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WO2007001283A2 - Synthese chimique de polyglucosamines et de polygalactosamines de faible masse moleculaire - Google Patents

Synthese chimique de polyglucosamines et de polygalactosamines de faible masse moleculaire Download PDF

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WO2007001283A2
WO2007001283A2 PCT/US2005/022116 US2005022116W WO2007001283A2 WO 2007001283 A2 WO2007001283 A2 WO 2007001283A2 US 2005022116 W US2005022116 W US 2005022116W WO 2007001283 A2 WO2007001283 A2 WO 2007001283A2
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product
solution
thioglycoside
polyhexose
added
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WO2007001283A3 (fr
Inventor
Subramaniam Sabesan
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EIDP Inc
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EI Du Pont de Nemours and Co
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Priority claimed from US11/154,457 external-priority patent/US7485718B2/en
Priority claimed from US11/154,193 external-priority patent/US20060286149A1/en
Application filed by EI Du Pont de Nemours and Co filed Critical EI Du Pont de Nemours and Co
Publication of WO2007001283A2 publication Critical patent/WO2007001283A2/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
    • C07H5/04—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to nitrogen
    • C07H5/06—Aminosugars
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0087—Glucomannans or galactomannans; Tara or tara gum, i.e. D-mannose and D-galactose units, e.g. from Cesalpinia spinosa; Tamarind gum, i.e. D-galactose, D-glucose and D-xylose units, e.g. from Tamarindus indica; Gum Arabic, i.e. L-arabinose, L-rhamnose, D-galactose and D-glucuronic acid units, e.g. from Acacia Senegal or Acacia Seyal; Derivatives thereof

Definitions

  • the present invention is directed processes for chemical synthesis of low molecular weight polymers of galactosamine and glucosamine. Also provided are a novel derivatized monomer building block that is useful for the processes, and a process for joining the monomer building block in beta linkage. The processes disclosed herein allow stepwise addition of a monomer to lengthen the polymer chain by one unit at a time.
  • Chitosan which is a ⁇ 1 ,4-linked glucosamine polymer, is known to provide antimicrobial activity useful in a wide range of applications including in food preparation and packaging, in personal hygiene such as in garments and personal care articles, and in locations with high potential for microbial contamination such as bathrooms and hospitals.
  • oligosaccharides of chitosan derivatives were found to have anti- inflamatory properties providing potential use as a pharmaceutical, dietary supplement, or cosmetic component for treatment of inflammation (WO 03026677).
  • Polygalactosamines with alpha linkages are found in nature and ⁇ -galactosamines in an acylated form are found as structural components of chondrointin sulfate and dermatan sulfate, compounds that are a part of the proteoglycan structure found in cartilage.
  • Low molecular weight polygalactosamines may have chondroprotective effects and/or other important biological properties, and thus may be useful pharmaceutically.
  • Chitosan is the commonly used name for poly-[1 -4]- ⁇ -D- glucosamine.
  • Chitosan is chemically derived from chitin, which is a poly- [1-4]-/?-N-acetyl-D-glucosamine, and which, in turn, is derived from the cell walls of fungi, the shells of insects and, especially, crustaceans.
  • Chitin is treated with strong alkalis to remove acetyl groups producing chitosan.
  • chitosan can vary in the degree of deacetylation.
  • Chitosan preparations that are obtained in this manner, and are commercially available at low cost (from, for example, Primex Corporation (Norway), Biopolymer Engineering, Inc. (St. Paul, MN), Biopolymer Technologies, Inc. (Westborough, MA), and CarboMer, Inc.
  • Thioglycosides are known to be shelf stable monomers for use in the synthesis of oligosaccharides (Fugedi et al., Glycoconjugate Journal, 1987, 4:97-108). However, thioglycosides generally have to be activated with strong electrophiles prior to coupling to sugar hydroxyl groups.
  • Thioglycoside activation procedures are known and are described in US 20040019198, but application of the activated thioglycosides for the efficient synthesis of multi-gram quantities of ⁇ -linked low molecular weight polymers of galactosamine and glucosamine is lacking.
  • One aspect of the present invention is a process for forming a glycosidic linkage between two hexoses, comprising: a. providing a suitably protected thioglycoside donor and a suitably protected glycosyl acceptor, both of which are hexoses; b. activating the thioglycoside donor using as activating agents an N-haloimide and at least about a 0.5 molar equivalent amount, to the glycosyl acceptor, of a perfluoroalkyl sulfonic acid in the presence of said acceptor; and c.
  • Another aspect of the present invention is a process of extending a chain of a polyhexose having a protecting group at a linkage position comprising: a removing the protecting group from the polyhexose linkage position to form a polyhexose acceptor; b providing a suitably protected thioglycoside donor; c activating the polyhexose and the thioglycoside using as activating agents an N-haloimide and at least a 0.5 molar amount of a perfluoroalkyl sulfonic acid; and d reacting the thioglycoside donor and polyhexose acceptor at a temperature from about -20° C to about -70° C; wherein the thioglycoside and the polyhexose form a beta linked polyhexose that has a length of x + 1 monomer units, wherein x is the length of the starting polyhexose.
  • R 1 and R 2 are each independently selected from H and Ci to C 2 0 alkyl, aryl, and aralkyl groups;
  • R 3 and R 4 are each independently selected from monofunctional acyl, bifunctional acyl, phthaloyl, trichloroacetyl, and tetrachlorophthaloyl groups;
  • R 5 , R 6 , R 7 and R 8 are each independently selected from Ci to C 20 alkyl, aryl, and aralkyl groups.
  • a further aspect of the present invention is a process of deprotecting a synthetic polyhexose product comprising treating the product at refluxing temperature with hydrazine and n-butanol such that substantially all protecting groups are removed in one step.
