Crystal structure of a lysozyme-tetrasaccharide lactone complex

The binding of a proposed transition-state analogue, the δ-lactone derived from tetra-N-acetylchitotetraose, to lysozyme in the crystal at pH 2.6 has been studied by X-ray diffraction techniques to a resolution of 2.5 Å. The tetrasaccharide lactone is bound in sites A, B, C, D with sugar residues located in sites A, B and C in similar positions to those observed previously in the complex with tri-N-acetylchitotriose. Analysis of the electron density map for site D, by direct model-building and with a computer model-building programme, indicates that the δ-lactone ring is in a conformation close to a sofa or a boat which brings the hydroxymethyl group C₍₆₎O₍₆₎ axial. These studies provide support for the role of strain in the proposed mechanism of lysozyme catalysis. The orientation of the lactone group in site D is slightly different from that originally derived by hypothetical model-building.     

Selective formation of glycosidic linkages of N-unsubstituted 4-hydroxyquinolin-2-(1H)-ones

A comparative study for selective glucosylation of N-unsubstituted 4-hydroxyquinolin-2(1H)-ones into 4-(tetra-O-acetyl-β-d-glucopyranosyloxy)quinolin-2(1H)-ones is reported. Four glycosyl donors including tetra-O-acetyl-α-d-glucopyranosyl bromide, β-d-glucose pentaacetate, glucose tetraacetate and tetra-O-acetyl-α-d-glucopyranosyl trichloroacetimidate were tested, along with different promoters and reaction conditions. The best results were obtained with tetra-O-acetyl-α-d-glucopyranosyl bromide with Cs₂CO₃ in CH₃CN. In some cases the 4-O-glucosylation of the quinolinone ring was accompanied by 2-O-glucosylation yielding the corresponding 2,4-bis(tetra-O-acetyl-β-d-glucopyranosyloxy)quinoline. Next, 4-(tetra-O-acetyl-β-d-glucopyranosyloxy)quinolin-2(1H)-ones were deacetylated into 4-(β-d-glucopyranosyloxy)quinolin-2(1H)-ones with Et₃N in MeOH. In some instances the deacetylation was accompanied by the sugar-aglycone bond cleavage. Structure elucidation, complete assignment of proton and carbon resonances as well as assignment of anomeric configuration for all the products under investigation were performed by 1D and 2D NMR spectroscopy.     

Purification of heparinase and heparitinase by affinity chromatography on glycosaminoglycan-bound ah-sepha-rose 4b

Heparinase and heparitinase were separated from an extract of Flavobacterium heparinum, induced with heparin by using column chromatography on hydroxylapatite. As the heparinase preparation contained chondroitinases B and C, chondroitinase B was removed by rechromatography on a hydroxylapatite column. Chondroitinase C was then eliminated by column chromatography on O-phosphono(“phospho”)-cellulose. The heparinase preparation thus obtained was free from sulfoamidase for 2-deoxy-2-sulfoamino-D-glucose (GlcN-2S), sulfatase for 2-amino-2-deoxy-6-O-sulfo D-glucose (GlcN-6S), as well as (δ_4,5glycosiduronase for the unsaturated disaccharides obtained from heparin. The remaining sulfatase for 4-deoxy-α-L-thero-hex-4-enopyranosyluronic acid 2-sulfate (δUA-2S) in the heparinase preparation was removed by affinity chromatography with dermatan sulfate-bound AH-Sepharose 4B coated with dermatan sulfate. The heparitinase preparation separated by column chromatography on hydroxyla patite was purified by affinity chromatography with heparin-bound AH-Sepharose 4B coated with heparin. Sulfatase for 2-amino-2-deoxy-6-O-sulfo-D-glucose (GlcN-6S) and δ_4,5glycosiduronase for the unsaturated disaccharides obtained from heparin were removed by this chromatography. Sulfatase for 4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid 2-sulfate (δUA-2S) remaining in the heparitinase preparation was finally removed by column chromatography on hydroxylapatite. The recoveries of the purified preparations of heparinase and heparitinase were estimated to be 39 and 50%, respectively, from the crude enzyme fractions obtained by the first column chromatography on hydroxyl- patite. The purified heparinase and heparitinase were free from all enzymes that could degrade the sulfated unsaturated disaccharides produced from heparin with heparinase.     

