Temperature Control of the Self-Assembly Process of 4-Aminoquinoline Amphiphile: Selective Construction of Perforated Vesicles and Nanofibers, and Structural Restoration Capability.

Invited for the cover of this issue is the group of Yosuke Hisamatsu, Naoki Umezawa, and co-workers at Nagoya City University and Nagoya Institute of Technology. The image depicts the selective construction of perforated vesicles and nanofibers, influenced by the heating temperatures during the self-assembly process of the 4-aminoquinoline amphiphile. Read the full text of the article at 10.1002/chem.202400134.

Temperature Control of the Self-Assembly Process of 4-Aminoquinoline Amphiphile: Selective Construction of Perforated Vesicles and Nanofibers, and Structural Restoration Capability.

The construction of diverse and distinctive self-assembled structures in water, based on the control of the self-assembly processes of artificial small molecules, has received considerable attention in supramolecular chemistry. Cage-like perforated vesicles are distinctive and interesting self-assembled structures. However, the development of self-assembling molecules that can easily form perforated vesicles remains challenging. This paper reports a lower critical solution temperature (LCST) behavior-triggered self-assembly property of a 4-aminoquinoline (4-AQ)-based amphiphile with a tetra(ethylene glycol) chain, in HEPES buffer (pH 7.4). This property allows to form perforated vesicles after heating at 80 °C (> LCST). The self-assembly process of the 4-AQ amphiphile can be controlled by heating at 80 °C (> LCST) or 60 °C (< LCST). After cooling to room temperature, the selective construction of the perforated vesicles and nanofibers was achieved from the same 4-AQ amphiphile. Furthermore, the perforated vesicles exhibited slow morphological transformation into intertwined-like nanofibers but were easily restored by brief heating above the LCST.

Fluorescence Response and Self-Assembly of a Tweezer-Type Synthetic Receptor Triggered by Complexation with Heme and Its Catabolites.

There is increasing interest in the development and applications of synthetic receptors that recognize target biomolecules in aqueous media. We have developed a new tweezer-type synthetic receptor that gives a significant fluorescence response upon complexation with heme in aqueous solution at pH 7.4. The synthetic receptor consists of a tweezer-type heme recognition site and sulfo-Cy5 as a hydrophilic fluorophore. The receptor-heme complex exhibits a supramolecular amphiphilic character that facilitates the formation of self-assembled aggregates, and both the tweezer moiety and the sulfo-Cy5 moiety are important for this property. The synthetic receptor also exhibits significant fluorescence responses to biliverdin and bilirubin, but shows very weak fluorescence responses to flavin mononucleotide, folic acid, and nicotinamide adenine dinucleotide, which contain smaller π-scaffolds.

Effects of Structural Isomers of Spermine on the Higher-Order Structure of DNA and Gene Expression.

Polyamines are involved in various biological functions, including cell proliferation, differentiation, gene regulation, etc. Recently, it was found that polyamines exhibit biphasic effects on gene expression: promotion and inhibition at low and high concentrations, respectively. Here, we compared the effects of three naturally occurring tetravalent polyamines, spermine (SPM), thermospermine (TSPM), and N4-aminopropylspermidine (BSPD). Based on the single DNA observation with fluorescence microscopy together with measurements by atomic force microscopy revealed that these polyamines induce shrinkage and then compaction of DNA molecules, at low and high concentrations, respectively. We also performed the observation to evaluate the effects of these polyamine isomers on the activity of gene expression by adapting a cell-free luciferase assay. Interestingly, the potency of their effects on the DNA conformation and also on the inhibition of gene expression activity indicates the highest for TSPM among spermine isomers. A numerical evaluation of the strength of the interaction of these polyamines with negatively charged double-strand DNA revealed that this ordering of the potency corresponds to the order of the strength of the attractive interaction between phosphate groups of DNA and positively charged amino groups of the polyamines.

Development of Cell-Penetration PG-Surfactants and Its Application in External Peptide Delivery to Cytosol.