  • Another aspect of the present invention is a process of isolating a synthetic polyhexose product comprising purifying the product by selective extraction.
  • FIGURES Figure 1 is the 750 MHz Proton NMR spectrum in CD 2 CI 2 of an undecasaccharide derivative (product 27 in Example 22).
  • Figure 2 is the MALDI Spectrum of the synthetic undecasaccharide derivative product 27.
  • the present invention provides processes for synthesizing multi- gram quantities of low molecular weight polymers of galactosamine and glucosamine that are scalable for commercial use.
  • the processes allow the use of simple purification procedures and do not require cost prohibitive chromatographic separation procedures.
  • the low molecular weight polymers, called oligoglucosamines are made by efficient coupling of monomers that are stable to storage. Stepwise addition of a specific type of monomer, described hereinbelow and in copending patent application number CL 3152, to a growing polymer chain results in the synthesis of a defined chain length polymer, providing the ability to produce different preparations of molecules that are each enriched for a specific size polymer.
  • the processes also permit large-scale preparations of low molecular weight polymers of galactosamine and glucosamine that are enriched in a single size species.
  • antibacterial means bactericidal as is commonly known in the art.
  • the number of bacteria present after contact with an antibacterial material is substantially reduced from the number initially present.
  • the number of bacteria present is normally measured as colony forming units.
  • antimicrobial means antibacterial as well as having fungicidal and antiviral activities as is commonly known in the art.
  • shelf stable means that the compound remains intact with storage at room temperature and when exposed to moisture and air of laboratory storage conditions.
  • large scale refers to tens of grams to kilogram quantities of material.
  • low molecular weight polymer refers to a chain of monomer units that is greater than one unit and up to about 50 units in length. Oligomers are polymers with two to about 10 units. Therefore an oligoglucosamine, for example, is a type of low molecular weight polymer.
  • ⁇ -linkages includes 1 ,3-, 1 ,4-, and 1 ,6- linkages.
  • linkage position means the position of the carbon that is a part of the glycosyl bond. In 1 ,3-, 1 ,4-, and 1 ,6- linkages, the linkage position is 3, 4, or 6, respectively, on one glycoside and 1 on the linked glycoside.
  • non-linkage position means the position of a carbon which is not a part of the glycosyl bond.
  • 2, 3 and 6 positions are non-linkage positions.
  • glycosyl molecule which participates at the C-1 position in the glycosyl bond.
  • glycosyl acceptor means the glycosyl molecule which has a hydroxyl group at the position (either 3, 4, or 6) that will participate in the glycosyl bond and which connects through its oxygen to the C-1 glycosyl residue from the donor.
  • the glycosyl acceptor may be a single unit or a multiple unit chain that is a low molecular weight polymer.
  • suitable thioglycoside donor means a thioglycoside that has protecting groups at the positions that become non- linkage positions following formation of the glycosidic linkage. Protecting groups are used to prevent reaction at those sites.
  • suitable glycoside acceptor means a glycoside that has protecting groups at the positions that become non-linkage positions following formation of the glycosidic linkage. Protecting groups are used to prevent reaction at those sites.
  • enriched population means a population of polymers containing at least 80% of a single chain length polymer. An enriched population may result from a process of low molecular weight hexose polymer synthesis according to the instant invention, or through other processes. An enriched population may have more than 80% of a single chain length polymer, such as 85%, or in particularly efficient reactions, an enriched population may include 90% or greater of a single chain length polymer.
  • thioglycoside monomer can be very efficiently coupled to a glycosyl acceptor with least reactive groups of type represented by formula (II) by using activating agents generated from N-haloimides and an approximately equimolar amount of a strong protic acid.
  • the thioglycoside monomer is a novel compound represented by formula (I):
  • R 1 and R 2 are each independently selected from H and C-] to C 2 O alkyl, aryl, and aralkyl groups;
  • R 3 and R 4 are each independently selected from monofunctional acyl, bifunctional acyl, phthaloyl, trichloroacetyl, and tetrachlorophthaloyl groups; and R 5 , R 6 , R 7 and R 8 are each independently selected from C- j to
  • R 1 and R 2 are phenyl groups.
  • R 3 and R 4 are acyl groups derived from a phthaloyl unit.
  • R 5 is a p-toluyl group.
  • R 6 and R 7 are methyl groups.
  • R 8 is a tertiary butyl group-
  • glycosyl acceptor is represented by the formula:
  • R 1 and R 2 are each selected from H and C-j to C 2 o alkyl, aryl, and aralkyl groups; R 3 and R 4 are each independently selected from monofunctional acyl, bifunctional acyl, phthaloyl, trichloroacetyl, and tetrachlorophthaloyl groups; and R 5 is selected from C 1 to C 2 o alkyl, aryl, and aralkyl groups.
  • R 1 and R 2 are phenyl groups.
  • R 3 and R 4 are acyl groups derived from a phthaloyl unit.
  • R 5 is a methyl group.
  • a polymerization process according to the present invention can be illustrated as follows.
  • a unit, e.g, molecule, of monomer (II), the glycosyl acceptor, provides an initial unit onto which units of a monomer (I), the thioglycoside donor, are added to extend the polymer chain.
  • Monomers (I) and (II) can be made from D-glucosamine hydrochloride or D- galactosamine hydrochloride, which are commercially available.
  • D-glucosamine hydrochloride is derivatized with a phthaloyl group using phthalic anhydride to protect the amine (product 2 in Example 1).
  • the hydroxyl groups are then protected by acetylation (product 3 in Example 2), and the product is purified by crystallization.
  • a benzenethiol group is added to the 1 position (product 4 in Example 2) and the product is purified by washing with protic solvents.
