Novel fluorescent asparaginyl-N-acetyl-d-glucosamines (Asn-GlcNAc) for the resolution of oligosaccharides in glycopeptides, based on enzyme transglycosylation reaction

Several fluorescent asparaginyl-N-acetyl-d-glucosamines (Asn-GlcNAcs), i.e., DBD-Asn-GlcNAc, NBD-Asn-GlcNAc, NDA-Asn-GlcNAc, PS-Asn-GlcNAc, FITC-Asn-GlcNAc, DMEQ-Asn-GlcNAc and DBD-PZ-Boc-Asn-GlcNAc, were synthesized as the acceptors for the resolution of oligosaccharides in glycopeptides. The resolution of oligosaccharides is based on the transglycosylation reaction with end-β-N-acetylglucosaminidase (Endo-M), isolated from Mucor hiemalis. The synthesis of each fluorescent acceptor was carried out in a one-pot reaction of Asn-GlcNAc and the corresponding fluorescent tagging reagent. The transglycosylation reaction using Endo-M proceeds at RT in neutral phosphate buffer (pH 6.0) and reached maxima at around 30 min. When Fmoc-Asn-GlcNAc (acceptor) was enzymatically reacted with Disialo-Asn (donor) in the presence of Endo-M, the ratio of Disialo-Asn-Fmoc (transglycosylation product) was almost comparable with the decreasing ratio of Fmoc-Asn-GlcNAc. Therefore, the transglycosylation activity of Endo-M from Disialo-Asn (donor) and fluorescent-Asn-GlcNAc (acceptor) was calculated from the decreasing ratio of fluorescent-Asn-GlcNAc. The order was NDA-Asn-GlcNAc > DBD-Asn-GlcNAc Fmoc-Asn-GlcNAc > NBD-Asn-GlcNAc DMEQ-Asn-GlcNAc > DNS-Asn-GlcNAc > PS-Asn-GlcNAc > FITC-Asn-GlcNAc. On the other hand, the activity with a fluorescent acceptor (DBD-PZ-Boc-Asn-GlcNAc), labeled to a carboxylic acid group in the Asn residue, was the strongest among the synthesized acceptors.     

Asymmetric Total Synthesis of ent‐Pyripyropene A

An asymmetric total synthesis of ent‐pyripyropene A was achieved by a convergent synthetic route. We used our originally developed Ti^III‐catalyzed radical cyclization to construct an AB‐ring portion that consisted of a trans‐decalin skeleton with five contiguous stereogenic centers. The coupling between the AB‐ring and the DE‐ring portions, and a subsequent C‐ring cyclization, led to the total synthesis of ent‐pyripyropene A. An evaluation of the insecticidal activity of ent‐pyripyropene A against two aphid species revealed that ent‐pyripyropene A was 35–175 times less active than naturally occurring pyripyropene A. This result indicated that the biological target of pyripyropene A recognizes the absolute configuration of pyripyropene A.     

Toward the racemic total synthesis of hederacines A and B: construction of an advanced tricyclic intermediate

Progress toward the total synthesis of hederacines A and B is described. Our approach involves an allylic cyanate-to-isocyanate rearrangement, eneyne ring-closing metathesis, and a transannular reaction between the C-5 amino group and the C-12 position of the perhydroazuleno[5,6-b]furanone intermediate.     

Organogold clusters protected by phenylacetylene

A new class of monolayer-protected Au clusters with Au-C covalent bonds (organogold clusters) was synthesized by ligating phenylacetylene (PhC≡CH) to PVP-stabilized Au clusters. Matrix-assisted laser desorption ionization mass spectrometry revealed for the first time a series of stable compositions of the organogold (Au:C(2)Ph) clusters.     

Oriented reconstitution of a membrane protein in a giant unilamellar vesicle: experimental verification with the potassium channel KcsA

We report a method for the successful reconstitution of the KcsA potassium channel with either an outside-out or inside-out orientation in giant unilamellar vesicles, using the droplet-transfer technique. The procedure is rather simple. First, we prepared water-in-oil droplets lined with a lipid monolayer. When solubilized KcsA was encapsulated in the droplet, it accumulated at monolayers of phosphatidylglycerol (PG) and phosphoethanolamine (PE) but not at a monolayer of phosphatidylcholine (PC). The droplet was then transferred through an oil/water interface having a preformed monolayer. The interface monolayer covered the droplet so as to generate a bilayer vesicle. By creating chemically different lipid monolayers at the droplet and oil/water interface, we obtained vesicles with asymmetric lipid compositions in the outer and inner leaflets. KcsA was spontaneously inserted into vesicles from the inside or outside, and this was accelerated in vesicles that contained PE or PG. Integrated insertion into the vesicle membrane and the KcsA orientation were examined by functional assay, exploiting the pH sensitivity of the opening of the KcsA when the pH-sensitive cytoplasmic domain (CPD) faces toward acidic media. KcsA loaded from the inside of the PG-containing vesicles becomes permeable only when the intravesicular pH is acidic, and the KcsA loaded from the outside becomes permeable when the extravesicular pH is acidic. Therefore, the internal or external insertion of KcsA leads to an outside-out or inside-out configuration so as to retain its hydrophilic CPD in the added aqueous side. The CPD-truncated KcsA exhibited a random orientation, supporting the idea that the CPD determines the orientation. Further application of the droplet-transfer method is promising for the reconstitution of other types of membrane proteins with a desired orientation into cell-sized vesicles with a targeted lipid composition of the outer and inner leaflets.     

A new tomato pregnane glycoside from the overripe fruits

A new pregnane glycoside has been isolated from the overripe fruits of Cherry tomato (Mini tomato), Lycopersicon esculentum var. cerasiforme (DUNAL) ALEF. The structure was determined to be 3-O-beta-lycotetraosyl 3beta-hydroxy-5alpha-pregn-16-en-20-one on the basis of spectroscopic analysis. The seasonal variation of the tomato saponin is discussed.     

Theoretical study of lanthanide-based in vivo luminescent probes for detecting hydrogen peroxide

The 4f-4f emissions from lanthanide trication (Ln³⁺) complexes are widely used in bioimaging probes. The emission intensity from Ln³⁺ depends on the surroundings, and thus, the design of appropriate photo-antenna ligands is indispensable. In this study, we focus on two probes for detecting hydrogen peroxide, for which emission intensities from Tb³⁺ are enhanced chemo-selectively by the H₂O₂-mediated oxidation of ligands. To understand the mechanism, the Gibbs free energy profiles of the ground and excited states related to emission and quenching are computed by combining our approximation—called the energy shift method—and density functional theory. The different emission intensities are mainly attributed to different activation barriers for excitation energy transfer from the ligand-centered triplet (T1) to the Tb³⁺-centered excited state. Additionally, quenching from T1 to the ground state via intersystem crossing was inhibited by intramolecular hydrogen bonds only in the highly emissive Tb³⁺ complexes. © 2018 Wiley Periodicals, Inc.     

10 GHz, 2.4 ps pulse generation using a single-stage dual-drive Mach-Zehnder modulator

This Letter reports on picosecond pulse generation at a repetition of 10 GHz by using a single-stage electro-optic LiNbO(3) Mach-Zehnder modulator, stressing the simplicity of its setup. It is analytically and experimentally proved that dual-arm modulation with in-phase sinusoidal signals having slightly different amplitudes generated a highly coherent optical comb with a great spectral flatness and a parabolic phase relationship in its spectrum. The generated comb was Fourier synthesized and shaped into an ultrashort pulse train with an optical bandpass filter and a dispersive fiber. The pulse source was highly stable, exhibiting an ultralow timing jitter of less than 130 fs.