Some chemical,enzymic, and physical properties of di-saccharides from beef-lung heparin

A study is reported of the reactivities of the disaccharides isolated after deamination of beef-lung heparin and reduction of the products by sodium borotritide: 2,5-anhydro-O-(α-l-idopyranosyluronic acid sulfate)-d-mannitol sulfate, SIMS; 2,5-anhydro-O-(α-l-idopyranosyluronic acid)-d-mannitol sulfate, IMS; 2,5-anhydro-O-(α-l-idopyranosyluronic acid sulfate)-d-mannitol, SIM; and 2,5-anhydro-O-(β-d-glucopyranosyluronic acid)-d-mannitol sulfate, GMS. Results for the non-sulfated disaccharides IM and GM, prepared by desulfation of SIMS and GMS, are also reported. SIMS and SIM were inert to purified α-l-iduronidase, showed unexpected resistance to periodate oxidation, and lost sulfate rapidly in 50mm hydrochloric acid at 100°. Hydrolysis of IM and of IMS was catalyzed by α-l-iduronidase, and of GM and GMS by β-d-glucuronidase; the radioactive products were identified as 2,5-anhydro-d-mannitol (aM) and its sulfate (aMS). The products SIM and IMS obtained by deamination of heparin and desulfation of SIMS (the major deamination product) are apparently identical. In heparin partially desulfated by methanolic hydrogen chloride, residual sulfate groups were mostly linked to l-iduronic acid residues. Chemical, chromatographic, and electrophoretic methods that are valuable for separation and characterization of the disaccharides are described.     

Heparin trisaccharides with nonreducing 2-amino-2-deoxy-α-d-glucopyranosyl end-groups suitable as substrates for catabolic enzymes

Heparin trisaccharides having the sequence O-(2-amino-2-deoxy-α-d-glucopyranosyl)-(1→4)-O-α-l-idopyranosyluronic acid-(1→4)-2,5-anhydro-d-[1-³H]mannitol have been prepared, as substrate models for studying sulfatases of heparan sulfate catabolism, by α-l-iduronidase cleavage of previously reported heparin tetrasaccharides, with additional chemical and enzymic modification as required. Three series are described, including isomeric sulfate esters of that trisaccharide with no N-substituent, with N-acetyl substitution, and with N-sulfate substitution. New features of the substrate specificity of the hydrolases used, including iduronate sulfatase, α-l-iduronidase, glucosamine 6-sulfate sulfatase, and heparin sulfamidase, were observed, and simple procedures for partial purification of these hydrolases are reported. The structures assigned to the trisaccharides are supported by the mode of preparation, reactions, regularities in electrophoretic behavior, and identities of the products of deamination.     

Two crystalline pentofuranosyl bromides: tri-O-(p-nitrobenzoyl)-β-D-ribofuranosyl bromide and tri-O-(p-nitrobenzoyl)-α,β-D-xylofuranosyl bromide

Methyl α,β-D-ribofuranoside was p-nitrobenzoylated to give methyl tri-O-(p-nitrobenzoyl)-β-D-ribofuranoside (2),and this was treated with HBr in acetic acid to give tri- O-(p-nitrobenzoyl)-β-D-ribofuranosyl bromide (3). Bromide 3 could be converted into 2,5-anhydro-3,4,6-tri-O-(p-nitrobenzoyl)-D-allononitrile (4) with Hg(CN)₂, or hydrolyzed to 2,3,5-tri-O-(p-nitrobenzoyl)-D-ribose (5). On p-nitro- benzoylation, 5 gave tetra-O-(p-nitrobenzoyl)-β-D-ribofuranose (6). The synthesis of tri-O-(p-nitrobenzoyl)-α-β-D-xylofuranosyl bromide (11) started with methyl 3,5-O-isopropylidene-β-D-xyldfuranoside (7), which was p-nitrobenzoylated to give ester 8; this was then hydrolyzed, and the product p-nitrobenzoylated to give methyl tri-O-(p-nitrobenzoyl)-β-D-xylofuranoside (10) which, on treatment with HBr in CH₂Cl₂, afforded the desired bromide (11). Nucleophilic replacement with Hg(CN)₂ afforded 2,5-anhydro-3,4,6-tri-O-(p-nitrobenzoyl)-D-gulononitrile (12).     

