Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-07-10
2001-05-08
Lee, Howard C. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S124000, C536S066000, C536S103000, C536S116000
Reexamination Certificate
active
06228997
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the enzyme-catalyzed acylation of insoluble polysaccharides.
BACKGROUND OF THE INVENTION
Selective acylation of polysaccharides is desirable to tailor their structural and functional properties, hydrophobicity and hydrophilicity, interfacial properties, and biocompatibility. E.g., fatty acid esters of saccharides and polysaccharides may be useful as bioerodable drug delivery matrices, and biodegradable emulsifiers, compatibilizers, and detergents.
1
However, selective acylation of polysaccharides by chemical reactions are difficult due to the lack of specificity, solubility, and the multifunctionality of the polymer.
2
Enzymes have been used to acylate saccharides (up to 5 glucose moiety) regioselectively under mild conditions in organic solvents.
2-4
Similar reactions with polysaccharides would be desirable. However, the lack of solubility of these polymers and the enzymes in organic solvents implies significant problems in carrying out these conversions. Accordingly, alternative methods of achieving a functionally significant degree of acylation are required.
5-7
Enzymes are powerful catalysts in organic solvents where they catalyze a wide variety of reactions that are difficult to perform in aqueous solutions. This is particularly evident in transesterification reactions catalyzed by lipases and proteases wherein a variety of nucleophiles act as substrates for enzyme-catalyzed acyl transfer in nearly anhydrous organic solvents. Unfortunately, many polyhydroxylated compounds are either sparingly soluble in only the most polar organic solvents, or are completely insoluble in organic media. For these substrates, conventional non-aqueous enzymology is unable to support catalytic transformations. The development of a suitable technique for the selective modification of polysaccharides in organic solvents, therefore, would represent both an opportunity for the synthesis of novel materials as well as a means to overcome a technical hurdle in the broader uses of enzymes in non-aqueous media.
Accordingly, it is an object of this invention to overcome the above illustrated inadequacies and problems of insoluble polysaccharides by providing an improved method of their transesterification.
It is another object of this invention to provide a method of acylating polysaccharides wherein their selective modification results in structural and/or functional benefits.
Yet another object of the present invention is to provide a method of enabling the use of enzymes to catalyze reactions in non-aqueous media for the synthesis of bioerodable and biocompatible compounds.
SUMMARY OF THE INVENTION
The present invention provides processes for the production of polysaccharides esterified regioselectively in position C6 or, for the case of cyclodextrins, in position C2 and C3. Such attribute are novel because chemical esterification is unable to modify regioselectively such macromolecules. The processes also emphasize recycling of the reaction media leading to waste minimization, mild reaction conditions, and minimal byproduct formation. Isooctane and the solubilized enzyme can be easily regenerated to minimize product cost and environmental hazard.
Recently, a method was developed to solubilize enzymes in hydrophobic organic solvents through the formation of enzyme-surfactant ion pairs.
8
These ion-paired, organic-soluble enzymes are extremely active in hydrophobic solvents, such as isooctane. The present invention demonstrates that polysaccharides and cyclic oligosaccharides such as amylose, chitosan, pullulan, maltodextrose, cellulose, hydroxypropyl cellulose (HPC), hydroxyethyl cellulose (HEC), hydroxymethyl cellulose (HMC), &agr;-, &bgr;-, &ggr;-cyclodextrins and similar polysaccharides when deposited as a thin film or cryogenically milled can be regioselectively acylated by catalysis in organic solvents using an organic-soluble enzyme preparation of subtilisin (from
Bacillus subtilis
). This represents the first attempt at catalyzing solvent-insoluble polymer modification using enzymes in organic solvents.
Proteases such as subtilisin (from
Bacillus subtilis
), ion paired with AOT, remained predominantly active and soluble in isooctane. To enhance the reactivity of insoluble substrates, thin films and cryogenically milled powders of amylose, chitosan, pullulan, maltodextrose, cellulose, hydroxypropyl cellulose (HPC), hydroxyethyl cellulose (HEC), hydroxymethyl cellulose (HMC), &agr;-, &bgr;-, &ggr;-cyclodextrins and similar polysaccharides were prepared to increase surface area. All these polysaccharides were esterified and some of these biopolymers were selectively transesterified using an ion-paired protease. &agr;-Cyclodextrins were also non-selectively transesterified. Chitosan, pullulan, maltodextrose, cellulose, hydroxypropyl cellulose (HPC), and hydroxymethyl cellulose (HMC) were also transesterified enzymatically with the same methodology.
Modified polysaccharides can be used for biodegradable emulsifiers, compatibilizers, and detergents. Of particular importance is the potential broad use of these low cost polymers for edible wrapping film utilized for food storage. In addition to the above applications, esterified polysaccharides can be of extreme interest to the paper industry in order to efficiently recycle paper and other compounds based on cellulose. Other application can be envisioned in the manufacturing of drug delivery systems, specific filters, high absorbance compounds.
Other objects, features and advantages will be apparent from the following detailed description of preferred embodiments thereof taken in conjunction with the accompanying drawings.
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Akkara Joseph A.
Bruno Ferdinando F.
Dordick Jonathan S.
Kaplan David L.
Lee Howard C.
Ranucci Vincent J.
The United States of America as represented by the Secretary of
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