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|Publication number
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|Publication date
|Jan 30, 1979
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|Jun 21, 1976
|Priority date
|Jul 8, 1975
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|Raymond U. Lemieux, David R. Bundle, Donald A. Baker
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|Chembiomed Limited
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|Patent Citations (5), Non-Patent Citations (1), Referenced by (81), Classifications (19), Legal Events (1)
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It is well known that carbohydrate structures of various complexities are the antigenic determinants for a wide range of substances. It has also been shown that it is possible to take relatively small molecules (haptens), which by themselves are devoid of antigenicity, and by linking them to a suitable high molecular weight carrier obtain an antigen which when administered to an animal under appropriate conditions will give rise to the production of antibodies having a specificity for the hapten. Thus, Goebel and Avery1,2,3 showed that antibody specific for carbohydrate structures could be generated in experimental animals by immunizing them with artificial antigens prepared by the coupling of p-aminophenyl or p-aminobenzyl glycosides to protein. Canadian Pat. No. 966778 discloses that opium alkaloids covalently bonded to a protein molecule through a carboxy lower alkyl linking group are antigenic and these when administered to animals will give rise to the production of antibodies specific for the alkaloid. In both of the above examples the haptens must be linked to a carrier before they will show any antigenicity.
Heretofore there were two serious obstacles hindering the production of artificial carbohydrate antigens. The first was the difficulty in obtaining the antigenic determinant as very often these materials are only available in small amounts from natural sources. The Lewis-a trisaccharide antigenic determinant 2-acetamido-2-deoxy-4-O-(α-L-fucopyranosyl)-3-O-(β-D-galactopyranosyl)-D-glucopyranose, is a case in point. Prior to this invention no methods were available for the synthesis of a structure of this complexity, therefore, to obtain this compound it was necessary to isolate it from a rare natural source such as ovarian cyst fluid. From a single ovarian cyst, V. P. Rege et al.4 were able to obtain 2.4 g of "dialysable products." After extensive manipulation about 10 mg of the Lewis-a trisaccharide antigenic determinant was isolated from this material. Similar difficulties have been encountered in obtaining the antigenic determinants of other human blood groups. At this point the second major difficulty arises, that is, properly functionalizing the carbohydrate antigenic determinant so that it can be utilized for the preparation of monospecific artificial antigens and immunoabsorbants.
Much ingenuity has been involved in the establishment of methods for the attachment of sugars to carrier polymers, however, all methods divulged to date have some serious drawbacks. Kabat and coworkers5 have oxidized the reducing saccharide to aldonic acid which was subsequently attached to protein amino groups by way of a mixed anhydride. Unavoidably, this procedure results in the destruction of the terminal saccharide.
Conversion of the terminal saccharide to 1-(m-nitrophenyl) flavazole followed by reduction to the amine, diazotization and coupling to the protein has been used by Westphal and coworkers.6 In addition to the destruction of the terminal saccharide, this method requires that the 2- and 3-positions of that saccharide be unsubstituted. Also, a new and highly antigenic structure is introduced in close proximity to the carbohydrate hapten and in addition, some sugars are sensitive to the conditions employed for diazotization. It is these last two difficulties which also limit the method of Goebel and Avery.1,3
It is the objective of the present invention to provide processes to chemically synthesize carbohydrate antigenic determinants in a form suitable for linkage to carrier molecules or solid supports. The invention further concerns the use of the products of the process for the following purposes:
(a) preparation of artificial carbohydrate antigens.
(b) preparation of antisera specific for the carbohydrate hapten of the artificial antigen.
(c) preparation of immunoabsorbants (affinity absorbants) capable of selectively absorbing antibodies raised to either the artificial antigen or to natural antigens possessing the same terminal oligosaccharide structure as the antigenic determinant.
Particular reference is made to the lower oligosaccharide antigenic determinants of the human blood groups e.g., Lewis-a, Lewis-b, B and H(O).
Thus, according to the present invention there is provided a process for preparing a β-glycosyl plus bridging arm compound comprising:
reacting an activated sugar (a) selected from a glycosyl halide, a 1,2-orthoacyl ester derivative of a sugar, or a 1,2-oxazoline derivative of a sugar, with a monohydroxy carboxylic acid (b) of the general structure I,
ho--r--coor' i
where R is an aliphatic hydrocarbon thereof having from 3 to 17 carbon atoms and R' is a protecting group for the acid, to form a β-glycoside of the general structure II, ##STR1## and recovering said β-glycoside. The preparation of such a glycoside is the first claim and product of this invention.
As a further embodiment, the process is expanded to include the subsequent step of glycosidically linking in either the α- or β-anomeric configuration a further sugar to the initial sugar moiety to form a disaccharide of the general structure ##STR2## and recovering said disaccharide. This latter process may be further expanded with additional steps for the preparation of trisaccharides and tetrasaccharides of general structure IV and V, respectively, and recovering said compounds as novel haptens.
In addition there is provided a process for the preparation of artificial antigens and immunoabsorbants by attaching the monosaccharide or lower oligosaccharide, plus bridging arm compound to a soluble carrier macromolecule or to an insoluble support. This is the final process step of this invention and yields the final products.
The production of antigenic determinants corresponding to structure types III, IV and V from the structure type II are outlined in typical reaction Schemes 1, 2 and 3 with reference to specific examples provided below. ##STR3##
A detailed description of the present invention will now be given. Special reference will be made to the synthesis of the antigenic determinants for the human blood groups Lewis-a, Lewis-b, B and H(O) as examples, but the same techniques apply to other carbohydrate antigens. The overall process begins with the attachment of an appropriately selected sugar (for example 2-acetamido-2-deoxy-D-glucose (D-glcNAc) for the Lewis-a, b and H(O) determinants and D-galactose (D-gal) for the B determinant) by way of a β-glycosidic linkage to a protected monohydroxycarboxylic acid of general structure I.
ho--r--coor' i
the attachment is accomplished following procedures for the synthesis of β-glycosides well known in the art of carbohydrate chemistry and may follow β-glycosidation procedures such as the Koenigs-Knorr-Helferich-type methods using O-acetylated α-glycosyl halide derivatives of the sugar13-16 or in the case of D-galactose by way of an O-acylated-1,2-orthoacyl ester derivative17 and in the case of D-glucosamine by way of an O-acylated 1,2-oxazoline derivative18 and, in each of the latter cases, the use of a suitable acid catalyst for promotion of the reaction. In this way the first products (structures II) of this invention are prepared. ##STR4##
The aglycon (--O--R--COOR') of structure II is selected under the following conditions. The group R' is selected from the lower alkyl groups such as methyl, ethyl, n-propyl, isopropyl and n-butyl with some preference given to the methyl group for reasons of economy and generally more convenient reactivity of the methoxycarbonyl group over other alkoxycarbonyl groups. The --R-- group provides a physical separation, a bridging arm, between the antigenic determinant and the carrier or support. This group should be inert towards the chemical reactions used for the further elaboration of the carbohydrate structure and is thereby effectively limited to structures that are aliphatic hydrocarbon in composition. The preferred structures correspond to that shown below, where n is in the range 3 to 17.
--O--C.sub.n H.sub.2n --COOR'
and the carbon chain is normal. Particularly useful are structures where the value of n is in the range 8-10 not only in view of the ready availability of the parent hydroxyacid but also because the inert rather featureless aliphatic chain appears to function as an internal adjuvant. Thus, higher antibody titres are obtained using bridging arms of this length as opposed to those of shorter length. Most examples in this invention utilize --R-- equal to --C8 H16 -- since with this grouping very high titer antisera were consistently achieved.
The β-glycoside derivative products will have the general structure ##STR5## where R is an aliphatic hydrocarbon moiety having 3 to 17 carbon atoms and R" is --H, --OH, --NH2, --NHNH2, --N3, --O--alkyl or --O--aryl, and n has a value of 1 to 4. Thus the (Sugar)n → O--R--COOR' where R' is a protecting group, may be converted to the product (Sugar)n → O--R--COR" where the R" may constitute other than a protecting group. The initial protecting group --OR' e.g. the methyl, ethyl, propyl and butyl esters, may be replaced with other R" groups either to form alternative haptens for a particular application or to facilitate coupling to a carrier or support.
The following four specific preparations exemplify the preparation of compounds of structure II. In all cases, the structures achieved were substantiated by modern methods of chemical analysis including carbon-13 and proton nuclear magnetic resonance spectra (cmr and pmr, respectively), elemental analysis and in many instances by high-resolution mass spectrometry.
8-Methoxycarbonyl-1-octanol (21.4 g, 0.115 mole) and dried mercuric cyanide (30.3 g, 0.119 mole) were dissolved in a 1:1 mixture of dry distilled benzene-nitromethane (850 ml). The solution was stirred and 200 ml of solvent was distilled. Mixed solvent (200 ml) was added and calcium sulfate (40 g) was added followed by 2,3,4,6-tetra-O-acetyl-α-D-galactosyl bromide (38.7 g, 0.094 mole). The mixture was stirred at room temperature for 36 h and then heated at 70� for 2 h. After cooling, the solution was filtered and the filtrate evaporated in vacuo to give a yellow syrup. This syrup was dissolved in distilled ethyl acetate (500 ml) and the solution was washed with an aqueous solution (10% w/w, 500 ml) of sodium iodide. The organic phase was then washed successively with a saturated solution of sodium thiosulphate (200 ml) and H2 O (2 � 250 ml). The organic phase was dried over sodium sulphate and then evaporated in vacuo after filtration. This syrup (˜50 g) after drying under high vacuum overnight was dissolved in dry distilled methanol (200 ml) and to this solution, 200 ml of methanolic sodium methoxide (150 mg of sodium) was added. After stirring at room temperature for 24 h, acid resin (prewashed a few times in methanol) was added and stirred until the pH was neutral. After filtration, the solution was evaporated and the waxy residue dissolved at room temperature in 100 ml of H2 O. This solution was extracted with ether (2 � 40 ml) and then placed in the fridge. Upon standing, crystallization occurred. The white crystals were filtered and dried. Weight 13.5 g (41%), mp 104�-105�, [α]D 25 - 13.4� (c 1, 95% ethanol).