Preparation and properties of biologically active fluorescent heparins

Hog mucosal heparin (N-sulfate, 0.84 mol; O-sulfate, 1.55 mol; N-acetyl, 0.12 mol; anticoagulant activity assayed by the method of U.S. Pharmacopeia, 161 USP units/mg) or its partially N-desulfated heparin (N-sulfate, 0.71 mol; O-sulfate, 1.47 mol; N-acetyl 0.12 mol; anticoagulant activity, 117 USP units/ mg) was reacted with 5-isothiocyanatofluorescein in 0.5M carbonate buffer (pH 8.5) at 35°C for 6 h to yield the corresponding N-fluoresceinylthiocarbamoyl heparins (λ_em 516 nm, λ_ex 491 nm; degree of substitution 0.006 and 0.013, respectively, anticoagulant activity, 174 and 140 USP units/mg, respectively).The fluorescent heparin (degree of substitution, 0.006; 174 USP units/mg) was injected into rabbits intravenously. The half-life of the fluorescent heparin determined by fluorometry was 24 min, that determined by the clotting time assay was 39 min. The time-course of concentration and the half-life of the fluorescent heparin and of the starting heparin obtained by the clotting the assay were virtually identical.     

Preparation of some acylated D-arabino-hexopyranosuloses and 4-deoxy-D-glycero-hex-3-enopyranosuloses

Oxidation of 1,3,4,6-tetra-O-benzoyl-α- and β-D-glucopyranose gave the tetra-O-benzoyl-α- and -β-D-arabino-hexopyranosuloses (3α and β), from which benzoic acid was readily eliminated to give the anomeric tri-O-benzoyl-4-deoxy-D-glycero-hex-3-enopyranosuloses (4α and β). The anomeric 1-O-acetyl-tri-O-benzoyl-D-arabino-hexopyranosuloses (7α and β) were obtained as very unstable syrups which readily lost benzoic acid. Treatment of tetra-O-benzoyl-2-O-benzyl-D-glucopyranose (1) with hydrogen bromide gave 3,4,6-tri-O-benzoyl-α-D-glucopyranosyl bromide (5) in one step.     

Oligosaccharides from “standardized intermediates”. Synthesis of a branched tetrasaccharide glycoside isomeric with the blood-group B,type 2 determinant

Building-block derivatives of the component monosaccharides were used to construct the tetrasaccharide glycoside 15, in which an α- d-Galp-(1→4)- d-Gal linkage replaces the α-(1→3) linkage of the human blood-group B, type 2, determinant structure. The initial coupling of 2-O-benzoyl-3,6-di-O-benzyl-4-O-(tetrahydropyran-2-yl)-α- d-galactopyranosyl chloride to allyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-β- d-glucopyranoside was followed by selective deprotection of the disaccharide product, either at O-4′ (to give 8) or O-2′ (to give 3). The conversion of 8 into 15 involved successive coupling with tetra-O-benzyl-α- d-galactopyranosyl bromide ( 8 → 11), O-debenzoylation at O-2′ ( 11 → 12), coupling with tri-O-benzyl-α- l-fucopyranosyl bromide ( 12 → 14), and O-debenzylation by hydrogenolysis ( 14 → 15). Alternatively, 3 was α- l-fucosylated to give 6, and 6 was selectively deprotected at O-4′ to give 7. However, attempts to α- d-galactosylate 7 were unsuccessful. The unsubstituted forms of the intermediate disaccharide ( 8) and trisaccharide ( 12) glycosides were obtained by appropriate deblocking procedures.     