  • the resulting product is deacetylated (product 5 in Example 2), benzoyl protecting groups are added at the 3 and 6 hydroxy! positions (product 6 in Example 2) and the product is purified by crystallization.
  • tBDMS t-butyldimethylsilyl
  • the combination of reactions and purifications is amenable to large scale preparation of the novel monomer (I), which is an embodiment of the instant invention.
  • the resulting monomer (I) provides a building block for the synthesis of low molecular weight polymers of glucosamine.
  • a galactosamine glycosyl donor can be used in the place of a glucosamine molecule as a starting point for synthesis of a galactosamine- monomer (I) in the same manner as described above for the glucosamine- monomer (I).
  • the resulting monomer provides a building block for the synthesis of low molecular weight polymers of galactosamine.
  • the amine can be protected with monofunctional acyl, bifunctional acyl, trichloroacetyl or tetrachlorophthaloyl groups and the hydroxy! groups can be protected with C 1 to C 2 O alkyl, aryl, or aralkyl groups as a part of an ester group.
  • the silyl group canbe any tri-substituted silicon, substituted with, for example, C 1 to C 2 o alkyl, aryl, and aralkyl groups.
  • Benzoyl groups are added to product 8 at the 3 and 6 positions creating monomer (II) ( Figure 1). Each of these individual steps is carried out using reaction conditions well known to one skilled in the art.
  • the resulting monomer (II) provides the initial unit onto which molecules prepared as for monomer (I) are added for the synthesis of a low molecular weight glucosamine.
  • the compound shown in Reaction 1 below (methyl 2-deoxy-3,6-di-O-benzoyl-2-phthalimido- ⁇ -D- glucopyraoside) represents one example of a monomer (II) type compound, which is a suitably protected glycosyl acceptor.
  • Galactosamine can be used in the place of the starting glucosamine molecule for synthesis of a galactose-monomer (II) in the same manner as described above for monomer (II).
  • the resulting galactose-monomer (II) provides the initial unit onto which molecules prepared as galactose- monomer (I) are added for the synthesis of a low molecular weight galactosamine.
  • glucosamine and galactosamine may be interchangingly used in the synthesis of monomer (I) and monomer (II) for the synthesis of a mixed composition oligosaccharide.
  • different monomers prepared from different hexosamines can be added alternately, or in any order, to prepare a mixed composition polysaccharide.
  • protecting groups can be used in the preparation of intermediates to monomer (II) or galactose- monomer (II).
  • the amine may be protected with monofunctional acyl, bifunctional acyl, trichloroacetyl or tetrachlorophthaloyl groups and the hydroxyl groups may be protected with with C 1 to C 2 O alkyl, aryl, or aralkyl groups as a part of an ester group.
  • the 4 and 6, or 3 and 4 positions are protected, respectively.
  • the intermediate products 2-8 leading to the synthesis of monomers (I) and (II) can be obtained by using the steps described above, which result in highly selective reactions.
  • the simplicity of the protecting groups used in the processes for synthesis of monomers (I) and (II) facilitate purification and chain extension, enabling the practical chemical synthesis of polyglucosamines.
  • benzoate groups are conventionally considered to be the least preferred protecting groups in oligosaccharide synthesis, due to their causing decreased reactivity of the glycosyl donor and acceptor (as disclosed, for example, in Zhang et al., supra), benzoate groups have been found to be ideally suited for the present synthesis methods.
  • Benzoate groups are used in the instant processes to permit ready crystallization of the product, thus facilitating simplified isolation of the product from impurities and allowing large-scale preparation of the products. It has been found that the use of increased levels of activating agents relative to the monomers improves coupling efficiency of the glycoside formation, thus making the synthesis commercially attractive.
  • the silicon protecting group in the monomer (I) serves as a convenient temporary protecting group that can be removed easily for chain extension.
  • glycosyl units can be conveniently added to the desired length, as described below.
  • the monomer based approach disclosed herein allows the use of low cost thioglycoside agents in excess amounts for near quantitative coupling efficiency in the glycosylation reaction and the ready removal of undesired by-products by simple solvent extraction, and makes it possible to prepare individual members of a family of low molecular weight polymers of glucosamine or galactosamine for biological testing and commercial use.
  • Each member of a family has a unique monomer unit length, with 1 unit being the smallest difference between two members.
  • Coupling of monomers (I) and (II), as well as coupling of an oligoglucosamine chain + monomer (I), is carried out using thioglycoside activating agents under saturating substrate concentration in the reaction.
  • the thioglycoside activating agents are generated from N-haloimides and strong protic acids.
  • N-halosuccinimides such as N- iodosuccinimide and N-bromosuccinimide can be used as activating agents in combination with strong protic acids such as triflic acid (trifluoromethanesulfonic acid) and other perfluroalkylsulfonic acids.
  • triflic acid trifluoromethanesulfonic acid
  • other perfluroalkylsulfonic acids such as triflic acid (trifluoromethanesulfonic acid) and other perfluroalkylsulfonic acids.
  • triflic acid alone is sufficient to activate the thioglycoside, the combined use of triflic acid and methyltriflate
  • thioglycosides are traditionally carried out with catalytic quantities of triflic acid or its salt in conjunction with a molar equivalent or excess of N-halosuccinimide. These conditions were found to be insufficient for efficient glycosylation of monomer (II), especially with a growing glucosamine chain, resulting in either incomplete reaction or no reaction occurring.
  • N-halosuccinimide at 1 to 1.5 molar equivalent to monomer (I) and approximately a molar equivalent (to monomer (II)) amount of any perfluoroalkyl sulfonic acid, of which triflic acid is an example, together with a molar equivalent (to monomer (II)) of methyltriflate provides efficient glycosylation.
