FR2854161A1 - Crystalline polysaccharide derivatives in the form of water-insoluble aggregates of microcrystals, for use e.g. as viscosity modifiers or super-absorbers, manufactured by controlled oxidation of primary alcohol groups - Google Patents

Abstract

Crystalline polysaccharide derivatives (I) in which the primary alcohol groups are at least partly oxidised to carboxylic acid (which may be in the form of salts or functionalised), in the form of water-insoluble aggregates of individual microcrystals and/or microfibrils with a width of 1-30 nm and a length of up to several tens of microns, say about 100 microns. An independent claim is also included for a method for the production of (I) by controlled radical oxidation of the -CH2OH groups in polysaccharides with an oxidising agent in presence of catalytic amounts of amine oxide radicals, the oxidising agent being added drop by drop so as to keep the mixture at pH 10 by addition of sodium hydroxide until the end of the reaction.

Description

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Derivatives of crystalline polysaccharides, processes for their production and their applications
The invention relates to crystalline polysaccharide derivatives of which at least a portion of the -CH 2 OH groups are oxidized to carboxyl-COOH groups and / or substituted with functional groups, the processes for obtaining them and their applications.

 The regioselective oxidation of the -CH 2 OH groups of crystalline polysaccharides has already been reported by several authors.

 Thus, Battista describes in US Pat. No. 3,111,513, a process for the preparation of polysaccharide crystallites comprising a prior step of acid hydrolysis, followed by oxidation with nitrogen dioxide or periodic acid, which leads to to a dialdehyde, which can be oxidized to carboxyls using hydrogen peroxide, sodium hypochlorite or potassium dichromate.

 This technique, however, leads to degradation of the treated polysaccharide.

 It has also been proposed to use the TEMPO-NaBr-NaOCl system for the oxidation step (see in particular Isogai A. and Kato Y., Cellulose, (1998) 153-164 and Nordic Pulp and Paper Research Journal, 14 (1999) 279-284, Tahiri C. and Vignon MR, Cellulose, 7 (2000) 177-188, and Araki J. et al, Langmuir, 17 (2001), 21-27).

 The conditions described however do not allow control of the characteristics of the oxidized crystallites formed, more particularly to prepare in a single step aggregates of carboxyl derivatives, more or less divided, insoluble in water and capable of forming stable colloidal suspensions.

 The inventors have now found that by carrying out this radical oxidation reaction in a controlled manner,

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 it was possible, in a single step, to obtain crystalline polysaccharides more satisfactorily satisfying the requirements of the technique, without the need for prior hydrolysis of the starting polysaccharide. In particular, the implementation of specific operating conditions makes it possible to oxidize the desired quantity of primary hydroxyls and to form polysaccharides in the form of microcrystals and / or microfibrils, of appropriate sizes for a given application, and sufficiently individualized to give stable colloidal suspensions. These polysaccharides also have the advantage of being functionalizable and thus giving access to several families of chemical derivatives particularly useful in various sectors of the industry, while maintaining the material in the crystalline state.

 However, a step of prior acid hydrolysis may be used, in order to obtain shorter-sized crystals, for example in the case of primary wall cellulose microfibrils, the length of these microfibrils may be reduced.

 By microcrystals is meant crystals of microscopic size, consisting of macromolecular chains oriented for example along the longest axis for a rod, or perpendicularly for platelets (for example in the case of starch).

 The term microfibrils refers to a long, more or less fine element consisting of macromolecular chains aligned in the same direction, having a length / width ratio of 10 to 1000 (for example, in the case of cellulose, the element has a diameter from 1.5 to 30 nm and a length of 0.2 to 10 μm). These polysaccharides also have the advantage of being functionalizable and thus giving access to

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 several families of chemical derivatives particularly useful in various sectors of the industry.

 The object of the invention is therefore to provide novel crystalline polysaccharide derivatives of various sizes and shapes (more or less rigid rods or crystals, of variable lengths, or platelets) of variable molecular masses with a controlled charge rate which can to be used if desired in coupling reactions for grafting given functional groups.

 It also relates to a process for obtaining polysaccharides substituted with COOH groups and / or functional groups thereof.

 The invention also relates to the applications of these derivatives.

 The polysaccharide derivatives of the invention are characterized in that they are in the form of aggregates comprising microcrystals and / or individualized microfibrils, with lateral sizes of microcrystals and microfibrils of 1 to 30 nm, their length being to reach a few tens of m, even 100 m, the aggregates, microcrystals and microfibrils being insoluble in water.

 These derivatives, given the presence of COO-surface charges, have the advantage of forming stable colloidal dispersions or suspensions in an aqueous medium. In addition, once dried, the aggregates, microcrystals and microfibrils can be redispersed in water, or in an organic solvent, in the case of coupling with hydrophobic groups, while retaining their properties related to the dispersion. They can also be in the form of gels, depending on their size and their OD.

 These microcrystals more or less elongated, of nanometric size form stable colloidal suspensions, which have the possibility of presenting a type organization

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 chiral nematic (this liquid crystal behavior particularly cholesteric is particularly advantageous in view of the applications envisaged).

 Preferably, at least a portion of the -CH 2 OH groups are oxidized to COOH groups, the latter groups, as well as the unoxidized CH 2 OH groups, which may be partially or completely functionalized.

 In a preferred embodiment of the invention, the polysaccharide derivatives are derivatives containing carboxylic groups, the COOH groups being on the surface and, where appropriate, in the amorphous phases, which makes it possible to modify the hydrophilicity and the reactivity. polysaccharides.

 In these derivatives, the average number of carboxyl groups per saccharide unit (that is, the degree of oxidation or OD) varies from 0 to 0.4 depending on the polysaccharide.

 The polysaccharide derivatives of the invention may have a weight-average molecular weight of from about 2500 to 200000 g / mol, preferably from 2500 to 100,000 g / mol.

 By way of example, in the case of cellulose monocrystals obtained from tunicier, which have a mean section of 20 nm, the number of cellulose molecules in a crystal is of the order of 400, ie 80 cellulose molecules. on the surface for 320 molecules of cellulose in the heart. Insofar as one has the order of 20% of surface chains and it is possible to oxidize only one hydroxyl out of two, one will oxidize at the most of the order of 10% of the set of primary hydroxyls, ie a maximum OD of 0.1.

 In the case of cotton linters, which have an average cross-section of 5 nm, the number of cellulose molecules in a crystal is of the order of 80 molecules, ie an average of 32 surface cellulose molecules for 48 cellulose molecules. c # ur. The number of surface chains is therefore

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 the order of 38-40%. As the oxidation can only concern one primary hydroxyl out of two, the oxidation will have a maximum of 20% of all the primary hydroxyls, ie a maximum OD of 0.2.

