Disaccharide Residues Research Articles

Total Synthesis of Clostrienose.

This paper considers the total synthesis of a cellular differentiation regulator of Clostridium acetobutylicum, clostrienose, which is a unique fatty-acid glycosyl ester consisting of clostrienoic acid, (3R,5E,8E,10E)-3-hydroxy-tetradeca-5,8,10-trienoic acid and α-d-galactofuranosyl-(1 → 2)-α-l-rhamnose. The key features of our synthesis include stereoselective construction of a skipped-triene system in clostrienoic acid and its esterification with a disaccharide residue. The partially protected clostrienoic acid employed for the coupling also served for the preparation of l-rhamnosyl clostrienoate, thus leading to confirmation of the proposed structure unambiguously.

Synthesis of the Key Saccharide Fragments of the Glucuronic Acid-Containing Repeat Unit of Pentosan Polysulfate, a Heparin Sulfate Mimetic

AbstractSuitably protected mono- and di-saccharide residues associated with the glucuronic acid-containing repeat unit related to pentosan polysulfate have been prepared. The stereo-controlled coupling, using trichloroacetimidate chemistry, of certain of these is also described and the structure of a disaccharide so-formed has been confirmed by single-crystal X-ray analysis.

Change of phytochemicals and bioactive substances in Lactobacillus fermented Citrus juice during the fermentation process

This study evaluated the conversion of bioactive substances and phytochemicals in the 96 h of fermented Citrus juice by L. brevis, L. plantarum, L. paracasei, and L. fermentium, results showed fermentation maintained a higher vitamin C, antioxidant activity, enhanced the contents of total phenolic, total flavonoid and amino acids in Citrus juice. The steady acidity and sweetness, as well as the greatest increase of total flavonoids were found in L. fermentium 252 fermented Citrus juices from 54.30 ± 11.41 mg/L to 127.82 ± 6.41 mg/L. Furthermore, a high level of phenyllactic acid was detected with 17.69 ± 1.26 mg/L, 53 kinds of flavonoids were identified in fermented samples with L. fermentium 252, the flavanones and flavones increased significantly (P < 0.05), such as naringenin-7-glucoside, hesperetin and nobiletin. Another interesting founding, L. fermentium 252 likely inclined to remove one glycosyl instead of the disaccharide residue. Hence, L. fermentium 252 may be the most promising Lactobacillus for fermentation of Citrus juice among the four strains.

Specific Binding of the α-Component of the Lantibiotic Lichenicidin to the Peptidoglycan Precursor Lipid II Predetermines Its Antimicrobial Activity.

To date, a number of lantibiotics have been shown to use lipid II-a highly conserved peptidoglycan precursor in the cytoplasmic membrane of bacteria-as their molecular target. The α-component (Lchα) of the two-component lantibiotic lichenicidin, previously isolated from the Bacillus licheniformis VK21 strain, seems to contain two putative lipid II binding sites in its N-terminal and C-terminal domains. Using NMR spectroscopy in DPC micelles, we obtained convincing evidence that the C-terminal mersacidin-like site is involved in the interaction with lipid II. These data were confirmed by the MD simulations. The contact area of lipid II includes pyrophosphate and disaccharide residues along with the first isoprene units of bactoprenol. MD also showed the potential for the formation of a stable N-terminal nisin-like complex; however, the conditions necessary for its implementation in vitro remain unknown. Overall, our results clarify the picture of two component lantibiotics mechanism of antimicrobial action.

Polymerization of chondroitin sulfate and its stimulatory effect on cartilage regeneration; a bioactive material for cartilage regeneration