Inhibition by heparin and dextran sulfate of stimulated rat pancreatic adenylate cyclase

Heparin inhibited the adenylate cyclase activity of semipurified rat pancreatic plasma membranes stimulated by hormones and by Gpp(NH)p but not by fluoride or when in the persistently active state. When observed, the inhibition was rapid and sustained. It was of a noncompetitive type and never exceeded 20% for secretin. The inhibition of Gpp(NH)p-stimulated activity was more pronounced (48% inhibition at a heparin concentration of 50 μg/ml). For the C-terminal octapeptide of pancreozymin (CCK-8)-stimulated adenylate cyclase, the inhibition amounted to 93% at 50 μg/ml. This inhibition was competitive at low heparin concentration and of a mixed type above 10 μg/ml. Besides, heparin inhibited (I₅₀ = 6 μg/ml) the binding of peptides of the CCK family to their specific receptors without affecting the apparent K_d value of binding. Taken together, these relatively specific effects of heparin gave evidence in favor of the existence of CCK spare receptors. Dextran sulfate was more potent than heparin as an inhibitor of adenylate cyclase activation while chondroitin-4-sulfate and chondroitin-6-sulfate were ineffective. Dansylated pancreatic plasma membranes exhibited characteristics of adenylate cyclase activation by CCK-8 which were similar to those found for untreated membranes exposed to heparin.     

Infrared absorption and raman scattering of sulfate groups of heparin and related glycosaminoglycans in aqueous solution

The i.r. spectra for aqueous solutions of sulfated glycosaminoglycans and model compounds in the transmittance “window” region of the solvent (1400-950 cm^?1) are dominated by the strong and complex absorption centered at ～1230 cm^?1 and associated with the antisymmetric stretching vibrations of the SO groups. Primary and secondary O-sulfate groups absorb at somewhat higher frequencies (1260-1200 cm^?1) than N-sulfates (～1185 cm^?1). Each sulfate band lends itself to quantitative applications, especially within a given class of sulfated polysaccharide. Laser-Raman spectra of heparin and model compounds have been obtained in aqueous solution and in the solid state. The most-prominent Raman peak (at ～1060 cm^?1) is attributable to the symmetrical vibration of the SO groups, with N-sulfates emitting at somewhat lower frequencies (～1040 cm^?1) than O-sulfates. The Raman pattern in the 950-800 cm^?1 region (currently used in the i.r. for distinguishing between types of sulfate groups) also involves vibrations that are not localized only in the COS bonds.     

Action of porcine-pancreatic amylase on oxidized-reduced amylose of low degree of modification

Amylose was oxidized with 0.1–0.2 mol of periodate per glucose residue (G), and then reduced with sodium borohydride or borotritide to give an oxidized-reduced amylose of low degree of modification. Mild acid hydrolysis gave erythritol, 2-O-α-d-glucosyl-l-erythritol, higher homologs, and other products. Extensive action of porcine-pancreatic amylase on the polymer gave, besides d-glucose and maltose, oligosaccharides containing one or more oxidized-reduced (modified, M), acyclic residues. The enzymic products containing only one oxidized-reduced residue were identified as a modified tetrasaccharide (MG₃) and a modified pentasaccharide (MG₄). Structures of MG₃ and MG₄ were identified by a combination of enzymic and chemical approaches. With glucoamylase, MG₄ was converted into MG plus d-glucose, whereas MG₃ was totally resistant. On mild acidic hydrolysis, MG₃ was converted into 2-O-α-d-glucosyl-d-erythritol plus maltose. These results indicate that MG₃ is G-M-G-G and that MG₄ is G-G-M-G-G. In principle, MG₄ could occupy the five d-glucose residue, substrate-binding site of porcine-pancreatic amylase in such a way that M, the acyclic structure replacing a d-glucose residue, is placed just to the “left” of the catalytic site. The modified structure, being very vulnerable to acidic hydrolysis, might then be expected to be very readily attacked by the amylase, but in fact, it is not.     