With microfibrils of primary walls (beet pulp for example), which have an average section of 1.5 to
At 3 nm, the number of cellulose chains at the surface is 16 to 9 inside the crystal, which corresponds to 64% of surface cellulose molecules for 36% of heart molecules. As the oxidation can only reach one surface hydroxyl in two, at most 32% of all the primary hydroxyls present will be oxidized, ie a maximum OD of 0.32.

 The invention also relates to the salts of the above derivatives.

 Alkali metal carboxylates, especially sodium, alkaline earth metal, heavy metal, radioactive isotope or ammonium (quaternary), acetates, propionates, aceto-propionates, acetobutylrates, sulfoacetates.

In another preferred embodiment of the invention, the polysaccharide derivatives comprise functional groups -COR1, and where appropriate -CH20R2, in which: R1 represents OX or NX, X being an alkyl radical
C1-C12, or alkylamino, the alkyl or alkylamino radical being optionally substituted by one or more -OH groups, or an aromatic group, in particular phenyl, or a polymer chain including alkyl polyethylene or polypropylene glycol or alkoxy polyethylene or polypropylene glycol; . R2 represents COX or X, X being as defined above.

 The basic polysaccharides are semi-crystalline or microfibrillar products, such as cellulose, chitin, starch, amylose, nigerane ((α (1 → 3) α (1-> 4) glucan), ss (1-> 3) glucan, (1-> 4) mannan, (1-> 3) xylan, inulin, agarose, carrageenans.

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 It is recalled that the native cellulose consists of D-glucopyranose units linked together by ss (1-> 4) bonds and exists in several crystalline forms (cellulose I form in the natural state; II, after a treatment in medium alkali or a derivatization, followed by regeneration / coagulation, cellulose III, after treatment of the natural cellulose in liquid NH3 medium, or in the presence of amines).

 Cellulose II has the advantage of being in the form of filaments of variable size according to the extrusion die (0.01 to 1 mm section) and which is in the form of crystalline zones and amorphous zones. It will be possible, depending on the operating conditions, either to introduce charges on the surface of these filaments while retaining this filament, or to oxidize on the surface and in the amorphous zones, which leads to crystalline aggregates of cellulose II of variable sizes. It will also be possible, depending on the operating conditions, to introduce fabric surface fillers made from these filaments while retaining the structure of these tissues, in order to obtain absorbent dressings, and / or applications of the hemostatic dressing type, or bioresorbable surgical wires.

 Cellulose III has the advantage of a high reactivity and a smaller crystallite size than that of the other forms.

 Chitin consists of N-acetyl-D-glucosamine units linked together by (3 (1 # 4) bonds.

 Chitosan consists of D-glucosamine units linked together by (3 (1 # 4) bonds.

 Ss (1 # 3) glucan consists of D-glucopyranose units linked together by (3 (1 # 3) bonds.

 The ss (14) mannan consists of Dmannopyranoses units linked together by 3 (1 # 4) bonds.

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 Nigerane consists of D-glucopyranose units linked together by alternating bonds a (1-3) and a (14).

 The starch consists of D-glucopyranose units linked together by α (1 # 4) linkages and which carry, in places, branches consisting of D-glucopyranose units linked together by α (16) bonds.

 Amylose consists of D-glucopyranose units linked together by α (1-4) linkages.

 Inulin consists of (3 (12) -D-fructofuranose units.

 The agarose consists of a ss (1-4) -D-galactopyranose unit that alternates with a (1 # 3) 3,6-anhydro-L-galactopyranose unit.

 Carrageenans consist of a unit (3 (1-4) -D-galactopyranose which alternates with a unit (1 # 3) 3,6-anhydro-D-galactopyranose, and carry a 1 to 3 sulfate groups (respectively K, i and 1 carrageenan).

 The invention also relates to a process for obtaining polysaccharides as defined above.

 This process, which comprises the radical oxidation of the -CH 2 OH groups of the polysaccharides in the presence of a catalytic amount of an aminoxide radical, and in the presence of an oxidizing agent, is characterized in that the oxidation step is carried out in a controlled manner by adding the oxidizing agent dropwise so as to maintain the pH at 10, the pH then being maintained at 10 by addition of sodium hydroxide until the end of the reaction.

 This step of regioselective oxidation of the primary OHs makes it possible to prepare in a single step aggregates of carboxylic derivatives, more or less divided, insoluble in water and capable of forming stable colloidal dispersions.

 The radical oxidation reaction is most generally carried out with, as amine oxide radical, the

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 2,2,6,6-tetramethylpiperidine-1-oxide (TEMPO) radical and, as an oxidizing agent, sodium hypochlorite.

 Other aminoxide radicals are however also usable, such as the 4-hydroxy TEMPO analogue, acylated derivatives such as 4-acetoxy, 4-phosphonooxy and 4-benzyloxyTEMPO, amino derivatives such as 4-amino, 4-acetamido and 4-maleimido-TEMPO. Catalytic quantities of these compounds are used in the order of 0.1 to 5 moles of reagent for 100 anhydroglucose units.

 Similarly, other oxidizing agents can be used, in particular those capable of reoxidizing reduced nitroxides, such as ozone, and in particular hypochlorites, especially sodium hypochlorite. Mention may also be made of hydrogen peroxide, organic hydroperoxides or the like. It is also possible to use enzymes such as peroxidases or oxidases, in particular polyphenol oxidases, laccases and lipases.

 The reaction can be carried out in the presence of sodium bromide. This compound is then advantageously used in a proportion of 1 to 70 moles per 100 anhydroglucose units.

 The oxidation reaction is carried out at a temperature of from -5 ° C. to 50 ° C., preferably from 0 ° to 25 ° C.

 To facilitate the reaction, if necessary, a controlled mechanical destructuring is carried out, for example using an ultraturax, ultrasound, MantonGaulin type homogenizer or others.

 The oxidation operation can be stopped for example by addition of an alcohol, such as methanol or ethanol.

 In order to improve the stability of the oxidized samples, a treatment with a reducing agent may be used, with a view to reducing the aldehyde and / or ketone functions optionally formed, as well as the residual hemiacetal functions of the chain ends (reducing terminal end). . A reducing agent such as an alkaline borohydride

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 is particularly indicated, the sodium borohydride (NaBH4) will be chosen preferentially.

 The medium can then be neutralized to a pH of about 7, the oxidized derivatives being recovered for example by filtration and / or centrifugation and several washes, especially with water.

 Depending on the OD and the size of the crystallites, it is found that the material swells more or less, or is hydrated and then behaves like a gel formed of crystallites coated with chains of polysaccharides high OD, highly hydrated, up to at an OD of 1, corresponding to soluble products.