Chondroitin sulfate (CS) is one of the major glycosaminoglycans (GAGs). GAGs are linear polymers comprising disaccharide residues and are found as the side chains of proteoglycans. CS has significant stimulatory effects on cell behavior and is widely used in tissue-engineered and drug delivery devices. However, it is difficult to incorporate a sufficient amount of CS into biopolymer-based scaffolds such as collagen to take full advantage of its benefit. In this study, CS has been polymerized to an 11 times higher molecular weight polymer (PCS) in an attempt to overcome this deficiency. We have previously shown that PCS was significantly more effective than CS in chondrogenesis. This study aimed to characterize the physicochemical properties of the manufactured PCS. PCS was characterized by Fourier transform infra-red (FTIR) spectroscopy together with X-ray photoelectron spectroscopy (XPS) to obtain information about its chemical structure and elemental composition. Its molecular size was measured using dynamic light scattering (DLS) and its viscoelastic properties were determined by rheology measurements. The average PCS diameter increased 5 times by polymerization and PCS has significantly enhanced viscoelastic properties compared to CS. The molecular weight of PCS was calculated from the rheological experiment to give more than an order of magnitude increase over CS molecular weight. Based on these results, we believe there is a great potential for using PCS in regenerative medicine devices.

Disaccharide Residues are Required for Native Antifreeze Glycoprotein Activity.

Antifreeze glycoproteins (AFGPs) are able to bind to ice, halt its growth, and are the most potent inhibitors of ice recrystallization known. The structural basis for AFGP’s unique properties remains largely elusive. Here we determined the antifreeze activities of AFGP variants that we constructed by chemically modifying the hydroxyl groups of the disaccharide of natural AFGPs. Using nuclear magnetic resonance, two-dimensional infrared spectroscopy, and circular dichroism, the expected modifications were confirmed as well as their effect on AFGPs solution structure. We find that the presence of all the hydroxyls on the disaccharides is a requirement for the native AFGP hysteresis as well as the maximal inhibition of ice recrystallization. The saccharide hydroxyls are apparently as important as the acetyl group on the galactosamine, the α-linkage between the disaccharide and threonine, and the methyl groups on the threonine and alanine. We conclude that the use of hydrogen-bonding through the hydroxyl groups of the disaccharide and hydrophobic interactions through the polypeptide backbone are equally important in promoting the antifreeze activities observed in the native AFGPs. These important criteria should be considered when designing synthetic mimics.

Characterisation of phenylethanoid glycosides by multiple-stage mass spectrometry.

Although phenylethanoid glycosides (PhGs) occur widely in plants, their characterisation by liquid chromatography/mass spectrometry (LC/MS) is less well studied than other phenolic glycosides such as flavonoid glycosides. The multiple-stage mass spectrometry (MSn ) experiments required to improve the annotation of common verbascoside-type PhGs are described here. Deprotonated, ammoniated and sodiated molecules of nine PhGs were subjected to low-energy collision-induced dissociation (CID) in a hybrid ion trap/orbitrap mass spectrometer. Most experiments were recorded at nominal mass using the linear ion trap analyser for wider applicability in the plant metabolomics community. Data interpretation was supported by high-resolution orbitrap scanning of product ions. Comparative data was acquired on the same instrument by performing higher-energy collisional dissociation (HCD) in the C-trap. Low-energy CID-MS2 of the deprotonated and ammoniated molecules generated diagnostic product ions from which the molecular masses of the phenolic acid and phenylethanoid moieties, respectively, could be determined. The sugar at C-3' of the core glucose was preferentially lost from the sodiated molecule following CID-MS2 , while CID-MSn produced a sodiated product ion from ring cleavage of the core glucose bearing the sugar at C-6'. Evidence of a disaccharide substitution came from a sodiated disaccharide residue in CID-MSn spectra. The consistency of PhG dissociation following low-energy CID-MSn of various ions is sufficient to enable annotation of verbascoside-type PhGs in LC/MS analyses of crude plant extracts. This can be achieved on a low-resolution instrument capable of MSn .

Bivalent glycoconjugates based on 1,5-diazabicyclo[3.3.0]octa-3,6-diene-2,8-dione (“bimane”) as a central scaffold

The heteroaromatic fused diazabicyclic “bimane” ring system, discovered four decades ago, is endowed with remarkable chemical and photophysical properties. No carbohydrate derivatives of bimanes have, however, been described thus far. Here we report on the syntheses of a range of bimanes decorated with various glycosyl residues. Mono- and disaccharide residues were attached to syn- or anti-bimane central cores via thio-, disulfido- or selenoglycosidic linkages to obtain novel fluorescent or nonfluorescent glycoconjugates. Cu(I)-catalyzed cycloaddition of glycosyl azides to a bimane diethynyl derivative furnished further bivalent glycoconjugates with sugar residues linked to the central bimane core via 1,2,3-triazole rings. We have determined the crystal and molecular structures of several glycosylated and non-glycosylated bimanes and report fluorescence data for the new compounds.