  • Use of triflic acid in amounts of between about 0.5 and about 1.0 molar equivalent amount can be employed with a lesser efficiency. The coupling efficiency is directly related to the ease of purification of the desired product from starting material.
  • the activating agents are added to the glycosides and the coupling reaction is carried out at a low temperature. Temperatures from about -20° C to about -70° C are desired for the reaction. It is preferred that the temperature for the reaction be less than about -50° C, with -60° C being a most preferred temperature.
  • the reaction time is from about 15 minutes to about 8 hours. The reaction is desirably allowed to run for a time sufficient for all potential glycosidic linkages to be formed. Preferred is a reaction time between about 4 and about 6 hours.
  • a general description of a process for coupling of monomer (I), a suitably protected thioglycoside donor, and monomer (II), a glycosyl acceptor, is as follows.
  • Monomer (II) (about 1.0 eq.) and monomer (I) (at least 1 and up to about 3 eq., with about 1-2 eq. being preferred) are dissolved in a minimum of an aprotic solvent, such as methylenechloride, diethylether, acetonitrile, and benzotrichloride.
  • the most preferred solvent is methylenechloride.
  • the solution is cooled to about -55° C to -60° C under nitrogen atmosphere with vigorous stirring.
  • Powdered N- lodosuccinimide (NlS) is added to the cold solution. After about 15 min, a solution of a perfluoroalkyl sulfonic acid, such as triflic acid (about 1.0 eq.) and methyltrifluromethanesulfonate (about 1.0 eq.), dissolved in minimum of aprotic solvent, e.g., methylenechloride, is added in drops, while maintaining the reaction temperature under about -60° C. After the addition, the reaction mixture is maintained at the same temperature with stirring, for about 6 hours and then poured directly over a 1 :1 mixture of saturated sodium thiosulfate and saturated sodium bicarbonate solution.
  • a perfluoroalkyl sulfonic acid such as triflic acid (about 1.0 eq.) and methyltrifluromethanesulfonate (about 1.0 eq.)
  • aprotic solvent e.g., methylenechloride
  • the efficiency of the described coupling reaction reduces the level of undesired by-products and starting materials in reaction mixture following coupling, thereby facilitating the removal of the existing minor impurities through selective solvent extraction methods.
  • Selective washing with organic solvents provides a simplified purification method that is useful for large-scale production.
  • Solvents useful for the washing during purification include diethylether and hexane-ethylacetate mixture. Any combination of solvents in which the product is insoluble, but the impurities and the by-products are soluble, may be used.
  • step B the silicon blocking group is removed from the polyhexose linkage position as shown in Reaction 2 below, step B.
  • the silicon group can be removed, for example, by dissolving in minimum anhydrous tetrahydrofuran (THF), then reacting with acetic acid (2-3 eq.) and n-tetrabutylammonim fluoride solution in THF (1 M, 2-3 eq.). The reaction progress may be monitored either by TLC or NMR of the reaction mixture.
  • reaction mixture is concentrated to dryness, the residue dissolved in solvent such as methylenechloride and washed with water, 1 M aqueous HCI solution, 0.6% bleach solution (to remove the dark brown color), and aqueous saturated sodium bicarbonate solution. The remaining organic layer is dried over anhydrous magnesium sulfate and concentrated to dryness. Purification of the product is typically accomplished by precipitation with, for example, diethylether or an n- hexane-ethyl acetate mixture, which ensures the removal of residual monomer from the previous step as well as the silicon impurity.
  • any combination of solvents in which the product is insoluble, but the impurities and the by-products are soluble may be used for precipitation.
  • Additional monomer (I), a suitably protected thioglycoside donor, is then added through a glycosyl bond to the unblocked disaccharide using activating agents as described above.
  • the disaccharide is used in place of monomer (II), as shown in Reaction 2, according to the general coupling procedure described above. Shown is an example reaction of a glucosamine dimer with removal of the silicon blocking group in step B and addition of a glucosamine-monomer (I) forming a trimer low molecular weight polyglucosamine.
  • the coupling of monomer (I) to the glycosyl acceptor in the example reaction, the glucosamine dimer
  • the steps can be repeated until a polysaccharide chain of desired length up to about 50 units is synthesized.
  • Preparation of a low molecular weight polymer of galactosamine or glucosamine can be completed by the removal of the remaining protective groups.
  • this is carried out in a two step procedure.
  • de-O-benzoylation can be accomplished by Zemplens' method, which is well known to those skilled in the art, using sodium methoxide in methanol.
  • the phthaloyl group can be removed by using an ethylenediamine-derivatized Merrifield resin (P.Stangier, O. Hindsgaul, Synlett.
  • removal of the benzoyl and the phthalimido groups can be accomplished in a single step by treating the protected product at refluxing temperature with hydrazine in n-butanol, followed by selective extraction of the product polyhexosamine with water.
  • the single step method is preferred for polymers of length greater than 4, due to their incomplete de-benzoylation under Zemplens' condition and their lack of solubility in methanol and n-butanol.
  • the monomer based glycosylation process disclosed herein for polyhexosamine may be suitable for the preparation of polyhexoses as well.
  • glucose and galactose can be used in the place of the starting glucosamine molecule.
  • a suitably protected thioglycoside of hexose with a silicon protecting group at the chain extension site and ester protecting groups at non-linkage positions replaces monomer (I).
  • the glycosylation using a molar equivalent of triflic acid/methyltriflate in combination with N-haloimides under saturating substrate concentration provides high yields of the desired glycoside. Once a high yield of glycosylation is achieved, purification by selective precipitation is effective.
  • An enriched population of a single anomer of beta linkage oligohexosamine molecules preferably contains at least 80% of chains of oligosaccharide having a single length of from 3 to about 50 units, more preferably from 3 to about 25 units, and even more preferably from 3 to about 11 units.