 The semi-crystalline polysaccharides used in the process of the invention are obtained directly from the biomasses, or by dissolution and recrystallization, or by precipitation. Crystallization can be controlled (in the case of amylose crystals crystallizing in the form of platelets), or uncontrolled (in the case of vegetable sponges obtained by the viscose process, or rayon fibers obtained by the viscose process or by dissolving in a solvent and regeneration, which precipitate in the form of amorphous phases and disorganized crystalline phases).

 Amylose platelets are also pre-existing in their natural state.

 According to the organization of the polysaccharides used, the oxidation will be on the surface and / or in the disorganized or amorphous phases. It is thus possible to create reactive sites on which may be attached molecules of interest by coupling. The sizes of the crystallites will be different.

 As regards, for example, cellulose, mention may be made among the substrates that can be used, without these indications being of a limiting nature, natural fibers such as ramie, cotton, jute, algae, bisulphite pastes.

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 bleached, sulphate pulp or bleached kraft pulp, microfibrils of primary or secondary walls (sugar beet pulp), citrus pulp (lemon, orange, grapefruit), fibers or films of regenerated cellulose (rayon, fortisan, lyocell), bacterial cellulose, animal cellulose, fungal cellulose, and microcrystalline cellulose.

 The microfibrils advantageously have a length / diameter ratio of between 100 and 1000 and a mean diameter of 1.5 to 3 nm. They are advantageously obtained from the cellulosic sources mentioned above, according to conventional methods.

 The cellulose microfibrils may be of any origin, for example of plant, animal, bacterial, fungal or amoebic origin, preferably of vegetable, animal or bacterial origin.

 Examples of animal sources of cellulose include animals of the tunicate family. The plant sources may be natural fibers as indicated above.

In another preferred embodiment of the invention, the polysaccharide derivatives comprise functional groups -COR1, and where appropriate -CH20R2, in which: RI represents OX or NX, X being a C1-C12 alkyl or alkylamino radical, the alkyl or alkylamino radical being optionally substituted by one or more -OH groups, or an aromatic group, in particular phenyl, or a chain; polymer in particular alkyl polyethylene or polypropylene glycol or alkoxy polyethylene or polypropylene glycol;
R2 represents COX or X, X being as defined above.

 The invention also relates to a process for obtaining polysaccharide derivatives having functional groups -COOR1, and where appropriate -CH20R2 as defined above.

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 This process comprises a coupling step, in solution in water, of the derivatives as obtained by the oxidation process defined above with an amine of formula HN (X), in which X is as defined above. .

 The coupling reaction is advantageously carried out in the presence of a water-soluble amidation reagent and a compound acting as esterifying agent.

 As the amidation reagent particularly suitable for carrying out the coupling reaction, mention will be made of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC). Compounds capable of playing the role of esterifying agent in the coupling reaction include N-hydroxysuccinimide sulfonate (NHS).

 The temperature of the coupling reaction is generally 5 to 100 ° C, preferably 20 to 60 ° C, most preferably 45 to 55 ° C.

 The reaction is carried out at a basic pH of from 7 to 10, preferably from 7.5 to 8.

 Preferably, the amounts of reagents used correspond to: 1 to 10 moles of amine for 1 mol anhydroglucose oxidized or otherwise, 1 to 10 moles of EDAC for 1 mol anhydroglucose oxidized or not,. 1 to 10 moles of NHS for 1 mol anhydroglucose oxidized or not.

 It is found that the grafted crystals have the same size as the ungrafted carboxylated crystals.

 The grafting of the cellulose crystals, and the resulting decrease in the carboxyl content, does not seem to affect the formation of a nematic chiral phase.

 After grafting and lyophilization, the crystals can be redispersed in water to give suspensions

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 birefringent stable, which can be observed under a microscope in polarized light.

 It will be noted with interest that the invention thus provides the means for accessing new polysaccharide derivatives, which retain the crystalline structure of the carboxylic derivative.

These derivatives can have various sizes and shapes, such as more or less rigid rods, and of varying lengths, or platelets.

 In the case where the coupling product has a terminal amino group, it is possible to crosslink the residual amine functions with for example glutaraldehyde and thus obtain crystalline substrates with a controlled degree of crosslinking.

 The invention thus provides the means for obtaining very different products and in a wide range.

 These are polysaccharide derivatives with: - anhydroglucose units (A) whose carbon in the C6 position carries a -COOH group, where appropriate in salt form, - anhydroglucose units (B) whose carbon in C6 position carries an ester function -COOR1 and, - optionally anhydroglucose units (C) whose carbon in the C6 position carries a group - CHzOH, - anhydroglucose units (D) whose carbon in the C6 position carries a -CH2OR2 group.

 The molecules thus formed may therefore contain: molecules consisting of a series of anhydroglucose units and which constitute the crystalline core of the crystallite (whisker) or the microfibril, molecules consisting of a series of anhydroglucose units and which are on the surface of the crystallite (whisker) or the microfibrilla, the anhydroglucose units which are on the surface of the crystallite (whisker) or the

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 microfibrous can be advantageously in the forms A to D described above.

 The level of units (A) represents more particularly from 3 to 33% of all the anhydroglucose units of the microfibril crystallite; rate of units (C) or (D) from 1 to 80% of the total quantity of units (A) and (C).

 The grafting of the cellulose crystals, and the resulting decrease in the carboxyl content, does not seem to affect the formation of a nematic chiral phase. In general, the coupling of the polysaccharide molecules decreases the OD and leads to products that are less and less soluble in water, all the more so when hydrophobic chains are grafted.

 After grafting and lyophilization, the crystals can be redissolved in water to give stable birefringent suspensions, which can be observed under a microscope in polarized light.

 The properties of the derivatives of the invention give them great interest in many applications.

 The presence on the surface of groups - COO-, at an appropriate level, with the resulting repulsion of the charges makes it possible to use the corresponding derivatives as metal chelating agents, viscosifiers or stabilizers, or even superabsorbent polymers.

 These products are of great importance in particular: - in the food industry, as thickeners, stabilizers of dispersions, emulsions and suspensions, as surface-active agents for low-caloric products, or in a low proportion of fats or cholesterol, - in the industry of paints, paper, textiles, as steric stabilizers of fillers, cosmetics (support of ointments, creams ..) - in agriculture, in pesticide applications,

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 - in the pharmaceutical industry, as an excipient of drugs, agents for controlling the release of active ingredients or biomolecules, support for ointments or creams, intestinal transit agents, dental pastes, flavoring support.

 in the medical or biomedical field (separation and purification of proteins, viruses by precipitation / affinity adsorption, hemostatic dressings, bioabsorbable strands).

 as complexing or sequestering agents, metal ions, such as calcium ion, which are particularly useful for detergents, cosmetics and surface treatment; heavy metals and radioactive elements in the nuclear industry.

 - to prepare stimulable polymers or stiff hair sticks that tend to self-organize.

 These crystallites of polysaccharides, and more particularly cellulose crystallites or microfibrils, can be used to modify the rheology of the media into which they are introduced.