Sequencing of chondroitin sulfate oligosaccharides using a novel exolyase from a marine bacterium that degrades hyaluronan and chondroitin sulfate/dermatan sulfate.

Glycosaminoglycans (GAGs) are a family of chemically heterogeneous polysaccharides that play important roles in physiological and pathological processes. Owing to the structural complexity of GAGs, their sophisticated chemical structures and biological functions have not been extensively studied. Lyases that cleave GAGs are important tools for structural analysis. Although various GAG lyases have been identified, exolytic lyases with unique enzymatic property are urgently needed for GAG sequencing. In the present study, a putative exolytic GAG lyase from a marine bacterium was recombinantly expressed and characterized in detail. Since it showed exolytic lyase activity toward hyaluronan (HA), chondroitin sulfate (CS), and dermatan sulfate (DS), it was designated as HCDLase. This novel exolyase exhibited the highest activity in Tris-HCl buffer (pH 7.0) at 30°C. Especially, it showed a specific activity that released 2-aminobenzamide (2-AB)-labeled disaccharides from the reducing end of 2-AB-labeled CS oligosaccharides, which suggest that HCDLase is not only a novel exolytic lyase that can split disaccharide residues from the reducing termini of sugar chains but also a useful tool for the sequencing of CS chains. Notably, HCDLase could not digest 2-AB-labeled oligosaccharides from HA, DS, or unsulfated chondroitin, which indicated that sulfates and bond types affect the catalytic activity of HCDLase. Finally, this enzyme combined with CSase ABC was successfully applied for the sequencing of several CS hexa- and octasaccharides with complex structures. The identification of HCDLase provides a useful tool for CS-related research and applications.

Click Glycosylation for the Synthesis of 1,2,3‐Triazole‐Linked Picropodophyllotoxin Glycoconjugates and Their Anticancer Activity

AbstractCu(I)‐catalyzed azide‐alkyne cycloaddition (CuAAC) reaction was employed successfully to prepare a series 4β‐triazole‐linked picropodophyllotoxin glycoconjugates (25 – 34). Maltose residue, 1,6‐β‐D‐diglucose residue, and several triazole‐linked disaccharide and trisaccharide residues were coupled to 4β‐azido‐podophyllotoxin derivatives through click glycosylation strategy. The initial click glycosylation products were treated with catalytic amount of NaOCH3 to facilitate global deacylation and epimerization at C‐2 position to yield the cis‐γ‐lactone moiety in the picropodophyllotoxin glycoconjugates. Most of these picropodophyllotoxin glycoconjugates show weak cytotoxicity (IC50 &gt; 40 μM) against a panel of five human cancer cell lines (HL‐60, SMMC‐7721, A‐549, MCF‐7, SW480) as indicated by in vitro MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] assay. However, compound 27 that contains a 1,6‐β‐D‐di‐glucose residue displays strong anticancer activity against all cancer cell lines tested, with IC50 values ranging from 0.67 to 7.41 μM, which is significantly more potent than the control drug etoposide against four of the five cancer cells tested. Structure activity relationship analysis suggests that the 4’‐O‐methyl group on the E ring of podophyllotoxin scaffold is perhaps important for the anticancer activity of glycosylated picropodophyllotoxin derivatives with a cis‐γ‐lactone moiety.

Synthesis and predetermined supramolecular chirality of carbohydrate-functionalized perylene bisimide derivatives.