Mercuric cyanide (31.8 g, 126 mmol) and anhydrous calcium sulfate were added to a solution of 8-ethoxycarbonyl-1-octanol (25 g, 124 mmol) in 100 ml of dry benzene. The mixture was protected from moisture while stirring for 1 h at room temperature prior to addition of 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-glucopyranosyl chloride (25 g, 68 mmol). The mixture was efficiently stirred for 4 days at room temperature. Dichloromethane (400 ml) was added, the solids were removed by filtration, and the filtrate was sequentially washed with 10% aqueous sodium chloride solution (50 ml), once with saturated aqueous sodium bicarbonate solution (25 ml), and twice with water (50 ml). In each case, the aqueous layer was back-extracted with a little dichloromethane. After drying over magnesium sulfate, the solvents were removed to leave a syrup which crystallized from a mixture of diethyl ether and n-hexane. The crude yield of 8-ethoxycarbonyloctyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside was 28.9 g (80%). A recrystallized sample showed mp 112�, [α]D 20 - 12.2� (c 2.4, chloroform). De-O-acetylation of this compound with triethylamine (27 ml) in methanol (500 ml) at 0�-5� for 18 h gave after evaporation of the solvent compound 2 as a chromatographically homogeneous powder which resisted crystallization and was therefore characterized as its hydrazide derivative mp 200�-201�, [α]D 26 - 22.5� (c 1, water), prepared by treating 2 with an 85% solution of hydrazine hydrate.
A mixture of p-toluene sulfonic acid (10 mg), 8-ethoxycarbonyl-1-octanol (1 g), 2-methyl-4,5-(3,4,6-triacetyl-2-deoxy-α-D-glucopyrano)-Δ2 -oxazoline (1 g) was dissolved in 10 ml of a 1:1 mixture of benzene and nitromethane and refluxed for 3 hours. The usual workup gave 8-ethoxycarbonyloctyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside (1.28 g, 79%). De-O-acetylation as described in example II gave the title antigenic determinant in 90% yield.
Compound 3 was prepared as described in example VI below.
Condensation of 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-glucopyranosyl chloride with 5-ethoxycarbonyl-1-pentanol under the same conditions as reported in example II gave a 74% yield of 5-ethoxycarbonylpentyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside, mp 101�-102�, [α]D 25 - 16.8� (c 1.06, chloroform). De-O-acetylation of this compound using sodium methoxide gave after crystallization a 92% yield of 4 mp 154�-155� (melts and resolidifies) remelts 167�, [α]D 18 - 25.5� (c 1.1, water).
The spirit and scope of this invention encompasses the use of compounds of general structure II for the elaboration of more complex antigenic determinants. In such processes, additional sugars are added to appropriately blocked derivatives of II using synthetic methodologies known to carbohydrate chemistry. In this fashion, structures of type III are achieved wherein a disaccharide unit is present in contrast to the simple monosaccharide units in II. ##STR6## Examples V, VI, and VII illustrate the preparation of compounds of general structure III.
To a colorless solution of 6.6 g of α,α-dimethoxytoluene (0.043 mole) in 60 ml of distilled acetonitrile, p-toluenesulfonic acid (0.285 g, recrystallized) was added. The solution instantaneously became yellow and immediately 8-methoxycarbonyloctyl β-D-galactopyranoside (10 g, 0.028 mole) was added. The yellow color disappeared within 5 minutes and dissolution took place in 30 minutes. The solution, protected from humidity, was stirred at room temperature for 44 hours. At this time, no starting material was detectable by thin layer chromatography (tlc) and a new product 8-methoxycarbonyloctyl 4,6-O-benzylidene-β-D-galactopyranoside was present. The presence of the isomeric 3,4-O-benzylidene analog of this compound could not be detected by tlc. A few drops of triethylamine were added (pH ˜7) and the solvent was evaporated. Toluene was added to the residual syrup and then evaporated in vacuo and this operation was repeated once more. The white waxy solid was then stirred overnight with hexane (distilled) to remove a fast running uv absorbing spot, and a very fine powder was obtained. After filtration, the solid was dissolved in dichloromethane (distilled, 500 ml) and the solution washed with a saturated sodium bicarbonate solution (100 ml) and then water (300 ml). After drying over sodium sulfate, the organic phase was evaporated to dryness to give the pure 4,6-benzylidene derivative. The product in solution in methanol was placed in a dessicator containing n-pentane for crystallization. The yield of recrystallized product was 83%, mp 127�-128�, [α]D 25 -33� (c 1, chloroform). A portion of the benzylidinated compound (7 g, 0.016 mole) was dissolved in 400 ml of dry distilled dichloromethane containing 10 ml of pyridine (distilled). The solution was cooled to -40� and 2 ml (0.017 mole) of distilled benzoyl chloride was added dropwise. After 2 h at that temperature tlc examination showed the presence of only one product, 8-methoxycarbonyloctyl 3-O-benzoyl-4,6-O-benzylidene-β-D-galactopyranoside. Ice chips were added and the solution was poured in a mixture of water and ice (300 ml) vigorously stirred. The organic phase was separated and the aqueous phase back-extracted with dichloromethane (2 � 100 ml). The combined organic phase was washed with a saturated sodium bicarbonate solution (200 ml) and then with water (2 � 300 ml). After drying over sodium sulphate, the organic phase was evaporated to dryness. The remaining pyridine was co-evaporated with toluene in vacuo to give 8.7 g (100%) of the desired blocked galactopyranoside. Recrystallization was achieved by dissolving the syrup in 2-propanol and the solution was placed in a desiccator containing n-pentane. The yield was quantitative, mp 96�-97�, [ α]D 25 + 60.5� (c 1, chloroform).
The above selectively blocked galactopyranoside (4 g, 7.38 mmol) was dissolved in a mixture of purified dichloromethane (10 ml) and distilled N,N-dimethylformamide (0.62 ml) containing 1.6 g (10.7 mmol) of tetraethylammonium bromide and 7.8 g of diisopropylethylamine. To the solution tri-O-benzyl-α-L-fucosyl bromide (freshly prepared from 20.8 mmol of tri-O-benzyl-l-p-nitrobenzoyl-α-L-fucopyranose) was added, followed by addition of 2 g of molecular sieves 4A. After 48 h the reaction mixture was diluted with dichloromethane (40 ml) and the solids were removed by filtration. The filtrate was washed with water and dried over sodium sulphate. After filtration, the solution was evaporated in vacuo to a pale yellow syrup. This syrup was dissolved in ether-ethyl acetate (3:2) and applied to an alumina column. The products were rapidly eluted with the same solvent system and obtained as a pale yellow syrup upon evaporation of the solvents. This syrup was dissolved in hexane-ethyl acetate (8:2) and applied to a silica gel (200 g) column. The elution was performed using the same solvent mixture. Two fast running spots were eluted first, then the desired compound. The weight (after being dried under high vacuum) was 6.1 g (86%).
De-O-benzoylation with methanolic sodium methoxide gave 8-methoxycarbonyloctyl 4,6-O-benzylidene-2-O-(tri-O-benzyl-α-L-fucopyranosyl)-β-D-galactopyranoside in 80% yeild, mp 104�-105�, [α]D 25 - 54� (c 1, chloroform). Hydrogenation over 5% palladium on charcoal gave the title compound 5 as a tlc homogeneous syrup which was characterized as its hydrazide derivative, mp 178� (ethanol-ether), [α]D 18 -87.4 (c 1.02, water).
Sodium (18 mg) was added to dry methanol (40 ml) and after the reaction was complete 8-ethoxycarbonyloctyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside (1.06 g, 2 mmol) was added. The solution was kept at room temperature for 24 h prior to deionization using the acid resin. Solvent removal left 753 mg of the de-O-acetylated product which was not characterized but used directly for the preparation of the title compound. The material, dried in vacuo over phosphorus pentoxide, was dissolved in DMF (5 ml) which contained α,α-dimethoxytoluene (2 ml) and p-toluenesulfonic acid (25 mg). After heating at 40� for 1.5 h, the solution was cooled and triethylamine added to neutralize the acid. After solvent removal, toluene (5 ml) was added and removed by evaporation. This procedure was repeated twice and the residue then triturated with hexane (10 ml). The solid was collected and dissolved in dichloromethane (25 ml). The solution was washed three times with water (5 ml), dried over magnesium sulfate, and evaporated to a residue which crystallized from ethanolpetroleum ether. The yield of 8-methoxycarbonyloctyl 2-acetamido-4,6-O-benzylidene-2-deoxy-β-D-glucopyranoside was 481 mg (50%), mp 219�. Recrystallization gave the analytical sample, mp 221�, [α]D 25 - 56� (c 1.3, dimethylformamide).