 In the case of aqueous or organic fluid media of the type intended in particular for obtaining cosmetic compositions, drilling fluids, etc., the microfibrils may modify the viscosity and / or the texture of the medium, or even its rheological profile.

 In highly viscous and solid media, such as those intended to be used in thermoplastic, thermosetting, elastomer and mastic materials, microfibrils can modify the mechanical properties and in particular serve as reinforcing filler.

 The interesting mechanical properties of the microfibrils are attributed to their particular structure they have an important hydrophilic character due to the presence of hydroxyl functions on the surface of the microfibril.

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 However, the use of these microfibrils is not without drawbacks. In fact, the hydrophilic nature that may be desirable for certain applications, for example in aqueous and / or hydrophilic media, may constitute an obstacle to the various desired applications in organic and / or hydrophobic media. For example, in organic and / or hydrophobic media, the microfibrils do not disperse and there are phenomena of agglomeration and flocculation, due to the incompatibility of the latter with the organic medium in which they are found; microfibrils having a strongly hydrophilic character will naturally tend to flocculate and agglomerate in an organic medium of hydrophobic nature. Following these phenomena, more particularly in an organic medium, the microfibrils will no longer be able to exert their function as a viscosifying texturing agent and / or a reinforcing filler.

 The cellulose microfibrils of the invention, while having retained their initial morphological and crystalline aspects, and therefore all the interesting mechanical properties that result from it, have a markedly attenuated hydrophilic character. The microfibrils thus become dispersible in an organic medium.

The measurements of the characteristics of the products obtained are carried out with the following apparatus:
X-ray photos
The X-ray diffraction patterns were recorded from the lyophilized samples and then shaped, usually by pressing between two disks in a press, so as to be mounted on a sample holder equipped with a circular aperture collimator. 0.02 inches (0.5mm) in diameter. They are exposed for a few hours, in a Warhus flat vacuum chamber, to an X-ray source produced by a Philips PW 1720 generator with a voltage of 30 kV and a current of 20 mA. The clichés, once

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 developed, are compared with each other before and after oxidation to check the crystal structure of the cellulose sample (cellulose I, cellulose II, cellulose III). These observations also confirm that the reaction was limited to the surface of microcrystals and did not propagate to the core of the crystal structure.

Transmission electron microscopy (TEM):
The suspended samples are deposited on a carbon grid. After drying, the grid is observed by transmission electron microscopy using a Philips CM 200 CRYO microscope subjected to an 80kV acceleration voltage. This technique makes it possible to observe the crystals, to control their state of association and / or individualization and to measure their sizes (length and section).

NMR soluble samples
The carbon-13 NMR spectra of the soluble samples were recorded on a BRUKER AC 300 spectrometer at a frequency of 75.468 MHz.

 The samples are solubilized in D20 (20 mg in 0.5 ml of solvent) and the acquisition is carried out at 30 ° C. (in a tube of 5 mm internal diameter).

 The degree of oxidation is determined by quantitative 13C-NMR (C6 carbon monoxide (CH20H), oxidized C6 carbons (COONa), C1 carbons and C4 carbons) using the BRUKER INVGATE sequence with a 6.5 s pulse (90). , spectral width 15,000 Hz, 16K data points, acquisition time of 0.54 s and a relaxation time of 3 s. 20,000 to 100,000 acquisitions are required depending on the solubility of the samples.

 In the case of coupling reactions, it can be verified that a covalent bond has indeed been formed by carbon-13 NMR. Indeed the formation of the amide bond (O = C-NH-) results in the appearance of a signal C = O

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 feature. The coupling rate of water-soluble oxidized samples can also be measured by the same carbon-13 NMR technique.

Degree of oxidation (DO)
In order to determine the degree of oxidation of the compounds obtained, particularly in the case of partially oxidized and therefore insoluble samples in water, the solid NMR appears to be a reliable method for determining the degree of oxidation, but unfortunately this The technique can not be used for routine measurements because it requires several hours of acquisition. There are other, more accessible methods, such as infrared spectroscopy and conductimetric assay, that can be used to determine the OD of both soluble and insoluble samples.

Infrared spectroscopy
The infrared spectra are recorded on a PERKIN-ELMER 1720X Fourier Transform spectrometer from KBr pellets (1% by mass). The spectra are recorded by making 16 acquisitions over a spectral range between 400 and 4000 cm-1 with a resolution of 2 cm-1.

 Infrared spectroscopy is a quick and easy method for obtaining qualitative information on chemical modifications of polysaccharides. In the case of quantitative studies, sample preparation and data processing must be very rigorous.

 The analyzes were carried out using KBr pellets at 1% by mass of analyte.

 The selective oxidation at the 6-position of the anhydroglucose units induces a significant change in the absorption bands of the infrared spectrum, notably the appearance of an intense elongation band at 1604 cm-1 due to the C = O bonds of the carboxyl groups. in ionized form (COO-Na +).

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 It is found that the bands which correspond to the adsorbed water (or to the hydrogen bonding) and to the elongation of the C = O bond of the carboxyl groups in ionized form are superimposed. The integration of this elongation band can not therefore be used for the quantification of the degree of oxidation.

 It is therefore preferable to carry out the analysis of these samples in acid form (COOH), by passage over cation exchange resin or preferably in the case of crystalline samples by acidification (pH = 2) with a 0.5M HCl solution. In fact, the elongation band of the C = O bond moves from 1604 cm -1 to 1730 cm -1 and is no longer superimposed on the adsorption water band. The infrared spectra of the starting cellulose and the oxidized cellulose are shown in FIG.

 In the case of coupling reactions, we can verify that we have reacted by the appearance of 2 low-intensity lines at 1657 cm-1 (vc = o) and 1544 cm-1 (8 NH) which correspond to the absorptions amide 1 and amide II, and the decrease of the C = O band of the residual acid at 1730 cm-1.

Conductivity method
Another very conventional method for determining the degree of oxidation is the conductimetric assay. This method has the advantage of being applicable to both soluble and insoluble samples in water.

 The sample in ionized form (40 mg) is acidified with a solution of hydrochloric acid (15 ml, 0.01 M). This solution is dosed with 0.01 M sodium hydroxide solution.

 The titration curve (Figure 2) shows two equivalence points. The first part of the curve corresponds to the neutralization of the excess hydrochloric acid, the second part corresponds to the neutralization of the carboxyl groups of the sample and the third part corresponds to

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 to excess soda added. As a result, the difference (V2-V1) corresponds to the volume of sodium hydroxide necessary to neutralize the carboxyl groups of the sample.

DO = [162 ncooh / (méchantillon - 36 ncooH )] nCOOH = (V2 - Vi ) x 0 , 01 avec V en mL et méchantillon en mg. The degree of oxidation of the sample is defined by the following formula:

OD = [162 ncooh / (Sample - 36 ncooH)] nCOOH = (V2 - Vi) x 0.01 with V in mL and sample in mg.

