Thermodynamic properties of tetraalkylammonium chondroitin-4-sulfates (R₄NChS), which have the hydrophilic sugar rings and the hydrophobic counterions, have been studied to elucidate the effects of the hydrophobic counterions on the properties of the polyelectrolyte solution. The transfer molar enthalpies, Δ_trH, from water to aqueous urea solution of ammonium and tetraalkylammonium chondroitin-4-sulfate were measured by means of a twin-type conduction microcalorimeter at 298.15 and 310.1 5 K and the urea concentration of 0.5-5.0 molkci^-1The Δ_trH values were shown to be negative for the NH4ChS salt and to decre ase with the increase of the carbon number of the counterion. After, reaching the minimum with the Et4NChS salt, the Δ_trH value increased with increase of the counterion's carbon number, a nd became positive for the Bu4NChS salt (Fig.2 and 4). The observed values of the R₄NChS salts were compared with those of the tetraalkylammonium bromides. The solution properties of R4NChS salts were found to depend considerably on the property of the R₄1N⁺ ion. The H value of ChS polymer ion itself was estimated to be ca.9kJmol^-1. The Δ^trH valuetr was interpreted in terms of the change of the water structure around ChS ions.

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Almond glycopeptidase which cleaved specifically the β-aspartylglucosylamine linkages in glycopeptides proved to be useful for the simple and rapid analysis of Asn-linked oligosaccharides derived from glycoproteins. The oligosaccharide fraction in each glycopeptidase digest was desalted by paper chromatography with the solvent composed of 1-butanol/ethanol/water (4: 41) for 2 days, and analyzed by thin-layer chromatography on silica gel 60. The solvent system was 1-propanol/acetic acid/water (3: 3: 2). Only one kind of the high-mannose-type oligosaccharide was present in a Taka-amylase A molecule (Fig.3). The human fibrinogen molecule was found to have the identical four biantennary oligosaccharides of complex-type (Fig.4). Fig.6 shows that several kinds of high-mannose-type oligosaccharides and hybrid-type oligosaccharides were attached to the same position in a peptide sequence of hen ovalbumin. We extended the application of the enzyme to histochemical examination, i. e., the distribution of Asn-linked oligosaccharides in umbilical artery, vein and matrix (Fig.7).

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Recently, Stuart et al. have reported that on heating with deuterated Raney nickel in D2O, hexopyranosides and oligosaccharides were deuterated selectively at carbon atoms bound to a free hydroxyl group.. For the application of this procedure to chemistry and biochemistry of more complex carbohydrates, the further study on this reaction was conducted.
On prolonged treatment with Raney nickel (24 h), methyl a-L-arabinopyranoside [4] and -D-xylopyranoside (5) were partially epimerized to give (5) and methyl α-L-lyxopyranopside ( 6 )from (4) and methyl β-D-ribopyranoside (7) and (4) from ( 5), respectively. Methyl -L-rharanopyranoside (8) was epimerized more slowly to yield methyl 6-deoxy-α-L- glaucopyranoside (9). It was revealed that the best conditions for the deuteration under minimizing the epimerization was refluxing with W-7 type deuterated Raney nickel in D2O-dioxane (1: 1)or methanol-d4-D2O (1: 1) for about.9 h.
Methyl β-sophoroside (13)m, ethyl β-cellobioside(2 3 ) and β-sophorosaynl d β-gentiobiosyl units of ginsenoside-Rb1( 2 4), a Ginseng-saponin, were deuterated as expected under these conditions, while C-2 and 4 of the inner β-glucopyranosyml oiety of methyl β-laminaribioside 189 and C -4 of the inner β-glucopyranosyml oiety of methyl 2, 3-di-O-β-D-glucopyranosyl-β-D-glucopyranoside (22) remained almost undeuterated. Deuteration of α-1, 6-glucan (25)proceededr ather slowly than those of mono- and oligosaccharideto obtain the expected deuteration after. treatment for 24 h. In the case of the deuteration of cis- and dl-trans-1, 2cyclohexanediols (11) and (12), both deuteration and epimerization proceeded very fast to give an equilibrated mixture of (11-d2) and (12-d2) within 9 h, while the isolated carbinyl carbon (C-12) of (24)was not deuterated.
Further, by means of the abo v e selective deuteration, ¹³C-NMRs ignal-assignments of the above oligo- and polysaccharides were substantiated or revised as shown in Tables.