This compound was acetylated using a 1:1 mixture of acetic anhydride and pyridine for 24 h. The product was isolated in the usual manner and de-O-benzylidinated using 50% acetic acid at 100� for 25 min to yield 2-acetamido-3-O-acetyl-2-deoxy-β-D-glucopyranoside, mp 122�-123�, [α]D 30 - 47.3 (c 1.1, chloroform).
The 6-position of this compound was selectively benzoylated in 88% yield by treatment with 1.1 equivalents N-benzoylimidazole. A mixture of the above 6 benzoate, 8-methoxycarbonyloctyl 2-acetamido-3-O-acetyl-6-O-benzoyl-2 2-deoxy-β-D-glucopyranoside, mp 105�, [α]D 32 -42.0� (c 1, chloroform), (1.55 g, 2.9 mmol) tetraethylammonium bromide (630 mg, 3 mmol), diisopropylethylamine (450 mg) and DMF (4 ml) in dichloromethane 20 ml was added to 2,3,4-tri-O-benzyl-α-L-fucopyranosyl bromide freshly prepared from 3.38 g, 5.8 mmol, of 2,3,4-tri-O-benzyl-1-O-p-nitrobenzoyl-β-L-fucopyranose. The solution was stirred at room temperature for three days after which time tlc examination no longer showed the presence of any starting material. Dichloromethane (150 ml) was added and the solution was washed in the usual manner with water and aqueous sodium bicarbonate solution prior to solvent removal.
The syrupy product was used directly for the preparation of 8-methoxycarbonyloctyl 2-acetamido-4-O-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-2-deoxy-.beta.-D-glucopyranoside. The crude disaccharide (3.78 g) was dissolved in 50 ml of dry methanol, and 5 ml of 0.2 N sodium methoxide in methanol was added. After 24 h at room temperature, the solution was deionized using the acid resin. Solvent removal left an oil which was dissolved in dichloromethane (100 ml). The solution was washed with water and dried over magnesium sulfate prior to evaporation to a residue (1.01 g, 85% yield) from the fully blocked disaccharide. Purification was by recyrstallization from ethyl acetate-hexane, mp 145�-146�, [α]D 25 - 33.6� (c 1.2, chloroform).
Hydrogenation of this compound in ethanol over 5% palladium on charcoal at 50 psi and 70� for 3 days gave after removal of the solvent and catalyst compound 6 as an amorphous solid, [α]D 25 - 95.5� (c 1.0, water) in 97% yield.
Compound 2 (6.3 g, 15.5 mmol) was added to a stirred solution of anhydrous zinc chloride (7.0 g) in freshly distilled benzaldehyde (100 ml) to which was added an anhydrous calcium sulfate (14 g). After 20 h at room temperature, the solids were removed by filtration. The addition of hexane (500 ml) to the filtrate caused the precipitation of a gummy precipitate which was triturated several times with hexane. The residue was then triturated with pyridine (7 ml) prior to the addition of dichloromethane (300 ml). The resulting solution was washed twice with water (25 ml), twice with saturated aqueous sodium bicarbonate solution (25 ml), and twice again with water (25 ml). Solvent removal, after drying over sodium sulfate, left a residue which crystallized from ethanol (4.68 g, 61% yield), mp 210�-220�. The melting point was unchanged by recrystallization from aqueous methanol, [α]D 24 -55.5� (c 1.3, chloroform).
A solution of this compound (12.1 g, 24. 5 mmol) and mercuric cyanide (7.35 g, 30 mmol) in 1600 ml of a 1:1 mixture of benzene and nitromethane was distilled at atmospheric pressure to remove 100 ml of solvent. Calcium sulfate (40 g) and tetra-O-acetyl-α-D-galactopyranosyl bromide (12.3 g, 29.6 mmol) were added, after cooling, and the temperature was then maintained at 50� for 20 hours. Tetra-O-acetyl-galactosyl bromide (10 g, 24.4 mmol), mercuric cyanide (6.15 g), and calcium sulfate (10 g) were then added, and the mixture was stirred a further 20 h at 50�. TLC examination indicated the absence of the monosaccharide alcohol. The solids were removed by filtration and washed with dichloromethane (800 ml). The combined filtrates were twice washed with 30% aqueous potassium iodide solution (50 ml), twice with saturated aqueous sodium bicarbonate solution (50 ml), and then twice with water (50 ml). After drying over sodium sulfate, the solvent was removed to leave a syrup. The syrup was dissolved in a little hot ethanol and diethyl ether then added to near turbidity. The addition of petroleum ether caused the precipitation of a solid which was crystallized from ethyl acetate-hexane (17.1 g, 85%, mp 108-109�). Recrystallization from ethyl acetate-diethyl ether afforded pure material, mp 110�-111�, [α]D 27 - 8.2� (c 1.4, chloroform). Removal of the hydroxyl protecting groups in the manner described in previous examples gave 7 in 82% yield as a white solid, mp 207�-208�. Recrystallization from methanol raised the melting point to 211�-212�, [α]D 17 - 22� (c 1, water).
Appropriately blocked derivatives of compounds of structure-type III and encountered in the preparations of compounds 5, 6 and 7 are used to elaborate by way of chemical synthesis structures of type IV, ##STR7## Thus, the terminal trisaccharide IV units of important human blood group antigens are obtained. This is exemplified by the following preparations of the antigenic determinants for the Lewis-a, O(H) and B Human blood groups, in Examples VIII, IX and X respectively.
8-Ethoxycarbonyloctyl 2-acetamido-3-O-(tetra-O-acetyl-β-D-galactopyranosyl)-4,6,-O-benzylidene-2-deoxy-β-D-glucopyranoside (7.65 g) (cf example VII) was de-O-benzylidinated using aqueous acetic acid under conditions similar to those used in example VI to give 5.6g (81%) of product, mp 153.5�, [α]D 16 + 9.6� (c 1.4, chloroform). A portion of this material (1.47 g, 2 mmol) was selectively acetylated at the 6-position using N-acetyl imidazole (2.2 mmol) to yield 1.10 g (71%) of 8-ethoxycarbonyloctyl 2-acetamido-6-O-acetyl-3-O-(tetra-O-acetyl-β-D-galactopyranosyl)-2-deoxy-β-D-glucopyranoside, as a tlc homogeneous syrup, [α]D 31 + 6.2� (c 1, chloroform).
A mixture of this compound (3.0 g, 3.86 mmol), tetraethylammonium bromide (840 mg, 4 mmol), diisopropylethylamine (600 mg, 4.64 mmol), dichloromethane (25 ml), and DMF (5 ml) was added to tri-O-benzyl-fucosyl bromide (2.56 g, 5.15 mmol) dissolved in dichloromethane (5 ml) and DMF (1 ml) was then added, and the solution was stirred for an additional 3 days. Dichloromethane (300 ml) was added, and the product was isolated in the usual manner. Purification was effected by chromatography on a silica gel column (75 � 3.5 cm). Development with 1:1 ethyl acetatehexane (300 ml) was followed by the same solvent but to which 5% ethanol was added. The first band to appear soon after changing solvent provided the blocked trisaccharide as a tlc homogeneous syrup (4.36 g, 94%), [α]D 22 - 54� (c 1.1, chloroform). Removal of the hydroxyl protecting groups by methods similar to those already described gave the desired antigenic determinant 8 as an amorphous solid, [α]D 25 - 73.5� (c 1, water).
A solution of 8-methyoxycarbonyloctyl 2-acetamido-4,6-O-benzylidene-2-deoxy-β-D-glucopyranoside (480 mg, 1 mmol) (cf example VI) and mercuric cyanide (304 mg, 1.2 mmol) in 50 ml of a 1:1 mixture of benzene and nitromethane was distilled at atmospheric pressure to remove 10 ml of solvent. Powdered calcium sulfate (1.5 g) and the bromide (prepared from 900 mg of 3,4,6-tri-O-benzyl-1,2-di-O-p-nitrobenzoyl-β-D-galactopyranose) in 5 ml of a 1:1 benzene nitromethane mixture were added after cooling, and the temperature then maintained at 40� for 36 h. Tlc examination at this point indicated that no bromide remained. The reaction mixture was diluted with dichloromethane (25 ml), filtered and the filter cake washed with dichloromethane (3 � 10 ml). The combined filtrate and washings were made up to 200 ml with dichloromethane and washed with 30% aqueous potassium iodide (10 ml), saturated sodium bicarbonate solution (10 ml) and water (2 � 10 ml). After drying over sodium sulfate the solvent was removed leaving a syrup which was dissolved in a small amount of dichloromethane and applied to a column of silica gel D-O (65 g). The column was first developed with a mixture of benzene-ethyl acetate (4:1) and then the product was eluted with a 2:1 benzene-ethyl acetate mixture. Solvent removal afforded 550 mg (52%) of the blocked disaccharide which was crystallized from ethanol, mp 152�-153�, [α]D 24 + 42.0� (c 1.1, chloroform).
This disaccharide (400 mg) was suspended in 40 ml anhydrous methanol containing 12 mg sodium and the reaction mixture stirred for 24 h at room temperature. The solution was de-ionized with acid resin and the residue obtained after evaporation of the solvent was dissolved in benzene and applied to a 4 g column of silica gel D-O. After washing the column with benzene to remove the methyl p-nitrobenzoate the product was eluted with benzene-ethyl acetate 2:1. Evaporation of the eluent afforded 260 mg (78%) of the de-O-p-nitrobenzoylated compound as a tlc homogeneous syrup.