Eight carbohydrate-modified perylene bisimides (PBI-4 lac-2 lac, PBI-4 lac-2 Man, PBI-4 lac-2 Gal, PBI-4 lac-2 Mal, PBI-4 Man-2 Man, PBI-4 Man-2 lac, PBI-4 Man-2 Gal and PBI-4 Man-2 Mal) were synthesized, and the following predetermined supramolecular chirality rule was found: perylene bisimides modified with disaccharides (D-lactose and D-maltose) at the imide position generated right-handed chirality, and those modified with monosaccharides (D-mannose and D-galactose) generated left-handed chirality, when D-lactose or D-mannose was substituted in the bay positions of perylene bisimides with amide bonds as the linking spacers. These results may be because of the difference in the stacking angle of the perylene bisimide backbones induced by the steric effect and the additional hydrogen bonds between the disaccharide residues. This study provides an important design rule for predetermined chiral self-assembly of perylene bisimides.

In vitro enzymatic treatment to remove O-linked mannose from intact glycoproteins

The methylotrophic yeast Pichia pastoris is an attractive expression system for heterologous protein production due to its ability to perform posttranslational modifications, such as glycosylation, and secrete large amounts of recombinant protein. However, the structures of N- and O-linked oligosaccharide chains in yeast differ significantly from those of mammalian cells. The most common O-linked glycan structures added by P. pastoris are typically polymers of between one and four α-linked mannose residues, with a subset of glycans being capped by a β-1,2-mannose disaccharide or phosphomannose residue. Such mannosylation of recombinant proteins is considered a key factor in immunomodulation, with mannose-specific receptors binding and promoting enhanced immune responses. As a result of engineering the N-linked glycosylation pathway of P. pastoris, the recombinant proteins expressed in this system are devoid of phospho- and β-mannose on O-linked glycans, leaving only α-mannose polymers. Here we screen a library of α-mannosidases for their ability to decrease the extent of O-mannosylation on glycoproteins secreted from this expression system. In doing so, we demonstrate the utility of the α-1,2/3/6-mannosidase from Jack bean in not only reducing extended O-linked mannose chains but also in specifically hydrolyzing the Man-α-O-Ser/Thr glycosidic bond on intact glycoproteins. As such, this presents for the first time a strategy to remove O-linked glycosylation from intact glycoproteins expressed in P. pastoris. We additionally show that this strategy can be used to significantly decrease the extent of O-mannosylation on commercial products produced in other similar expression systems.

Heparan sulfate in angiogenesis: a target for therapy

Heparan sulfate (HS), a long linear polysaccharide of alternating disaccharide residues, interacts with a wide variety of proteins, including many angiogenic factors. The involvement of HS in signaling of pro-angiogenic factors (e.g. vascular endothelial growth factor and fibroblast growth factor 2), as well as interaction with anti-angiogenic factors (e.g. endostatin), warrants its role as an important modifier of (tumor) angiogenesis. This review summarizes our current understanding of the role of HS in angiogenic growth factor signaling, and discusses therapeutic strategies to target HS and modulate angiogenesis.

Exposure to common food additive carrageenan leads to reduced sulfatase activity and increase in sulfated glycosaminoglycans in human epithelial cells

The commonly used food additive carrageenan, including lambda (λ), kappa (κ) and iota (ι) forms, is composed of galactose disaccharides linked in alpha-1,3 and beta-1,4 glycosidic bonds with up to three sulfate groups per disaccharide residue. Carrageenan closely resembles the endogenous galactose or N-acetylgalactosamine-containing glycosaminoglycans (GAGs), chondroitin sulfate (CS), dermatan sulfate (DS), and keratan sulfate. However, these GAGs have beta-1,3 and beta-1,4 glycosidic bonds, in contrast to the unusual alpha-1,3 glycosidic bond in carrageenan. Since sulfatase activity is inhibited by sulfate, and carrageenan is so highly sulfated, we tested the effect of carrageenan exposure on sulfatase activity in human intestinal and mammary epithelial cell lines and found that carrageenan exposure significantly reduced the activity of sulfatases, including N-acetylgalactosamine-4-sulfatase, galactose-6-sulfatase, iduronate sulfatase, steroid sulfatase, arylsulfatase A, SULF-1,2, and heparan sulfamidase. Consistent with the inhibition of sulfatase activity, following exposure to carrageenan, GAG content increased significantly and showed marked differences in disaccharide composition. Specific changes in CS disaccharides included increases in di-sulfated disaccharide components of CSD (2S6S) and CS-E (4S6S), with declines in CS-A (4S) and CS-C (6S). Specific changes in heparin-heparan sulfate disaccharides included increases in 6S disaccharides, as well as increases in NS and 2S6S disaccharides. Study results suggest that carrageenan inhibition of sulfatase activity leads to re-distribution of the cellular GAG composition with increase in di-sulfated CS and with potential consequences for cell structure and function.

