Bifidobacterial GH146 ß-L-arabinofuranosidase for the removal of ß1,3-L-arabinofuranosides on plant glycans.

L-Arabinofuranosides with ß-linkages are present in several plant molecules, such as arabinogalactan proteins (AGPs), extensin, arabinan, and rhamnogalacturonan-II. We previously characterized a ß-L-arabinofuranosidase from Bifidobacterium longum subsp. longum JCM 1217, Bll1HypBA1, which was found to belong to the glycoside hydrolase (GH) family 127. This strain encodes two GH127 genes and two GH146 genes. In the present study, we characterized a GH146 ß-L-arabinofuranosidase, Bll3HypBA1 (BLLJ_1848), which was found to constitute a gene cluster with AGP-degrading enzymes. This recombinant enzyme degraded AGPs and arabinan, which contain Araf-ß1,3-Araf structures. In addition, the recombinant enzyme hydrolyzed oligosaccharides containing Araf-ß1,3-Araf structures but not those containing Araf-ß1,2-Araf and Araf-ß1,5-Araf structures. The crystal structures of Bll3HypBA1 were determined at resolutions up to 1.7 Å. The monomeric structure of Bll3HypBA1 comprised a catalytic (α/α)6 barrel and two ß-sandwich domains. A hairpin structure with two ß-strands was observed in Bll3HypBA1, to extend from a ß-sandwich domain and partially cover the active site. The active site contains a Zn2+ ion coordinated by Cys3-Glu and exhibits structural conservation of the GH127 cysteine glycosidase Bll1HypBA1. This is the first study to report on a ß1,3-specific ß-L-arabinofuranosidase. KEY POINTS: • ß1,3-l-Arabinofuranose residues are present in arabinogalactan proteins and arabinans as a terminal sugar. • ß-l-Arabinofuranosidases are widely present in intestinal bacteria. • Bll3HypBA1 is the first enzyme characterized as a ß1,3-linkage-specific ß-l-arabinofuranosidase.

Synthesis of Sucrose-Mimicking Disaccharide by Intramolecular Aglycone Delivery.

Rare sugars are known for their ability to suppress postprandial blood glucose levels. Therefore, oligosaccharides and disaccharides derived from rare sugars could potentially serve as functional sweeteners. A disaccharide [α-d-allopyranosyl-(1→2)-ß-d-psicofuranoside] mimicking sucrose was synthesized from rare monosaccharides D-allose and D-psicose. Glycosylation using the intermolecular aglycon delivery (IAD) method was employed to selectively form 1,2-cis α-glycosidic linkages of the allopyranose residues. Moreover, ß-selective psicofuranosylation was performed using a psicofuranosyl acceptor with 1,3,4,6-tetra-O-benzoyl groups. This is the first report on the synthesis of non-reducing disaccharides comprising only rare d-sugars by IAD using protected ketose as a unique acceptor; additionally, this approach is expected to be applicable to the synthesis of functional sweeteners.

Bifidobacterial GH146 ß-l-Arabinofuranosidase (Bll4HypBA1) as the Last Enzyme for the Complete Removal of Oligoarabinofuranosides from Hydroxyproline-Rich Glycoproteins.

In plant cell walls, the hydroxyproline-rich glycoproteins (HRGPs) such as extensin contain oligoarabinofuranoside linked to a hydroxyproline (Hyp) residue. The mature arabinooligosaccharide was revealed to be a tetrasaccharide (α-l-Araf-(1→3)-ß-l-Araf-(1→2)-ß-l-Araf-(1→2)-ß-l-Araf, l-Araf4 ), whose linkages are targets of the bifidobacterial and Xanthomonas arabinooligosaccharide-degrading enzymes. The l-Araf4 motif was cleaved by GH43 α-l-arabinofuranosidase (Arafase) and converted to an l-Araf3 -linked structure. The latter is then cleaved by GH121 ß-l-arabinobiosidase (HypBA2), producing ß-l-Araf-(1→2)-l-Ara (ß-l-arabinobiose) and mono-ß-l-Araf linked to the HRGP backbone. In bifidobacteria, the ß-l-arabinobiose is then hydrolyzed by GH127 ß-l-Arafase (Bll1HypBA1), a mechanistically unique cysteine glycosidase. We recently identified the distantly related homologue from Xanthomonas euvesicatoria as GH146 ß-l-Arafase along with paralogues from Bifidobacterium longum, one of which, Bll4HypBA1 (BLLJ_0089), can degrade l-Araf1 -Hyp in a similar way to that of GH146. As the chemical synthesis of the extensin hydrophilic motif 1 a, which possesses three distinct linkages that connect four oligoAraf residues [Hyp(l-Arafn ) (n=4, 3, 1)], was achieved previously, we precisely monitored the step-wise enzymatic cleavage of 1 a in addition to that of potato lectin. The results unequivocally revealed that this enzyme specifically degrades the Hyp(l-Araf1 ) motif.