To a mixture of this compound (1.03 g, 1.13 mmol), tetraethylammonium bromide (317 mg, 1.5 mmol), diisopropylethylamine (194 mg, 1.5 mmol) molecular sieves 4A (1 g), dichloromethane (8 ml) and dimethylformamide (0.6 ml) was added 2,3,4-tri-O-benzyl-α-L-fucopyranosyl bromide freshly prepared from 2,3,4-tri-O-benzyl-l-O-p-nitrobenzoyl-β-L-fucopyranose (1.459 g, 2.5 mmol) in dichloromethane (2 ml). After 4 days tlc examination showed that no starting material remained, 200 μl of methanol were then added and after 2 h the reaction mixture was diluted with 25 ml dichloromethane and filtered. The filtrate was made up to 100 ml with dichloromethane and washed in the usual manner with water and aqueous saturated sodium bicarbonate prior to solvent removal. Chromatography of the crude product on silica gel D-O using ethyl-acetate-hexane 1:1 as eluent gave the fully blocked trisaccharide (1.116 g, 74% of theoretical) as a tlc (benzene-ethyl acetate 2:1) homogeneous syrup.
A solution of this compound (890 mg) dissolved in 9 ml of dichloromethane containing 1.0 ml of 90% trifluoroacetic acid was allowed to stand at room temperature for 10 minutes. The reaction mixture was diluted with toluene (10 ml) and water (0.5 ml) and the solvents evaporated. Chromatography of the crude product on silica gel gave after crystalliaztion from ethyl acetate-pentane 530 mg (64% of theoretical yield) of the de-O-benzylidinated trisaccharide, mp 125�-126�, [α]D 24 - 37.4� (c 1.1, chloroform).
To a solution of this compound (452 mg) in ethanol (7 ml) was added 300 mg of 5% palladium on charcoal and the mixture shaken under a 100 psi atmosphere of hydrogen for 36 hr. The catalyst was removed by filtration and washed with three 10 ml portions of hot ethanol. The combined filtrate and washings were evaporated and the product was dissolved in a small amount of water and freeze dried. The crude product 9, (255 mg, quantitative yield) was homogeneous by tlc (isopropanol-ammonium hydroxide-water 7:1:2) and crystallized from a mixture of methanol and ether, mp 195�, [α]D - 60.3� (c, 1.1, water).
8-Methoxycarbonyloctyl 3-O-benzoyl-4,6-O-benzylidene-2-O-(tri-O-benzyl-α-L-fucopyranosyl)-.beta.-D-galactopyranoside (5 g, 5.2 mmol) (cf example V) in 250 ml of a freshly prepared methanolic sodium methoxide solution (1%) was stirred at room temperature for 2 h at which time the reaction was complete as judged by tlc. Sufficient acid ion exhange resin, prewashed with methanol, was added to neutralize the solution (pH ≃7). The resin was removed by filtration and the filtrate evaporated in vacuo to give a pale yellow syrup (≃4.3 g). Crystallization from ethanol hexane gave 3.55 g (80%) of crystalline de-O-benzolyated compound, mp 104�-105�, [α]D 25 - 54� (c 1, chloroform).
This compound (0.6 g, 0.7 mmol) was dissolved in dichloromethane (0.66 ml) and N,N-dimethylformamide (0.06 ml) containing tetraethylammonium bromide (0.16 g, 1 mmol), Hunigs base (0.27 g) and molecular sieve 4A (1 g). Freshly prepared tetra-O-benzyl-α-D-galactopyranosyl bromide (1.04 g, 1.7 mmol) in a small amount of dichloromethane was then added and the reaction mixture was then stirred at room temperature for 48 hours. An additional portion (0.5 g, 0.85 mmol) of the bromo sugar was then added and the reaction mixture stirred for a further 24 hours. The reaction mixture was diluted with dichloromethane and the solids were removed by filtration. The filtrate was washed with water and dried over sodium sulphate. After filtration the solution was evaporated to a syrup. This syrup was purified by rapid chromatography on an alumina column before being applied to a silica gel column. Elution was obtained with ethyl acetate-hexane (2:8) and the desired blocked trisaccharide obtained pure by pooling the appropriate fraction. The yield was 0.686 g (71%), mp 82�-83� (recrystallized from heptane-ether).
To a solution of the above compound (600 mg, 0.43 mmol) in ethanol (10 ml) containing ethyl acetate (1 ml) was added 350 mg of 5% palladium on charcoal and the mixture was shaken under hydrogen at a pressure of 100 psi (gauge pressure) for 5 days. The catalyst was filtered off and washed with hot ethanol (3 � 5 ml). The combined filtrates were evaporated in vacuo to give compound 10 (177 mg, 62%). A portion was recrystallized from methanol-ether, mp 147�-150�.
Elaboration of appropriately blocked derivatives of structure type IV give tetrasaccharides of general structure type V. ##STR8## Thus, the terminal tetrasaccharide antigenic determinant of the human blood group Lewis-b is obtained as illustrated by Example XI.
8-Methoxycarbonyloctyl 2-acetamido-3-O-(3,4,6-tri-O-benzyl-2-O-[2,3,4-tri-O-benzyl-α-L-fucopyranosyl]-α-D-galactopyranosyl)-2-deoxy-β-D-glucopyranoside (620 mg, 0.5 mmol) (cf example IX) was selectively acetylated at the 6-position of the N-acetyl glucosamine residue by treatment with N-acetyl imidazole (0.6 mmol). α-Fucosylation of the compound as in example VIII gave the desired blocked tetrasaccharide in 70% yield as a tlc homogeneous syrup. Removal of the hydroxyl protecting groups by the methods previously described (cf example VIII) gave tetrasaccharide 11 in 80% yield.
The products obtainable through the general procedures embodied in this invention arise from the novel concept of exploring applications of synthetic carbohydrate chemistry toward the elaboration of antigenic determinants in the form of structure-types II, III, IV and V for use not only as inhibitors of antibody-antigen interactions but, and especially, for use as reagents for the preparation of highly effective artificial antigens and immunoabsorbants which show promise of a wide range of useful application.
The demonstration that synthesis can lead to large scale preparations of important natural carbohydrate determinants means that mankind will no longer be dependent on rare and expensive sources of minute amounts of these determinants. The demonstration that these determinants can be produced in such a fashion and that these are readily employable for the preparation of effective highly immunogenic antigens promises to be of great utility for the provision of antibodies which are monospecific for the selected determinant. Thus, applications for the provision of typing sera for both blood sera and cells are indicated and demonstrated. Extension to the typing of tissues is evident and is expected to be of major utility for the proper matching of tissues for tissue transplantation operations. Antibodies raised against the wide range of carbohydrate determinants arising from the disclosure of this invention can serve as diagnostic reagents and in the preparation of affinity columns as now widely practiced using so-called lectins which are natural proteins possessing specific affinity for a carbohydrate structure. Of major importance is the provision of antisera specific to minor human blood groups such as Lea and Leb. Effective typing sera for such minor groups are presently rare and expensive but their diagnosis is of increasing importance due to increasing likelihood in modern medicine of multiple blood transfusions and the hazard thus raised of a recipient having, unknowingly, been previously sensitized.
The demonstrations contained in this invention show that the synthesized determinants can be used for the preparation of highly effective monospecific immunoabsorbants. These immunoabsorbants promise to have important and wide utility for the removal of undesired antibodies from blood sera and for the isolation and purification of specific antibodies through absorption-desorption procedures. in this latter regard, the utility of incomplete determinants for the preparation of affinity chromatograms is of substantial significance since the use of complete determinants can lead to binding of the antibodies that is so strong that the desorption cannot be accomplished without extensive denaturation of the antibody and, thereby, loss of its specificity.
The above-mentioned benefits accruing as the result of the discoveries leading to this invention are demonstrated by Examples XII to XIX presented below. These examples were selected from a large list of similar investigations leading to this invention and were chosen so as to well establish the spirit and scope of the invention in terms of the utility of determinants and carrier molecules to prepare artificial antigens and of determinants and solid supports to prepare immunoabsorbants.
The attachments of the determinants beginning with the ester grouping contained in the structure-types II, III, IV and V can be made following several procedures well known to this area of investigation. The most direct way is by straightforward aminolysis of the ester on the bridge-in arm using dry methanol as solvent,
--COOCH.sub.3 + H.sub.2 N-- → --CONH-- + CH.sub.3 OH
the amide grouping serving as the linkage between hapten and carrier or solid support.
A second method comprises first converting the ester to the free carboxylic acid followed by condensation of the acid with amino groups using suitable condensing agents such as dicyclohexyl and other carbodiimides or 1-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), ##STR9##
A third method (and that preferred) involves conversion of the ester to hydrazide followed by nitrous acid oxidation of the hydrazide to form the acyl azide which serves as acylating agent, ##STR10## Le.sup.
In these various ways artifical antigens are prepared by coupling the synthesized determinants to soluble carrier macromolecules such as the proteins bovine serum albumin (BSA) human serum albumin (HSA) and polylysine, to red blood cells, and to polysaccharides such as aminated dextran. Also, immunoabsorbants are prepared using solid supports such as aminated sepharose, aminated polystyrene, polyvinylamine, aminated polyvinylalcohol, aminated polyacrylamide and aminated glass. These solids normally are employed as beads or latex particles but may be used on surfaces of tubes and plates depending on the use for the antigenic surface; that is, as an insoluble absorbant for the extraction of antibodies, for the preparation of an affinity chromatographic column, for the detection of agglutination-type phenomena as for spot tests on surfaces, etc.
