Quantitative and Nondestructive Colorimetric Amine Detection Method for the Solid-Phase Peptide Synthesis as an Alternative to the Kaiser Test.

Solid-phase peptide synthesis (SPPS) is an essential technique for the synthesis of peptide. For half a century, many amine detection methods have been developed to monitor coupling reactions during SPPS. Despite such efforts, to the best of our knowledge, a nondestructive and quantitative colorimetric method has not been developed. Here, we developed the first quantitative and nondestructive colorimetric amine detection method based on an acid-base reaction between HCl salt of electron donor-acceptor type dyes and amino groups on the resin. In this method, a noncolored solution of HCl salt consisting of dyes, whose pKBH+ value was carefully tuned, was deprotonated by amines, allowing the appearance of a yellow color. A good linear relationship (R2 = 0.999) between the absorption of the colored solution and the amine group quantities was confirmed. For all tested proteinogenic and nonproteinogenic amino acids, we achieved quantitative colorimetric analysis with a small relative standard deviation (RSD < 3.6%). Furthermore, during the practical synthesis of an octapeptide containing undetectable amino acids with the Kaiser test, our amine detection allowed for detailed monitoring of the coupling reaction, resulting in a significantly purer peptide in the crude form than that obtained using the Kaiser test.

Naphthyridine-Based Electron Push-Pull-Type Amine-Reactive Fluorescent Probe for Sensing Amines and Proteins in Aqueous Media.

In bioengineering, fluorescent amine-reactive probes are invaluable for the detection of amine species. In particular, targeting probes for lysine, which has a free amino group in amino acids, are a valid method for protein detection. For this purpose, many fluorescent "turn-on type" probes with amine reactivity have been developed; however, they require improvements. In the typical florescence probes, BODIPY and NBD analogs have small Stokes shifts based on absorption and emission and lability in an aqueous environment, respectively. In this study, a new class of fluorescent probes, 1,8-Nap-F, based on the electron push-pull-type 1,8-naphthyridine framework, was designed and investigated as an amine-reactive probe. Generally, electron push-pull-type fluorophores exhibit a large Stokes shift at the expense of fluorescent enhancement in aqueous media; thus, there is a trade-off between possessing a large Stokes shift and intense emission. However, 1,8-Nap-F reacts with primary amines, yielding emissive amine products with a large Stokes shift (>70 nm) without fluorescence quenching and side products, even in an aqueous environment, thereby overcoming the disadvantages of electron push-pull-type fluorophores and lability in aqueous conditions. By applying the specific features of 1,8-Nap-F, we achieved selective lysine detection and fluorescence bioimaging, such as endoplasmic reticulum-selective protein labeling and organelle staining, in living cells by utilizing amine-substituted derivatives.

Photoisomerization of "Partially Embedded Dihydropyridazine" with a Helical Structure.

Invited for the cover of this issue are the groups of Kazuteru Usui and Satoru Karasawa at Showa Pharmaceutical University and Yasuhiro Kobori of Kobe University. The image depicts chirality control of helical compounds through cycles of photocleavage and recombination under sunlight with a "Jack and the Beanstalk" motif. Read the full text of the article at 10.1002/chem.202302413.

Photoisomerization of "Partially Embedded Dihydropyridazine" with a Helical Structure.

Herein, we report the synthesis of two "partially embedded fused-dihydropyridazine N-aryl aza[5]helicene derivatives" (PDHs) and the demonstration of their intrinsic photo-triggered multi-functional properties based on a Kekulé biradical structure. Introducing bulky electron-withdrawing trifluoromethyl or pentafluoroethyl groups into the aza[5]helicene framework (PDH-CF3 and -C2 F5 ) gives PDH axial chirality based on the helicity of the P and M forms, even at room temperature. Upon photo-irradiation of PDH-CF3 in a frozen solution, an ESR signal from the triplet biradical with zero-field splitting values, generated by N-N bond dissociation, was observed. However, when the irradiation was turned off, the ESR signal became silent, thus indicating the existence of two equilibria: between the biradical and quinoidal forms based on the Kekulé structure, and between N-N bond cleavage and recombination. The observed photo- and thermally induced behaviors indicate that T-type photochromic molecules are involved in the photoisomerization mechanism involving the two equilibria. Inspired by the photoisomerization, chirality control of PDH by photoracemization was achieved. Multiple functionalities, such as T-type photochromism, photo-excitation-mediated triplet biradical formation, and photoracemization, which are attributed to the "partially embedded dihydropyridazine" structure, are demonstrated.

