Synthesis and molecular recognition studies of the HNK-1 trisaccharide and related oligosaccharides. The specificity of monoclonal anti-HNK-1 antibodies as assessed by surface plasmon resonance and STD NMR

The human natural killer cell carbohydrate, HNK-1, plays function-conducive roles in peripheral nerve regeneration and synaptic plasticity. It is also the target of autoantibodies in polyneuropathies. It is thus important to synthesize structurally related HNK-1 carbohydrates for optimizing its function-conducive roles, and for diagnosis and neutralization of autoantibodies in the fatal Guillain-Barré syndrome. As a first step toward these goals, we have synthesized several HNK-1 carbohydrate derivatives to assess the specificity of monoclonal HNK-1 antibodies from rodents: 2-aminoethyl glycosides of selectively O-sulfated trisaccharide corresponding to the HNK-1 antigen, its nonsulfated analogue, and modified structures containing 3-O-fucosyl or 6-O-sulfo substituents in the N-acetylglucosamine residues. These were converted, together with several related oligosaccharides, into biotin-tagged probes to analyze the precise carbohydrate specificity of two anti-HNK-1 antibodies by surface plasmon resonance that revealed a crucial role of the glucuronic acid in antibody binding. The contribution of the different oligosaccharide moieties in the interaction was shown by saturation transfer difference (STD) NMR of the complex consisting of the HNK-1 pentasaccharide and the HNK-1 412 antibody.     

Synthetic sulfated glucuronosyl paragloboside (SGPG) and its use for the detection of autoimmune peripheral neuropathies

The sulfated glucuronosyl paragloboside SO₄-3-GlcA(β1-3)Gal-(β1-4)GlcNAc(β1-3)Gal-(β1-4)Glcβ(1-1′)Cer (SGPG) is a specific constituent of the peripheral nervous system involved in human dysimmune neuropathies. Although it is highly immunogenic in human pathology, it is not a major constituent of peripheral nerve in humans. Pure SGPG is very difficult to obtain from natural sources, but is needed for the design of efficient and reproducible immunoassays. We describe a new chemical synthesis of this important glycolipid with a stearic tail, including its complete characterization by mass spectrometry and its use in the detection of autoimmune neuropathies.     

Natural Sphingomonas glycolipids vary greatly in their ability to activate natural killer T cells

Mouse natural killer T (NKT) cells expressing an invariant T cell antigen receptor (TCR) recognize glycosphingolipids (GSLs) from Sphingomonas bacteria. The synthetic antigens previously tested, however, were designed to closely resemble the potent synthetic agonist alpha-galactosyl ceramide (alphaGalCer), which contains a monosaccharide and a C18:0 sphingosine lipid. Some Sphingomonas bacteria, however, also have oligosaccharide-containing GSLs, and they normally synthesize several GSLs with different sphingosine chains including one with a cyclopropyl ring-containing C21:0 (C21cycl) sphingosine. Here we studied the stimulation of NKT cells with synthetic GSL antigens containing natural tetrasaccharide sugars, or the C21cycl sphingosine. Our results indicate that there is a great degree of variability in the antigenic potency of different natural Sphingomonas glycolipids, with the C21cycl sphingosine having intermediate potency and the oligosaccharide-containing antigens exhibiting limited or no stimulatory capacity.     

Linear aglycones are the substrates for glycosyltransferase DesVII in methymycin biosynthesis: analysis and implications

The two essential structural components of macrolide antibiotics are the polyketide aglycone and the appended sugars. The aglycone formation is catalyzed by polyketide synthase (PKS), and glycosylation is catalyzed by an appropriate glycosyltransferase. Although it has been shown that glycosylation occurs after the cyclic aglycone is released from PKS, it is not known whether the acyl carrier protein (ACP)-bound linear polyketide chain can also be processed by the corresponding glycosyltransferase. To explore this possibility, the aglycone, 10-deoxymethynolide, which is the precursor of methymycin and neomethymycin, was chemically synthesized in the linear form as a N-acetylcysteamine (NAC) thioester. Subsequent incubation with TDP-d-desosamine in the presence of the dedicated glycosyltransferase, DesVII, and activator, DesVIII, produces a more polar product whose high-resolution mass is consistent with the anticipated glycosylated product. This study demonstrated for the first time that a macrolide glycosyltransferase can also recognize and process the linear precursor of its macrolactone substrate with a reduced but measurable activity.     