Recent Progress in 1,2-cis glycosylation for Glucan Synthesis.

Controlling the stereoselectivity of 1,2-cis glycosylation is one of the most challenging tasks in the chemical synthesis of glycans. There are various 1,2-cis glycosides in nature, such as α-glucoside and ß-mannoside in glycoproteins, glycolipids, proteoglycans, microbial polysaccharides, and bioactive natural products. In the structure of polysaccharides such as α-glucan, 1,2-cis α-glucosides were found to be the major linkage between the glucopyranosides. Various regioisomeric linkages, 1→3, 1→4, and 1→6 for the backbone structure, and 1→2/3/4/6 for branching in the polysaccharide as well as in the oligosaccharides were identified. To achieve highly stereoselective 1,2-cis glycosylation, including α-glucosylation, a number of strategies using inter- and intra-molecular methodologies have been explored. Recently, Zn salt-mediated cis glycosylation has been developed and applied to the synthesis of various 1,2-cis linkages, such as α-glucoside and ß-mannoside, via the 1,2-cis glycosylation pathway and ß-galactoside 1,4/6-cis induction. Furthermore, the synthesis of various structures of α-glucans has been achieved using the recent progressive stereoselective 1,2-cis glycosylation reactions. In this review, recent advances in stereoselective 1,2-cis glycosylation, particularly focused on α-glucosylation, and their applications in the construction of linear and branched α-glucans are summarized.

Identification of difructose dianhydride I synthase/hydrolase from an oral bacterium establishes a novel glycoside hydrolase family.

Fructooligosaccharides and their anhydrides are widely used as health-promoting foods and prebiotics. Various enzymes acting on ß-D-fructofuranosyl linkages of natural fructan polymers have been used to produce functional compounds. However, enzymes that hydrolyze and form α-D-fructofuranosyl linkages have been less studied. Here, we identified the BBDE_2040 gene product from Bifidobacterium dentium (α-D-fructofuranosidase and difructose dianhydride I synthase/hydrolase from Bifidobacterium dentium [αFFase1]) as an enzyme with α-D-fructofuranosidase and α-D-arabinofuranosidase activities and an anomer-retaining manner. αFFase1 is not homologous with any known enzymes, suggesting that it is a member of a novel glycoside hydrolase family. When caramelized fructose sugar was incubated with αFFase1, conversions of ß-D-Frup-(2→1)-α-D-Fruf to α-D-Fruf-1,2':2,1'-ß-D-Frup (diheterolevulosan II) and ß-D-Fruf-(2→1)-α-D-Fruf (inulobiose) to α-D-Fruf-1,2':2,1'-ß-D-Fruf (difructose dianhydride I [DFA I]) were observed. The reaction equilibrium between inulobiose and DFA I was biased toward the latter (1:9) to promote the intramolecular dehydrating condensation reaction. Thus, we named this enzyme DFA I synthase/hydrolase. The crystal structures of αFFase1 in complex with ß-D-Fruf and ß-D-Araf were determined at the resolutions of up to 1.76 Å. Modeling of a DFA I molecule in the active site and mutational analysis also identified critical residues for catalysis and substrate binding. The hexameric structure of αFFase1 revealed the connection of the catalytic pocket to a large internal cavity via a channel. Molecular dynamics analysis implied stable binding of DFA I and inulobiose to the active site with surrounding water molecules. Taken together, these results establish DFA I synthase/hydrolase as a member of a new glycoside hydrolase family (GH172).

