates can be used as molecular rulers to estimate the location of the hydrophobic contact point on the surface of GPCR. We focused on lysophosphatidylserine (LysoPS) receptor GPR34 and prepared a series of simplified membrane-lipid-surrogate-conjugated lysophospholipid analogues by attaching alkoxy amine chains of varying lengths to the hydrophobic tail of a potent GPR34 agonist. As expected, the activity of these lipid-conjugated LysoPS analogues was dependent on chain length. The predicted contact position matches the position of the terminal benzene ring of a nonlipidic ligand that protrudes between TMs 4 and 5 of the receptor. We further found that the nature of the terminal hydrophilic functional group of the conjugated membrane lipid surrogate strongly influences the activity, suggesting that lateral hydrophilic contact of LysoPS analogues with the receptor's surface is also crucial for ligand-GPCR binding.

Peptide-based short single ß-strand mimics without hydrogen bonding or aggregation.

Generation of short peptides with a single ß-strand structure in solution is difficult. Herein, we design a new class of single ß-strand peptidic mimics that are stable without self-aggregation in protic and non-protic solvents. Introduction of one present ß-strand mimic can induce and propagate the ß-strand structure for at least a penta-peptide sequence.

Non-naturally Occurring Helical Molecules Can Interfere with p53-MDM2 and p53-MDMX Protein-Protein Interactions.

We have discovered that ß-amino acid homooligomers with cis- or trans-amide conformation can fold themselves into highly ordered helices. Moreover, unlike α-amino acid peptides, which are significantly stabilized by intramolecular hydrogen bonding, these helical structures are autogenous conformations that are stable without the aid of hydrogen bonding and irrespective of solvent (protic/aprotic/halogenated) or temperature. A structural overlap comparison of helical cis/trans bicyclic ß-proline homooligomers with typical α-helix structure of α-amino acid peptides reveals clear differences of pitch and diameter per turn. Bridgehead substituents of the present homooligomers point outwards from the helical surface. We were interested to know whether such non-naturally occurring divergent helical molecules could mimic α-helix structures. In this study, we show that bicyclic ß-proline oligomer derivatives inhibit p53-MDM2 and p53-MDMX protein-protein interactions, exhibiting MDM2-antagonistic and MDMX-antagonistic activities.

Overall Shape Constraint of Alternating α/ß-Hybrid Peptides Containing Bicyclic ß-Proline.

Our NMR, IR/Raman, CD spectroscopic, and X-ray crystallographic studies, as well as accelerated molecular dynamics simulations, showed that alternating hybrid α/ß-peptides containing a bicyclic ß-proline surrogate form unique extended curved folds, regardless of the peptide length and solvent environment. It is suggested that extended ß/PPII structures are preferred in the insulating α-alanine moieties between the rigid bicyclic ß-proline structures. These hybrid peptides inhibit p53-MDM2 and p53-MDMX protein-protein interactions.

Uncovering the Networks of Topological Neighborhoods in ß-Strand and Amyloid ß-Sheet Structures.

Although multiple hydrophobic, aromatic π-π, and electrostatic interactions are proposed to be involved in amyloid fibril formation, the precise interactions within amyloid structures remain poorly understood. Here, we carried out detailed quantum theory of atoms-in-molecules (QTAIM) analysis to examine the hydrophobic core of amyloid parallel and antiparallel ß-sheet structures, and found the presence of multiple inter-strand and intra-strand topological neighborhoods, represented by networks of through-space bond paths. Similar bond paths from side chain to side chain and from side chain to main chain were found in a single ß-strand and in di- and tripeptides. Some of these bond-path networks were enhanced upon ß-sheet formation. Overall, our results indicate that the cumulative network of weak interactions, including various types of hydrogen bonding (X-H-Y; X, Y = H, C, O, N, S), as well as non-H-non-H bond paths, is characteristic of amyloid ß-sheet structure. The present study postulated that the presence of multiple through-space bond-paths, which are local and directional, can coincide with the attractive proximity effect in forming peptide assemblies. This is consistent with a new view of the van der Waals (vdW) interactions, one of the origins of hydrophobic interaction, which is updating to be a directional intermolecular force.

Amide nitrogen pyramidalization changes lactam amide spinning.

Although cis-trans lactam amide rotation is fundamentally important, it has been little studied, except for a report on peptide-based lactams. Here, we find a consistent relationship between the lactam amide cis/trans ratios and the rotation rates between the trans and cis lactam amides upon the lactam chain length of the stapling side-chain of two 7-azabicyclo[2.2.1]heptane bicyclic units, linked through a non-planar amide bond. That is, as the chain length increased, the rotational rate of trans to cis lactam amide was decreased, and consequently the trans ratio was increased. This chain length-dependency of the lactam amide isomerization and our simulation studies support the idea that the present lactam amides can spin through 360 degrees as in open-chain amides, due to the occurrence of nitrogen pyramidalization. The tilting direction of the pyramidal amide nitrogen atom of the bicyclic systems is synchronized with the direction of the semicircle-rotation of the amide.