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Highly sensitive fluorometric detection and determination of carbohydrates in thin-layer chromatography (TLC) and iiigh-performance liquid chromatography (HPLC) have been developed using, t aurine-borate reagent- which gives intense fluorescence on reaction with carb ohydrates by heating in neutral or slightly alkaline media.,
The flnorescence development on thin-layer plate was performed by spraying an 8% taurine solution in 0.05 m ol. d m^-3 Na₂B₄O₇-0.1m oldm^-3 KH₂PO₄ buffer, pH 7.5, on a thin -layer plate which was then heated at 130°C for 10 m in and by measuring the fluorescence intensity with a scaning, fluorophotometer. Detection limits for reducing sugars were in th e range between 0.1 nmol and 0.2 nmol. Standard curves for glucose and galactose were linear i n the range from 0.05 μg/spott o 1.0μg/spot and for lactose from 0.1 μg/spotto 1.0 μg/spot.
HPLC was carried out by eluting a sample solution through an anion Hexchange Aminex A-27 column or a reversed phase Lichrosorb-NH₂ column, heating the eluate after mixing with an 8% taurine solution or an 8% taurine solution containing 0.05 m ol. dm^-3 borate adjusted to pH 8.7 with NaOH pellets and measuring the fluorescence intensity with a fluor omonitor. The present method facilitated excellent separation of mono- and oligosaccha ride mixtures within a remarkably short period. Since this reaction proceeds without the use of str ong acid or alkali, the chromatographic methods do not require acid or alkali resistant equipm ents.

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¹H- and ¹³C-NMR studies have been carried out on the anomeric pairs of glycopyranosides and 1-thioglycopyranosides (Fig.1) for which the ring oxygen helicity rule was previously proposed based on the sign and rotational strength of the ring oxygen band.
The ¹H chemical shift and spin coupling data were analyzed by mean s of the conventional rules relating these data with structural parameters.¹H chemical shifts showed a linear relation with the rotational strength of the ring oxygen band (in Fig.4--6), suggesting that both methods can be applicable to the determination of the anomeric configuration and conformation.
In ¹³C-NMR sum of the chemical shifts (∑¹³Ca)g reed with the conventional rule (in Table 4, β-anomer gave the larger ∑¹³C than α-one). However, phenyl 1-thioglycopyranosides formed the exception in the δ_β>δ_α, r, ule of C-1 chemical shifts (Table 2). The δ_β-δ_α. values decreased in the order: (in Table 3), suggesting that this tendency was due to the positive anisotropy effect (deshield effect) of the equatorial aglycons on the C-1. The 4C1 ring conformations were suggested in all the compounds except for the phenyl 1-thio-β-mannosides (⁴C₁=Boat).

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Structural characterization of formoses has been carried out by chemical ionization mass spectrometry (CIMS) which is reputed to be useful for molecular weight determination afid structural characterization of relatively involatile compounds. While no protonated molecule (MH⁺) was found in the CI mass spectra of (1a), (1b)and (1c) with isobutan e as a reagent gas, the ammonia-mediated CI mass spectra showed definitely the formation of the ammonium adduct ion (M. NH⁴⁺) from which reliable information on the molecular weight was obtained. Ammonia-d₃ was found to be effective for the hydrogen-deuterium (H-D)exchange reaction. The shifting technique using this reagent gas confirmed the ion structures and the number of active hydrogens in formoses. Accordingly, CIMS using a series of three reagent gases, isobutane, ammonia and ammonia-d₃, promises to be useful for the structural characterization of unknown formose samples.

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A novel method for the preparation of haloacyl derivatives of chitosan under mild conditions is described. To a solution of chitosan (0.16 g) in a halocarboxylic acid (-7 ml) was added a halocarboxylic anhydride (3, -5 ml). The mixture was allowed to stand at 75-80°C for a few minutes to two hours or at room temperature for 18 h. N, O-Acyl derivatives (d. s.1.2-3.0/G1cN) were produced and isolated in 72-97% yield Their O-deacylation with alk ali afforded N-trifluoroacetyl, N-chloroacetyl and N-formyl derivatives (d. s, =1.0/G1cN) in 7586% yield. The N-trifluoroacetyl gorup was unstable to alkali. All the products were insoluble in water and in common organic solvents.