The artifical antigens reported in Examples XII to XVI were tested by administration to experimental animals; mainly rabbits and goats. For rabbits the immunization protocol was that described by Martineau8 and for goats that of Marcus and Grollman.9 The course of the immunization was followed by quantitative precipitin tests on samples of blood withdrawn at various times. The procedures used were all standard to the field of immunology.10
Compound 1 (1 g) was dissolved in a mixture of ethanol (5 ml) and 85% hydrazine hydrate (2 ml). After 24 h the solvent was evaporated and residual traces of hydrazine removed by co-evaporation with toluene to give 8-hydrazinocarbonyloctyl β-D-galactopyranoside as a white solid which was recrystallized from ethanol, mp 194�, [α]D 18 - 2.16� (c 1.1, water) 92% yield.
The hydrazide (88 mg, 0.25 mmol) was dissolved in DMF (3 ml) and the solution cooled to -25�. A 3.3 N solution of hydrogen chloride in dioxane (0.33 ml) was added and then tert-butyl nitrite (36 mg, 0.35 mmol) in DMF (0.1 ml). After 30 min, sulfamic acid (20 mg) was added and the stirring was contained for 15 minutes. The acyl azide solution was then added directly to a 0� solution of bovine serum albumin (BSA) (300 mg) dissolved in an aqueous solution 0.08 M in Na2 B4 O4 and 0.35 M in KHCO3. The pH of the solution remained between 9.05 and 9.30 during the course of the addition. After 16 h the solution was dialyzed against water and freeze dried to provide antigen 12 (310 mg) as a white powder. Incorporation of galactose was determined by the phenol sulfuric acid method and calculated to be 24 moles galactose per mole of BSA.
A group of six San Juan rabbits were immunized with antigen 12 incorporated into Freund's complete adjuvant (FCA). The amount of conjugate administered and the immunization schedule followed the protocol described by Martineau et al.8 Antibody levels upon completion of the schedule ranged from 178 to 396 μg per 50 μl of sera with the average being 277 μg per 50 μl sera.
That the antibodies raised to this conjugate are directed mainly toward the β-galactopyranoside portion of the antigen was demonstrated by measuring the maximum amount of antibody precipitated by three antigens 12, (α-D-gal-O(CH2)8 CONH)17 -BSA, and BSA. Typically for 50 μl of crude sera the results were as follows: antigen 12, 396 μg; (α-D-gal-O(CH2)8 CONH)17 -BSA, 87 μg; and BSA, 87 μg. Thus, it is possible to conclude that the greatest portion of the antibody population recognizes specifically the β-D-gal portion of the hapten. This conclusion is reinforced by inhibition experiments which show that while methyl β-D-galactopyranoside gives 50% inhibition of precipitation [using (β-D-gal-O(CH2)8 CONH)23 -HSA as the precipitating antigen] at a concentration of 0.83 μM/ml, compounds having similar structures require much larger concentrations to be effective; i.e. galactose require 6.6 μM/ml; methyl α-D-galactopyranoside require 33 μM/ml; lactose require 70 μM/ml and methyl β-D-glucopyranoside gives approximately no inhibition.
This antigen was prepared from the hydrazide derivative of compound 2 using the acyl azide coupling method described in example XII. Sera obtained from rabbits immunized with 13 showed a high specificity for antigens containing the β-D-glcNAc hapten. Proof of the hapten specific nature of the response comes from hapten specific inhibition of precipitation between 13 and antisera raised to 13. Typically, methyl β-D-glcNAc gave inhibition of precipitation in the range of 60-80%, while methyl β-D-galNAc and methyl β-D-glc virtually no inhibition at the concentration that methyl β-D-glcNAc was effective.
Compound 8 (1 g) was dissolved in 85% hydrazine hydrate (4 ml) and allowed to stand for 4 h at ambient temperature. The reaction mixture was diluted with 95% ethanol (20 ml) and the solvents evaporated. The residue was dissolved in 10 ml water and dialyzed against 5 changes of water in an ultrafiltration cell. Lyophilization of the water gave 8-hydrazinocarbonyloctyl 2-acetamido-2-deoxy-4-O-(α-L-fucopyranosyl)-3-O-(β-D-galactopyranosyl)-β-D-glucopyranoside (14) in quantitative yield as an amorphous solid, [α]D - 72.8� (c 1.0, water). This compound and the 5-hydrazinocarbonylpentyl analog were used to prepare the following Lewis-a antigens [Lea -O(CH2)8 CONH]n -BSA where n can have any of the following values 11, 22, 30, or 45; [Lea -O(CH2)8 CONH]28 -HSA; [Lea -O(CH2)8 CONH]15 -polylysine; [Lea -O(CH2)8 CONH(CH2)2 NH].sub. 10 -dextran; [Lea -O(CH2)5 CONH]n -BSA where n was 10 or 30; [Lea -O(CH2)5 CONH]25 -HSA by the acyl azide method (cf example XIII). The abbreviation Lea as used above stands for the Lewis-a trisaccharide determinant: ##STR11## Goat antiserum prepared against the natural Lewis-a blood group substance was found to form a precipitate with the above Lea antigens.
Using the above Lea antigens it was possible to examine the effect of factors such as length of the bridging arm and level of hapten incorporation on the immune response to raise antisera specific for the Lewis-a blood group. Antibody levels of sera raised in rabbits to the Lea -BSA antigens possessing C6 and C9 aglyconic "bridging arms" suggest very little variation due to the 3 methylene difference between the two antigens, especially at incorporations of 30 haptens/mole of BSA. At 11 haptens/mole of BSA the Lea -BSA antigen with a C9 bridging arm gave titers averaging about 50% higher than the C6 analog. The level of hapten incorporation also affected antibody production. For rabbits it appeared that an incorporation of about 22 moles of hapten/mole of BSA gave an optimum response in terms of antibody level and specificity. At lower levels of hapten incorporation (˜11 moles/mole BSA) increasing amounts of antibody in the sera had a specificity for the carrier, while at higher levels of incorporation (˜30 moles/mole BSA) antibody titers were generally lower.
The specificity of the antisera was demonstrated by immunodiffusion reactions. Rabbit antisera raised to Lea -BSA antigens showed a strong line of precipitation with all the artificial Lea antigens and the natural Lewis-a blood group substance and a very weak line of precipitation was shown with BSA. No line of precipitation was seen with HSA, polylysine or dextran.
To further explore the extent of cross reactions between antibodies raised to the artificial Lea antigens and human Lewis-a blood group substance and to obtain larger amounts of anitsera, a goat was immunized with [Lea -O(CH2)8 CONH]30 -BSA. The antisera obtained showed the expected cross-reaction with human Lewis-a blood group substance and after appropriate treatment effectively agglutinated human blood group Lewis-(a+b-) red cells and did not agglutinate Lewis (a-) red cells. The goat preimmune sea treated in the same manner did not agglutinate Lewis-a red cells.
Artificial antigens bearing the H(O) determinant ##STR12## were prepared from compound 9 by the methods described in example XIV. Five rabbits underwent immunization with the anitgen [H(O)]22 -BSA. On immunodiffusion, the antisera reacted weakly with BSA but strongly with the artificial antigens and human H substance.
Goat antisera were prepared against the natural blood substance H. The artificial antigen was found to form a precipitate with the resulting antiserum when tested by immunodiffusion. Thus, it is apparent antiserum specific for the terminal trisaccharide unit of the type I H-determinant was successfully raised.
Artificial antigens bearing the B determinant ##STR13## were prepared from compound 10 by the methods described in example XIV. It was found that the artificial B antigens strongly inhibited agglutination of B cells by human anti-B sera. Goat antisera prepared against the natural blood group B substance was found to form a precipitate with the artificial B antigens.
Porous glass beads (100-200 mesh, nominal exclusion limit 1000 A) were "aminated" using 3-aminopropyltriethoxysilane under standard conditions.11 The amine content was 69 μM per gram as estimated by the procedure of Esko et al.12
The Lewis-a hapten precursor (10 mg, 14.3 μM) (8-hydrazinocarbonyloctyl 2-acetamido-2-deoxy-4-O-(α-L-fucopyranosyl)-3-O-(β-D-galactopyransoyl)-β-D-glucopyranoside) was converted to acyl azide (cf example XIV) for coupling to 5 g of the "aminated" glass beads. The excess amino groups were then acetylated by treatment for 20 min at room temperature with 5% acetic anhydride in aqueous saturated sodium bicarbonate. The beads were then washed with water and air dried to provide immunoabsorbant GB-Lea -1.
When GB-Lea -1 (50 mg) was added to 250 μl of anti-Lea -BSA rabbit serum, the absorption of Lea antibody was complete within 30 min as estimated by immunodiffusion. Using 25 mg of the absorbant, the antibody removal took near 3 hours. With 10 mg, a small amount of residual antibody could be detected after 5 hours.
The above antiserum had been raised against the artificial Lewis-a anitgen [Lea -O(CH2)8 CONH]30 -BSA. The maximum precipitation against [Lea -O(CH2)8 CONH]28 -HSA as test antigen was 277 μg of antibody per 50 μl of antiserum.
The immunoabsorbant GB-Lea -1 was also tested against an anti-Lewis-a serum raised in a goat using natural Lewis-a blood group substance. The presence of 60 mg of the absorbant in 250 μl of this antiserum caused disappearance of the antibody in the supernatent serum in less than 45 min.
No discernible antibody absorption occurred when N-acetylated "aminated" glass beads were used as control.
An immunoabsorbant specific for anti-H antibodies was prepared by coupling compound 9 to glass beads (cf example XVII). This immunoabsorbant was able to absorb from sera antibodies raised to both artificial H antigens and natural H substance.