Facile preparation of polycyclic halogen-substituted 1,2,3-triazoles by using intramolecular Huisgen cycloaddition.

When 1-(ω-azidoalkyl)-2-(2,2-dihalovinyl)arenes were heated in DMF, the intramolecular Huisgen cycloaddition of an azido group with a 1,1-dihalovinyl group afforded 5-halo-1,2,3-triazole-fused tricyclic benzo compounds. Based on the remaining bromo groups, carbon elongation by the Mizoroki-Heck or Suzuki-Miyaura coupling reactions, followed by an intramolecular Friedel-Crafts reaction, afforded polycyclic compounds with fused triazole rings. Thereafter, the bromo groups were converted into 2-nitrophenyl groups via the Suzuki-Miyaura coupling reaction, which was followed by the Cadogan reaction; a fluorescent pentacyclic compound was obtained.

Basic Fluorescent Protonation-Type pH Probe Sensitive to Small ΔpKa of Methanol and Ethanol.

An optical pH probe is a simple and effective tool for determining an accurate pH value in its localized area. However, basic pH probes with pKBH+ values above 8 have rarely been reported, although many components with high pKa such as arginine play important roles in vivo. Herein, we introduce novel colorimetric and fluorescent basic probes 1-5, which are designed using push-pull-type aminoquinoline and aminobenzoquinoline fluorophores, with pKBH+ values ranging from 8.4 to 9.9. After the basicity of the remarkably sensitive basic probe 4 was tuned, it was able to successfully distinguish between the pKa values of MeOH (15.5) and EtOH (15.9), thus displaying selective protonation and fluorescence enhancement in MeOH over EtOH. Our pH probes can be used to detect MeOH poisoning in commercial EtOH products such as hand sanitizers, providing an effective solution to this problem observed during the COVID-19 pandemic.

Effect of Alkynyl Group on Reactivity in Photoaffinity Labeling with 2-Thienyl-Substituted α-Ketoamide.

Minimalist photo-reactive probes, which consist of a photo-reactive group and a tag for detection of target proteins, are useful tools in chemical biology. Although several diazirine-based and aryl azide-based minimalist probes are available, no keto-based minimalist probe has yet been reported. Here we describe minimalist probes based on a 2-thienyl-substituted α-ketoamide bearing an alkyne group on the thiophene ring. The 3-alkyne probe showed the highest photo-affinity labeling efficiency.

Oxidation of an Internal-Edge-Substituted [5]Helicene-Derived Phosphine Synchronously Enhances Circularly Polarized Luminescence.

Invited for the cover of this issue are the groups of Kazuteru Usui and Satoru Karasawa at Showa Pharmaceutical University, and Yoshitane Imai at Kindai University. The image depicts how a phosphine-oxide-bearing helicene exhibits markedly enhanced CPL response in the excited state compared with that of one with a corresponding phosphine. Read the full text of the article at 10.1002/chem.202202922.

Oxidation of an Internal-Edge-Substituted [5]Helicene-Derived Phosphine Synchronously Enhances Circularly Polarized Luminescence.