Triterpene glycosides from the roots of Codonopsis lanceolata

In the course of the development of new designer foods using the roots of Codonopsis lanceolata, we found that hot-water extracts of C. lanceolata recovered decreased testosterone levels in the blood and accelerated the restoration of reproductive dysfunction induced by hyperthermic treatment in male mice. Thus we studied the constituents of the polar fraction of the roots of C. lanceolata and identified six new triterpene saponins, lancemasides B (2), C (3), D (4), E (5), F (6), and G (7), along with the known saponin lancemasaide A (1) and phenylpropanoid glycosides 8-10. The structures of the new compounds 2-7 were determined by means of spectral data including 2D-NMR studies and chemical reactions to be oleanan-type bisdesmoside with sugars at C-3 and C-28. Compounds 2-6 have echinocystic acid as an aglycone, and compound 7 has asterogenic acid as an aglycone. Identification of the sugars and determination of their D,L-chiralities were carried out by application of the exciton chirality method to the per-O-p-bromobenzoylmethyl sugar derived from saponins.     

Structural characterization of the GM1 ganglioside by infrared multiphoton dissociation, electron capture dissociation, and electron detachment dissociation electrospray ionization FT-ICR MS/MS

Gangliosides play important biological roles and structural characterization of both the carbohydrate and the lipid moieties is important. The FT-ICR MS/MS techniques of electron capture dissociation (ECD), electron detachment dissociation (EDD), and infrared multiphoton dissociation (IRMPD) provide extensive fragmentation of the protonated and deprotonated GM1 ganglioside. ECD provides extensive structural information, including identification of both halves of the ceramide and cleavage of the acetyl moiety of the N-acetylated sugars. IRMPD provides similar glycan fragmentation but no cleavage of the acetyl moiety. Cleavage between the fatty acid and the long-chain base of the ceramide moiety is seen in negative-ion IRMPD but not in positive-ion IRMPD of GM1. Furthermore, this extent of fragmentation requires a range of laser powers, whereas all information is available from a single ECD experiment. However, stepwise fragmentation by IRMPD may be used to map the relative labilities for a series of cleavages. EDD provides the alternative of electron-induced fragmentation for negative ions with extensive fragmentation, but suffers from low efficiency as well as complication of data analysis by frequent loss of hydrogen atoms. We also show that analysis of MS/MS data for glycolipids is greatly simplified by classification of product ion masses to specific regions of the ganglioside based solely on mass defect graphical analysis.     

C-glycoside derivatives. XXVI. New ways of approach to thiophene and isoxazole C-nucleosides]

3-O-benzyl (and 3-O-methyl)-1,2-O-isopropylidene-α-D-xylo-dialdo-1,4-furanoses treated with bromocyanomethylidene- or bromoacylmethylidene-triphenylphosphoranes gave in good yields the corresponding olefinic sugars. These compounds, which bear three adjacent electrophilic carbon atoms (C(5), C(6), C(7)) constitute useful synthetic intermediates in carbohydrate chemistry. They represent, for example, good starting materials for the preparation of 3-glycosylisoxazoles, 5-glycosylisoxazoles and 2-glycosylthiophenes.     

Synthesis of selected LeY and KH-1 analogues: a medicinal chemistry approach to vaccine optimization

[structure: see text] As part of our ongoing anticancer vaccine program, we recently found that antibodies generated in response to the KH-1-KLH construct recognized not only KH-1 antigen but also the Lewis Y (Le(y)) antigen as well, with antibody titer levels much higher than those observed after immunization with individual Le(y)-KLH vaccine constructs. In an attempt to explore the structure-antigenic relationship of these carbohydrate epitopes, several analogues of both KH-1 and Le(y) were synthesized. A convergent synthetic approach to the analogues was designed on the basis of well-established glycal methodology, employing a minimum number of building blocks to generate competent antigens with high stereoselectivity and reasonable yield.     