  • Such enriched populations of single anomers of beta linked low molecular weight galactosamine or glucosamine polymer can be used as precursors for synthesizing other compounds, which may be biologically active, for use in, e.g., antimicrobial compositions, anti-inflammatory compositions, and other pharmacological applications.
  • an enriched population of single anomer can be used in preparing certain plant growth regulators related to chitin oligomers, such as the nod factors, which are involved in inducing nodulation in legumes, and contain a tetra- 1 ,4- ⁇ -aminoglucoside unit that is acylated with the same or different fatty acids at the amino groups.
  • the monomer based approach disclosed herein not only allows the synthesis of such tetra-aminoglucoside derivatives, but also enables the selective introduction of a desired fatty acid molecule.
  • Another naturally occurring ⁇ 1-6 linked glucosamine is the endotoxic Lipid A component of the outer membrane of most gram- negative bacteria, which triggers mediators of inflamation in mammalian cells.
  • Lipid A molecules which are aminoacylated derivatives of ⁇ 1-6 linked glucosamine units, can be synthesized through the amine derivatives and can be made using the processes disclosed herein.
  • An enriched population of a single anomer of beta linked low molecular weight hexose or hexosamine polymer can be a component, along with a carrier, of a composition that is designed to deliver properties provided by the polyhexose or polyhexosamine.
  • the carrier in the composition can include, for example, dissolving agents and inactive ingredients, In addition, the composition can include other active ingredients.
  • the composition is prepared such that it may be applied to a surface by methods such as spraying, dipping, soaking, and wiping or rubbing.
  • the low molecular weight polyglucosamines can be used as antimicrobial additives, including as anti-dandruff agents in shampoos as disclosed in EP 1384404, and in inhibiting the growth or propagation of spoilage or pathogen microorganisms in foods as disclosed in WO
  • Food products can be treated with an enriched population of a single anomer of beta linkage low molecular weight polyglucosamines such as by adding dry powder or a solution comprising the enriched population to the food.
  • Food that can be treated includes, for example, fresh or processed fruits and vegetables.
  • the food can be coated with a composition containing an enriched population of a single anomer of beta linked low molecular weight hexosamine polymer. The coating need not completely cover the food, but the food is coated to an extent adequate to provide antimicrobial activity for preservation of the food.
  • Polygalactosamines can also provide antimicrobial activity due to their negative charge.
  • the low molecular weight polyglucosamines of the present invention may be used in pharmaceutical compositions for treatments against inflammatory activity, including treatment of joint disorders such as of rheumatoid arthritis and osteoarthritis as disclosed in WO 2003026677.
  • An enriched population of a single anomer of beta linkage low molecular weight polyglucosamine may be included in the manufacture of a medicament for treatment of these conditions.
  • the low molecular weight polygalactosamines are potentially useful as chondroprotective pharmaceuticals. Initially, preparations of synthetic low molecular weight polygalactosamines are useful in biological testing applications to identify specific applications.
  • D-Glucosamine hydrochloride (compound 1, 1.0 Kg) was suspended in methanol (5.0 L) and vigorously stirred. NaOH (184.8 g) was dissolved in minimum deionized water and added to the D- Glucosamine/Methanol suspension. The suspension was stirred for 15 min and the insoluble material (sodium chloride) was filtered off by vacuum filtration. The theoretical amount of NaCI formed should be about 270 g.
  • phthalic anhydride (342 g) was added and the solution was stirred until most of the solid dissolved (about 30 min). This was then followed by the addition of triethylamine (468 g) and stirred for 10 to 15 min. To the resulting clear solution, another portion of phthalic anhydride (342 g) was added and the mixture was allowed to stir overnight at room temperature. Product usually began to precipitate out after two hours. The precipitated product was filtered and the residue was washed with minimum ice-cold methanol so as to remove the yellow color from the product. The residue was then washed three times with acetonitrile, with enough solvent added to the filter to completely immerse the solid, and dried at room temperature under high vacuum.
  • the weight of the white solid, product 2 was 954 g.
  • the product 2 from above (1.01 Kg, made from two batches) was placed in a 10 liter 3 neck round bottom flask set up with an overhead electric stirrer, an N 2 inlet and an addition funnel.
  • Acetic anhydride (3 L) and N, N-dimethylaminopyridine (1.0 g) were added to the flask and stirred vigorously.
  • Pyridine (2.8 L) was added slowly and the reaction mixture was stirred for 2 days at room temperature.
  • the reaction mixture was quenched with ice-water (4 L) and the product was extracted with methylenechloride.
  • the organic layer was repeatedly washed with aqueous hydrochloric acid solution, and then with saturated sodium bicarbonate solution.
  • Product 3 (464 g) was dissolved in toluene and the solvent was evaporated. This was repeated and the remaining solid was placed on a high vacuum line overnight.
  • Product 5 Product 6 Product 5 (295 g; 638; mmol) was suspended in dry toluene (1 L) and evaporated under vacuum. This procedure was repeated once more to ensure the removal of methanol contaminant that is detrimental to the reaction. 265 grams total was recovered. The residue after toluene evaporation was suspended in methylenechloride (3 L) in a 3-neck flask fitted with an overhead stirrer and the suspendion was stirred under dry nitrogen atmosphere.
  • Product 8 (crude; 105.3), after being evaporated with toluene-DMF, was suspended in CH2CI2 (500 ml). Pyridine (61.8 g; 782 mmol; 2.5 eq.) was added first, followed by the drop-wise addition of benzoyl chloride (88 g; 626 mmol; 2.0 eq.) to the mixture. The reaction mixture was allowed to stir at room temperature for 24 h. It was then diluted with CH2CI2 and washed sequentially with H2 ⁇ ,1 M HCI (2X), then aqueous saturated sodium bicarbonate solution, dried with MgSO ⁇ filtered, and concentrated.