Small chiral organic molecules with CD properties are in high demanded due to their potential use in promising electronic and biological applications. Herein, we reveal a system in which the oxidation of a phosphino group to the corresponding phosphine oxide on the inner rim of a helicene derivative induces a CPL response. Laterally π-extended 7,8-dihydro[5]helicenes bearing phosphine and phosphine oxide groups on their inner helical rims (i. e., the C1 position) were synthesized, and their helical structures were unambiguously determined by X-ray crystallography. The photophysical (UV/visible and emission) and chiroptical properties of these compounds were investigated in various solvents. Despite their structural similarities, phosphine oxide showed a significantly better CPL response than phosphine, with a high dissymmetry factor for emission (|glum |=(1.3-1.9)×10-3 ) that can be attributed to structural changes in the interior of the helicene helix.

Acid responsiveness of emissive morpholinyl aminoquinolines and their use for cell fluorescence imaging.

Herein, we report emissive aminoquinoline derivatives (TFMAQ) containing alkylmorpholine and arylmorpholine groups and their photophysical properties, acid-responsiveness, and organelle targeting. The alkylmorpholine group is well-known to favour accumulation in lysosomes and be acid-responsive, but, counterintuitively, the TFMAQ derivatives containing ethylmorpholine groups showed limited accumulation in lysosomes and, instead, preferential accumulation in lipid droplets. The findings reported here will aid the development of organelle/tissue specific dyes for cell imaging and diagnosis.

Artificial Host Molecules to Covalently Capture 8-Nitro-cGMP in Neutral Aqueous Solutions and in Cells.

New 1,3-diazaphenoxazine derivatives (nitroG-Grasp-Guanidine, NGG) have been developed to covalently capture 8-nitro-cGMP in neutral aqueous solutions, which furnish a thiol reactive group to displace the 8-nitro group and a guanidine unit for interaction with the cyclic phosphate. The thiol group was introduced to the 1,3-diazaphenoxazine skeleton through a 2-aminobenzylthiol group (NGG-H) and its 4-methyl (NGG-pMe) and 6-methyl (NGG-oMe) substituted derivatives. The covalent adducts were formed between the NGG derivatives and 8-nitro-cGMP in neutral aqueous solutions. Among the NGG derivatives, the one with the 6-methyl group (NGG-oMe) exhibited the most efficient capture reaction. Furthermore, NGG-H showed a cell permeability into HEK-293 and RAW 264.7 cells and reduced the intracellular 8-nitro-cGMP level. The NGG derivatives developed in this study would become a valuable tool to study the intracellular role of 8-nitro-cGMP.

Push-Pull Bisnaphthyridylamine Supramolecular Nanoparticles: Polarity-Induced Aggregation and Crystallization-Induced Emission Enhancement and Fluorescence Resonance Energy Transfer.

Emissive push-pull-type bisnaphthyridylamine derivatives (BNA-X: X=Me, Et, Bzl, Ph, BuBr, and BuTEMPO) aggregate in aqueous methanol. Furthermore, a two-step emission and aggregation process is controllable by varying the methanol-to-water ratio. At 2:3 MeOH/H2 O, crystallization-induced emission enhancement (CIEE) occurs via formation of an emissive crystal phase, whereas, at 1:9 MeOH/H2 O, aggregation-induced emission enhancement (AIEE) occurs, induced by emissive supramolecular nanoparticles (NPs). For BNA-Ph, the emission quantum yield was 25 times higher in aqueous methanol than that in pure methanol. Despite the high hydrophobicity of BNA-X (C log P=6.1-8.0), the spherical NPs were monodisperse (polydispersity indices <0.2). Moreover, the emissive NPs exhibited fluorescence resonance energy transfer (FRET) with pyrene; however, for BNA-X bearing the TEMPO radical (BNA-BuTEMPO), no FRET was observed because of quenching. In particular, the BNA-BuTEMPO NPs have a slow rotational correlation time (1.3 ns), suggesting applications as magnetic resonance imaging contrast agents with large relaxivity.

Characterization and Water-Proton Longitudinal Relaxivities of Liposome-Type Radical Nanoparticles Prepared via a Supramolecular Approach.