Determination of the nitrogen content
Nitrogen analysis by elemental analysis is carried out by the central analysis department of the CNRS, analysis office, BP22, Vernaison, France.

Other characteristics and advantages of the invention will appear in the following examples and with reference to the appended figures:
Figure 1: Infrared spectra of cellulose: (a) unoxidized cotton linters; (b) surface oxidized cotton linters; (c) fully oxidized cotton linters or polyglucuronic acid.

 Figure 2: NaOH conductimetric titration curve of an oxidized cellulose suspension.

 Figure 3: Transmission electron microscopy of cotton linters (cellulose I) (a) starting sample, (b) Example 2 (0.52 eq NaOCl / eq anhydroglucose), (c) Example 4 (0, 73 eq NaOCl / eq.anhydroglucose), (d) Example 5 (2 eq NaOCl / eq.anhydroglucose).

 Figure 4: Transmission electron microscopy of cellulose microfibrils of beet pulps (cellulose I): (a) Example 12 (0.18 eq NaOCl / eq anhydroglucose), (b) Example 14 (0.36 eq NaOCl) anhydroglucose), (c) Example 16 (0.75 eq NaOCl / eq anhydroglucose).

 Figure 5: Comparative IR spectra of tunicate whiskers: (a) starting sample; (b) Example 25 (0.06

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 eq. NaOCl / eq. anhydroglucose); (c) Example 26 (0.12 eq.

NaOCl / eq. anhydroglucose); (d) Example 27 (0.20 eq.

NaOCl / eq. anhydroglucose); (e) Example 28 (0.5 eq NaOCl / eq anhydroglucose).

 Figure 6: X-ray diffraction patterns of tunicate whiskers (a) unoxidized, (b) completely surface-oxidized (Example 28).

 Figure 7: Tunicate whiskers observed by transmission electron microscopy: (a) after acid hydrolysis; (b) completely surface oxidized (Example 28).

 Figure 8: Tunicate Whiskers observed between crossed polarizer: (a) unoxidized, (b) totally surface oxidized (Example 28).

 Figure 9 Transmission electron microscopy of starch platelets: Example 34 (0.6 eq NaOCl / eq anhydroglucose).

 Figure 10: Transmission electron microscopy of chitin whiskers (a) starting sample after acid hydrolysis, (b) Example 36 (0.5 eq NaOCl / eq.anhydroglucose).

 Figure 11: Carbon-13 NMR spectra; oxidized cellulose sample (OD = 1); (b) oxidized cellulose sample coupled with 2-methoxyethylamine (Example 37).

 Figure 12: Comparative IR spectra: (a) a partially oxidized cellulose sample; (b) a partially oxidized cellulose sample coupled with 2-methoxyethylamine (Example 50).

Example 1
510 mg (3.15 mmol) of cotton linters are dispersed in 100 ml of distilled water using an ultraturate, the TEMPO (8 mg, 0.051 mmol) and NaBr (162 mg, 1 57 mmol). The solution is maintained at 20 ° C. and the commercial solution NaOCl 1.47M (0.55 ml, 0.81 mmol, ie a ratio eq NaOCl / eq anhydroglucose = 0.26) is added in small volumes to maintain the pH. to 10. After adding all

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 hypochlorite (generally in 5 to 60 minutes, more particularly in less than 30 minutes), the pH is constantly maintained at 10 by adding a sodium hydroxide solution (2% w / v NaOH). We continue to add sodium hydroxide until the pH no longer varies.

 Finally, the reaction was stopped by adding MeOH and then neutralized (pH7) with 1M HCl. It is possible to add a tip of NaBH4 spatula and stir for a few hours at room temperature, in order to reduce the ketone and aldehyde functions that may be present.

Separation
The cellulosic residue can be separated either by centrifugation or by filtration. at. Centrifugation: The suspension is centrifuged (20,000 g) for 30 minutes and the supernatant (soluble fraction, which is concentrated, dialyzed and lyophilized) and the insoluble material are recovered. The latter is redispersed in excess water (100 ml) and stirred for 1 hour, then centrifuged to separate the insoluble fraction which is dispersed in water and lyophilized. The fraction which remains in suspension (gel fraction) is concentrated, dialyzed and lyophilized. b. Filtration: Sartorius SM 162 49 stainless steel filter pressurized filter, cellulose nitrate membrane with a porosity of 5 m is filtered to recover the insoluble part. The soluble part is concentrated, dialyzed and lyophilized (soluble fraction). The water-insoluble fraction is redispersed and stirred for 1 hour. It is centrifuged to separate the insoluble fraction which is redispersed in water and freeze-dried. The fraction which remains in suspension (gel fraction) is concentrated, dialyzed and lyophilized.

<Desc / Clms Page number 22>

 16 mg of soluble fraction are recovered (OD = 100%); 4.7 mg gel fraction and 506 mg insoluble fraction (OD = 12%).

 In the tables below, the oxidation conditions used in Example 1, as well as in Examples 2 to 36, which relate to oxidation reactions conducted according to similar protocols, were summarized.

Example 1

<Tb>
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> NaBr <SEP> Temperature <SEP> NaOCl <SEP> pH
<tb> departure
<tb> Linters <SEP> of <SEP> TEMPO <SEP>: <SEP> 8 <SEP> 20 C <SEP> Addition <SEP> by <SEP> Hold
<tb> cotton <SEP>: <SEP> mg <SEP>;<SEP> 0.051 <SEP> small <SEP> volumes, <SEP> of <SEP> pH <SEP> to
<tb> 510mg <SEP> (3.15 <SEP> mmol <SEP> for <SEP> maintain <SEP> 10 <SEP> with
<tb> mmol) <SEP> NaBr <SEP>: <SEP> 162 <SEP><SEP> pH <SEP> to <SEP> 10, <SEP> addition
<tb> mg <SEP>;<SEP> 1.57 <SEP> of a <SEP> solution <SEP> of <SEP> soda
<tb> mmol <SEP> of <SEP> NaOCl <SEP> 1,47M <SEP> 2 <SEP>%
<tb> (0.55 <SEP> mL <SEP>;<SEP> (weight /
<tb> mmol <SEP> or <SEP> a <SEP> volume
<tb> report <SEP> eq
<tb> NaOCl / eq
<tb> anhydroglucose
<Tb>

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~= 0,26) On récupère 16 mg de fraction soluble (DO = 1) ; mg de fraction gel et 506 mg de fraction insoluble (DO = 0,12).

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ = 0.26) 16 mg of soluble fraction (OD = 1) is recovered; mg gel fraction and 506 mg insoluble fraction (OD = 0.12).