Analysis of Density‐Dependent Binding of Glycans by Lectins Using Carbohydrate Microarrays

To investigate the density‐dependent binding of glycans by lectins using carbohydrate microarrays, a number of C‐terminal hydrazide‐conjugated neoglycopeptides with various valences and different spatial arrangements of the sugar ligands were prepared on a solid support. The synthetic strategy includes (1) assembly of alkyne‐linked peptides possessing C‐terminal hydrazide on a solid support, (2) coupling of azide‐linked, unprotected sugars to the alkyne‐linked peptides on the solid support utilizing click chemistry, and (3) release of the neoglycopeptides from the solid support. By using this synthetic methodology, sixty five neoglycopeptides with a valency ranging from 1 to 4 and different spatial arrangements of the carbohydrate ligands were generated. Carbohydrate microarrays were constructed by immobilizing the prepared neoglycopeptides on epoxide‐derivatized glass slides and were used to analyze the density‐dependent binding of glycans by lectins. The results of binding property determinations show that lectin binding is highly dependent on the surface glycan density.     

Design and synthesis of a bimodal target-specific contrast agent for angiogenesis

[structure: see text] A bimodal target-specific contrast agent based on a cyclic peptide containing the target-specific NGR sequence, gadolinium(III) diethylenetriaminepentaacetic acid (Gd(III)DTPA), and Oregon Green 488 (OG488) suitable for both MR imaging and optical imaging of angiogenesis is developed. The synthetic strategy for this target-specific contrast agent exploits the use of highly efficient, chemoselective reactions, such as native chemical ligation, and gives a straightforward approach for double labeling of peptides in general.     

On-line cut-off technique and organic modifier addition aided signal enhancement for trace analysis of carbohydrates in cellulase hydrolysate by ion exclusion chromatography-electrospray ionization mass spectrometry

Paper cellulose has been hydrolyzed with calcium alginate immobilized cellulase to produce carbohydrate products and the three trace sugars, galactose, arabinose, and mannose in the cellulase hydrolysate have been analyzed by HPIEC/ESI-MS. Applying the on-line cut-off technique to the HPIEC/ESI-MS can cut the high concentration glucose off to eliminate its interference on the peaks of minor sugars and enhance their signals from 1.1- to 1.6-fold. However, the on-line post column addition of 15% ethanol to the eluate can increase the signal of the three trace sugars, galactose, arabinose, and mannose up to 17-, 23-, and 11-fold, respectively, and make the corresponding detection limits as 0.04, 0.04, and 0.03 ppm. The accuracies of the quantitative analysis for the three trace sugars with the signal enhanced HPIEC/ESI-MS by the two enhancement methods were larger than 95%. The precisions of the analytical results were also greatly improved by the assistance of the two techniques and were less than 6.5%. The quantitative analysis of the three trace sugars was performed with the internal standard method and the internal standard (IS) was sorbitol. Overall, the signal enhancement of HPIEC/ESI-MS and quantification of the three trace sugars by the on-line cut-off technique and organic modifier addition was successful.     

Development of Spectrophotometric Method for the Analysis of Multi-component Carbohydrate Mixture of Different Moieties

Present study is a critical analysis and subsequent development of an analytical tool to measure the total sugar concentration in a carbohydrate mixture comprising both hexose and pentose. For this purpose, individual sugars were measured and standardized with anthrone reagent prepared in an ice-cold 98 % sulphuric acid followed by 3 min of boiling. Furthermore, regression analysis was performed after mathematical manipulation with the individual standards to formulate a linear relation between the absorbance of the mixture and its concentration, which satisfies Beer’s law. It was found that the correlation coefficient for the equation is 0.973, when confidence interval was set at 0.95. The validation was done with a synthetic mixture of concentrations at 0.17 and 0.22 g/L (as range was ensured between 0.1 and 0.3 g/L) and also with the carbohydrate mixture as the prehydrolyzate obtained after the pretreatment of banana stem, which showed around 94.1 % accuracy and higher sensitivity with the cellulose present in the mixture. Thus, the method is evident to quantify the total sugars accurately obtained from hydrolyzed lignocellulosic biomass.     

Total Synthesis of Tiacumicin B: Study of the Challenging β-Selective Glycosylations**

We give a full account of the total synthesis of tiacumicin B (Tcn-B), a natural glycosylated macrolide with remarkable antibiotic properties. Our strategy is based on our experience with the synthesis of the tiacumicin B aglycone and on unique 1,2-cis-glycosylation steps. We used sulfoxide anomeric leaving-groups in combination with a remote 3-O-picoloyl group on the donors that allowed highly β-selective rhamnosylation and noviosylation that rely on H-bond-mediated aglycone delivery. The rhamnosylated C1–C3 fragment was anchored to the C4–C19 aglycone fragment by a Suzuki–Miyaura cross-coupling. Ring-size-selective Shiina macrolactonization provided a semiglycosylated aglycone that was engaged directly in the noviolysation step with a virtually total β-selectivity. Finally, a novel deprotection method was devised for the removal of a 2-naphthylmethyl ether on a phenol, and efficient removal of all the protecting groups provided synthetic tiacumicin B.     