  • the weight of the purified product was 116.1 g.
  • the product was about 90% pure as determined by NMR.
  • a portion (21.1 g) of this product was crystallized from dietylether-hexane to obtain pure crystalline material (13.8 g) of monomer (II).
  • Monomer (I) Monomer (II) Product 9 Monomer (II) (80.6 g, 109.3 mmol, 1.2 eq.) and monomer (II) (48.4 g, 91.1 mmol), both previously evaporated with toluene once, were dissolved in CH2CI2 (150 ml_) in a 3-necked, 500 ml flask. 4A Molecular sieve was added (5 g). The mixture was cooled to -60° C under nitrogen atmosphere with vigorous stirring.
  • N-lodosuccinimide N-lodosuccinimide (NIS; 44.3 g; 196.7 mmol; 2.2 eq.) was added as a dry powder, followed by the drop-wise addition of a solution of triflic acid (TfOH; 13.7 g, 91.1 mmol, 1.0 eq.) and methyltriflate (14.9 g, 54.8 mmol, 1.0 eq.) in methylenechloride. The reaction mixture was left at -55° C for an additional 4 hr. An additional 100 ml of the triflic acid/lmethyltriflate solution was added to the reaction mixture dropwise to reduce of the viscosity.
  • TfOH triflic acid
  • methyltriflate 14.9 g, 54.8 mmol, 1.0 eq.
  • the reaction mixture was filtered cold over a celite pad into a filter flask containing 1 :1 saturated sodium thiosulfate-sodium bicarbonate solution that was stirred thoroughly during the filtration.
  • the flask and the residue on the filter were rinsed with methylenechloride and the combined filtrate was worked up as follows.
  • the filtrate was poured into a separatory funnel.
  • the contents were thoroughly mixed, the aqueous solution separated, and the organic layer washed one more time with saturated aqueous sodium thiosulfate solution, followed by water, and aqueous saturated sodium bicarbonate solution.
  • the solution was then dried with magnesium sulfate, filtered and concentrated. Weight of the crude product was 111.1 g.
  • Fraction B product had about 5% silicon impurity (peak around 0 ppm) along with the major desired disaccharide.
  • Fraction A was contaminated about 10% with tBDMS impurities and a tetrabutylammonium derivative. Therefore, Fraction A was resuspended in 600 ml of ether, mixed for about 10 minutes, filtered and the process was repeated once more (weight of the solid recovered was 77.3 g). This solid was purified once more by dissolving the product in ethyl acetate and precipitating the product with the aid of hexane (weight of the product recovered was 71.7 g).
  • reaction mixture was poured over saturated sodium bicarbonate and saturated sodium thiosulfate aqueous solution (1 :1 , 400 ml) contained in an Erlenmeyer flask and thoroughly stirred. Additional methylenechloride (200 ml) was added and the contents were thoroughly mixed for 10 min, the aqueous solution separated, and the organic layer washed with 0.6% aqueous bleach solution, de-ionized water, and aqueous saturated sodium bicarbonate solution. The solution was then dried with MgSO4 , filtered and concentrated.
  • the crude product was suspended in diethylether (600 ml), the solid thoroughly mixed and the supernatent filtered. This process was repeated three times and the residue finally dissolved in methylenechloride, then concentrated to dryness giving 93.5 g of product 11. To the filtrate, about 40% volume of hexane was added and the precipitated material filtered, redissolved in methylenechloride and concentrated to dryness under vacuum to obtain an additional amount of compound 11 (26.0 g).
  • Product 11 was dissolved in minimum THF (500 ml). To this solution, 1 M solution of acetic acid (150 ml) and a 1 M solution of n- tetrabutylammonium fluoride in THF (150 ml) were added and the reaction mixture was stirred at room temperature for 3 days. The reaction mixture was evaporated to dryness, the residue redissolved in methylenechloride, washed sequentially with deionized water, 1 M HCI, 1 % aqueous bleach solution (to remove the dark brown color), and saturated sodium bicarbonate solution, then concentrated to dryness.
  • the solid was dissolved in minimum ethyl acetate. Hexane was added in drops (the final solvent ratio EtOAc-Hexane was 17:14). This resulted in a gluey material. The liquid was filtered and the gluey material redissolved in EtOAc (200 ml) and precipitated with hexane (100 ml) as described above. Finally, diethylether was added to solidify the gluey material and the solid was filtered. The solid was redissolved in methylenechloride and concentrated to dryness giving 81.4 g of product 12.
  • Tetrasaccharide product 13 (60 g) was dissolved in minimum THF. This was followed with the addition of a solution of 1 M acetic acid in THF (60 ml) and a 1 M solution of tetrabutylammonium fluoride in THF (60 ml) and stirred at room temperature for 48 h. The reaction mixture was evaporated to dryness, redissolved in methylenechloride, washed sequentially with deionized water, 1M HCI, 1% aqueous bleach solution (to remove the dark brown color), saturated sodium thiosulfat ⁇ solution, and saturated sodium bicarbonate solution, then concentrated to dryness giving 56.5 g of material.
  • reaction mixture was diluted with another 50 ml of CH2CI2 and then worked up as follows.
  • reaction mixture was poured over saturated sodium bicarbonate and saturated sodium thiosulfate aqueous solution (1 :1 , 500 ml) contained in an Erlenmeyer flask and thoroughly stirred. Additional methylenechloride (400 ml) was added and the contents were thoroughly mixed for 10 min, the aqueous solution separated, and the organic layer washed sequentially with 1% aqueous bleach solution, 10% aqueous sodium thiosulfate solution, and aqueous saturated sodium bicarbonate solution. The solution was then dried with MgSO ⁇ ., filtered and concentrated giving 171.5 g of material.