Substrate complex structure, active site labeling and catalytic role of the zinc ion in cysteine glycosidase.

ß-l-Arabinofuranosidase HypBA1 from Bifidobacterium longum belongs to the glycoside hydrolase family 127. At the active site of HypBA1, a cysteine residue (Cys417) coordinates with a Zn2+ atom and functions as the catalytic nucleophile for the anomer-retaining hydrolytic reaction. In this study, the role of Zn2+ ion and cysteine in catalysis as well as the substrate-bound structure were studied based on biochemical and crystallographic approaches. The enzymatic activity of HypBA1 decreased after dialysis in the presence of EDTA and guanidine hydrochloride and was then recovered by the addition of Zn2+. The Michaelis complex structure was determined using a crystal of a mutant at the acid/base catalyst residue (E322Q) soaked in a solution containing the substrate p-nitrophenyl-ß-l-arabinofuranoside. To investigate the covalent thioglycosyl enzyme intermediate structure, synthetic inhibitors of l-arabinofuranosyl haloacetamide derivatives with different anomer configurations were used to target the nucleophilic cysteine. In the crystal structure of HypBA1, ß-configured l-arabinofuranosylamide formed a covalent link with Cys417, whereas α-configured l-arabinofuranosylamide was linked to a noncatalytic residue Cys415. Mass spectrometric analysis indicated that Cys415 was also reactive with the probe molecule. With the ß-configured inhibitor, the arabinofuranoside moiety was correctly positioned at the subsite and the active site integrity was retained to successfully mimic the covalent intermediate state.

Mechanism of Cooperative Degradation of Gum Arabic Arabinogalactan Protein by Bifidobacterium longum Surface Enzymes.

Gum arabic is an arabinogalactan protein (AGP) that is effective as a prebiotic for the growth of bifidobacteria in the human intestine. We recently identified a key enzyme in the glycoside hydrolase (GH) family 39, 3-O-α-d-galactosyl-α-l-arabinofuranosidase (GAfase), for the assimilation of gum arabic AGP in Bifidobacterium longum subsp. longum. The enzyme released α-d-Galp-(1→3)-l-Ara and ß-l-Arap-(1→3)-l-Ara from gum arabic AGP and facilitated the action of other enzymes for degrading the AGP backbone and modified sugar. In this study, we identified an α-l-arabinofuranosidase (BlArafE; encoded by BLLJ_1850), a multidomain enzyme with both GH43_22 and GH43_34 catalytic domains, as a critical enzyme for the degradation of modified α-l-arabinofuranosides in gum arabic AGP. Site-directed mutagenesis approaches revealed that the α1,3/α1,4-Araf double-substituted gum arabic AGP side chain was initially degraded by the GH43_22 domain and subsequently cleaved by the GH43_34 domain to release α1,3-Araf and α1,4-Araf residues, respectively. Furthermore, we revealed that a tetrasaccharide, α-l-Rhap-(1→4)-ß-d-GlcpA-(1→6)-ß-d-Galp-(1→6)-d-Gal, was a limited degradative oligosaccharide in the gum arabic AGP fermentation of B. longum subsp. longum JCM7052. The oligosaccharide was produced from gum arabic AGP by the cooperative action of the three cell surface-anchoring enzymes, GAfase, exo-ß1,3-galactanase (Bl1,3Gal), and BlArafE, on B. longum subsp. longum JCM7052. Furthermore, the tetrasaccharide was utilized by the commensal bacteria. IMPORTANCE Terminal galactose residues of the side chain of gum arabic arabinogalactan protein (AGP) are mainly substituted by α1,3/α1,4-linked Araf and ß1,6-linked α-l-Rhap-(1→4)-ß-d-GlcpA residues. This study found a multidomain BlArafE with GH43_22 and GH43_34 catalytic domains showing cooperative action for degrading α1,3/α1,4-linked Araf of the side chain of gum arabic AGP. In particular, the GH43_34 domain of BlArafE was a novel α-l-arabinofuranosidase for cleaving the α1,4-Araf linkage of terminal galactose. α-l-Rhap-(1→4)-ß-d-GlcpA-(1→6)-ß-d-Galp-(1→6)-d-Gal tetrasaccharide was released from gum arabic AGP by the cooperative action of GAfase, GH43_24 exo-ß-1,3-galactanase (Bl1,3Gal), and BlArafE and remained after B. longum subsp. longum JCM7052 culture. Furthermore, in vitro assimilation test of the remaining oligosaccharide using Bacteroides species revealed that cross-feeding may occur from bifidobacteria to other taxonomic groups in the gut.