Recently, development of techniques to deliver pharmacologically active biomacromolecules such as peptides and proteins to cytosol has gained much interest. Here, we applied the peptide gemini (PG)-surfactants to a novel platform to design cell penetration lipopeptides (CP-PGs), which can deliver exogenous peptides and proteins to cytosol. Among the number of candidate CP-PGs having different peptide sequences at the X-, Y-, and Z-positions, we focused on those having two C12 alkyl chains appended to the side chain of two Cys residues, the betaine sequence -Asp-Lys-Asp-Lys- between the alkylated Cys residues (i.e., at the X-position), and having different cationic peptide sequences of oligo-Lys or oligo-Arg at the Y- and/or Z-positions. With respect to cytotoxicity for mammalian cells such as NIH3T3 cells upon 1 h exposure, those having (Lys)3 (K3-DKDKC12 and DKCK12-K3) showed lower cytotoxicity (IC50 = 241 and 198 µM) among those having oligo-Lys, (Lys)n (n = 1, 3, 5; IC50 = 88-197 µM). Similar lower cytotoxicity was also observed for the CP-PG having two (Lys)3 at both N- and C-terminal sides (K3-DKDKC12-K3) (IC50 = 225 µM). In contrast, the CP-PG having (Arg)3 at the N-terminal side (R3-DKDKC12) showed higher cytotoxicity (IC50 = 88 µM). Carrier abilities of the CP-PGs for exogenous peptides were evaluated using the proapoptotic domain (PAD) peptide, which induces apoptosis by disturbing mitochondrial membranes after delivery into cytosol. As a result, the CP-PGs of K3-DKDKC12, DKCK12-K3, K3-DKDKC12-K3, DKCK12-K5, and R3-DKDKC12 exhibited micromolar range carrier ability (the necessary half concentration to induce cell death (EC50) by delivering PAD peptide to cytosol was 10, 6.2, 8.5, 5.8, and 11.5 µM, respectively). Especially, the carrier abilities of DKCK12-K3 and DKCK12-K5 were superior to the well-established cell penetration Arg-rich R8 peptide (EC50 = 6.8 µM). Together, our results indicate that the PG-surfactant molecular framework could be a potential new platform to design efficient cell penetration carrier materials.

Hydrogen sulfide bypasses the rate-limiting oxygen activation of heme oxygenase.

Discovery of unidentified protein functions is of biological importance because it often provides new paradigms for many research areas. Mammalian heme oxygenase (HO) enzyme catalyzes the O2-dependent degradation of heme into carbon monoxide (CO), iron, and biliverdin through numerous reaction intermediates. Here, we report that H2S, a gaseous signaling molecule, is part of a novel reaction pathway that drastically alters HO's products, reaction mechanism, and catalytic properties. Our prediction of this interplay is based on the unique reactivity of H2S with one of the HO intermediates. We found that in the presence of H2S, HO produces new linear tetrapyrroles, which we identified as isomers of sulfur-containing biliverdin (SBV), and that only H2S, but not GSH, cysteine, and polysulfides, induces SBV formation. As BV is converted to bilirubin (BR), SBV is enzymatically reduced to sulfur-containing bilirubin (SBR), which shares similar properties such as antioxidative effects with normal BR. SBR was detected in culture media of mouse macrophages, confirming the existence of this H2S-induced reaction in mammalian cells. H2S reacted specifically with a ferric verdoheme intermediate of HO, and verdoheme cleavage proceeded through an O2-independent hydrolysis-like mechanism. This change in activation mode diminished O2 dependence of the overall HO activity, circumventing the rate-limiting O2 activation of HO. We propose that H2S could largely affect O2 sensing by mammalian HO, which is supposed to relay hypoxic signals by decreasing CO output to regulate cellular functions. Moreover, the novel H2S-induced reaction identified here helps sustain HO's heme-degrading and antioxidant-generating capacity under highly hypoxic conditions.

Stable Iron Porphyrin Intramolecularly Coordinated by Alcoholate Anion: Synthesis and Evaluation of Axial Ligand Effect of Alcoholate on Spectroscopy and Catalytic Activity.

We synthesized intramolecularly aliphatic alcoholate-coordinated iron porphyrins (1a, 1b) that retain their axial coordination in the presence of another ligand or oxidant. The electron-donative character of alcoholate was less than that of thiolate, and the coordination ability of a sixth ligand to 1a and 1b was very much lower than in the case of the thiolate-coordinated compounds. Density functional theory calculations indicated that the marked difference in coordination ability could be explained in terms of thermodynamic and steric factors. The catalytic oxidizing ability of the thiolate-coordinated compound, SR complex, was much higher than that of 1a.

Distinct modulation of group I ribozyme activity among stereoisomers of a synthetic pentamine with structural constraints.