Example 2

<Tb>
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> NaBr <SEP> temperature <SEP> NaOCl <SEP> pH
<tb> departure
<tb> Same <SEP> ex. <SEP> 1 <SEP> Same as <SEP> ex.1 <SEP> 20 C <SEP> Addition <SEP> by <SEP> Same as <SEP> ex. <SEP> 1
<tb> small <SEP> volumes
<tb> for <SEP> maintain
<tb> the <SEP> pH <SEP> to <SEP> 10,
<tb> of a <SEP> solution
<tb><SEP> NaOCl <SEP> 1.47M
<tb> (1.12 <SEP> mL,
<tb> 1.65mmol <SEP>) <SEP> either
<tb> a <SEP> report <SEP> eq
<tb> NaOCl / <SEP> eq
<tb> anhydroglucose
<tb> = 0.52
<Tb>

30.7 mg (4.9% eq / eq) of soluble fraction and 475 mg (90.1% eq / eq) of insoluble fraction (OD = 0.15) are recovered.

<Desc / Clms Page number 23>

 The sample of cotton linters and the insoluble fraction suspended in water are monitored by transmission electron microscopy (Figure 3a and 3b). The starting sample (FIG. 3a) is in the form of agglomerates of crystals and a beginning of individualization of the oxidized cotton linters is noted according to the conditions of example 2 (FIG. 3b).

Example 3

<Tb>
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> NaBr <SEP> Temperature <SEP> NaOCl <SEP> pH
<tb> departure
<tb> Same <SEP> ex. <SEP> 1 <SEP> Same <SEP> ex.l <SEP> 0-4 C <SEP> Same as <SEP> ex. <SEP> 2 <SEP> Same as <SEP> ex.l
<Tb>

7.2 mg (1.15% eq / eq) of soluble fraction (OD = 1) are recovered; 6.3 mg of gel fraction and 500 mg of insoluble fraction (OD = 0.12).

Example 4

<Tb>
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> NaBr <SEP> temperature <SEP> NaOCl <SEP> pH
<tb> departure
<tb> Same <SEP> ex. <SEP> 1 <SEP> Same as <SEP> ex.1 <SEP> 20 C <SEP> Addition <SEP> by <SEP> Idem
<tb> small <SEP> volumes <SEP> ex.l
<tb> for <SEP> maintain
<tb> the <SEP> pH <SEP> to <SEP> 10,
<tb> of a <SEP> solution
<tb> of <SEP> NaOCl <SEP> 1.25
<tb> M,
<tb> (1.85 <SEP> mL <SEP>;
<tb> mmol, <SEP> or <SEP> a
<tb> report <SEP> eq
<tb> NaOCl / eq
<tb> anhydroglucose
<Tb>

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~= 0,73)
On sépare le résidu insoluble, la fraction soluble et la fraction gel, comme décrit dans l'exemple 1.

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ = 0.73)
The insoluble residue, the soluble fraction and the gel fraction are separated as described in Example 1.

 57 mg of soluble fraction (OD = 1) is recovered; 10.6 mg of gel fraction and 450 mg of insoluble fraction (OD = 0.22). The insoluble fraction suspended in water is monitored by transmission electron microscopy (FIG. 3c). It is found that the cotton linters that are initially

<Desc / Clms Page number 24>

 in the form of agglomerates are relatively well individualized.

Example 5

<Tb>
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> temperature <SEP> NaOCl <SEP> pH
<tb> departure <SEP> NaBr
<tb> Same <SEP> ex. <SEP> 1 <SEP> Same as <SEP> ex.1 <SEP> 20 C <SEP> Addition <SEP> by <SEP> Same as <SEP> ex.1
<tb> small <SEP> volumes
<tb> for <SEP> mantenir
<tb> the <SEP> pH <SEP> to <SEP> 10,
<tb> of a <SEP> solution
<tb> of <SEP> NaOCl <SEP> 1.47
<tb> M,
<tb> (4.3 <SEP>mL;<SEP> 6.32
<tb> mmol, <SEP> (either <SEP> one
<tb> report <SEP> eq
<tb> NaOCl / eq
<tb> anhydroglucose
<tb> = 2)
<Tb>

61 mg of soluble fraction (OD = 1) is recovered; 8.9 mg of gel fraction and 427 mg of insoluble fraction (OD = 23%). The insoluble fraction suspended in water is monitored by transmission electron microscopy (Figure 3d). It can be seen that the cotton linters which are initially in the form of agglomerates are relatively well individualized.

 Example 6 a. Preparation of cotton linters by acid hydrolysis.

 A sample of cotton linters (20 g) is hydrolysed with 2.5N hydrochloric acid under reflux for 1 hour, or with a 22.5 N solution of 280 ml of H 2 SO 4 (65%) for 45 minutes at 45.degree. C. The suspension is then filtered on a frit of porosity N 4 and washed abundantly with water until neutrality of the washing water. The residue is resuspended, diluted and homogenized in small amounts with ultratrope, then filtered rapidly on a sinter of porosity N 1. The suspension of cotton linters thus recovered is either lyophilized or concentrated so as to bring it to dry matter rate desired and kept cool.

<Desc / Clms Page number 25>

b. Oxidation
The conditions of Example 1 are reproduced using prehydrolysed cotton linters and a ratio of NaOCl to anhydroglucose = 0.2.

<Tb>
<Tb>

Product <SEP> of <SEP> TEMPO <SEP> + <SEP> NaBr <SEP> temperature <SEP> NaOCl <SEP> pH
<tb> departure
<tb> Linters <SEP> of <SEP> Same <SEP> ex. <SEP> 1 <SEP> 20 C <SEP> Addition <SEP> with <SEP> Same as <SEP> ex. <SEP> 1
<tb> cotton <SEP> small <SEP> volumes,
<tb> Prehydrolysed <SEP> for <SEP> maintain
<tb>: <SEP> 510 <SEP> mg <SEP><SEP> pH <SEP> to <SEP> 10,
<tb> (3,15mmol) <SEP> of a <SEP> solution
<tb> Dispersion <SEP> of <SEP> NaOCl <SEP> 1,47M
<tb> with <SEP> one <SEP>(0.43mL;<SEP> 0.63
<tb> sonicator <SEP> mmol <SEP>) <SEP> is <SEP> a
<tb> (5min) <SEP> in <SEP> report <SEP> eq
<tb> cooling <SEP> NaOCl / eq
<tb> the <SEP> container <SEP> anhydroglucose =
<tb> in <SEP> of <SEP> the <SEP> 0.2
<tb> ice cream
<Tb>

25.6 mg of soluble fraction (OD = 1) and 496 mg of insoluble fraction (OD = 0.1) are recovered.
Example 7

<Tb>
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> temperature <SEP> NaOCl <SEP> pH
<tb> departureNaBr
<tb> Same <SEP> ex. <SEP> 6 <SEP> Same as <SEP> ex. <SEP> 1 <SEP> 20 C <SEP> Addition <SEP> with <SEP> Same as <SEP> ex. <SEP> 1
<tb> small
<tb> volumes, <SEP> for
<tb> maintain <SEP> the
<tb> pH <SEP> to <SEP> 10, <SEP> of a
<tb> solution <SEP> of
<tb> NaOCl <SEP> 1.47M
<tb>(0.73mL;<SEP> 1, <SEP> 07 <SEP>
<tb> mmol <SEP>) <SEP> be <SEP> a
<tb> report <SEP> eq
<tb> NaOCl / eq
<tb> anhydroglucose
<tb> = <SEP> 0, <SEP> 34 <SEP>
<Tb>
a recovery of 39.2 mg of soluble fraction (CO = 1) and 488 mg of insoluble fraction (OD = 0.17).