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The formose reaction, which was found to give 2, 4-bis(hydroxymethyl)-3-pentulose (2, 4BH-3-P) with high selectivity, was carried out in methanol and/or water in the presence of various alkaline earth salts and potaslium hydroxide at 60°C, monitoring the reaction wi th continuous measurements of redox potential and pH of the reaction mixture.
The effects of these factors on the formose reaction and the com ponent of products were discussed in detail. No influence of anions of barium salts on the product's distribution was observed, but the concentration of the dissolved barium was strongly affected by the kind of anion and the formose reaction rate increased with an increase in its concentration. In the. formose reaction, catalyzed with BaBr2-KOH, below 15 vol% of the amount of water, the formose reaction was not influenced by the amount of water and the main product was 2, 4 (cez.4 5 g lc%), and above 25 v ol% the reaction rate decreased with an increase in th Be H-3-P amount of water and the yield of 2-(hydroxymethyl)glycerol, 3- (hydroxymethyl)pentitol, and 2, 4-bis(hydroityinethy1)pentitol increased up to 54 glc%.
The maintenance of pH of the reaction mixture to a constant value during the reaction or the ratio of [Catalyzer]/[HCHO] is an important factor for the formose reaction to procee d smoothly in water with the alkaline earth salts-KOH catalyst. In methanol, strontium salt was found to give 3-(hydroxymethyl)pentofuranose and 2, 4-BH-3-P with high selectivity. Formdse Reactions. XVII.

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A new monomer, 1, 6-anhydro-2, 4-di-O-benzy1-3-O-dodecyl-β-D-glucopyranose (2), was prepared from 1, 6-anhydro-2, 4-di-O-benzyl-β-D-glucopyranose (1) and 1-bromododecane, and was polymerized in the presence of a PF⁵ initiator at 60°C. The polymerization reactivity of (2 ) was high and a high molecular weight polymer with intrinsic viscosity (η) of 1.29was obtained in a quantitative yield in a short time. The 1H- and ¹³C-NMR spectra indicated that the polymer was stereoregular 2, 4-di-O-benzy1-3-O-dodecyl-(1→6)-α-D-glucopyrana (3), which would serve as a precursor for synthesizing a polymer model of glycolipids. Regioselectively Modified Stereoregular Polysaccharides. V.

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Cationic ring-opening polymerizations of 1, 6-anhydro sugars which were derived from levglucosan(1, 6-anhydro-*β-D-glucopyranose) and levomannosan (1, 6-anhydro-*β-D-mannopyranose)by protecting the hydroxyl groups with various substituents were investigated. The structures and physical properties of the polymers obtained were also examined. Tri-O-benzyllevoglucosan was polymerized with such catalysts as PF₅, NbF₅, TaF₅, and 2-iodopropane. AgPF₆ complex to give stereoregular dextran derivatives. Although tri-O- (methyl and ethyl)levoglucosans showed high polymerizabilities, tri-O-(2-butenyl)levoglucosan did not polymerize under these conditions. A levoglucosan derivative in which the C-2 carbon had a tosylamino group instead of a hydroxyl group gave oligomers even by the action of strong Lewis acid. Although triO-(methyl and ethyl)levomannosans showed high polymerizabilities, a 2, 3-O-benzylidenelevominnosan derivative showed very low reactivity. As a result, a large influence of substituent of C-2 carbon of the 1, 6-anhydro sugars on the polymerizability was deduced. Polymers obtained from tri-O-methyllevoglucosan and -levomannosan and tri-O-ethyllevoglucosan were f ound to have high crystallinity by X-ray diffractiometry. The methylated polysaccharides were insoluble in all the solvents examined.¹H-NMR spectra (400 MHz) of 2, 3, 4-tri-O-benzyl-(1+6) α-D-glucopyranan and -mannopyranan revealed highly stereoregular stru ctures with α-configuration,