Synthesis of naturally occurring ß-l-arabinofuranosyl-l-arabinofuranoside structures towards the substrate specificity evaluation of ß-l-arabinofuranosidase.

Methyl ß-l-arabinofuranosyl-(1 â†’ 2)-, -(1 â†’ 3)-, and -(1 â†’ 5)-α-l-arabinofuranosides have been stereoselectively synthesized through 2-naphthylmethyl ether-mediated intramolecular aglycon delivery (NAP-IAD), whose ß-linkages were confirmed by NMR analysis on the 3JH1-H2 coupling constant and 13C chemical shift of C1. The NAP-IAD approach was simply extended for the synthesis of trisaccharide motifs possessing ß-l-arabinofuranosyl-(1 â†’ 5)-l-arabinofuranosyl non-reducing terminal structure with the branched ß-l-arabinofuranosyl-(1 â†’ 5)-[α-l-arabinofuranosyl-(1 â†’ 3)]-α-l-arabinofuranosyl and the liner ß-l-arabinofuranosyl-(1 â†’ 5)-ß-l-arabinofuranosyl-(1 â†’ 5)-ß-l-arabinofuranosyl structures in olive arabinan and dinoflagellate polyethers, respectively. The results on the substrate specificity of a bifidobacterial ß-l-arabinofuranosidase HypBA1 using the regioisomers indicated that HypBA1 could hydrolyze all three linkages however behaved clearly less active to ß-(1 â†’ 5)-linked disaccharide than other two regioisomers including the proposed natural degradation product, ß-(1 â†’ 2)-linked one from plant extracellular matrix such as extensin. On the other hand, Xanthomonas XeHypBA1 was found to hydrolyze all three disaccharides as the substrate with higher specificity to ß-(1 â†’ 2)-linkage than bifidobacterial HypBA1.

Mechanism-based inhibition of GH127/146 cysteine glycosidases by stereospecifically functionalized l-arabinofuranosides.

To understand the precise mechanism of the glycoside hydrolase (GH) family 127, a cysteine ß-l-arabinofuranosidase (Arafase) - HypBA1 - has been isolated from Bifidobacterium longum in the human Gut microbiota, and the design and synthesis of the mechanism-based inhibitors such as l-Araf-haloacetamides have been carried out. The α-l-Araf-azide derivative was used as the monoglycosylamine equivalent to afford the l-Araf-chloroacetamides (α/ß-1-Cl) as well as bromoacetamides (α/ß-1-Br) in highly stereoselective manner through Staudinger reaction followed by amide formation with/without anomerization. Against HypBA1, the probes 1, especially in the case of α/ß-1-Br inhibited the hydrolysis. Conformational implications of these observations are discussed in this manuscript. Additional examinations using l-Araf-azides (α/ß-5) resulted in further mechanistic observations of the GH127/146 cysteine glycosidases, including the hydrolysis of ß-5 as the substrate and oxidative inhibition by α-5 using the GH127 homologue.

Novel 3-O-α-d-Galactosyl-α-l-Arabinofuranosidase for the Assimilation of Gum Arabic Arabinogalactan Protein in Bifidobacterium longum subsp. longum.