<Desc / Clms Page number 26>

Example 8

<Tb>
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> temperature <SEP> NaOCl <SEP> pH
<tb> departure <SEP> NaBr
<tb> Same <SEP> ex. <SEP> 6 <SEP> Same as <SEP> ex. <SEP> 1 <SEP> 20 C <SEP> Addition <SEP> with <SEP> Same as <SEP> ex. <SEP> 1
<tb> small
<tb> volumes, <SEP> for
<tb> maintain <SEP> the
<tb> pH <SEP> to <SEP> 10, <SEP> of a
<tb> solution <SEP> of
<tb> NaOCl <SEP> 1.47M
<tb> (1.07 <SEP> mL <SEP>;<SEQ> 1.57
<tb> mmol <SEP>) <SEP> be <SEP> a
<tb> report <SEP> eq
<tb> NaOCl / eq
<tb> anhydroglucose
<Tb>

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~= 0, 5
On récupère 49,8 mg de fraction soluble (DO = 1) et 454 mg de fraction insoluble (DO = 0,17).

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ = 0, 5
49.8 mg of soluble fraction (OD = 1) and 454 mg of insoluble fraction (OD = 0.17) are recovered.

Example 9 a. Transformation of cellulose I into cellulose III
2 g of cotton linters are weighed into a 250 ml flask in which liquid ammonia (obtained by liquefaction) is added. The cellulose is completely covered with liquid ammonia. The flask is cooled by an acetone bath maintained between -50 and -60 ° C. by addition of liquid nitrogen. These conditions are maintained for about 5 hours.

Ammonia then causes the cellulose crystals I to swell, which progressively transform into cellulose III. We then formed crystals of much smaller size. The ammonia is allowed to evaporate slowly (in a few hours).

Residual ammonia is removed by vacuum stripping for 24 hours.

 It is also possible to use samples of cellulose treated with ammonia under pressure and decompressed rapidly according to the Rhodia Acetow process (T. Karstens, German Patent No. 19951011061).

<Desc / Clms Page number 27>

b. Oxidation
The conditions of Example 2 are reproduced using crystallized cotton linters in the form of cellulose III (ie, anhydroglucose ratio / eq NaOCl = 1 / 0.5).

<Tb>
<Tb>

Product <SEP> of <SEP> TEMPO <SEP> + <SEP> NaBr <SEP> Temperature <SEP> NaOCl <SEP> pH
<tb> departure
<tb> Linters <SEP> of <SEP> Same <SEP> ex. <SEP> 1 <SEP> 20 C <SEP> Same as <SEP> ex.2, <SEP> or <SEP> Same as <SEP> ex.l
<tb> cotton <SEP> a <SEP> report <SEP> eq
<tb> crystallized <SEP> NaOCl / <SEP> eq
<tb> under <SEP> form <SEP> anhydroglucose
<tb> of <SEP> cellulose <SEP> = 0.52
<tb> III <SEP>: <SEP> 510 <SEP> mg
<tb> (3, <SEP> 15mmol) <SEP>
<Tb>

25 mg of soluble fraction (OD = 1) are recovered; 12 mg of gel fraction and 457 mg of insoluble fraction (OD = 0.16).

Example 10

<Tb>
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> NaBr <SEP> Temperature <SEP> NaOCl <SEP> pH
<tb> departure
<tb> Same <SEP> ex. <SEP> 9 <SEP> Same <SEP> ex.l <SEP> 0-4 C <SEP> Same as <SEP> ex. <SEP> 2, <SEP> Idem
<tb> either <SEP> a <SEP> report <SEP> ex. <SEP> 1
<tb> eq <SEP> NaOCl / <SEP> eq
<tb> anhydroglucose
<tb> = 0.52
<Tb>

12 mg of soluble fraction (OD = 1) is recovered; 10 mg of gel fraction and 496 mg of insoluble fraction (OD = 0.16).

Example 11

<Tb>
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> NaBr <SEP> Temperature <SEP> NaOCl <SEP> pH
<tb> departure
<tb> Same <SEP> ex. <SEP> 9 <SEP> Same <SEP> ex.l <SEP> 20 C <SEP> Same as <SEP> Ex.4, <SEP> Same as <SEP> ex.1
<tb> be <SEP> one
<tb> report <SEP> eq
<tb> NaOCl / <SEP> eq
<tb> anhydroglucose
<tb> = 0.73
<Tb>

45 mg of soluble fraction (OD = 1) are recovered; 10 mg of gel fraction and 485 mg of insoluble fraction (OD = 0.20).

<Desc / Clms Page number 28>

Example 12

<Tb>
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> Temperature <SEP> NaOCl <SEP> pH
<tb> departure <SEP> NaBr
<tb> microfibrils <SEP> Same <SEP> 20 C <SEP> Addition <SEP> with <SEP> Same <SEP> ex.
<tb> of <SEP> cellulose <SEP> ex. <SEP> 1 <SEP> small <SEP> volumes, <SEP> 1
<tb> got <SEP> to <SEP> to <SEP> maintain
<tb> from <SEP> of <SEP> pulp <SEP><SEP> pH <SEP> to <SEP> 10,
<tb> of <SEP> beets <SEP> of a <SEP> solution
<tb> sugar <SEP>: <SEP> from <SEP> NaOCl <SEP> 1.47
<tb> 510 <SEP> mg <SEP> M, <SEP> (0, <SEP> 38 <SEP>mL;
<tb> (3.15mmol) <SEP> 0.56 <SEP> mmol,
<tb> (ie <SEP> one
<tb> report <SEP> eq
<tb> NaOCl / eq
<tb> anhydroglucose =
<tb> 0.18)
<Tb>

In the case of cellulose microfibrils, during the purification step, it is advantageous to add sodium chloride so as to screen the charges, which causes flocculation and thus allows separation by centrifugation, as in the case cotton linters.

 It is also possible to filter on a 5 μm or 1 μm porosity membrane to recover the insoluble part, which is washed with distilled water in order to remove the salts. The soluble portion is concentrated, dialyzed and lyophilized.