Development of a fluorescent probe for the study of the sponge-derived simplexide immunological properties

Simplexide is a glycolipid of marine origin, endowed with immunological properties, composed of a long chain secondary alcohol glycosylated by an α- d-glucosyl-(1→4)-β- d-galactosyl disaccharide residue. Herein we describe the preparation of a fluorescent derivative of simplexide, labeled at position 6 of the distal glucose with a dansyl group, as a probe for future studies on the mechanism by which simplexide affects the immune system. Fluorescent simplexide was prepared from a 6″-amino functionalized compound, which in turn was obtained through a highly efficient glycosylation between the preformed activated disaccharide and the long chain secondary alcohol 18-pentatriacontanol.

Conjugates of polyhedral boron compounds with carbohydrates 8. Synthesis and properties of nido-ortho-carborane glycoconjugates containing one to three β-lactosylamine residues

β-Lactosylamine derivatives containing one to three disaccharide residues with the bromine atom at the terminal position of aglycon were synthesized. Conjugation of these derivatives with 1-mercapto-1,2-dicarba-closo-dodecaborane was carried out in DMSO, which was accompanied by deboronation to form nido-ortho-carborane (7,8-dicarba-nido-undecaborate) glycoconjugates in up to 70% yield. The hydrolytic stability of glycoconjugates and the binding efficiency of their triethylammonium salts to castor-bean galectin (Ricinus communis aggluinin, RCA120) were estimated. Glycoconjugates are decomposed in an aqueous solution by ∼15% at 37 °C in three days. The glycoconjugate salt with one β-lactosylamine residue binds to galectin analogously to lactose; after indroduction of the second β-lactosylamine residue into glycoconjugate, the binding efficiency increases ∼6-fold due to the cluster effect.

α-Rhamnosyl-β-glucosidase-Catalyzed Reactions for Analysis and Biotransformations of Plant-Based Foods

Most aroma compounds exist in vegetal tissues as disaccharide conjugates, rutinose being an abundant sugar moiety in grapes. The availability of aroma precursors would facilitate analytical analysis of plant-based foods. The diglycosidase α-rhamnosyl-β-glucosidase from Acremonium sp. DSM 24697 efficiently transglycosylated the rutinose moiety from hesperidin to 2-phenylethanol, geraniol, and nerol in an aqueous-organic biphasic system. 2-Phenethyl rutinoside was synthesized up to millimolar level with an 80% conversion regarding the donor hesperidin. The hydrolysis of the synthesized aroma precursors was not detected in an aqueous medium. However, in the presence of ethanol as a sugar acceptor, the enzyme was able to transfer the disaccharide residue forming the alkyl-rutinoside. The aroma precursors were significantly hydrolyzed (up to 3-4% in 2 h at 30 °C), which indicated the potential use of the enzyme for biotechnological applications, for example, in aroma modulation of fermented foods.

Identification of common glycosyl groups of flavonoid O‐glycosides by serial mass spectrometry of sodiated species