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Polycondensation of D-glucose has been carried out in dimethyl sulfoxide (DMSO) and N, N-dimethylformamide (DMF) with polyphosphoric acid (PPA) and polyphosphoric ester (PP E)as condensation agents. A colorless synthetic glucan was obtained in high yield by the reaction of 20 g of anhydrous ID-glucose with 30 g of the condensation agent (PPA or PPE) in 50ml of solvent (DMSO or DMF) at 50°C for 7 h. DMSO proved to be better than DMF for obtaining the colorless synthetic glucan in high yield. For example, the characteristics of the synthetic glucan which was obtained in the reaction mixture of DMSO-PPE were as follows: the degree- of polymerization; 28, [α]_D²²+26 8.0° (c=1, H₂O), combined phosphorus; 0.70%and yield.; 63%. The structure of the synthetic glucan was studied by the periodate oxi dation gnd-the Smith degradation. The amount of the formic acid formed was relatively hi gh when compared with the amount of the consumed periodate and the amount of the glycerol fo rmed was also high in the Smith degradation. The results suggest that the synthetic glucan has many nonreducing end groups and, therefore, many branched chains. The polycondensation reactions with the use of PPA and PPE in DMSO were found to be fair different from each other. Studies on the Synth esis of Polysaccharide. II.

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Octa-O-acetylcellobiose, -lactose, and -laminaribiose were converted into the corresponding hepta-O-benzyl derivatives with a free hydroxyl at the C-1 position.6-O-β-Cellob iosyl-, -β-lactosyl-, and -α-laminaribiosyl-D-glucopyranoses were synthesized through the biosylation of benzyl- 2, 3; 4-tri-O-benzyl-α-n-glucopyranoside with these biose 1-OH derivatives and th e mixture of p-nitrobenzenesulfonyl chloride, silver trifluoromethanesulfonate, and triethylamine in dichloromethane, followed by catalytic O-debenzylation. In this paper, the glycosylation with a hep ta-O-benzylglucobiose derivative bearing a free hydroxyl at C-1 position is abbreviated to biosylation.

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Two routes have been developed for the synthesis of pentasaccharide, methyl O-(β-D-glucoranosl)-(1→4)-O-β-D-glucopyranosyl-(1→3)-O-β-D-glucopyranosyl-(1→4)-Op-β-D-glucopyranosyl, (1→4)-β-O-D-glucopyranoside which constitutes the repeating unit of the lichen (Getreiria islandica) polysaccharide lichenan having antitumor activity.

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A homologous series of α, α-trehalose 6, 6'-diesters involving five new compounds, acetate, octanoate, decanoate, laurate, and myristate, in addition to the known palmitate, stearate, and behenate were synthesized according to the pathway reported in our previous paper. These esters, except the clecanoate, were tested for their in vitro antitumor activity against two leukemia cell lines, L-5178 Y and L-1210, by the cell culture method and found to be. somewhat effective in inhibiting the growth of these tumor cells. In either case, the lowest ID₅₀ value was observed with the laurate; the increasing or decreasing acyl chain-length gave higher ID50. values. When in vivo antitumor activity of the esters, with one exception of the behenate, was evaluated against mouse Ehrlich ascites carcinoma by the total packed cell volame method, it was found that the compounds having the acyl moieties longer than deca, _ noyl were, all highly effective, while the two shorter analogs, acetate and octanoate, were completely inactive. Chemical and Biochemical Studies on Carbohydrate Esters. XIV.

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N-(4-L-Asparty1)-β-gentiobiosyl-, -isomaltosyl-, and -manninotriosylamines were synthesized in order, . t o provideu sefulmodelc ompoundfso r studieso n nephritogenosidieso latedb y Shibata et al from the glomeruler, basement membrane of rats.
The desired compounds were synthesized from the corresponding fully acetylated glycosyl bromides via the following steps: aide formation, reduction 'to glycosylamine, condensation with: protected L-aspartic acid, followed by removal of the protecting groups.