For the construction of metal-free magnetic resonance imaging (MRI) contrast agents, radical-based nanoparticles (RNPs) are promising materials because they allow the water-proton longitudinal relaxivity (r1) to be enhanced not only by paramagnetic resonance effects but also by prolonging the rotational correlation times (τR). However, the τR effect is limited because the radical units are often located within the central hydrophobic core of oil-in-water (o/w) emulsions, resulting in a lack of water molecules surrounding the radical units. In this study, to construct supramolecular RNPs that have high r1 values, we designed a liposome-type RNP in which the radical units are located at positions with sufficient surrounding water molecules. Using this strategy, PRO1 with a PROXYL framework was prepared by introducing hydrophilic groups on both sides of the radical unit. The RNP composed of PRO1 formed spherical nanoparticles approximately 100 nm in size and yielded a higher r1 value (0.26 mM-1 s-1) compared to those of small radical species and similar supramolecular o/w emulsion-type nanoparticles (0.17 mM-1 s-1 in PRO2).

Characterization of Push-Pull-Type Benzo[X]quinoline Derivatives (X = g or f): Environmentally Responsive Fluorescent Dyes with Multiple Functions.

Benzo[X]quinoline (X = g or f: BQX) derivatives bearing bis-trifluoromethyl and amine groups have been designed as push-pull-type fluorescent dyes. Through the synthesis of BQX derivatives from 2,7-diaminonaphthalene, linear-type (BQL) and angular-type (BQA) structural isomers were obtained. X-ray structures of single crystals from six given BQX derivatives revealed that the BQL and BQA series adopt planar- and bowl-shaped structures. In the fluorescence spectra, interestingly, the BQL series emitted in the near-infrared region over 700 nm in polar solvents. Based on the visible absorptions and base properties related to the amine moiety, the ammonia responsiveness was investigated using an ion-exchange reaction by the BQX-HCl salt. By exploiting the environmentally responsive fluorescence probe, cell imaging through confocal laser microscopy was conducted using HeLa and 3T3-L1 cells, emitting specific lipid droplets. The results indicate that BQX derivatives have multiple functions and may be applied in materials chemistry and biochemistry.

A fully synthetic 6-aza-artemisinin bearing an amphiphilic chain generates aggregates and exhibits anti-cancer activities.

Installation of a nitrogen at the C6 position of artemisinin facilitates the addition of a functional unit on the cyclohexane moiety (C-ring). In this study, conjugation of an amphiphilic chain, composed of sequentially connected hydrophilic oligoethylene glycol, hydrophobic alkyl chain, urea, and 4,4'-disubstituted biphenyl linker, imparted self-assembling properties. The fully synthetic mid-molecular weight 6-aza-artemisinin 6 bearing the amphiphilic moiety formed aggregates (approx. 200 nm) at ambient temperature and exhibited increased in vitro anti-cancer activities compared to the N-benzylated aza-artemisinin 5.

Fluorescence Properties and Exciplex Formation of Emissive Naphthyridine Derivatives: Application as Sensors for Amines.

Water-soluble donor-acceptor-type fluorophore 15Nap-Cl having two trifluoromethyl groups and a Cl group on a 1,5-aminonaphthyridine framework was prepared. Fluorophore 15Nap-Cl showed strong solvatochromic fluorescence, and, as the solvent polarity increased, a bathochromic shift was observed accompanied by an increase in the fluorescence quantum yield. In addition, in the presence of amines such as ethylamine, diethylamine, and aniline, further considerable bathochromic shifts in the fluorescence were observed. Density functional calculations identified the source of the fluorescence behavior as exciplex formation between 15-Nap-Cl and the corresponding amine. The fluorescence behavior was exploited to fabricate a sensor that can identify various primary, secondary, and tertiary amines.

Development of Turn-On Probes for Acids Triggered by Aromaticity Enhancement Using Tricyclic Amidine Derivatives.