Gum arabic arabinogalactan (AG) protein (AGP) is a unique dietary fiber that is degraded and assimilated by only specific strains of Bifidobacterium longum subsp. longum Here, we identified a novel 3-O-α-d-galactosyl-α-l-arabinofuranosidase (GAfase) from B. longum JCM7052 and classified it into glycoside hydrolase family 39 (GH39). GAfase released α-d-Galp-(1→3)-l-Ara and ß-l-Arap-(1→3)-l-Ara from gum arabic AGP and ß-l-Arap-(1→3)-l-Ara from larch AGP, and the α-d-Galp-(1→3)-l-Ara release activity was found to be 594-fold higher than that of ß-l-Arap-(1→3)-l-Ara. The GAfase gene was part of a gene cluster that included genes encoding a GH36 α-galactosidase candidate and ABC transporters for the assimilation of the released α-d-Galp-(1→3)-l-Ara in B. longum Notably, when α-d-Galp-(1→3)-l-Ara was removed from gum arabic AGP, it was assimilated by both B. longum JCM7052 and the nonassimilative B. longum JCM1217, suggesting that the removal of α-d-Galp-(1→3)-l-Ara from gum arabic AGP by GAfase permitted the cooperative action with type II AG degradative enzymes in B. longum The present study provides new insight into the mechanism of gum arabic AGP degradation in B. longumIMPORTANCE Bifidobacteria harbor numerous carbohydrate-active enzymes that degrade several dietary fibers in the gastrointestinal tract. B. longum JCM7052 is known to exhibit the ability to assimilate gum arabic AGP, but the key enzyme involved in the degradation of gum arabic AGP remains unidentified. Here, we cloned and characterized a GH39 3-O-α-d-galactosyl-α-l-arabinofuranosidase (GAfase) from B. longum JCM7052. The enzyme was responsible for the release of α-d-Galp-(1→3)-l-Ara and ß-l-Arap-(1→3)-l-Ara from gum arabic AGP. The presence of a gene cluster including the GAfase gene is specifically observed in gum arabic AGP assimilative strains. However, GAfase carrier strains may affect GAfase noncarrier strains that express other type II AG degradative enzymes. These findings provide insights into the bifidogenic effect of gum arabic AGP.

Zinc(II) Iodide-Directed ß-Mannosylation: Reaction Selectivity, Mode, and Application.

A direct, efficient, and versatile glycosylation methodology promises the systematic synthesis of oligosaccharides and glycoconjugates in a streamlined fashion like the synthesis of medium to long-chain nucleotides and peptides. The development of a generally applicable approach for the construction of 1,2-cis-glycosidic bond with controlled stereoselectivity remains a major challenge, especially for the synthesis of ß-mannosides. Here, we report a direct mannosylation strategy mediated by ZnI2, a mild Lewis acid, for the highly stereoselective construction of 1,2-cis-ß linkages employing easily accessible 4,6-O-tethered mannosyl trichloroacetimidate donors. The versatility and effectiveness of this strategy were demonstrated with successful ß-mannosylation of a wide variety of alcohol acceptors, including complex natural products, amino acids, and glycosides. Through iteratively performing ZnI2-mediated mannosylation with the chitobiosyl azide acceptor followed by site-selective deprotection of the mannosylation product, the novel methodology enables the modular synthesis of the key intermediate trisaccharide with Man-ß-(1 → 4)-GlcNAc-ß-(1 → 4)-GlcNAc linkage for N-glycan synthesis. Theoretical investigations with density functional theory calculations delved into the mechanistic details of this ß-selective mannosylation and elucidated two zinc cations' essential roles as the activating agent of the donor and the principal mediator of the cis-directing intermolecular interaction.

Cysteine Nucleophiles in Glycosidase Catalysis: Application of a Covalent ß-l-Arabinofuranosidase Inhibitor.

The recent discovery of zinc-dependent retaining glycoside hydrolases (GHs), with active sites built around a Zn(Cys)3 (Glu) coordination complex, has presented unresolved mechanistic questions. In particular, the proposed mechanism, depending on a Zn-coordinated cysteine nucleophile and passing through a thioglycosyl enzyme intermediate, remains controversial. This is primarily due to the expected stability of the intermediate C-S bond. To facilitate the study of this atypical mechanism, we report the synthesis of a cyclophellitol-derived ß-l-arabinofuranosidase inhibitor, hypothesised to react with the catalytic nucleophile to form a non-hydrolysable adduct analogous to the mechanistic covalent intermediate. This ß-l-arabinofuranosidase inhibitor reacts exclusively with the proposed cysteine thiol catalytic nucleophiles of representatives of GH families 127 and 146. X-ray crystal structures determined for the resulting adducts enable MD and QM/MM simulations, which provide insight into the mechanism of thioglycosyl enzyme intermediate breakdown. Leveraging the unique chemistry of cyclophellitol derivatives, the structures and simulations presented here support the assignment of a zinc-coordinated cysteine as the catalytic nucleophile and illuminate the finely tuned energetics of this remarkable metalloenzyme clan.