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Introduction of the isothiocyanate group into anomeric position of sugar moiety is effective for the syntheses of nucleoside (thymine, thiothymine, 5-azacytidine, and 5-azathiocytidine)and glycoproteins.
In this paper, we report here on convenient methods for the syntheses of modified nucleoside analogs utilizing disaccharide isothiocyanates [7 a-c]. The compounds of [7a-c] were reacted with acyl hydrazine to give thiocarboxamides (8), which afforded 1, 2, 4-triazole disaccharide [9 ] by treatment of Ac₂O-H₃PO₄ through a cyclodehydration reaction. Reactions of [7a-c] with 6-amino-1, 3-dimethyluracil gave disaccharide aminoisothiazolo [3, 4-d] pyrimi -[dines [11] in good yields. Treatments of [7 a-c] with T-amino-2'-deoxy-β-D-glucopyranose yielded diglycosylthioureas [12]. Reactions of [7 a-c] with chloroethylamine hydrochloride under basic conditions afforded disaccharide imidazoline-2-thiones [13] instead of N-glycosy12-c N'-chloroeth ylthioureas.

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Two procedures for the synthesis of the title compound, i. e., the coupling reaction of 5, 6dimethylbenzimidazolew ith 2, 3, 5 -tri-O-benzyl-β-D-ribofuranosyblr omide, which was prepared from methyl 2, 3, 5 -tri-O-benzyl-α-o r -β-D-riboluranosideb y treatment with excess acetyl bromide in the presence of a catalytic amount of methanol, and the reaction of 5, 6dimethylbenzimidazolew ith N, N '-diisopropy1-O-(23, - O-isopropylidene-5-O-trityl-D-ribofuranosyl)isourea, which was prepared from diisopropylcarbodiimidaen d the correspongng 1 -OH sugar derivative with the catalytic amount of copper( I) chloride, are described in addition to some basic aspects of the above chemistry. t Partial Protection of Carbohydrate Derivatives. X.

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Two new pseudodisaccharides, istamycins X₀ and Y_o have been isolated from culture broth of Streptomyces tenjimariensis producing istamycins A and B, which are potent aminoglycoside antibiotics. The isolation, characterization and structural elucidation of these compounds are reported. They have a weak antimicrobial activity. The structures of istamycins X_o and Y_o have been determined by their methanolysis and spectral analyses to be 1_D-4-0-(2, 6diamino-2, 3, 4, 6-tetradeoxy-α-D-erythro-hexopyranosyl)-1-0-methyl-(1, 2, 4/3, 5)-5-amino-2methylamino-1, 3, 4-cyclohexanetriol and 1L-4-0-(2, 6-diamino-2, 3, 4, 6-tetradeoxy-α-D-erythrohexopyranosyl)1-0-methyl (1, 3, 5/2, 4)-5-amino-2-methylamino-1, 3, 4-cyclohexanetriol, respectively.

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Recently, new amino glycoside antibiotics having 1, 4-diaminocyclitol have been found successively. We reported that Streptomyces sannanensis KC-7038 produces sannamycins A, B and C which belonged to this group. Upon further investigation, we have isolated seven new components of sannamycin named sannamycins E [4], F[5], G [6], H [7], J[8], K [9]and L [10], and elucidated the structures of these components using chemical degradation and various spectrometric methods.

The ¹H-NMR spectrum of [5] i ndicated the presence of a N-formylglycyl group, and the alkaline hydrolysis of the component gave sannamycin C. These results suggested that the structure of [5] is 2'-N-(N-formylglycyl)sannamycin C. The structure of [8] was decid ed to be 6'-N-demethyl compound of sannamycin C [ 3 ]by comparing the ¹H-NMR spectrum of [8] with that of [3]. Tetra-N-acetyl compound of [6] was methanolyzed with 5.5 m ol [dm^-3 methanolic hydrogen chloride to give methyl di-N-acetyl α- and β-purpurosaminides C 12] and [13] and a bis(acetylamino)cyclitol [14]. From the spectrometric data of [6] and [the N-deacetyl compound of [14], the structure of [ 6 ] was determined to be 6'-N-demethy1 4-epi-sannamycin B. The structure of [7]was determind to be 6'-N-demethyl-4-epi-sannamycin C in the same manner as described for [6]. The structure of 4 was determined to be 3-O-demethyl compound of [7] by O-demethylation of [7] with concentrated hydriodic acid. The MS and the ¹H-NMR data of [9] indicated the presence of 4', 5'-unsaturated purpurosamine C, and the structure of [9] was decided to be 4', 5'-didehydro compound of [4] The structure of [10] was decided to be 4-N-demethyl compound of [4] by comparing the 1HNMR spectrum of [10] with that of [4].