Among cationic molecules that can modulate ribozyme activities, polyamines act as both activator and inhibitor of ribozyme reactions partly due to their structural flexibility. Restriction of structural flexibility of polyamines may allow them to emphasize particular modulation effects. We examined eight stereoisomers of a synthetic pentamine bearing three cyclopentane rings. In the reaction of a structurally unstable group I ribozyme, three stereoisomers exhibited distinct effects as inhibitor, an additive with a neutral effect, and also as an activator.

Biogenic triamine and tetraamine activate core catalytic ability of Tetrahymena group I ribozyme in the absence of its large activator module.

Group I intron ribozymes share common core elements that form a three-dimensional structure responsible for their catalytic activity. This core structure is unstable without assistance from additional factors that stabilize its tertiary structure. We examined biogenic triamine and tetraamine and also their fragments for their abilities to stabilize a structurally unstable group I ribozyme, ΔP5 ribozyme, derived from the Tetrahymena group I intron ribozyme by deleting its large activator module. Biogenic triamine (spermidine) and tetraamine (spermine) efficiently activated the ΔP5 ribozyme under conditions where the ribozyme was virtually inactive. These observations suggested that polyamines are promising small molecule modulators to activate and possibly inhibit the core catalytic ability of group I ribozymes.

Branched-Chain Polyamine Found in Hyperthermophiles Induces Unique Temperature-Dependent Structural Changes in Genome-Size DNA.

A pentavalent branched-chain polyamine, N4 -bis(aminopropyl)spermidine 3(3)(3)4, is a unique polycation found in the hyperthermophilic archaeon Thermococcus kodakarensis, which grows at temperatures between 60 and 100 °C. We studied the effects of this branched-chain polyamine on DNA structure at different temperatures up to 80 °C. Atomic force microscopic observation revealed that 3(3)(3)4 induces a mesh-like structure on a large DNA (166 kbp) at 24 °C. With an increase in temperature, DNA molecules tend to unwind, and multiple nano-loops with a diameter of 10-50 nm are generated along the DNA strand at 80 °C. These results were compared to those obtained with linear-chain polyamines, homocaldopentamine 3334 and spermidine, the former of which is a structural isomer of 3(3)(3)4. These specific effects are expected to neatly concern with its role on high-temperature preference in hyperthermophiles.

Comparative study of polyethylene polyamines as activator molecules for a structurally unstable group I ribozyme.

Polyamines are a promising class of molecules that can modulate RNA enzyme activities. To analyze the effects of the number of amine moieties systematically, we employed four polyamines sharing dimethylene units to connect amine moieties. As a model RNA enzyme, we used a structurally unstable group I ribozyme, which was activated most and least efficiently by tetraethylenepentamine and diethylenetriamine respectively.

Role of Thiolate Ligand in Spin State and Redox Switching in the Cytochrome P450 Catalytic Cycle.

The catalytic cycle of cytochrome P450 involves a change from the resting-state, water-bound, six-coordinated form (1, low-spin state) to a five-coordinated form (2, high-spin state) upon binding of a hydrophobic substrate. Here, we used a heme-thiolate model complex (SR complex) with THF as a model of nonionic H2O to address the question of whether or not coordination of nonionic water is sufficient to induce the low-spin state. Measurements of electronic absorption spectra and magnetic properties confirmed that five-coordinated SR complex has a high-spin state, and THF-bound, six-coordinated SR has a low-spin state in dichloromethane at ambient temperature. The redox potential E1/2 (FeII/FeIII) of THF-bound SR was 80-90 mV more negative than that of five-coordinated SR. These properties indicate SR is a good model of P450. Our results suggest that thiolate coordination plays a key role in setting the low energy barrier between the high-spin and low-spin states.

Activation of lysine-specific demethylase 1 inhibitor peptide by redox-controlled cleavage of a traceless linker.

We have previously employed cyclization of a linear peptide as a strategy to modulate peptide function and properties, but cleavage to regenerate the linear peptide left parts of the linker structure on the peptide, interfering with its activity. Here, we focused on cyclization of a linear peptide via a "traceless" disulfide-based linkage that would be cleaved and completely removed in a reducing environment, regenerating the original linear peptide without any linker-related structure. Thus, the linker would serve as a redox switch that would be activated in the intracellular environment. We applied this strategy to a lysine-specific demethylase 1 (LSD1) inhibitor peptide 1. The resulting cyclic peptide 2 exhibited approximately 20 times weaker LSD1-inhibitory activity than peptide 1. Upon addition of reducing reagent, the linker was completely removed to regenerate the linear peptide 1, with full restoration of the LSD1-inhibitory activity. In addition, the cyclic peptide was far less susceptible to proteolysis than the linear counterpart. Thus, this switch design not only enables control of functional activity, but also improves stability. This approach should be applicable to a wide range of peptides, and may be useful in the development of peptide pharmaceuticals.