  • the solid was dissolved in ethyl acetate (400 ml). Hexane (400 ml) was added in drops with stirring of the precipitated material. The precipitated solid became a gluey material during the course of the addition and became a solid at the end of the addition. The precipitate was filtered and the process was repeated once more, followed by a final washing of the solid with 1 :1 EtOAc-Hexane and then dried giving100.7 g of product 16.
  • reaction mixture was poured over saturated sodium bicarbonate and saturated sodium thiosulfate aqueous solution (1 :1 , 500 ml) contained in an Erlenmeyer flask and thoroughly stirred. Additional methylenechloride (400 ml) was added and the contents were thoroughly mixed for 10 min, the aqueous solution separated, and the organic layer washed sequentially with 1 % aqueous bleach solution, 10% aqueous sodium thiosulfate solution, and aqueous saturated sodium bicarbonate solution. The solution was then dried with MgSO ⁇ filtered and concentrated. The residual solid was dissolved in EtOAc (400 ml), followed by the slow addition of hexane (400 ml).
  • Purified heptasaccharide product 19 (35.6 g) was dissolved in minimum THF followed by the addition (45 ml each) of 1 M solution of acetic acid in THF and 1 M solution of tetrabutylammoniumfluoride in THF and stirred at room temperature. After 3 days, the reaction mixture was evaporated to dryness, redissolved in methylenechloride, washed sequentially with saturated sodium thiosulfate solution, 1 M HCI, and saturated sodium bicarbonate solution, then concentrated to dryness. To remove nonpolar silicon impurities, the solid was dissolved in ethyl acetate (210 ml) and hexane was added slowly in drops (200 ml).
  • reaction mixture was left at -6O 0 C for an additional 5 hr.
  • An additional 100 ml of the triflic acid/methyl triflate solution was added to the reaction mixture dropwise to reduce of the viscosity and then worked up as follows.
  • the reaction mixture was poured over saturated sodium bicarbonate and saturated sodium thiosulfate aqueous solution (1 :1 , 200 ml) contained in an Erlenmeyer flask and thoroughly stirred.
  • Purified octasaccharide product 21 (29.0 g) was dissolved in minimum THF followed by the addition (25 ml each) of 1 M solution of acetic acid in THF and 1 M solution of tetrabutylammoniumfluoride in THF and stirred at room temperature. After 3 days, the reaction mixture was evaporated to dryness, redissolved in methylenechloride, washed sequentially with saturated sodium thiosulfate solution, 1M HCI, and saturated sodium bicarbonate solution, then dried and concentrated to dryness.
  • the reaction mixture was allowed to stir five minutes, before addition of a solution of triflic acid (0.8 g, 5 mmol) and methyl triflate (0.82 g, 5 mmol), dissolved together in CH2CI2 (20 ml), was added in drops (over 40 minutes). The reaction mixture was left at -60° C for an additional 5 hr. After 4 h, an additional 50 ml of the triflic acid/methyl triflate solution was added to the reaction mixture dropwise to reduce of the viscosity.
  • the reaction mixture was poured over saturated sodium bicarbonate and saturated sodium thiosulfate aqueous solution (1 :1 , 150 ml) contained in an Erlenmeyer flask and thoroughly stirred. Additional methylenechloride (100 ml) was added and the contents were thoroughly mixed for 10 min, the aqueous solution separated, and the organic layer washed sequentially with 10% aqueous sodium thiosulfate solution, 1% aqueous bleach solution, and aqueous saturated sodium bicarbonate solution. The solution was dried and concentrated. The resultant solid was dissolved in ethylacetate (200 ml) forming a colloidal suspension and hexane (200 ml) was slowly added.
  • ethylacetate 200 ml
  • Nonasaccharide (19.9 g, 4.3 mmol) was dissolved in minimum THF containing AcOH (15 mmol) and NBU4F (15 mmol) and stirred at room temperature. After 24 h, the reaction mixture was evaporated to dryness, the residue redissolved in methylenechloride and washed sequentially with deionized water, 1 M HCI, 1 % aqueous bleach solution (to remove the light brown color), and saturated sodium bicarbonate solution, then dried and concentrated to dryness. To remove nonpolar silicon impurities, the solid was suspended in ethyl acetate (150 ml) producing a milky solution, and hexane was added slowly in drops (150 ml).
  • Reaction mixture was allowed to stir five minutes, before addition of TfOH (0.62 g, 4.1 mmol) and methyl triflate (0.70 g, 4.1 mmol), both dissolved together in CH2CI2 (5 ml), was added to the cold solution in drops (over 40 minutes). The reaction mixture was left at -6O 0 C for an additional 5 h. After 4 h, an additional 50 ml of TfOH/methyl triflate solution was added to the reaction mixture dropwise to reduce of the viscosity. The reaction mixture was poured over saturated sodium bicarbonate and saturated sodium thiosulfate aqueous solution (1 :1 , 150 ml) contained in an Erlenmeyer flask and thoroughly stirred.
  • Decasaccharide product 25 (18.5 g) was dissolved in minimum THF containing AcOH (15 mmol) and NBU4F (15 mmol) and stirred at room temperature. After 24 h, the reaction mixture was evaporated to dryness, the residue redissolved in methylenechloride and washed sequentially with deionized water, 1M HCI, 1% aqueous bleach solution (to remove the light brown color), and saturated sodium bicarbonate solution, then dried and concentrated to dryness.