LC Enantioseparation of β-Lactam and β-Amino Acid Stereoisomers and a Comparison of Macrocyclic Glycopeptide- and β-Cyclodextrin-Based Columns

Direct reversed-phase high-performance liquid chromatographic methods were developed for the separation of the enantiomers of tricyclic β-lactams, cis-3,4-benzo-6-azabicyclo[3.2.0]heptan-7-one, cis-4,5-benzo-7-azabicyclo[4.2.0]-octan-8-one, cis-5,6-benzo-8-azabicyclo[5.2.0]nonan-9-one and new bicyclic β-amino acids, the six- and seven-membered homologues of cis-1-amino-4,5-benzocyclopentane-2-carboxylic acid (benzocispentacin), cis-1-amino-5,6-benzocyclohexane-2-carboxylic acid and cis-1-amino-6,7-benzocycloheptane-2-carboxylic acid. The direct separations of the analytes were performed on chiral stationary phase (CSP) columns containing the macrocyclic glycopeptide antibiotic teicoplanin (Chirobiotic T), teicoplanin aglycone (Chirobiotic TAG), vancomycin (Chirobiotic V), vancomycin aglycone (Chirobiotic VAG), ristocetin A (Chirobiotic R) or a new dimethylphenyl carbamate-derivatized β-cyclodextrin-based Cyclobond DMP. The results achieved with the different methods were compared in systematic chromatographic examinations. The effects of an organic modifier and of the mobile phase composition on the separation and the separation efficiency of different columns were investigated. The difference in enantioselective free energy between the aglycone CSP and the teicoplanin CSP for these β-lactams and β-amino acids ranged between 0.3 and −1.1 kJmol⁻¹. Better enantioseparations were attained in most cases on the aglycone CSP.

     

Solid-Phase Enzymatic Synthesis of a Lewis a Trisaccharide Using an Acceptor Reversibly Bound to Sepharose

Abstract

The disaccharide 2-aminoethyl O-β-D-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside was reacted with thiobutyrolactone to give a disaccharide with a thiol group on the aglycone. This disaccharide was reacted with activated Thiopropyl Sepharose, which gave a disaccharide bound to Sepharose via a disulphide bond. Enzymatic fucosylation, using GDP-fucose and partially purified human milk fucosyltransferase, gave a trisaccharide in good yield, which was cleaved from Sepharose by treatment with mercaptoethanol or dithiothreitol.     

Influence of cation adduction on the separation characteristics of flavonoid diglycoside isomers using dual gate-ion mobility-quadrupole ion trap mass spectrometry

An electrospray ionization-dual gate-ion mobility-quadrupole ion trap mass spectrometer was used to evaluate the separation characteristics of isomeric flavonoid diglycosides adducted with sodium, potassium, and silver. This instrumental configuration allows ions to be selectively accumulated within the ion trap on the basis of their gas phase conformation prior to mass analysis. For the metal cations examined, silver produced the most compact adducts with flavonoid diglycosides. Listed in order of increasing size, the trend of flavonoid diglycoside ion-neutral cross sections adducted with Na+, K+, and Ag+ was narirutin < naringin < hesperidin < neohesperidin < rutin. To examine the separation contribution of the carbohydrate group, hesperetin, the aglycone of hesperidin, and neohesperin were compared to quercetin, the aglycone of rutin. Separation of the flavonoid diglycosides indicated that quercetin-derived diglycosides drifted longer than their hesperetin-derived isomers. Combined with the observed collision assisted dissociation (CAD) data, these findings suggest that carbohydrate moiety plays a significant role in both the separation and metal chelating characteristics of flavonoid diglycosides.     