Development and crystallographic evaluation of histone H3 peptide with N-terminal serine substitution as a potent inhibitor of lysine-specific demethylase 1.

Lysine-specific demethylase 1 (LSD1/KDM1A) is a flavoenzyme demethylase, which removes mono- and dimethyl groups from histone H3 Lys4 (H3K4) or Lys9 (H3K9) in complexes with several nuclear proteins. Since LSD1 is implicated in the tumorigenesis and progression of various cancers, LSD1-specific inhibitors are considered as potential anti-cancer agents. A modified H3 peptide with substitution of Lys4 to Met [H3K4M] is already known to be a potent competitive inhibitor of LSD1. In this study, we synthesized a series of H3K4M peptide derivatives and evaluated their LSD1-inhibitory activities in vitro. We found that substitutions of the N-terminal amino acid with amino acids having a larger side chain were generally not tolerated, but substitution of Ala1 to Ser unexpectedly resulted in more potent inhibitory activity toward LSD1. X-ray crystallographic analysis of H3K4M derivatives bound to the LSD1·CoREST complex revealed the presence of additional hydrogen bonding between the N-terminal Ser residue of the H3 peptide derivative and LSD1. The present structural and biochemical findings will be helpful for obtaining more potent peptidic inhibitors of LSD1.

Design of New Extraction Surfactants for Membrane Proteins from Peptide Gemini Surfactants.

The development of additional extraction surfactants for membrane proteins is necessary for membrane protein research, since optimal combinations for the successful extraction of target membrane proteins from biological membranes that minimize protein denaturation are hard to predict. In particular, those that have a unique basal molecular framework are quite attractive and highly desired in this research field. In this study, we successfully constructed a new extraction surfactant for membrane proteins, NPDGC12KK, from the peptide-gemini-surfactant (PG-surfactant) molecular framework. The PG-surfactant is a U-shaped lipopeptide scaffold, consisting of a short linker peptide (-X-) between two long alkyl-chain-modified Cys residues and a peripheral peptide (Y-) at the N-terminal side of long alkyl-chain-modified Cys residues. Using photosystem I (PSI) and photosystem II (PSII) derived from Thermosynecoccus vulcanus as representative membrane proteins, we evaluated whether NPDGC12KK could solubilize membrane proteins while maintaining structure and functions. Neither the membrane integral domain nor the cytoplasmic domain of PSI and PSII suffered any damage upon the use of NPDGC12KK based on detailed photophysical measurements. Using thylakoid membranes of T. vulcanus as a representative biological membrane sample, we performed experiments to extract membrane proteins, such as PSI and PSII. Based on the extraction efficiency and maintenance of protein supramolecular structure established using clear native-PAGE analyses, we proved that NPDGC12KK functions as a novel class of peptide-containing extraction surfactants for membrane proteins.

Naturally occurring branched-chain polyamines induce a crosslinked meshwork structure in a giant DNA.

We studied the effect of branched-chain polyamines on the folding transition of genome-sized DNA molecules in aqueous solution by the use of single-molecule observation with fluorescence microcopy. Detailed morphological features of polyamine/DNA complexes were characterized by atomic force microscopy (AFM). The AFM observations indicated that branched-chain polyamines tend to induce a characteristic change in the higher-order structure of DNA by forming bridges or crosslinks between the segments of a DNA molecule. In contrast, natural linear-chain polyamines cause a parallel alignment between DNA segments. Circular dichroism measurements revealed that branched-chain polyamines induce the A-form in the secondary structure of DNA, while linear-chain polyamines have only a minimum effect. This large difference in the effects of branched- and linear-chain polyamines is discussed in relation to the difference in the manner of binding of these polyamines to negatively charged double-stranded DNA.

Structurally Diverse Polyamines: Solid-Phase Synthesis and Interaction with DNA.

