Masashi Ohmae

Linker Effects on Monolayer Formation and Long-Range Electron Transfer in Helical Peptide Monolayers

Helical peptides carrying a ferrocene unit at the C-terminus were immobilized on gold at the N-terminus via three different linkers to form self-assembled monolayers, and the long-range electron transfer from the ferrocene unit to gold was electrochemically studied. The linkers are 4-thiobenzoic acid, 3-fluoro-4-thiobenzoic acid, and 2-methoxy-4-thiobenzoic acid. All the peptides formed a monolayer with vertical orientation but some differences in monolayer packing and ferrocene surface density as they formed. However, the treatment with dodecanethiol in a gas phase uniformed to show similar monolayer physical parameters, and the electron-transfer rate constants were reproducibly obtained as well. These three peptide monolayers exhibited the same electron-transfer rate constants despite three linkers with different oxidation potentials. On the other hand, the electron transfer was decelerated seemingly by reducing the ferrocene surface density. Theoretical calculations with simple models demonstrated that the experimental result supports a hopping mechanism rather than electron tunneling though it cannot be fully excluded.

Keratanase II-catalyzed synthesis of keratan sulfate oligomers by using sugar oxazolines as transition-state analogue substrate monomers : A novel insight into the enzymatic catalysis mechanism

Keratan sulfate (KS) oligomers with well‐defined structures were synthesized by keratanase II (KSase II)‐catalyzed transglycosylation. N‐Acetyllactosamine [Galβ(1→4)GlcNAc; LacNAc] oxazoline derivatives with sulfate groups at the C‐6 (1 a) and both the C‐6 and the C‐6′ (1 b) were prepared as transition‐state analogue substrate monomers for KSase II. Monomer 1 a was effectively oligomerized by the enzyme under weak alkaline conditions, to give alternating 6‐sulfated KS oligomers (2 a) in good yields, and with total control of regioselectivity and stereochemistry. KSase II also recognized 1 b, which provided fully 6‐sulfated KS oligomers (2 b) in good yields under similar conditions. Nonsulfated LacNAc oxazoline was difficult to oligomerize enzymatically. These results imply that the catalysis mechanism of KSase II involves a sugar oxazolinium ion that requires the 6‐sulfate group in the GlcNAc residue not only in hydrolysis of KS chains, but also in oligomerization of oxazoline monomers. This is the first report of KSase II‐catalyzed transglycosylation to form β(1→3)‐glycosidic bond through a substrate‐assisted mechanism.

Immobilization of His‐Tagged Endoglucanase on Gold via Various Ni‐NTA Self‐Assembled Monolayers and Its Hydrolytic Activity

A genetically modified His-tagged endoglucanase, EGII(core2), with two active sites in series was immobilized on gold via three different kinds of anchor molecules, and its hydrolytic activity was studied. Immobilization of EGII(core2) was influenced by the chain length and hydrophilicity of anchor molecules. The hydrolytic activity of the immobilized EGII(core2) was nearly the same on either anchor molecule. Interestingly, the immobilized EGII(core2) apparently retained the inherent hydrolytic activity similarly to free EGII(core2). It is therefore considered that the local high concentration of EGII(core2) on the surface should promote the successive hydrolysis of the transient products to show the high hydrolytic activity despite of immobilization.

Enzymatic activities of novel mutant endoglucanases carrying sequential active sites

Novel mutant enzymes of endoglucanase II (EGII) from fungus Trichoderma viride were prepared and their hydrolysis and enzymatic polymerization activities were studied. EGII(core)2 and EGII(core)2-His, which possess sequential two active sites of EGII with a His-tag probe at the N-terminal and with His-tag probes at the N and C terminals, respectively, showed higher hydrolysis activities than EGIIcore with a single active site even in comparison on the active-site concentration basis. These mutant enzymes were applied to the enzymatic polymerization to afford artificial cellulose. The polymerization rates with using EGII(core)2 and EGII(core)2-His were also higher than that with using EGIIcore. The polymerization products were identified as highly crystalline cellulose of type II. The mutant enzymes were also effective to prepare spherulites. EGII(core)2 and EGII(core)2-His are considered to possess higher hydrolysis and polymerization activities than EGIIcore mainly due to the suitably stabilized conformation with the sequential arrangement.

Immune activation with peptide assemblies carrying Lewis y tumor-associated carbohydrate antigen.

