Yoshiyuki Manabe

The high performance of 3XFLAG for target purification of a bioactive metabolite: A tag combined with a highly effective linker structure

Affinity purification using immunoprecipitation (IP) is an extremely useful method for target profiling of bioactive natural products. We examined IP purification of CMetE, which is a molecular target for potassium isolespedezate (1), a leaf-opening factor of Cassia plant. We studied IP efficiency using a panel of FLAG-connected molecular probes (2-8), including probes with varying structures and lengths of the linker moiety. The results suggest that not only the length, but the chemical nature of the linker moiety, strongly affect the IP efficiency. 3XFLAG, a tag combined with a linker moiety of charged amino acids, gave the best results and was most useful for IP purification of the molecular target.

Revisiting the Bromination of CH Bonds with Molecular Bromine by Using a Photo‐Microflow System

The photobromination of C-H bonds by using molecular bromine was reinvestigated under microfluidic conditions. The continuous-flow method suppressed the production of dibrominated compounds and effectively produced the desired monobrominated products with high selectivity. Rapid bromination of benzylic substrates containing a photoaffinity azide group was achieved without any decomposition.

Cassia obtusifolia MetE as a Cytosolic Target for Potassium Isolespedezate, a Leaf‐Opening Factor of Cassia plants: Target Exploration by a Compact Molecular‐Probe Strategy

Affinity chromatography by using ligand-immobilized bead technology is generally the first choice for target exploration of a bioactive ligand. However, when a ligand has comparatively low affinity against its target, serious difficulties will be raised in affinity-based target detection. We report here that the use of compact molecular probes (CMP) will be advantageous in such cases; it enables the retention of moderate affinity between the ligand and its target in contrast to immobilizing the ligand on affinity beads that will cause a serious drop in affinity to preclude target detection. In the CMP strategy, a CMP containing an azide handle is used for an initial affinity-based labeling of target, and subsequent tagging by CuAAC with a large FLAG tag will give a tagged target protein. By using the CMP strategy, we succeeded in the identification of Cassia obtusifolia MetE as a cytosolic target protein of potassium isolespedezate (1), a moderately bioactive ligand.

Efficient Synthesis of the Disialylated Tetrasaccharide Motif in N-Glycans through an Amide-Protection Strategy.

A disialylated tetrasaccharide, Neu5Ac(α2,3)Gal(β1,3)[Neu5Ac(α2,6)]GlcNAc (1), which is found at the termini of some N-glycans, has been synthesized. Compound 1 was obtained through an α-sialylation reaction between a sialic acid donor and a trisaccharide that was synthesized from the glycosylation of a sialylated disaccharide with a glucosaminyl donor. This synthetic route enabled the synthesis of the as-described disialylated structure. A more-convergent route based on the glycosylation of two sialylated disaccharides was also established to scale up the synthesis. Protection of the amide groups in the sialic acid residues significantly increased the yield of the glycosylation reaction between the two sialylated disaccharides, thus suggesting that the presence of hydrogen bonds on the sialic acid residues diminished their reactivity.

Practical and Efficient Method for α-Sialylation with an Azide Sialyl Donor Using a Microreactor

Efficient α-sialylation with a C5 azide sialyl donor was investigated under microfluidic conditions using a microreactor composed of a Comet X-01 micromixer and a tube reactor. Precise temperature control realized excellent α-selectivity, and Siaα(2,6)Gal, Siaα(2,3)Gal, and Siaα(2,9)Sia disaccharides were obtained in moderate to good yields.

Recent advances on bioorganic chemistry of plant metabolites controlling nyctinasty

Leguminous plants open their leaves during the daytime and close them at night as if sleeping, a type of movement that follows circadian rhythms, and is known as nyctinastic movement. This phenomenon is controlled by two endogenous bioactive substances that exhibit opposing activities: Leaf-Opening Factor (LOF), which opens the leaves, and Leaf-Closing Factor (LCF), which closes them. The authors have carried out chemical biological research using these bioactive substances as molecular probes in order to clarify the mechanisms of nyctinastic movement. Here, we report on the detection and identification of the target proteins of these compounds using original methodology.

Convergent Synthesis of a Bisecting N-Acetylglucosamine (GlcNAc)-Containing N-Glycan

The chemical synthesis of a bisecting N-acetylglucosamine (GlcNAc)-containing N-glycan was achieved by a convergent synthetic route through [4+2] and [6+2] glycosylations. This synthetic route reduced the number of reaction steps, although the key glycosylations were challenging in terms of yields and selectivities owing to steric hindrance at the glycosylation site and a lack of neighboring group participation. The yields of these glycosylations were enhanced by stabilizing the oxocarbenium ion intermediate through ether coordination. Glycosyl donor protecting groups were explored in an effort to realize perfect α selectivity by manipulating remote participation. The simultaneous glycosylations of a tetrasaccharide with two disaccharides was investigated to efficiently construct a bisecting GlcNAc-containing N-glycan.

Expanding the Applicability of the Metal Labeling of Biomolecules by the RIKEN Click Reaction: A Case Study with Gallium-68 Positron Emission Tomography

Radiolabeled biomolecules with short half‐life times are of increasing importance for positron emission tomography (PET) imaging studies. Herein, we demonstrate an improved and generalized method for synthesizing a [radiometal]‐unsaturated aldehyde as a lysine‐labeling probe that can be easily conjugated into various biomolecules through the RIKEN click reaction. As a case study, 68Ga‐PET imaging of U87MG xenografted mice is demonstrated by using the 68Ga‐DOTA‐RGDyK peptide, which is selective to αVβ3 integrins.

Single-step Per-O-sulfonation and 1,6-Anhydro-bridge Formation of Sugar Oligomers for Fibroblast Growth Factor Interactions

Many circulating cancer‐related proteins, such as fibroblast growth factors (FGFs), associate with glycosaminoglycans—particularly heparan sulfate—at the cell surface. Disaccharide analogues of heparan sulfate had previously been identified as the shortest components out of the sugars that bind to FGF‐1 and FGF‐2. Taking note of the typical pose of l‐iduronic acid, we conceived of per‐O‐sulfonated analogues of such disaccharides, and devised a single‐step procedure for per‐O‐sulfonation of unprotected sugars with concomitant 1,6‐anhydro bridge formation to achieve such compounds through direct use of SO3⋅Et3N as sulfonation reagent and dimethylformamide as solvent. The synthesized sugars based on the oligomaltose backbone bound FGF‐1 and FGF‐2 mostly at the sub‐micromolar level, although the tetrasaccharide analogue achieved low‐nanomolar binding with FGF‐2.

Syntheses and Immunological Evaluation of Self‐Adjuvanting Clustered N‐Acetyl and N‐Propionyl Sialyl‐Tn Combined with a T‐helper Cell Epitope as Antitumor Vaccine Candidates

Sialyl-Tn (STn) is a tumor-associated carbohydrate antigen (TACA) rarely observed on healthy tissues. We synthesized two fully synthetic N-acetyl and N-propionyl STn trimer (triSTn) vaccines possessing a T-helper epitope and a TLR2 agonist, since the clustered STn antigens are highly expressed on many cancer cells. Immunization of both vaccines in mice induced the anti-triSTn IgG antibodies, which recognized triSTn-expressing cell lines PANC-1 and HepG2. The N-propionyl triSTn vaccine induced the triSTn-specific IgGs, while IgGs induced by the N-acetyl triSTn vaccine were less specific. These results illustrated that N-propionyl triSTn is a valuable unnatural TACA for anticancer vaccines.