A versatile solid-phase approach based on peptide chemistry was used to construct four classes of structurally diverse polyamines with modified backbones: linear, partially constrained, branched, and cyclic. Their effects on DNA duplex stability and structure were examined. The polyamines showed distinct activities, thus highlighting the importance of polyamine backbone structure. Interestingly, the rank order of polyamine ability for DNA compaction was different to that for their effects on circular dichroism and melting temperature, thus indicating that these polyamines have distinct effects on secondary and higher-order structures of DNA.

Identification of a novel aminopropyltransferase involved in the synthesis of branched-chain polyamines in hyperthermophiles.

Longer- and/or branched-chain polyamines are unique polycations found in thermophiles. N(4)-aminopropylspermine is considered a major polyamine in Thermococcus kodakarensis. To determine whether a quaternary branched penta-amine, N(4)-bis(aminopropyl)spermidine, an isomer of N(4)-aminopropylspermine, was also present, acid-extracted cytoplasmic polyamines were analyzed by high-pressure liquid chromatography, gas chromatography (HPLC), and gas chromatography-mass spectrometry. N(4)-bis(aminopropyl)spermidine was an abundant cytoplasmic polyamine in this species. To identify the enzyme that catalyzes N(4)-bis(aminopropyl)spermidine synthesis, the active fraction was concentrated from the cytoplasm and analyzed by linear ion trap-time of flight mass spectrometry with an electrospray ionization instrument after analysis by the MASCOT database. TK0545, TK0548, TK0967, and TK1691 were identified as candidate enzymes, and the corresponding genes were individually cloned and expressed in Escherichia coli. Recombinant forms were purified, and their N(4)-bis(aminopropyl)spermidine synthesis activity was measured. Of the four candidates, TK1691 (BpsA) was found to synthesize N(4)-bis(aminopropyl)spermidine from spermidine via N(4)-aminopropylspermidine. Compared to the wild type, the bpsA-disrupted strain DBP1 grew at 85°C with a slightly longer lag phase but was unable to grow at 93°C. HPLC analysis showed that both N(4)-aminopropylspermidine and N(4)-bis(aminopropyl)spermidine were absent from the DBP1 strain grown at 85°C, demonstrating that the branched-chain polyamine synthesized by BpsA is important for cell growth at 93°C. Sequence comparison to orthologs from various microorganisms indicated that BpsA differed from other known aminopropyltransferases that produce spermidine and spermine. BpsA orthologs were found only in thermophiles, both in archaea and bacteria, but were absent from mesophiles. These findings indicate that BpsA is a novel aminopropyltransferase essential for the synthesis of branched-chain polyamines, enabling thermophiles to grow in high-temperature environments.

Synthesis of the carbon framework of scholarisine A by intramolecular oxidative coupling.

Scholarisine A, isolated from the leaves of Alstonia scholaris, is a monoterpene indole alkaloid with an unprecedented cage-like structure. In this paper, preparation of the distinctive cage-like core skeleton of scholarisine A is described. The key feature of this synthetic strategy is an intramolecular oxidative coupling reaction at the late stage to construct a 10-oxa-tricyclo[5.3.1.0(3, 8)]undecan-9-one structure fused with indolenine. Intramolecular oxidative coupling by using N-iodosuccinimide gave the carbon framework of scholarisine A in moderate yield, which is the first example of intramolecular oxidative-coupling reaction between non-activated enolate and indole. This study lays the foundation for continued investigations towards the total synthesis of scholarisine A.

Manganese salen complexes with acid-base catalytic auxiliary: functional mimetics of catalase.

Antioxidant therapies have been considered for a wide variety of disorders associated with oxidative stress, and synthetic catalytic scavengers of reactive oxygen species would be clinically superior to stoichiometric ones. Among them, salen-manganese complexes (Mn(Salen)) seem promising, because they exhibit dual functions, i.e. superoxide dismutase- and catalase-mimetic activities. We have been developing enzyme-mimetic Mn(Salen) complexes bearing a functional group that enhances their catalytic activity. Here, we describe the design and synthesis of novel Mn(Salen) complexes with general acid-base catalytic functionality, inspired by the reaction mechanism of catalase. As expected, these Mn(Salen) complexes showed superior catalase-like activity and selectivity, while retaining moderate SOD-like activity. An unsubstituted pyridyl group worked well as a functionality to promote catalase-like activity. The introduced functionality did not alter the redox potential suggesting that the auxiliary-modified complex acted as an acid-base catalyst analogous to catalase. We believe that our approach provides a new design principle for sophisticated catalyst design. Further, the compounds described here appear to be good candidates for use in antioxidant therapy.