  • reaction mixture was allowed to stir five minutes, before addition of TfOH (0.45 g, 3 mmol) and methyl triflate (0.50 g, 3 mmol), both dissolved together in CH2CI2 (5 ml), was added to the cold solution in drops (over 40 minutes). The reaction mixture was left at -60° C for an additional 5 hr. After 4 h, additional 50 ml of TfOH/methyl triflate solution was added to the reaction mixture dropwise to reduce of the viscosity. The reaction mixture was poured over saturated sodium bicarbonate and saturated sodium thiosulfate aqueous solution (1 :1 , 150 ml) contained in an Erlenmeyer flask and thoroughly stirred.
  • Step 2 The product from Step 1 was dissolved in 250 ml of n-butanol. Polystyrene-ethylenediamine resin (26.0 g) was added and the slurry was heated to 105° C with stirring for 24 h. It was then filtered, concentrated to dryness, and the resulting material was redissolved in water and washed with methylenechloride. The aqueous layer was concentrated to dryness.
  • Fractions A and B were separately suspended in n-butanol (200 ml), treated with MR-ethylenediamine resin (20 g) and heated to 100° C for 24 h. The hot solution from fraction A was filtered over a celite pad and washed with 1 :1 methanol-water. The combined filtrate was concentrated to dryness (Fraction C, 853 mg). Proton NMR of the product indicated it to be the desired triglucosamine product 29.
  • 1 H-NMR (D 2 O) ⁇ : 4.54, 4.52, and 4.37 (3 x H-1), 4.01 , 3.98, 3.87, 3.80 (6 x H-6), 3.63 (OCH 3 ), 2.78, 2.74, and 2.70 (3 x H-2).
  • MR-Ethylenediamine resin (9 g) was added and the reaction was continued for two days.
  • the hot reaction mixture was filtered over a celite pad, washed with 1 :1 methanol water (30 ml), and then with water (2 x 15 ml). The combined filtrate was concentrated to dryness.
  • Fraction F Fraction F The residue was dissolved in water and lyophilized (Fraction F). The NMR spectrum of Fraction F showed that though the major product was the desired triglucosamine product 29, it was contaminated with some incompletely deprotected trisaccharide.
  • Step 1 The derivatized tetrasaccharide product 14 (7.7 g, 3.6 mmol) was dissolved in anhydrous methanol (450 ml). Sodium methoxide solution (0.5 M, 7.5 ml) was added and the solution was stirred at room temperature for 7 days. This was then refluxed for 2 days. The clear solution deposited lots of solid after two days of refluxing. The solution was cooled and the deposited solid was filtered over a 0.1 ⁇ filter. The residue was redissolved in dimethylformamide (100 ml) and concentrated to dryness (Fraction B, 3.53 g). The filtrate was neutralized with acidic resin and concentrated to dryness (Fraction C).
  • Step 2 Fraction B (3.53 g) was suspended in 100 ml of n-butanol and heated to 100° C. MR-Ethylenediamine resin (30 g) was added and the reaction was continued for 18 h. The hot solution was filtered over a celite pad and the residue further washed with water. The filtrate was concentrated to dryness (Fraction D). The proton NMR analysis of Fraction D confirmed the identity and purity of the product as the desired tetraglucosamine product 30.
  • the Merrifield resin was washed with hot DMF (100 ml) and the filtrate was concentrated to dryness. A white solid was obtained. NMR examination of this solid in DMF-d7 showed that it was the starting material from Step 1. This was suspended in n-butanol (250 ml) containing MR-ethylenediamine resin (15 g) and heated to 104° C for two days. It was then filtered over a celite pad and the residue washed with 1 :1 methanol-water. The combined filtrate was concentrated to dryness and lyophilized to obtain more of the desired tetraglucosamine product 30 (381 mg).
  • the hexasaccharide product (9.0 g, 3.0 mmol) was dissolved in anhydrous methanol (500 ml). Sodium methoxide solution (0.5 M, 7.5 ml) was added. Anhydrous THF (40 ml) was added to dissolve any suspended material and the solution was stirred at room temperature for 1 day. This was then refluxed for 2 days. The reaction mixture was brought down to room temperature, the solid (Fraction A) in the flask filtered over a celite pad and the filtrate neutralized with sulfonic acid resin, refiltered and concentrated to dryness (Fraction B). The residue B was suspended in EtOAc-Hexane (1 :1 ) and filtered, and the process repeated twice.
  • fraction A was suspended in methanol (100 ml).
  • Sodium methoxide solution (0.5 M, 2 ml) was added and the solution was stirred at room temperature for 2 days and then refluxed for 2 days. NMR indicated most of the benzoates had been removed, but not quantitatively.
  • Heptasaccharide (8.0 g) was dissolved in n-Butanol (200 ml) and heated to 105 °C. Hydrazine (14 g) was added to the hot suspension and stirred. Initially, the heptasaccharide in n-butanol did not dissolve despite heating. After the addition of hydrazine, the solution became clear, then cloudy (10 min) and precipitate started to appear in the reaction flask. The reaction mixture was refluxed overnight and the hot suspension was filtered over a celite pad. The precipitate was washed with hot n-butanol and hot methanol (3 x 40 ml). The residue on the celite pad was extracted with water and concentrated to dryness to give product 33.

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Abstract

L'invention concerne un procédé de synthèse de polymères de faible masse moléculaire à liaison bêta de galactosamine et de glucosamine. Un couplage efficace de monomères stables est obtenu au moyen de taux élevés d'agents activants. Dans ce procédé, l'allongement de chaîne se fait par l'addition de monomères uniques, de façon à produire des populations de polyhexosamines à une seule longueur de chaîne.
PCT/US2005/022116 2005-06-16 2005-06-21 Synthese chimique de polyglucosamines et de polygalactosamines de faible masse moleculaire Ceased WO2007001283A2 (fr)

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