An immunoabsorbant specific for anti-B antibodies was prepared by coupling compound 10 to glass beads (cf example XVII). This immunoabsorbant (GB-B-1) was able to absorb from sera antibodies raised to both artificial B antigens and natural B substance. This immunoabsorbant also removed anti-B antibodies from typing sera.
Using a panel of fresh B cells suspended in saline the test serum gave positive agglutination to a dilution of 1 to 32. Under the same conditions H(O) cells showed no agglutination. The immunoabsorbant, GB-B-1, (50 mg) was added to 250 μl of sera after 45 min 150 μl of sera was withdrawn and tested against B cells suspended in saline. There was no agglutination of the cells indicating the anti-B antibodies had been removed.
No discernible antibody absorption occurred when N-acetylated "aminated" glass beads were used as control.
To prepare a B immunoabsorbant having a polyacrylic support, a resin containing carboxylic acid groups on an acrylic polymer lattice (10 ml) was treated with a mixture of ethylene diamine (21 g), 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate (15 g) in 100 ml of water for 24 h (the pH of the solution was maintained at 5 by the addition of concentrated hydrochloric acid). The resin was filtered off, washed with water and air dried.
N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline mediated condensation of 8-hydroxycarbonyloctyl 2-O-(α-L-fucopyranosyl)-3-O-α-D-galactopyranosyl-β-D-galactopyranoside (10 μM/ml resin) (prepared from 10 by hydrolysis of the ester) with the "aminated" resin, followed by N-acetylation of the unreacted amino groups gave the desired B immunoabsorbant. This immunoabsorbant also selectively removed anti-B antibodies from B antisera.
A B immunoabsorbant having an agarose matrix was also prepared by condensing 10 with "aminated" agarose.
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|Dec 30, 2014
|Glycorex Transplantation Ab
|Device for bio-affinity material
|Feb 7, 2001
|Feb 5, 2004
|Kurt Nilsson
|Oligosaccharide supports for e.g. removal of antibodies from blood
|Dec 15, 2000
|May 6, 2004
|Karl-Anders Karlsson
|Novel helicobacter pylori-binding substances and use thereof
|Oct 13, 2004
|Apr 13, 2006
|Dominique Charmot
|Toxin binding compositions
|Dec 15, 2005
|May 4, 2006
|Nilsson Kurt G L
|Device for bio-affinity material
|Oct 13, 2005
|May 11, 2006
|Ilypsa, Inc.
|Toxin binding compositions
|Aug 15, 2007
|Nov 29, 2007
|Glycorex Transplantation Ab
|Device for bio-affinity material
|Dec 23, 1996
|Aug 22, 2006
|Procur Ab
|Galactopyranosides and their use
|Dec 5, 2013
|Mar 26, 2014
|Synthetic method for regioselectively-acetylated methyl-D-pyranosides
|Dec 5, 2013
|Nov 4, 2015
|Aug 13, 2012
|Feb 19, 2014
|Fundaci� Institut d'Investigaci� Biom�dica de Bellvitge
|Methods and reagents for prevention and/or treatment of transplant rejection
|Mar 5, 1981
|Sep 17, 1981
|G Kaellenius
|Compositions for therapeutic or diagnostic use in connection with bacterial infections,and their use
|Oct 15, 1992
|Apr 29, 1993
|The Scripps Research Institute
|Production of fucosylated carbohydrates by enzymatic fucosylation synthesis of sugar nucleotides; and in situ regeneration of gdp-fucose
|May 24, 1993
|Dec 9, 1993
|Alberta Research Council
|Reducing inflammation by time dependent administration of oligosaccharides glycosides related to blood group determinants
|Jun 16, 1995
|Dec 28, 1995
|USE OF βGalNac(1-4)βGal CONJUGATES TO TREAT CANDIDA INFECTION
|Jun 16, 1995
|Mar 14, 1996
|Synthetic Peptides Inc
|USE OF βGalNac(1-4)βGal CONJUGATES TO TREAT CANDIDA INFECTION
|Dec 23, 1996
|Jul 3, 1997
|Procur Ab
|Galactopyranosides and their use
|Feb 7, 2001
|Aug 16, 2001
|Glycorex Transplantation Ab
|Oligosaccharide supports for e.g. removal of antibodies from blood
|U.S. Classification
|536/116, 436/543, 530/413, 536/18.2, 536/119, 530/807, 435/7.25, 536/17.9, 536/4.1, 536/53, 530/387.5, 536/115
|International Classification
|Cooperative Classification
|European Classification
|Apr 26, 1996
Owner name: ALBERTA RESEARCH COUNCIL, CANADA
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:CHEMBIOMED LTD.;REEL/FRAME:007908/0202
Effective date: 19951207


text:
Digital radio has been marketed for home use for a couple of years now, but it's only just making its way into cars sold in Australia.
As with mobile phones – all of which are now digital – the promise is of clearer reception and better sound quality. Digital radio also allows users to record snippets of programs to allow it to be rewound, much the same as modern TV tuners allow recording of programs. It's also handy if your favourite song comes on as your phone rings or one of your passengers decides to divert your ears.
Depending on your location there are also up to 22 additional, digital-only stations, many of which are themed, such as '80s or chill. Some established stations are also using it to effectively rebrand themselves to different target markets.
But digital radio is currently only available in Sydney, Melbourne, Brisbane, Adelaide and Perth, with Canberra and Darwin also running trials.
Digital radio has a fight on its hands, though. Streaming of radio stations from anywhere in the world is increasingly popular thanks to easy-to-use smartphone apps and new digital audio players, such as Sonos.
Digital radio DAB+: Availability
While some luxury models have offered it for a year or so, Toyota has become the first to put it in a mainstream car, with top models of its new Camry fitted with a so-called DAB+ (Digital Audio Broadcasting Plus, with the + an improvement on DAB previously used in places like Europe) tuner. Digital radio is also making its way into the Lexus range, starting with the new GS, which has a fantastic sound system to show it all off.
But its progress into other luxury marques is slower; it's offered as an option on some top-end models and not available yet from some brands. Audi, for example, has it standard on some A8 limousines but makes it a packaged option on A6 and A7s, while BMW makes it a standalone option on some of its newer models, with prices ranging from $385 to $900. BMW has found take up rates of between 4 and 13 per cent on its latest 1- and 3-Series models.
Aftermarket digital radio systems are also sneaking into auto accessory stores and audio retailers, providing options for the vast majority of cars without digital radio.
Digital radio DAB+: Sound quality and features
When in range (more on that later) the audio quality of the DAB+ tuner is noticeably better. The Toyota and Lexus models with DAB+ also have traditional AM and FM radio tuners, allowing a direct comparison between old and new.
On the regular FM station sound quality is respectable, but it takes a noticeable leap when you flick over to digital radio. Higher frequencies are much clearer and better defined, while bass tones are smoother and more pronounced. And when there's a break in a song or talking there's none of the hissing and turntable-like scratching that we've learned to tolerate on regular radio.
It won't take an audiophile to spot the very noticeable difference between regular and digital radio, provided your car's speakers are up to the task.
When running on AM the difference is even more pronounced. The muffled sound associated with the frequencies dominated by talkback stations gives a much crisper, clearer sound that makes presenters feel closer and more life-like.
Sound quality is helped by the overall system in the Camry and Lexus GS, which are a step up on many more expensive luxury cars. It's an impressive audio set up that's topped off by digital radio.
It's also worth noting that the coding and decoding of the signals leads to a delay of a few seconds when relying on the digital signal. So if you're alongside a car listening to live sport in AM or FM your oohs and ahhs might be fractionally behind the times.
One nifty feature is the Time Shift function. Press a button and you can use the tuner knob to rewind the program in 10 second blocks. The only trick is you have to be already tuned in to the station to perform the time shifting – and you can't record another channel, at least not on the Toyota and Lexus systems we tested. Still, it's handy if you want to replay a song, pick up a news snippet you missed or if you want to chat to someone but still hear the full radio broadcast.
Digital radio DAB+: Reception
This is the big test for digital radio, particularly in Australia where we have vast distances and much of the country with little or no radio reception. Many imported cars (Volkswagens spring to mind) don't have great reception in Australia, with crackling and scratchiness in even major centres.
Local manufacturers such as Ford and Holden, on the other hand, have traditionally placed more emphasis on long distance radio reception – and with good results.
Granted, most motorists spend time closer to major centres and near the coast, but even that poses challenges with reception.
With digital radio, because it's transmitting in digital (lots of 1s and 0s), you've either got full reception or nothing at all. So when you're hearing something sound quality is at near-CD levels, albeit depending on the quality of the rest of the system – and how it is transmitted.
Around town the DAB+ generally offers crystal clear reception. However, in carparks and tunnels it can cut out completely, which is frustrating if you're listening to the news or the final stages of a sports broadcast. Tunnels currently retransmit AM and FM radio frequencies but are not yet geared up for digital radio. It'll be up to individual tunnel operators as to when (or if) they upgrade their repeaters.
We also found digital radio also performed well in the heart of the city. We didn't drive down every Sydney laneway, but reception was clear during a few runs through town, while our Melbourne counterparts reported similarly impressive reception. There have apparently been some issues with "shielding" of the signal, particularly in Melbourne, but repeaters have been put in to reduce the chances.
The suburbs
Reception in even hilly suburbs proved almost flawless. Only occasionally did the reception skip briefly, similar to a CD skipping. It was mildly annoying but by no means a common occurrence during my time with the cars, and it was far better than the more frequent crackles from regular radio.