Catalytic and biochemical features of a monoclonal antibody heavy chain, JN1-2, raised against a synthetic peptide with a hemagglutinin molecule of influenza virus

It has long been an important issue to produce a catalytic antibody that possesses the ability to lose the infectivity of a bacteria or virus. The monoclonal antibody JN1-2 was generated using a synthetic peptide (TGLRNGITNKVNSVIEKAA) conjugated with human IgG. The peptide sequence includes the conserved region of the hemagglutinin molecule (HA(1) and HA(2) domains), which locates on the envelope of the influenza virus and plays an important role in influenza A virus infection. The monoclonal antibody specifically reacted with the HA2 domain, not only of H2 but also of an H1 strain of the H1N1 subtype (H1 strain). The heavy chain (JN1-2-H) isolated from the parent antibody showed catalytic activity cleaving the above antigenic peptide with very high turnover (kcat = 26 min(-1)), and it could slowly degrade the recombinant HA(2) domain by the catalytic function. Interestingly, the heavy chain exhibited the ability to reduce the infectivity of type A H1N1 but not type B, indicating specificity to type A. This characteristic monoclonal catalytic antibody heavy chain could suppress the infection of the influenza virus in vitro assays.     

Pregnane glycosides, gymnepregosides G-Q from the roots of Gymnema alternifolium.

The structural elucidation of eleven new related polyoxypregnane glycosides, gymnepregosides G (1), H (2), I (3), J (4), K (5), L (6), M (7), N (8), O (9), P (10) and Q (11), from the roots of Gymnema alternifolium (Asclepiadaceae) was achieved by a detailed study of 1H- and 13C-NMR spectral data and chemical means. The results obtained for new compounds, 1-11, show that they are (20S)-pregn-6-ene-3 beta,5 alpha,8 beta,12 beta,14 beta,17 beta,20-heptaol 3-O-glycosides, and all the sugars at C-3 are beta(i-->4)-linked. Some of them possess benzoyl, (E)- and (Z)-cin-namoyl, and tigloyl residues as the ester linkages located at C-12 and/or C-20 of the aglycone.     

Field-desorption mass spectra of flavonoid acylglycosides. II. Natural cinnamoyl and benzoyl derivatives

The field-desorption mass spectra of 14 flavonoid glycosides esterified with p-coumaric, ferulic, caffeic, p-hydroxybenzoic, and gallic acids have been studied. For all the compounds the molecular ions and the ions of the aglycone (A) were obtained as the main peaks. The spectra also contained a number of other fragmentary ions, including the ion of the acylated anhydrosugar. For flavonol glycosides in which the carbohydrate is esterified by cinnamic acids, the (A + Ac), and for the diacylglycosides the (A + 2 Ac), ions are diagnostic. The formation of these strong ions corresponding in mass to the molecules of the mono- and diacylated aglycone permits the assumption of the existence of acyl migration in the breakdown of the initial molecular ion.All-Union Scientific-Research Institute of Medicinal Plants, Moscow. L. Ya. Karpov Scientific-Research Institute of Physical Chemistry. Irkutsk Institute of Organic Chemistry, Siberian Branch, Academy of Sciences of the USSR. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 582–589, September–October, 1984.     

Triterpene glycosides from the sea cucumber Eupentacta fraudatrix. Structure and biological action of cucumariosides A1, A3, A4, A5, A6, A12 and A15, seven new minor non-sulfated tetraosides and unprecedented 25-keto, 27-norholostane aglycone

Seven new minor triterpene glycosides, cucumariosides A1 (1), A3 (2), A4 (3), A5 (4), A6 (5), A12 (6) and A15 (7) have been isolated from the Far Eastern sea cucumber Eupentacta fraudatrix. Structures of the glycosides were elucidated by 2D NMR spectroscopy and MS. Glycosides 1-7 belong to the group of cucumariosides A, having linear tetrasaccharide carbohydrate moieties without any sulfate group and possessing 3-O-methyl-D-xylose as a terminal monosaccharide unit. The latter peculiarity is rare in sea cucumber glycosides, but typical for the glycosides from E. fraudatrix. Glycosides 1-7 differ from each other by side chain structures in the aglycone moieties; three of them have unique structural features. The first is the presence of a 25-butoxy-group in the side chain of cucumarioside A3 (2), the second is a 23E,25-diene system in cucumarioside A6 (5) and the third is a 25-keto-27-nor-holostane aglycone in cucumarioside A12 (6); these were never previously found in sea cucumber glycosides. Cytotoxic activity of glycosides 1-7 against mouse spleen lymphocytes and the cells of the ascite form of mouse Ehrlich carcinoma, along with hemolytic activity against mouse erythrocytes and antifungal activity were studied. Glycosides 1 and 5 were the most active in all the tests.