Flavonoid O-glycosides are a ubiquitous and important group of plant natural products in which a wide variety of sugars are O-linked to an aglycone. Determining the identity of the sugars, and the manner in which they are linked, by mass spectrometry alone is challenging. To improve the identification of common O-linked di- and trisaccharides when analysing mixtures of flavonoid O-glycosides by liquid chromatography/mass spectrometry (LC/MS), the fragmentation of electrosprayed sodium adducts in an ion trap mass spectrometer was investigated. The sodium adducts [M + Na](+) of kaempferol 3-O-glycosides generated sodiated glycosyl groups by the neutral loss of kaempferol. The product ion spectra of these sodiated glycosyl groups differed between four isomeric kaempferol 3-O-rhamnosylhexosides and four isomeric kaempferol 3-O-glucosylhexosides in which the primary hexose was either glucose or galactose and bore the terminal glucose or rhamnose at either C-2 or C-6. Fragmentation of sodiated glycosyl groups from linear O-triglucosides and branched O-glucosyl-(1 → 2)-[rhamnosyl-(1 → 6)]-hexosides produced sodiated disaccharide residues, and the product ion spectra of these ions assisted the identification of the complete sugar. The product ion spectra of the sodiated glycosyl groups were consistent among flavonoid O-glycosides differing in the position at which the sugar was O-linked to the aglycone, and the nature of the aglycone. The abundance of sodiated species was enhanced by application of a pre-trap collision voltage, without the need to dope with salt, allowing automated LC/MS methods to be used to identify the glycosyl groups of common flavonoid O-glycosides, such as rutinosides, robinobiosides, neohesperidosides, gentiobiosides and sophorosides.

RB4CD12 epitope expression and heparan sulfate disaccharide composition in brain vasculature

RB4CD12 is a phage display antibody that recognizes a heparan sulfate (HS) glycosaminoglycan epitope. The epitope structure is proposed to contain a trisulfated disaccharide, [-IdoA(2-OSO(3))-GlcNSO(3) (6-OSO(3))-], which supports HS binding to various macromolecules such as growth factors and cytokines in central nervous tissues. Chemically modified heparins that lack the trisulfated disaccharides failed to inhibit the RB4CD12 recognition of HS chains. To determine the localization of the RB4CD12 anti-HS epitope in the brain, we performed an immunohistochemical analysis for cryocut sections of mouse brain. The RB4CD12 staining signals were colocalized with laminin and were detected abundantly in the vascular basement membrane. Bacterial heparinases eliminated the RB4CD12 staining signals. The RB4CD12 epitope localization was confirmed by immunoelectron microscopy. Western blotting analysis revealed that the size of a major RB4CD12-positive molecule is ∼460 kDa in a vessel-enriched fraction of the mouse brain. Disaccharide analysis with reversed-phase ion-pair HPLC showed that [-IdoA(2-OSO(3))-GlcNSO(3) (6-OSO(3))-] trisulfated disaccharide residues are present in HS purified from the vessel-enriched brain fraction. These results indicated that the RB4CD12 anti-HS epitope exists in large quantities in the brain vascular basement membrane.

Anti-proliferative effects of O-acyl-low-molecular-weight heparin derivatives on bovine pulmonary artery smooth muscle cells.

Heparin (HP) inhibits the growth of several cell types in vitro including bovine pulmonary artery (BPA) smooth muscle cells (SMCs). In initial studies we discovered that an O-hexanoylated low-molecular-weight (LMW) HP derivative having acyl groups with 6-carbon chain length was more potent inhibitor of BPA-SMCs than the starting HP. We prepared several O-acylated LMWHP derivatives having 4-, 6-, 8-, 10-, 12-, and 18- carbon acyl chain lengths to determine the optimal acyl chain length for maximum anti-proliferative properties of BPA-SMCs. The starting LMWHP was prepared from unfractionated HP by sodium periodate treatment followed by sodium borohydride reduction. The tri-n-butylammonium salt of this LMWHP was O-acylated with butanoic, hexanoic, octanoic, decanoic, dodecanoic, and stearyl anhydrides separately to give respective O-acylated LMWHP derivatives. Gradient polyacrylamide gel electrophoresis (PAGE) was used to examine the average molecular weights of those O-acylated LMWHP derivatives. NMR analysis indicated the presence of one O-acyl group per disaccharide residue. Measurement of the inhibition of BPA-SMCS as a function of O-acyl chain length shows two optima, at a carbon chain length of 6 (O-hexanoylated LMWHP) and at a carbon chain length 12-18 (O-dodecanoyl and O-stearyl LMWHPs). A solution competition SPR study was performed to test the ability of different O-acylated LMWHP derivatives to inhibit fibroblast growth factor (FGF) 1 and FGF2 binding to surface-immobilized heparin. All the LMWHP derivatives bound to FGF1 and FGF2 but each exhibited slightly different binding affinity.