Two fluorophores consisting of tricyclic amidine derivatives (DHIm and DHPy) were prepared as selective turn-on probes for acids, which were triggered by an aromaticity enhancement. Both amidine derivatives were expanded rings prepared by condensed reactions between the corresponding dibromoalkanes and an aminonaphthyridine analogue. In X-ray analyses, DHIm, in which the dihydroimidazole ring was condensed into aminonaphthyridine, showed high planarity, compared to DHPy, with condensed dihydropyrimidine. The fluorescence properties of DHIm exhibited a higher quantum yield than DHPy due to the difference in planarity. Under acidic conditions, such as in the presence of H+ and M(II), protonations and complexations occurred, exhibiting a higher quantum yield than the neutral DHX (X = Im or Py). The nucleus-independent chemical shift values from the density functional theory calculations suggested that the protonations and complexations caused an enhancement of the aromaticity within the frameworks. These aromaticity changes led to intense fluorescence, and DHX behaved as a selective turn-on probe for acids and metal ions. Interestingly, this fluorescence turn-on system triggered by the aromaticity-based enhancement is not a typical system, such as the photoinduced electron transfer, aggregation-induced enhanced emission, and twisted intramolecular charge transfer systems, but is classified as a novel turn-on system.

Internal-Edge-Substituted Coumarin-Fused [6]Helicenes: Asymmetric Synthesis, Structural Features, and Control of Self-Assembly.

π-Conjugated helicenes containing heteroatoms have attracted significant attention due to their diverse chemical and electronic structures, as well as tunable physical properties. It was rationally anticipated that the self-assembly of coumarin-fused helicenes would be controlled by the effects of a substituent on the internal edge of the helix. Here, this work reports the efficient syntheses of coumarin-fused helicenes 1 a,b (R=Ph, Me), and the enantioselective synthesis of 1 a (R=Ph) by chiral AuI -catalyzed hydroarylation. The helical structure of 1 was unambiguously determined by X-ray crystallography. Of particular note, the enantiomerically pure crystal of 1 a adopted a one-dimensional columnar structure based on π-π stacking interactions, as expected. Furthermore, a significant difference between the fluorescence quantum yields of the enantiomerically pure form and racemate of 1 a was observed.

Self-Assembly Behavior of Emissive Urea Benzene Derivatives Enables Heat-Induced Accumulation in Tumor Tissue.

In this study we describe the construction of a system composed of thermally responsive molecules that can be induced to accumulate in tumor tissues by heating. EgX molecules consisting of an urea-benzene framework and oligoethylene glycol (OEG) functional groups with an emissive aminoquinoline formed nanoparticles (NPs) ∼10 nm in size at 23 °C with a fluorescence quantum yield of 7-10%. At higher temperatures, additional self-assembly occurred as a result of OEG dehydration, and the NPs grew to over 1000 nm in size; this was accompanied by low critical solution temperature behavior. EgXs accumulated in tumor tissues of mice at a body temperature of around 33-35 °C, an effect that was accelerated by external heating around the tumor to approximately 40 °C as a result of increased particle size and enhanced retention in tissue. These EgX NPs can serve as a tool for in vivo monitoring of tumor progression and response to treatment.

Bis-Copper(II)/π-Radical Multi-Heterospin System with Non-innocent Doubly N-Confused Dioxohexaphyrin(1.1.1.1.1.0) Ligand.

A contracted doubly N-confused dioxohexaphyrin(1.1.1.1.1.0) complex consisting of two paramagnetic copper metals and open-shell π-radical ligand was synthesized as a new multi-heterospin motif. X-ray spectroscopy supported the divalent character of the inner copper centers, and electron paramagnetic resonance and magnetometric studies suggested the presence of unpaired d electrons strongly antiferromagnetically coupled with π-radicals delocalized on the macrocycle. The 25 π non-innocent dioxohexaphyrin ligand allowed the facile interconversion between antiaromatic 24 π and aromatic 26 π species, respectively, upon redox reactions.