However we also found some holes in reception, usually only lasting for a few seconds. So every time you drove down that particular street the reception cut out. These cases were rare, though, and the overall clear reception was a bigger bonus.
Out of town
This is where digital radio loses points. Even on the outskirts of Sydney (Richmond in the north-west was our test bed, while we also had a car head south on the Hume Highway) the digital tuner fumbled, completely cutting out for a few seconds on a couple of occasions.
It would be more frustrating had I been listening to live sport at a crucial moment in the game, although if it were that important you could also flick back to FM when you're in dodgy range.
Once you reach the far outskirts of town it cuts out completely.
Digital radio is also useless in the country – for now. On a longer run out of Sydney the signal cut out completely and that was it. There are plans to extend the current licence spread beyond the major centres, although don't expect changes overnight.
There's life left in the wireless yet, at a price. It's great around major cities but loses its lustre in tunnels and out of town.

subset: commoncrawl


text:
MURDER AT THE GALLOP, 1963. Original 1 Sheet (27 x 40" / 69 x 104 cm) folded Movie Poster starring Margaret Rutherford, Robert Moreley and Flora Robson. Directed by George Pollock this classic "whodunnit" murder mystery was based on Agatha Christie's After the Funeral and stars Margaret Rutherford as Miss Marple. Some of England's most refined and dry witted actors star in this movie which was the second of four movies featuring Miss Marple."The funniest woman alive – Time magazine".
This folded poster is in very good C8 original condition with no significant condition issues; there is a censor stamp in the upper section of the poster. Linen backing for an additional $80 upon request. For more information on linen backing please click here
Condition C8. For Sale $125
To see this poster and others visit our gallery the Limited Edition in old town Katy, Texas. Providing poster sales, Linen Backing, Restoration and Framing services to the Houston, San Antonio, Austin and Dallas regions since 1994.
Note: Important if you are an international buyer please Contact Us for an exact shipping quote before you make payment. International buyers expect to pay approximately $20 for a folded poster and $65 for a linen backed one sheet movie poster.
The Limited Edition Art and Antiques was established in 1994 in Historic Downtown Katy, Texas and features an eclectic variety of antiques, ephemera and original folk art created by the proprietor Elizabeth Proctor.
We specialize in the sales, linen backing, restoration and framing of vintage posters, We purchase collections and offer consignment services for your collections.
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"About Limited Edition"
Gallery Phone: 281-391-1993
EMail: [email protected]
Location: 5717 Second Street Katy, TX. 77493
Find Us – Google Map


text:
0800 043 2242
0333 577 2242
You´re ready to start learning to drive in Alvechurch and want to the best chance of passing your driving test on the 1ST attempt? That pretty much goes without saying! But how you going to give yourself the best chance of achieving this?
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Our driving instructors are selected to ensure they have the right training skills and techniques to give every pupil expert training. Everyone learns at a different pace and will require different training techniques in order to get the most from their driving lessons.
At The John Michael Driving School West Midlands, your lessons are tailor fitted to best suit you to ensure you get the absolute most from every single driving lesson. This ultimately results in fewer lessons required and saves you money.
What learning style are you? Why not try our questionnaire to find out?
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text:
A COUNCIL chief will give a talk at a residents' meeting.
Park Residents' Association meets at 7.15pm on Wednesday, March 28, at Hartlepool Cricket Club, in Park Drive.
Nicola Bailey, Hartlepool Borough Council's acting chief executive, will give a presentation about what is happening within the council and will also answer questions.


text:
With Ovum analysts forecasting that ITU's ultra-fast copper technology, G.fast, will support nearly 29 million subscribers by 2021, interoperability between chipsets (for Customer Premises Equipment and Distribution Point Units) is a complex but absolutely vital component for the industry when deploying the technology.
This is the message that I will be delivering on behalf of the Broadband Forum at next week's ITU Telecom World 2016 in the panel discussion: "Gearing up for Ultra-High Speed Networks," where I will be joined by fellow panelists Reinhard Scholl, Deputy Director of the ITU Telecommunication Standardization Bureau, and David Bessonen, Senior G.fast Engineer at Telebyte, Inc.
The discussion we will have is taking place at a time when consumer demand for high-speed networks, including gigabit access, is skyrocketing.
It is all very well having the technology, but if standards are not implemented from the start, there will always be limitations. That is why the work of ITU's Telecommunication Standardization Sector (ITU-T) and the Broadband Forum is so vital as we enter the Gigabit Era.
Why G.fast interoperability is so important
As the industry continues to embrace G.fast, its success in broadband networks absolutely depends on service providers having a wide choice of equipment and this is where interoperability comes in.
RELATED: First commercial G.fast launch in Europe
The Broadband Forum's collaboration with the University of New Hampshire InterOperability Laboratory aims to address exactly this issue and a huge amount of work is going on at the moment to achieve true G.fast interoperability.
The Forum's work on G.fast builds upon the progress made by the ITU-T, which approved its broadband standard designed to deliver access speeds of up to 1Gbit/s over existing telephone wires in 2014, in conjunction with the Forum's FTTdp architecture project. ITU-T recently doubled the access speeds achievable with G.fast, raising the bar to 2 Gbit/s.
RELATED: G.fast broadband doubles in speed with ITU standard, raising the bar to 2 Gbit/s
With collaboration like this, it will be entirely possible for the industry to achieve the high-speed connectivity being sought after by consumers and we are already beginning to see evidence of this.
Demos of cross-chipset interoperability
At Broadband World Forum, in London, the world's first cross-chipset interoperability demo took place.
A similar demonstration will be held at ITU Telecom World, where Forum member Telebyte, the leader in G.fast Physical Layer testing, will provide a live demonstration of G.fast in a real-world test environment, including products from its ID-337 solution group – based on the Broadband Forum's ID-337 G.fast Certification Test plan.
Such work pays testament to the work of the Forum and the ITU and with this continued approach, I see no reason why Ovum's forecast of 29 million by 2021 cannot be surpassed.
Mark Fishburn
Mark Fishburn is Director, Strategic Marketing at the Broadband Forum. Mark is also President of strategic marketing company, MarketWord formed in 2006. Previously, he was Chairman of the Board, Metro Ethernet Forum, Chairman of the Board of the 10 Gigabit Ethernet Alliance, and board member of the Gigabit Ethernet alliance and he was instrumental in the creation of the MEF's Carrier Ethernet, CE 2.0 and 2014 strategies. He has won more than 25 industry awards and studied BSc. Special Mathematics at the University of London.


text:
What dya reckon??(44 Posts)
Surreymommy Mon 08-Apr-13 23:52:23
openerofjars Tue 09-Apr-13 11:49:45
wendybird77 Tue 09-Apr-13 12:05:43
Surreymommy Tue 09-Apr-13 13:24:09
nipersvest Tue 09-Apr-13 13:28:46
myron Tue 09-Apr-13 13:33:34
myron Tue 09-Apr-13 13:38:46
7to25 Tue 09-Apr-13 13:39:30
Surreymommy Tue 09-Apr-13 14:38:18
MammyKaz Tue 09-Apr-13 14:56:47
kitsmummy Tue 09-Apr-13 15:04:34
Surreymommy Tue 09-Apr-13 16:25:13
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bonzoed Tue 09-Apr-13 18:02:17
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13 Reasons to Use (and keep using) Visual Schedules and 4 Reasons Why They Work
We all like to have an idea of what is going to happen in our day. Children with autism miss many of the language and social/behavioral cues that help the rest of us understand what is happening. Children can become anxious, resistant and may misbehave simply because they have no way to anticipate the events that come upon them each day. Here are some ideas that may help with that.
Visual Schedules Help the Staff:
- In the long run, providing a child with autism a visual schedule and helping him/her learn to use it almost always makes the teacher's job easier, not harder. Once the child has a visual schedule, his or her behavior will very often settle. Thus, a schedule does not make the child appear to be different; it helps him appear to be more the same as other children. Visual schedules increase on-task behavior and therefore increase academic learning time.
- Visual schedules quickly inform the child about the day's and/or activity's expectations. Visual schedules can help provide independence by removing the need for the student to ask others (particularly adults) about what schoolwork has to be done. The fact that the student functions more independently ("normally") can increase his or her acceptance by peers.
- Because they provide a means for the child to anticipate upcoming events, visual schedules help to ease the student through transitions. Visual schedules provide a means by which adults can systematically introduce the element of change into a student's day. Thus, a schedule is a means of reducing rigid behavior. In fact, used properly, they promote flexible behavior. Once the student knows how the visual schedule works, he or she will be less dependent upon consistency of staffing in order to function. Teachers or support people can be absent and the day won't "fall apart" for the student.
- Visual schedules (written or pictured) work so effectively that you may be tempted to offer their advantages to other students in the class who don't have an autism spectrum disorder, but who also could use help in learning to plan and organize themselves and their day.
- Many academically capable children, helped in their young years by the use of visual schedules, will later be able to transfer their understanding of agendas to computerized day planner systems.
Visual Schedules Help the Kid:
- The use of visual schedules capitalizes on the visual learning/memory strength that most children with autism have. The child will often remember what s/he has seen on the schedule, although what they have been told may be forgotten.
- The use of visual schedules is one support strategy that can provide the organization, structure, and predictability that are so essential to autistic individuals. When they lack such supports, autistic individuals find the world to be highly unpredictable and confusing.
- Visual schedules teach the importance of organization in a day. The child who learns to help prepare a visual schedule is developing a habit that will promote good organizational methods throughout life. Often helping to prepare the schedule daily gives the child a sense of some autonomy and control of the daily events in a world of confusing demands.