ESR evidence of the formation of a new superoxide complex of tetra-p-tolyporphyrinatocobalt(II) in aprotic solvents

The reactions of the superoxide ion (O₂⁻) with tetra-p-tolyporphyrinatocobalt(II) [Co(II)TTP] in dimethyl sulfoxide(DMSO) have been investigated by use of electron spin resonance (ESR) spectroscopy. In the absence of oxygen, Co(II)TTP in DMSO gives the DMSO adduct, Co(II)(TPP)(DMSO). When this DMSO adduct is exposed to air, an oxygen complex, Co(II)(TTP)(DMSO)(O₂), is formed in which the binding state between Co(II) and O₂ has been considered formally as Co(III)O₂⁻. When the superoxide ion (O₂⁻ is added to this oxygen complex, a new superoxide complex, Co(II)(TTP)(O₂⁻)₂, is formed. The same superoxide adduct is formed by the reaction of O₂⁻ with Co(II)TTP in the absence of oxygen.     

Aurothionein formation from Zn,Cd-thionein and Et3PAuCl,but not Et3PAuSATg (auranofin)

Et₃PAuCl reacts in vitro with horse-kidney zinc, cadmium-metallothionein (Zn, CdTh) to displace zinc, but not cadmium. The ratio of gold-bound to zinc displaced is larger than that observed for gold sodium thiomalate (AuSTm), suggesting a different mode of binding. Et₃PAuSATg (Auranofin: 2,3,4,6- tetra-O-acetyl-1-thio-β-D-glucopyranosato(triethyl- phosphine)gold(I)), a new antiarthritic compound, does not react with Zn, CdTh. The significance of the nonreaction of Et₃PAuSATg, in contrast to Et₃PAuCl and AuSTm (previously studied), for the in vivo pharmacology of gold is discussed.     

Nitrous acid deamination of conformationally inverted aminodeoxyhexopyranoses

Nitrous acid deamination of 2-amino-1,6-anhydro-2-deoxy-β-D-glucopyranose (1) in the presence of weakly acidic, cation-exchange resin gave 1,6:2,3-dianhydro-β-D-mannopyranose (3) and 2,6-anhydro-D-mannose (6), characterized, respectively, as the 4-acetate of 3 and the per-O-acetylated reduction product of 6, namely 2,3,4,6- tetra-O-acetyl-1,5-anhydro-D-mannitol, obtained in the ratio of 7:13. Comparative deaminatior of the 4-O-benzyl derivative of 1 led to similar qualitative results. Deamination of 3-amino-1,6-anhydro-3-deoxy-β-D-glucopyranose gave 1,6:2,3- and 1,6:3,4-dianhydro-β-D-allopyranose (13 and 16), characterized as the corresponding acetates, obtained in the ratio of 31:69, as well as the corresponding p-toluenesulfonates. Deamination of 4-amino-1,6-anhydro-4-deoxy-β-D-glucopyranose and of its 2-O-benzyl derivative gave the corresponding 1,6:3,4-D-galacto dianhydrides as the only detectable products. 2,5-Anhydro-D-glucose, characterized as the 1,3,4,6-tetra-O- acetyl derivative of the corresponding anhydropolyol, was obtained in 39% yield from the same deamination reaction performed on 2-amino-1,6-anhydro-2-deoxy-β-D- mannopyranose (24). In 90% acetic acid, the nitrous acid deamination of 24, followed by per-O-acetylation, gave only 1,3-4-tri-O-acetyl-2,5-anhydro-α-D-glucoseptanose. In the case of 1,6-anhydro-3,4-dideoxy-3,4-epimino-β-D-altropyranose, only the corresponding glycosene was formed, namely, 1,6-anhydro-3,4-dideoxy-β-D-threo--hex-3-enopyranose.     

