Chemoenzymatic Synthesis of Sialyl Sulfo-Oligosaccharides as Potent Siglec-8 Ligands via Transglycosylation Catalyzed by Keratanase II.

Sialyl type-II sulfo-oligosaccharides are gaining much attention as bioactive ligands for Siglecs. In this study, we have achieved the first synthesis of sialyl type-II sulfo-oligosaccharides chemoenzymatically by utilizing the transglycosylation activity of keratanase II. The oxazoline derivative of α(2→3)-sialylated 6,6'-di-sulfo-LacNAc (3) was newly designed as the glycosyl donor for enzymatic transglycosylation. Keratanase II efficiently catalyzed the transglycosylation of 3 with two kinds of glycosyl acceptors, 6-sulfo-Lewis X and 6,6'-di-sulfo-LacNAc derivatives, providing sialyl sulfo-hexasaccharide (1) and sialyl sulfo-pentasaccharide (2) with 86 and 95% yields, respectively. The products 1 and 2 showed higher affinity to Siglec-8 with KD 70 and 25 µmol·L-1, respectively, compared to the known ligand of the α(2→3)-sialylated 6,6'-di-sulfo-Lewis X with KD 185 µmol·L-1. Thus, this study will advance not only the study of Siglec-8 biology but also the exploration of functions of sialyl sulfo-oligosaccharides having various microstructures.

Immune responses against Lewis Y tumor-associated carbohydrate antigen displayed densely on self-assembling nanocarriers.

Immune responses against Lewis y (LY) displayed on nanocarriers at different surface densities were studied. The high surface density of LY was obtained by the A2B-type amphiphilic polypeptides having LY at the two terminals [LY-poly(sarcosine)2-b-(l- or d-Leu-Aib)6]. The equimolar mixture of these two amphiphilic polypeptides formed interdigitated planar sheet-like molecular assemblies densely displaying LY (G4). G4 seemed to induce the anti-LY IgM upon immunization to BALB/c mice by only a single administration. However, the amount of anti-LY IgM produced was moderate and significantly less than that induced by two administrations of the other molecular assembly (G1) with the average surface density of LY at a 1/4 of that of G4. Further, the anti-LY IgM produced after two administrations of G4 lowered the avidity more than after one administration.

Reaction specificity of keratanase II in the transglycosylation using the sugar oxazolines having keratan sulfate repeating units.

The reaction specificity of the transglycosylation catalyzed by keratanase II from Bacillus circulans KsT202 (KSase II) was studied by using the oxazoline derivatives having keratan sulfate repeating units. The addition of 10% organic cosolvent reduced the activity for the enzymatic transglycosylation. The oxazoline derivative of 6-O-sulfonato-N-acetyllactosamine (su-LacNAc) was processively oligomerized to the corresponding hexamer or longer by the enzyme. This result strongly implies that the enzyme has the large positively numbered subsites. In contrast, the transglycosylation of the su-LacNAc oxazoline donor with the 6-O-sulfonato-Lewis X (su-LeX) acceptor solely gave the su-LacNAc-su-LeX pentasaccharide. In addition, both the oxazoline derivatives of su-LeX and 6,6'-di-O-sulfonato-LacNAc have been exclusively oligomerized to the corresponding dimers respectively. These results strongly suggest that the steric hindrance exists around the (+3)(+4) subsites in KSase II. Furthermore, KSase II-catalyzed reaction of the excess su-LeX oxazoline with the su-LacNAc gave the su-LeX-su-LacNAc pentasaccharide as the sole transglycosylation product, also implying the steric hindrance at the catalytic center hampering processive shift of this pentasaccharide. Thus, KSase II has the sterically crowded structures at the catalytic center and around the (+3)(+4) subsites, which are all expected to be tunnel-like.

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 © 2016 European Peptide Society and John Wiley & Sons, Ltd.

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-ß(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.

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 (Galß(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 ß(1→3) glycosidic bond.

Synthesis of type 2 Lewis antigens via novel regioselective glycosylation of an orthogonally protected lactosamine diol derivative.