Unified Strategy toward Stereocontrolled Assembly of Various Glucans Based on Bimodal Glycosyl Donors.

Polymers of glucose, the most abundant and one of the biologically important natural products, named glucans are widely present in fungi, bacteria, mammals, and plants with various anomeric configurations and glycosidic linkages. Because of their structural diversity, the unified strategy for the assembly of pure glucans is yet to be developed. Herein, we describe a general strategy that is applicable to construction of all types of glucans by exploiting a bimodal glycosyl donor equipped with C2-o-TsNHbenzyl ether (TAB), which enables stereocontrolled synthesis of both α- and ß-glycosides by switching reaction conditions.

N-Glycosylation with synthetic undecaprenyl pyrophosphate-linked oligosaccharide to oligopeptides by PglB oligosaccharyltransferase from Campylobacter jejuni.

The oligosaccharyltransferase PglB from Campylobacter jejuni catalyses the N-glycosylation reaction with undecaprenyl-pyrophosphate-linked Glc1 GalNAc5 Bac1 (Und-PP-Glc1 GalNAc5 Bac1 ). Experiments using chemically synthesized donors coupled to fluorescently tagged peptides confirmed that biosynthetic intermediate Und-PP-Bac1 and Und-PP-GalNAc2 Bac1 are transferred efficiently to the Asn residue in the consensus sequence (D/E-X'-N-X-T/S, X',X≠P). The products were analyzed in detail by tandem MS to confirm their chemical structures.

Crystal structure of glycoside hydrolase family 127 ß-l-arabinofuranosidase from Bifidobacterium longum.

Enzymes acting on ß-linked arabinofuranosides have been unknown until recently, in spite of wide distribution of ß-l-arabinofuranosyl oligosaccharides in plant cells. Recently, a ß-l-arabinofuranosidase from the glycoside hydrolase family 127 (HypBA1) was discovered in the newly characterized degradation system of hydroxyproline-linked ß-l-arabinooligosaccharides in the bacterium Bifidobacterium longum. Here, we report the crystal structure of HypBA1 in the ligand-free and ß-l-arabinofuranose complex forms. The structure of HypBA1 consists of a catalytic barrel domain and two additional ß-sandwich domains, with one ß-sandwich domain involved in the formation of a dimer. Interestingly, there is an unprecedented metal-binding motif with Zn(2+) coordinated by glutamate and three cysteines in the active site. The glutamate residue is located far from the anomeric carbon of the ß-l-arabinofuranose ligand, but one cysteine residue is appropriately located for nucleophilic attack for glycosidic bond cleavage. The residues around the active site are highly conserved among GH127 members. Based on biochemical experiments and quantum mechanical calculations, a possible reaction mechanism involving cysteine as the nucleophile is proposed.

Synthesis of the highly glycosylated hydrophilic motif of extensins.

Extensin, the structural motif of plant extracellular matrix proteins, possesses a unique highly glycosylated, hydrophilic, and repeating Ser1Hyp4 pentapeptide unit, and has been proposed to include post-translational hydroxylation at proline residue and subsequent oligo-L-arabinosylations at all of the resultant hydroxyprolines as well as galactosylation at serine residue. Reported herein is the stereoselective synthesis of one of the highly glycosylated motifs, Ser(Galp1)-Hyp(Araf4)-Hyp(Araf4)-Hyp(Araf3)-Hyp(Araf1). The synthesis has been completed by the application of 2-(naphthyl)methylether-mediated intramolecular aglycon delivery to the stereoselective construction of the Ser(Galp1) and Hyp(Araf(n)) fragments as the key step, as well as Fmoc solid-phase peptide synthesis for the backbone pentapeptide.