Mice Deficient in N-Acetylgalactosamine 4-Sulfate 6-O-Sulfotransferase Are Unable to Synthesize Chondroitin/Dermatan Sulfate containing N-Acetylgalactosamine 4,6-Bissulfate Residues and Exhibit Decreased Protease Activity in Bone Marrow-derived Mast Cells

Chondroitin sulfate (CS) and dermatan sulfate (DS) containing N-acetylgalactosamine 4,6-bissulfate (GalNAc(4,6-SO₄)) show various physiological activities through interacting with numerous functional proteins. N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from 3′-phosphoadenosine 5′-phosphosulfate to position 6 of N-acetylgalactosamine 4-sulfate in CS or DS to yield GalNAc(4,6-SO₄) residues. We here report generation of transgenic mice that lack GalNAc4S-6ST. GalNAc4S-6ST-null mice were born normally and fertile. In GalNAc4S-6ST-null mice, GalNAc(4,6-SO₄) residues in CS and DS disappeared completely, indicating that GalNAc4S-6ST should be a sole enzyme responsible for the synthesis of GalNAc(4,6-SO₄) residues in both CS and DS. IdoA-GalNAc(4,6-SO₄) units that account for ∼40% of total disaccharide units of DS in the liver of the wild-type mice disappeared in the liver DS of GalNAc4S-6ST-null mice without reduction of IdoA content. Bone marrow-derived mast cells (BMMCs) derived from GalNAc4S-6ST-null mice contained CS without GlcA-GalNAc(4,6-SO₄) units. Tryptase and carboxypeptidase A activities of BMMCs derived from GalNAc4S-6ST-null mice were lower than those activities of BMMCs derived from wild-type mice, although mRNA expression of these mast cell proteases was not altered. Disaccharide compositions of heparan sulfate/heparin contained in the mast cells derived from BMMCs in the presence of stem cell factor were much different from those of heparan sulfate/heparin in BMMCs but did not differ significantly between wild-type mice and GalNAc4S-6ST-null mice. These observations suggest that CS containing GalNAc(4,6-SO₄) residues in BMMCs may contribute to retain the active proteases in the granules of BMMCs but not for the maturation of BMMCs into connective tissue-type mast cells.     

Reaction of acylated methyl arabinosides with hydrogen bromide

Treatment of methyl tri-O-acetyl-β-D-arabinopyranoside (1a) with hydrogen bromide in benzene or in acetic acid gave, in addition to the pyranosyl bromide (2a), a considerable proportion of tri-O-acetyl-D-arabinofuranosyl bromide (5). Similar treatment of methyl tri-O-benzoyl-β-D-arabinopyranoside (1b) gave a good yield of the pyranosyl bromide (2b); no furanoid derivative was formed. Ring contraction also took place when methyl 4-O-acetyl-2,3-di-O-benzoyl-β-D-arabinopyranoside (7) was treated with hydrogen bromide, whereas the isomeric 3-O-acetyl-2,4-di-O-benzoyl compound (12) gave the pyranosyl bromide 13 in high yield. Thus, methyl pyranosides with an O-acetyl group at C-4 undergo ring contraction on treatment with hydrogen bromide. The corresponding compounds with O-benzoyl groups at C-4 gave pyranosyl bromides only.     

Debenzylation of carbohydrate benzyl ethers and benzyl glycosides via free-radical bromination