The novel and efficient synthesis of type 2 Lewis antigens is reported in this study. The rationally designed lactosamine-3,2'-diol derivative with an orthogonal set of protecting groups is efficiently glycosylated with a benzyl protected 1-thio-l-fucoside donor in a unique regioselective manner to produce Lewis x (Le(x)) and Lewis y (Le(y)) derivatives in good yields. These derivatives can be prepared not only exclusively but also synchronously by choosing the appropriate reaction temperature and donor-acceptor molar ratio. The Le(x) derivatives are easily converted into sulfated or non-sulfated Le(x) bearing a terminal azido functionalized oligo-(ethyleneoxide) linker; the Le(y) derivative having the same linker can also be prepared, all of which can be further used for the chemical modification of other compounds and materials.

Enzymatic polymerization to an alternating N-L-cysteinyl chitin derivative: a novel class of multivalent glycopeptidomimetics.

A novel chitin derivative with an alternating N-L-cysteinyl group was successfully prepared. The precursor chitin derivative with an alternating N-L-thioprolinyl group could be obtained via chitinase-catalyzed polymerization of the N'-L-thioprolinyl chitobiose oxazoline derivative as a transition state analog substrate monomer under weak alkaline conditions. Conversion of the L-thioprolinyl group to an L-cysteinyl group in the product chitin derivative proceeded in a dose-dependent manner by methoxyamine hydrochloride, and the reaction could be completed almost quantitatively. The obtained chitin derivative is a uniformly amino acid-branching polysaccharide, which is categorized into a novel class of multivalent glycopeptidomimetics.

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.

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.

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.

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.

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 [Galbeta(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 beta(1-->3)-glycosidic bond through a substrate-assisted mechanism.

Enzymatic copolymerization to hybrid glycosaminoglycans: a novel strategy for intramolecular hybridization of polysaccharides.

Hybrid glycosaminoglycans (GAGs) having an intramolecularly hybridized structure of hyaluronan-chondroitin (3a) and hyaluronan-chondroitin 4-sulfate (3b) have been synthesized via enzymatic copolymerization catalyzed by hyaluronidase (HAase). N-Acetylhyalobiuronate (GlcAbeta(1-->3)GlcNAc)-derived oxazoline (1) was copolymerized with N-acetylchondrosine (GlcAbeta(1-->3)GalNAc)-derived oxazoline (2a) by HAase catalysis at pH 7.5 and 30 degrees C, giving rise to copolymer 3a with Mn 7.4 x 103 in a 50% yield. Also, HAase-catalyzed copolymerization of monomer 1 with N-acetylchondrosine oxazoline having a sulfate group at C4 on GalNAc (2b) was carried out to produce copolymer 3b with Mn 1.4 x 104 in a 60% yield. The copolymer compositions were controllable by varying the comonomer feed ratio. These hybrid GAGs were successfully digested by the catalysis of hyaluronan lyase, clearly exhibiting that the products are not a blend of different homopolymers but an intramolecularly hybridized GAG.

Hyaluronidase-catalyzed copolymerization for the single-step synthesis of functionalized hyaluronan derivatives.

Hyaluronidase-catalyzed copolymerization was carried out with monomer combinations of 2-methyl (1a)/2-vinyl (1b), 2-methyl (1a)/2-ethyl (1c), 2-methyl (1a)/2-n-propyl (1d), and 2-vinyl (1b)/2-ethyl (1c) oxazoline derivatives of hyalobiuronate [GlcAbeta(1-->3)GlcN]. All copolymerization reactions proceeded successfully in a regio and stereoselective manner, giving rise to hyaluronan derivatives bearing different N-acyl groups at the C2 position of the glucosamine unit in the polymer chain. The composition of the N-acyl groups was controlled by varying the comonomer feed ratio. The copolymerization mechanism was also discussed.

Chitinase-catalyzed synthesis of an alternatingly N-sulfonated chitin derivative.