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3'-Deoxykanamycin B (tobramycin) [19] was synthesiz ed from kanamycin B by a sequence of reactions involving 6'-N-benzyloxycarbonylation, N-tosylation, 4", 6"-O-cyclohexylidena tion, 4'-O-carbonylation, 3'-O-sulfonylation, and 3'-halogenation. Selective sulfonyla tion of [5 ] gave T-O-sulfonyl derivatives ([6] [9], [12]) as major products and 2", 3'di-O-sulfonyl derivatives ([7], [10], [13]) as minor products (Table 1). Therefore, the reactivity order of hydroxyl groups of [ 5 ] was 3'→2"→5-. Halogenation of the 3'-O-sul fonyl derivatives was easily performed with sodium iodide or lithium chloride in N, N-dimethyl formamide in spite of a bulky axial group at 1'. The 2", 3'-di-O-benzylsulfonyl derivativ[7] gave 3_eq'-chloro-2''-O-benzylsulfonyl derivative [17] via 3_ax'-chloro-2''-O-benzylsulfony derivative [16]by double S_N, 2 mechanism. Treatment of the 3'-halogeno derivatives wits sodium in liquid ammonia gave the 3'-deoxy derivative, the N-tosyl groups being simultaneous ly removed. Subsequent removal of the other protecting groups gave [19] in =30% of overall yield from kanamycin B, via [1 ]→[ 2]→[3]→[5]→([6], [7])→([15], [17]).

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Neomycin B [1], an aminoglycoside antibiotic, was chemically converted to 6'-deamino-6'hydroxyneomycin B [12] and 6'''-deamino-6'''-hydroxyneomycin B [13] via 5 steps (j)selective blocking of the 6'- or 6"-u-amino group with a benzyloxycarbonyl group (Cbz), ( ii)blocking of the remaining amino groups by ethoxycarbonylation (Cbe), (iii) selective deblocking of the N-Cbz group to generate the 6'- or 6'''-amino group, (iv) conversion of the 6'- or 6'''-amino function to a hydroxyl group via diazotization with sodium nitrite, and (v) removal of the Cbe groups. The sites of deamination in [12] and [13] were determined largely on the basis of ¹³C-NMR spectral data. Compound [12] was identified as paromomycin I [2] by direct comparison of both compounds. Antibacterial spectra of [1], [2], and [13] were similar to each other, whereas in terms of antibacterial activity, [1]was most active followed by [2] and [13]. The compound [13] was least toxic, being about one-fifth as toxic as [1 ].

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Validamycin A [1] is a main component of antibiotic validamycin complex, which is produced by Streptomyces hygroscopicus var. limoneus and is used to control sheath blight in rice plants. The structure of [ 1 ] is assigned as the β-D-glucopyranoside of validoxylamine A 2], which is composed of two branched-chain cyclitols bonded by way of an imino linkage. [In order to elucidate the structure-activity relationship of this kind of antibiotics, a racemic isomer of [2] was synthesized. Reaction of the DL-epoxycyclohexene [ 8], a precursor of unsaturated branched-chain cyclitol portion, with a blocked DL-validamine [16] yielded the blocked 6'-epimer [3 a, b] of [2 ]. Separation of racemic diastereomers of [3] was achieved by converting it into the corresponding octaacetates [20 a, b], followed by fractionation by column chromatography on silica gel. Synthetic Studies on the Validamycins. VI.

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Indirect electrochemical oxidation of δ-D-gluconolactone to D-arabinose in aqueous sulfuric acid has been carried out by the use of cerium sulfate as a mediator and platinum plates ao an anode and a cathode. At low temperatures and low current densities, D-arabinose was obtained in a 65% yield.

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Selective 2''-O-benzoylation of 4'-O-tetrahydropyranyl-4'', 6''-O-c yclohexylidene-tetrakis(N-ethotycarbonyl)kanamycin A with the aid of phase transfer catalysis and synthesis of 3'-deoxy2'-epikanamycin A vici-O-tetrallydropyranyl-2''-O-benzoyl-4'', 6''-O-cyclohexylidene-tetrakis (N-pthoxycarbonyl)kanamycin A are described.

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