The dealkylation of benzylated carbohydrates by free-radical bromination and hydrolysis has been further examined. Free-radical bromination of methyl 2,3,4,6-tetra-O-benzyl-α-D-glucopyranoside (1) methyl 2,3-di-O-benzyl-α-D-glucopyranoside (2) 6-O-benzyl-3,5-O-benzylidene-1,2-O-isopropylidene-α-D-glucofuranose (4) and 6-O-benzyl-D-glucose (3) appears to be quantitative. Spectroscopic evidence of a CBr bond indicates that an α-bromobenzyl ether is the product. Alkaline hydrolysis yielded methyl α-D-glucopyranoside from 1 and 2 and D-glucose from 3 and 4. A benzyl group present as an aglycon could be removed in the same way. Reaction of benzyl α-D-glucopyranoside tetraacetate (6) with bromine and chlorine under free-radical conditions and examination of the products by t.l.c. and i.r. spectrophotometry indicated that the first product was an α-halobenzyl glycoside and that the aglycon could be displaced by Br^- or Cl^- to form the tetra-O-acetyl-D-glucopyranosyl halide, undoubtedly with anomerization. Treatment of the mixture of products with moist ether and silver carbonate yielded only 2,3,4,6-tetra-O-acetyl-D-glucopyranose.     

Quantitation of the sulfated disaccharides of heparin by high performance liquid chromatography

Heparin was converted by treatment with nitrous acid primarily into sulfated disaccharides. The mixture of disaccharides was reduced with sodium boro[³H]hydride and the disaccharides were purified by preparative paper electrophoresis and paper chromatography. Four disaccharides were obtained. On the basis of their paper electrophoretic mobilities and the products formed at intermediate stages of their acid hydrolysis, the disaccharides were identified as 4-O-(2-O-sulfo-α-l-idopyranosyluronic acid)-6-O-sulfo-2,5-anhydro-d-mannitol, 4-O-(2-O-sulfo-α-l-idopyranosyluronic acid)-2,5-anhydro-d-mannitol, 4-O-(α-l-idopyranosyluronic acid)-6-O-sulfo-2,5-anhydro-d-mannitol, and 4-O-(β-d-glucopyranosyluronic acid)-6-O-sulfo-2,5-anhydro-d-mannitol. The purified disaccharides were used as standards in the development of a high-performance liquid chromatography procedure for their separation and quantitation on a Partisil-10 SAX anion-exchange column. The three monosulfated disaccharides were resolved by isocratic elution with 40 mm KH₂PO₄. The KH₂PO₄ concentration was tehn increased to 400 mm to elute the disulfated disaccharide. Column effluents were collected in $\text{1}\text{2}\text{-}\text{ml}$ fractions, and the recovery of each ³H-labeled product was determined by scintillation counting. When sodium boro-[³H]hydride with a specific activity of 315 mCi/mmol was used in the reduction of the heparin deamination products, the disaccharides gave 28,500 cpm/nmol in the effluent peaks. Quantitative recoveries of the ³H-disaccharides were obtained. It was demonstrated that the method developed using the purified disaccharides gave reproducible and quantitative results in direct assays of aliquots of boro[³H]hydride-reduced heparin deamination mixtures.     

Preparation,properties, crystal and molecular structure of uranyl(VI) complexes with a new cyclic schiff base compartmental ligand

Some uranyl(VI) complexes with new acyclic and cyclic Schiff base compartmental ligands have been prepared and characterized. The ligands have been obtained by reaction of 4-chloro-2,6-diformylphenol and polyamines of the type NH₂(CH₂)₂X (CH₂)₂NH₂ (X= NH, S). The structure of the uranyl(VI) complex with the ligand 1,7,15,21-tetra- aza-4,18-dithia-11,25-dichloro 8,22-bis-metadiphenyl cyclophane-gb-7,14,21,28 has been determined by X-ray crystallography. The compound crystallizes in the orthorhombic space group Pbca with eight formula units in a cell of dimensions a = 26.654(3), b = 22.871(3), c = 8.875(5) Å. The structure was solved by standard methods and refined by full- matrix least squares to the conventional R index of 4.6% for 2678 independent observed reflexions. Five donor atoms (including sulphur) of the ligand are equatorially bonded to the uranyl group to form discrete monomeric molecules with the seven-coordinated metal in the usual distorted pentagonal bipyramidal coordination geometry. Selected bond distances are: UO (equatorial), 2.22(1) and 2.25(1) Å; UN, 2.60(1) and 2.59(1) Å; US, 3.018(4) Å.