Molecular assemblies varying morphologies in a wide range from spherical micelle, nanosheet, curved sheet, nanotube and vesicle were prepared and loaded with Lewis y (Ley) tumor‐associated carbohydrate antigen on the assembly surface. The molecular assemblies were composed of poly(sarcosine)m‐block‐poly(L‐lactic acid)30 (m = 15 or 50, Lactosome), poly(sarcosine)m‐block‐(D/L‐Leu‐Aib)n (m = 22 or 30, n = 6 or 8) and their combinations. The molecular assemblies carrying Ley on the surface were administered in BALB/c nu/nu mice. The major epitopes of the molecular assemblies are commonly Ley and poly(sarcosine). IgM productions upon administrations of the molecular assemblies were assayed by ELISA, showing that anti‐poly(sarcosine) IgM was highly produced by Lactosome of spherical micelle but with a negligible amount of anti‐Ley IgM. On the other hand, the nanosheet of the interdigitated monolayer triggered the production of anti‐Ley IgM but with less anti‐poly(sarcosine) IgM production. Taken together, IgM specificity differs according to the molecular environment of the epitopes in the molecular assemblies. The antigenicity of poly(sarcosine) was augmented in polymeric micelle providing loose environment for B cells to penetrate in, whereas a high density of Ley on the molecular assembly was required for anti‐Ley IgM production. The antigenicity of Ley is therefore dependent on the molecular assemblies on which Ley is displayed on the surface. Copyright

Preparation of fibrous cellulose by enzymatic polymerization using cross-linked mutant endoglucanase II

A cross-linked mutant endoglucanase II was prepared for enzymatic polymerization to cellulose. The cross-linked enzyme is composed of three mutant enzymes showing polymerization activity. A characteristic feature of the polymerization with this cross-linked enzyme is formation of cellulose fibrils in contrast to plate-like crystals obtained by using a free enzyme.

Enzymatic polymerization to artificial hyaluronic acid using a transition state analogue monomer

Artificial hyaluronic acid has been synthesized in vitro via enzymatic polymerization catalyzed by testicular hyaluronidases, which is the first successful example of the hyaluronic acid synthesis via non-biosynthetic pathways. The novel GlcAβ(1→3)GlcNAc oxazoline derivative was designed and synthesized as a transition state analogue monomer for the hyaluronidase catalysis. The oxazoline monomer was efficiently tecognized by the enzymes at pH 7.1 to 9.0 and the polymerization reaction proceeded in a regio- and stereo-selective manner to give rise to artificial hyaluronic acid with molecular weight higher than 15000. These results strongly suggest that the transition state of these testicular hyaluronidases catalysis corresponds to a sugar oxazolinium ion.

A Novel Chemoenzymatic Synthesis of Sulfated Type 2 Tumor-Associated Carbohydrate Antigens by Transglycosylation of Sulfated Lewis X Oxazoline Catalyzed by Keratanase II.

Sulfated type 2 carbohydrate chains are known tumor‐associated carbohydrate antigens (TACAs). Many reports on cancer vaccines employing TACAs as specific antigens have been published, but structurally specified sulfated TACAs have not been used because of the low natural abundance and difficulties in chemical synthesis. We demonstrate for the first time the synthesis of the sulfated type 2 TACAs with an l‐fucose branch by keratanase‐II‐catalyzed transglycosylation of the sulfated Lewis X (Galβ(1→4)[Fucα(1→3)]GlcNAc(6‐OSO3−); su‐Lex) oxazoline derivative. Two keratanase IIs (from Bacillus sp. Ks36 and Bacillus circulans KsT202) efficiently catalyzed the transglycosylation reaction of the su‐Lex oxazoline derivative, thereby giving the su‐Lex dimer as the main product in good yields. Structural analysis of the oligomers confirmed exclusive formation of the β(1→3) glycosidic bond.

Reduced immune response to polymeric micelles coating sialic acids

Effects of sialic acid coatings on polymeric micelle consisting of poly(sarcosine)-block-poly(l-lactic acid) (Lactosome) in the aim of prevention of the accelerated blood clearance (ABC) phenomenon are studied. Two kinds of the sialic acid-presenting Lactosomes targeting the immunosuppressive receptors of Siglec-G and CD22 have been successfully prepared. Lactosome presenting 5-N-acetylneuraminic acid or 5-N-acetylneuraminyl-α(2→6)-galactosyl-β(1→4)-N-acetylglucosamine at the nanocarrier surface diminished the ABC phenomenon due to the reduction of the anti-poly(sarcosine) IgM production. Further, the sialic acid moieties could interact possibly with Siglec-E on immune cell to suppress phagocytosis of the opsonized nanocarriers.