Metallic radionuclide-labeled tetrameric 2,6-diisopropylphenyl azides for cancer treatment.

This study proposes a new method for radionuclide therapy that involves the use of oligomeric 2,6-diisopropylphenyl azides and a chelator to form stable complexes with metallic radionuclides. The technique works by taking advantage of the endogenous acrolein produced by cancer cells. The azides react with the acrolein to give a diazo derivative that immediately attaches to the nearest organelle, effectively anchoring the radionuclide within the tumor. Preliminary in vivo experiments were conducted on a human lung carcinoma xenograft model, demonstrating the feasibility of this approach for cancer treatment.

B(C6F5)3-Catalyzed Stereoselective 1,2-cis Arabinofuranosylation with a Conformationally Constrained Donor.

Compared with stereoselective glycosylation methods mainly addressed on the preparation of pyranose glycosides, the furanosylation has been more limited, especially for the 1,2-cis arabinofuranosylation. Herein, we report a novel stereoselective 1,2-cis-arabinofuranosylation strategy using a conformationally restricted 3,5-O-xylylene-protected arabinofuranosyl donor on activation with B(C6F5)3 for desired targets in moderate to excellent yields and ß-stereoselectivity. The effectiveness of the 1,2-cis-arabinofuranosylation strategy was demonstrated successfully with various acceptors, including carbohydrate alcohols.

Genetic and functional diversity of ß-N-acetylgalactosamine-targeting glycosidases expanded by deep-sea metagenome analysis.

ß-N-Acetylgalactosamine-containing glycans play essential roles in several biological processes, including cell adhesion, signal transduction, and immune responses. ß-N-Acetylgalactosaminidases hydrolyze ß-N-acetylgalactosamine linkages of various glycoconjugates. However, their biological significance remains ambiguous, primarily because only one type of enzyme, exo-ß-N-acetylgalactosaminidases that specifically act on ß-N-acetylgalactosamine residues, has been documented to date. In this study, we identify four groups distributed among all three domains of life and characterize eight ß-N-acetylgalactosaminidases and ß-N-acetylhexosaminidase through sequence-based screening of deep-sea metagenomes and subsequent searching of public protein databases. Despite low sequence similarity, the crystal structures of these enzymes demonstrate that all enzymes share a prototype structure and have diversified their substrate specificities (oligosaccharide-releasing, oligosaccharide/monosaccharide-releasing, and monosaccharide-releasing) through the accumulation of mutations and insertional amino acid sequences. The diverse ß-N-acetylgalactosaminidases reported in this study could facilitate the comprehension of their structures and functions and present evolutionary pathways for expanding their substrate specificity.

Stereoselective synthesis of Arabidopsis CLAVATA3 (CLV3) glycopeptide, unique protein post-translational modifications of secreted peptide hormone in plant.

The unique hydroxylproline (Hyp)-linked O-glycan modification is a common process in hydroxyproline-rich glycoproteins (HRGPs). The modification occurs through post-translational hydroxylation at 4-position of proline residues some of which are followed by O-glycosylation at the resulting Hyp which is also found in some secreted peptide hormones such as CLAVATA3 (CLV3) of Arabidopsis thaliana plants. An active mature CLV3 is a tridecapeptide linked to ß-L-Araf-(1→2)-ß-L-Araf-(1→2)-ß-L-Araf at a Hyp residue in the center of the peptide sequence such as Arg-Thr-Val-Hyp-Ser-Gly-Hyp(L-Arafn)-Asp-Pro-Leu-His-His-His (n = 3). We report here the synthesis of the secreted and modified CLV3 glycopeptide with all glycoforms (Araf0-3CLV3) of A. thaliana plants. A highly stereoselective ß-arabinofuranosylation of Hyp derivatives as the key step of the synthesis of CLV3 glycopeptide was achieved by NAP ether-mediated IAD, which was effectively applied to the synthesis of oligoarabinosylated hydroxylproline [Hyp(L-Araf1-3)] derivatives. Fmoc-solid phase peptide synthesis was carried out using COMU as the coupling reagent for the introduction of [Hyp(L-Araf0-3)] derivatives as well as further elongation to the CLV3 glycopeptides.