- Visual schedules may minimize the need to write as the daily agenda is being established; handwriting is often difficult for children who experience autism.
- Many academically capable children, helped in their young years by the use of visual schedules, will later be able to transfer their understanding of agendas to computerized day planner systems.
- The use of the visual schedule can be extended to teach the child long-range planning formats for multi-stage or multi-task assignments such as term papers or other semester length projects.
- The schedule can help the child learn patience and persistence. The alternating pattern of choice and work items within the visual schedule teaches the child that there are some required tasks within the day that are followed by more preferred activities. This "delayed gratification" is widely expected in our society, especially in work settings. The visual display may also serve as a reminder to a child that a preferred activity is only a few steps away after the completion of a non-preferred task.
- When the student learns to remove icons or check off completed tasks he is able to see his progress. This representation of accomplishment helps to build a sense of success and, thus, to improve self-esteem.
And Here are a Few Reasons Why Visual Schedules Work:
- Children with autism spectrum disorders often have difficulty sequencing ideas and procedures. The visual schedule demonstrates sequencing and promotes an understanding of it on a daily basis.
- The use of visual schedules capitalizes on the visual learning/memory strength that most autistic children have. Visual schedules are tangible and non-transitory; auditory language is fleeting. A child may not have heard/processed what the teacher said, but still has the schedule for reference.
- Some children with autism have obvious difficulties with language. Others may appear to have good or even excellent language skills. However, even the capable child with autism can have difficulties understanding spoken language for the following reasons:
- Slow processing time may interfere with the child's ability to really "take in" verbal instructions and much of what is said may be missed.
- Children on the often spectrum are often dealing with sensory malfunction which may make it almost impossible for the child to attend to what is being said.
- Children with autism are almost always highly stressed by their lack of understanding of the social milieu in which they must function. The child may be so overwhelmed with attempting to deal with social events and expectations, that he or she may entirely miss language-based input. Stress can even be brought on by the experience of being physically close to other children. The child is least likely to process auditory verbal information when he or she is upset, worried or angry. There is a greater likelihood that s/he will be able to process visual information.
- Children with autism often have poor sleep patterns. Their sleep maybe brief or interrupted and they will, therefore, often be too tired to be attentive.
- Children on the autism spectrum may have great challenges in understand the pragmatics of communication. Gestures, vocal intonation, facial expressions, conversations pauses and emphases may be lost on these children.
- There is a tendency among children on the spectrum to take words literally. What they hear may not be mediated by an intrinsic understanding of the social situation, nor by an understanding of the personality and intent of the speaker. In attempting to understand something that has just been said, even the bright child may miss other parts of the communication.
- Many children with autism have a poor sense of time and have difficulty understanding the vocabulary of time and order (e.g. before, after, then, yesterday, first, second). These words are used daily however as the schedule is set up. Thus, use of schedules teaches time and time concepts and vocabulary directly, routinely and systematically.
This material is adapted from Brown, Margaret 30 Reasons to Use and to Keep Using a Visual Schedule or "Please Don't Take Away My Visual Schedule." (2002) Provincial Outreach Program for Autism and Related Disorders in British Columbia.
©Project ACCESS – 2017 – Edna Smith, Ph.D.
This fact sheet and other #AutismResources, #AutismTraining and #AutismSupportServices information may be found on our website: http://education.missouristate.edu/access/


text:
A new killer is stalking the streets of London's East End. Though newspapers have dubbed him 'the Torso Killer', this murderer's work is overshadowed by the hysteria surrounding Jack the Ripper's Whitechapel crimes. The victims are women too, but their dismembered bodies, wrapped in rags and tied up with string, are pulled out of the Thames - and the heads are missing….
©2013 Sarah Pinborough (P)2013 W F Howes Ltd
"Pinborough really knows how to drag the reader into her books." (Simon Kernick, bestselling author of Relentless)
"This is deliciously inventive stuff, delivered with the narrative nous and sense of atmosphere that lifts Mayhem above the raft of many similar novels." (Crime Time)
"Just Wow."
I'm not normally one to write a review, but I have to say, I was just blown away by the combination of Sarah Pinborough's clever spin on the Ripper folklore and Steven Crossley's spot on narration. Seriously, if you like a story full of murder and mayhem and a little bit of the supernatural, do yourself a favor and read and/or listen to this one.
"A Parallel Ripper"
After reading other reviews I approached this book with interest but low expectations. The book began well and grew more interesting the more I listened.
As often as I've read books written set in this time and place I was pleased to find the author set the mood and events with a fresh eye. She skillfully sidesteps a host of clichés. The characters are complex and interesting. The protagonist set with all manner of conflicts. Yes, well done. It is an excellent read/listen. I will listen/read the sequel to this book and other books by this author.
"A Supernatural Mystery"
If you like stories about the supernatural you will love this book, if you think you are getting a mystery story set in 19th Century with Jack the Ripper thrown in - you will be very disappointed. The book focuses on another set of real murders of the same period: The Thames Torso Murders, also called the Embankment murders. These murders, however, were overshadowed by Jack the Ripper and have never really entered into stories about the period as Jack the Ripper did, like those of Jack the Ripper they were never solved.
The Author uses real people from the period, chiefly Dr. Thomas Bond, who was the police surgeon at the time. He is the main character in the book. While Dr. Bond does give some description of the Ripper Murders and the Ripper victims, his real focus is on the Torso Murders.
The Torso murders took place between 1887-1889. Torsos of young women were washing up along the Thames embankment. The bodies were headless and their limbs were hacked off. The limbs were found separately packaged, also washing up along the Thames. These murders were never solved, and since the heads were missing, the victims were never identified.
This sounds like the start of a really good book - but alas it is not, at least for me. Yes it is very atmospheric in describing the London of the period. But I am not a fan of the supernatural and the author attributes the Torso murders to a man who has been "taken over" by a supernatural being. So a lot of the book is consumed not with the mystery, but of finding the individual who was taken over by this supernatural being and when did it happen.
The writing style does not lend itself to a smooth, flowing story. The author uses several different characters to tell the story, although Thomas Bond is the main figure. The chapters in the book alternate between the point of view of different characters -- some written in the first person, other in the third person, and chapters which are newspapers accounts of the crimes. Also the book does not flow chronologically - but tends to skip around between 1887 and 1889, depending on the POV of the character in the chapter. The listener will need to pay close attention to the chapter titles in order to follow this book, as the date is always given as part of the chapter heading.
The Narration is excellent and helps carry the reader along through the story. However, the narrator cannot overcome the long periods of boredom as we explore a character's thoughts and internal musings. Again if you like supernatural/fantasy mysteries you will love this book. I did not.
"Doesn't make me want to start reading this genre"
This is a dark, grim, paranormal, mystery taking place in London during the late 19th century. consciously parallels Jack the Ripper series of murders as if demonic forces are at work.
As a period piece it works well showing how police investigations worked back then and understand why Jack the Ripper was hard to catch.
However, I have issue with the structure or narrative of the story. Starts off as a mystery but reveals the villain before you have any real chance of guessing. The whole story is slow and drawn out which makes the sudden ending feel anti-climatic. A lot of side questions were left unanswered and not in a prepare for sequel kind of way.
I don't normally read paranormal or horror. This book does not make me want to start.
As a side note: The main character is named Dr. Bond? Seriously, Every time the name is mentioned I was taken out of the story. Kept waiting for him to say "The name's Bond, Dr. Bond.
"Incredible read."
Mayhem is an excellent book. The narrator is excellent very well read with such enthusiasm and respect for the book.Sarah Pinborough is an amazing and excellent writer. You must read this book.
"Great narrator with a really tough job"
This was not a good book
I was not a fan of the book, the characters were one dimensional, uninteresting and annoying. The supernatural bit was unnecessary, not well formed. I relistened to several passages over a few times, because it was so boring, my mind kept drifting off . I kept at it, thinking it was going to get more interesting. It didn't, the ending was abrupt, predictable, and a dissapoointmenr
I listened to this book because he was the narrator-- I usually like his work, but I don't think there was much he could do with this one
All of them
Total waste of a credit, can't believe she wrote more in this series
"Stick with it"
Glad I stuck with this one through the slow but necessary setup. Pinborough has added a supernatural twist to the well-travelled trail of the Ripper murders (actually the lesser-known concurrent "Torso Murders" which also took place near and around Whitechapel). She does a nice job of setting us up with a reliable, skeptical witness/protagonist who resists the spooky truth, bringing to mind the best of Bram Stoker and Steven King. Steven Crossley does a fine job narrating.. Good enough to get me to check out the sequel.
"beware the evil spirit"
This a good story, well written and well narrated. The only drawback, greater for some, is the prominent role of an evil spirit inhabiting the body of a man. As far as evil spirits go, it's not over done. If you don't mind, or indeed, if you like, some fantasy in your books, buy it.
"A great listen"
This was the first time I've 'read' anything by Sarah Pinborough or heard Steven Crossley read and I will be looking out for both of them again - a great combination of gripping story and excellent narration.
"Silly/totally incomprehensible 'Monster'"
This is quite enjoyable at times, however, the supernatural element is vague and just too weird to truly be a great novel. Think of a very weakly described Dr Jeckyl type, it spoils what could have been a great idea. Better than some but by no means Stoker!
Listened to Murder as well. Pretty much the same. Never again, though!
"good book"
quite dark, but well read. great characters. looking forward to the next one. A+++ thanks
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