An alternatingly N-sulfonated chitin derivative (2) was synthesized via ring-opening polyaddition of an N-sulfonated chitobiose oxazoline derivative (1) catalyzed by chitinases from Bacillus sp. and Serratia marcescens. The polymerization proceeded homogeneously, providing 2 as a water-soluble polysaccharide in good yields with total control of regioselectivity and stereochemistry. M(n) of 2 reached 1900 and 4180 by use of chitinases from Bacillus sp. and Serratia marcescens, which correspond to 8-10 (n = 4-5) and 18-20 (n = 9-10) saccharide units, respectively. These results indicate that M(n) of 2 is controllable by selecting chitinases from different origins. It is considered that the C-2 position of the nonreducing unit in the oxazoline-type monomer is not deeply involved in the catalysis of chitinase.

Synthesis of fluorinated chitin derivatives via enzymatic polymerization.

Synthesis of fluorinated chitin derivatives has been achieved using chitinase from Bacillus sp. as a catalyst. 6'-Fluoro- (1a), 6-fluoro- (1b) and 6,6'-difluoro- (1c) chitobiose oxazoline derivatives were newly prepared as TSAS monomers for chitinase. Ring-opening polyaddition of these monomers proceeded effectively at pH 8.0-9.0 and 30-40 degrees C, giving rise to alternatingly 6-fluorinated chitin derivatives (2a and 2b) from 1a and 1b, and fully 6-fluorinated chitin derivative (2c) from 1c under total control of regioselectivity and stereochemistry. XRD measurements revealed that polysaccharides 2a and 2b had crystalline structures similar to that of alpha-chitin. [reaction: see text]

Chemical reaction at specific sites and reaction-induced self-assembly as observed by in situ and real time SANS: enzymatic polymerization to synthetic cellulose.

We have investigated the self-assembling process of cellulose artificially synthesized via enzymatic polymerization by means of in situ and time-resolved SANS (small-angle neutron scattering). The results elucidated the following: (i) Cellulose molecules synthesized at a special reaction site of the enzyme (cellulase) located on or near the smooth surface of self-assembled enzymes formed in the reaction medium. (ii) The synthesized molecules associated themselves via DLA (diffusion-limited association) and crystallized into fibrils. (iii) The fibrils formed the aggregates, which had surface fractal dimension D(s) increasing from 2 to 2.3 with the reaction time, on the smooth surface of the enzyme aggregates.

A hyaluronidase supercatalyst for the enzymatic polymerization to synthesize glycosaminoglycans.

Hyaluronidase (HAase) catalyzes multiple enzymatic polymerizations with controlling regio- and stereoselectivity perfectly. This behavior, that is, the single enzyme being effective for multireactions and retaining the enzyme catalytic specificity, is not usual, and hence, HAase is a supercatalyst. Various sugar oxazoline monomers prepared based on the concept "transition-state analogue substrate" were successfully polymerized and copolymerized with HAase catalysis, yielding natural and unnatural glycosaminoglycans.

Enzymatic polymerization to novel polysaccharides having a glucose-N-acetylglucosamine repeating unit, a cellulose-chitin hybrid polysaccharide.

A cellulose-chitin hybrid polysaccharide having alternatingly beta(1-->4)-linked D-glucose (Glc) and N-acetyl-d-glucosamine (GlcNAc) was synthesized via two modes of enzymatic polymerization. First, a sugar oxazoline monomer of Glcbeta(1-->4)GlcNAc (1) was designed as a transition-state analogue substrate (TSAS) monomer for chitinase catalysis. Monomer 1 was recognized by chitinase from Bacillus sp., giving rise to a cellulose-chitin hybrid polysaccharide (2) via ring-opening polyaddition with perfect regioselectivity and stereochemistry. Molecular weight (M(n)) of 2 reached 4030, which corresponds to 22 saccharide units. Second, a sugar fluoride monomer of GlcNAcbeta(1-->4)Glc (3) was synthesized for the catalysis of cellulase from Trichoderma viride. The enzyme catalyzed polycondensation of 3, providing a cellulose-chitin hybrid polysaccharide (4) in regio- and stereoselective manner. M(n) of 4 reached 2840, which corresponds to 16 saccharide units. X-ray diffraction measurements revealed that these hybrid polysaccharides did not form any characteristic crystalline structures. Furthermore, these unnatural hybrids of 2 and 4 were successfully digested by lysozyme from human neutrophils.