 The insoluble fraction is redispersed in water and stirred for one hour, then centrifuged for separation and lyophilized. The fraction which remains in suspension (gel fraction) is concentrated, dialyzed and lyophilized.

 21 mg of soluble fraction (OD = 1) is recovered; 15 mg of gel fraction and 435 mg of insoluble fraction (OD = 0.06). The insoluble fraction suspended in water is monitored by transmission electron microscopy (Figure 4a). It is noted that the cellulose microfibrils are relatively associated with each other in strips and have a length of the order of one micron or greater.

<Desc / Clms Page number 29>

Example 13

<Tb>
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> NaBr <SEP> Temperature <SEP> NaOCl <SEP> pH
<tb> departure
<tb> Same <SEP> ex. <SEP> 12 <SEP> Same as <SEP> ex.l <SEP> 20 C <SEP> Addition <SEP> by <SEP> Same as <SEP> ex.l
<tb> small <SEP> volumes,
<tb> for <SEP> maintain
<tb> the <SEP> pH <SEP> to <SEP> 10,
<tb> of a <SEP> solution
<tb> of <SEP> NaOCl <SEP> (in <SEP> 5
<tb> min) <SEP> from <SEP> NaOCl
<tb> 1.47 <SEP> M, (0.55
<tb> mL <SEP>;<SEP> 0.81 <SEP> mmol,
<tb> either <SEP> a <SEP> report
<tb> eq <SEP> NaOCl / eq
<tb> anhydroglucose =
<tb> 0.26)
<Tb>

33 mg of soluble fraction (OD = 1) is recovered; 39 mg of gel fraction and 447 mg of insoluble fraction (OD = 0.07).

Example 14

<Tb>
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> NaBr <SEP> Temperature <SEP> NaOCl <SEP> pH
<tb> departure
<tb> Same <SEP> ex. <SEP> 12 <SEP> Same as <SEP> ex.1 <SEP> 20 C <SEP> Addition <SEP> by <SEP> Same as <SEP> ex.l
<tb> small <SEP> volumes,
<tb> for <SEP> maintain
<tb> the <SEP> pH <SEP> to <SEP> 10,
<tb> of a <SEP> solution
<tb> of <SEP> NaOCl <SEP> 1.47
<tb> M, (0.76 <SEP>mL;
<tb> 1.12 <SEP> mmol, <SEP> either
<tb> a <SEP> report <SEP> eq
<tb> NaOCl / eq
<tb> anhydroglucose =
<tb> 0.36)
<Tb>

44 mg of soluble fraction (OD = 1) are recovered; 29 mg of gel fraction and 449 mg of insoluble fraction (OD = 0.12). The insoluble fraction suspended in water is monitored by transmission electron microscopy (Figure 4b). It is found that the cellulose microfibrils are no longer associated with each other in strips. They are in the form of crystallites relatively well individualized due to the presence of surface charges and variable length between 100 nm and 1 micron.

<Desc / Clms Page number 30>

Example 15

<Tb>
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> Temperature <SEP> NaOCl <SEP> pH
<tb> departure <SEP> NaBr
<tb> Same <SEP> ex. <SEP> 12 <SEP> Same as <SEP> ex. <SEP> 1 <SEP> 20 C <SEP> Addition <SEP> with <SEP> Same as <SEP> ex. <SEP> 1
<tb> small
<tb> volumes, <SEP> for
<tb> maintain <SEP> the
<tb> pH <SEP> to <SEP> 10, <SEP> of a
<tb> solution <SEP> of
<tb> NaOCl <SEP> 1.47 <SEP> M,
<tb> (1.26 <SEP>mL;<SEP> 1.86
<tb> mmol, <SEP> or <SEP> a
<tb> report <SEP> eq
<tb> NaOCl / eq
<tb> anhydroglucose
<tb> = <SEP> 0.6)
<Tb>

55 mg of soluble fraction (OD = 1) is recovered; 31 mg gel fraction and 442 mg insoluble fraction (OD = 0.18).

Example 16

<Tb>
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> Temperature <SEP> NaOCl <SEP> pH
<tb> departure <SEP> NaBr
<tb> Same <SEP> ex. <SEP> 12 <SEP> Same as <SEP> ex. <SEP> 1 <SEP> 20 C <SEP> Addition <SEP> with <SEP> Same as <SEP> ex. <SEP> 1
<tb> small <SEP> volumes,
<tb> for <SEP> maintain
<tb> the <SEP> pH <SEP> to <SEP> 10,
<tb> of a <SEP> solution
<tb> of <SEP> NaOCl <SEP> 1.47
<tb> M, <SEP> (1, <SEP> 58 <SEP> mL <SEP>;
<tb> 2.34 <SEP> mmol,
<tb> (ie <SEP> one
<tb> report <SEP> eq
<tb> NaOCl / eq
<tb> anhydroglucose =
<tb> 0.75)
<Tb>

63 mg of soluble fraction (OD = 1) are recovered; 24 mg of gel fraction and 428 mg of insoluble fraction (OD = 0.24). The insoluble fraction suspended in water is monitored by transmission electron microscopy (FIG. 4c). It can be seen that the cellulose microfibrils are no longer associated with each other in strips, but are in the form of relatively well-crystallized crystallites with a length of between 100 nm and 1 μm.

<Desc / Clms Page number 31>

Example 17

<Tb>
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> NaBr <SEP> Temperature <SEP> NaOCl <SEP> pH
<tb> departure
<tb> Same <SEP> ex. <SEP> 12 <SEP> Same as <SEP> ex.1 <SEP> 20 C <SEP> Addition <SEP> by <SEP> Same as <SEP> ex. <SEP> 1
<tb> small <SEP> volumes,
<tb> for <SEP> maintain
<tb> the <SEP> pH <SEP> to <SEP> 10,
<tb> of a <SEP> solution
<tb> of <SEP> NaOCl <SEP> 1.47
<tb> M, (1.9 <SEP>mL;<SEP> 2.8
<tb> mmol, <SEP> (either <SEP> one
<tb> report <SEP> eq
<tb> NaOCl / eq
<tb> anhydroglucose =
<tb> 0.9)
<Tb>

112 mg of soluble fraction (OD = 1) is recovered; 33 mg gel fraction and 402 mg insoluble fraction (OD = 0.24).

Example 18

<Tb>
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> NaBr <SEP> Temperature <SEP> NaOCl <SEP> pH
<tb> departure
<tb> Same <SEP> ex.12 <SEP> Same <SEP> ex.l <SEP> 0-4 C <SEP> Addition <SEP> by <SEP> Same <SEP> ex. <SEP> 1
<tb> small <SEP> volumes
<tb> for <SEP> maintain
<tb> the <SEP> pH <SEP> to <SEP> 10,
<tb> of a <SEP> solution
<tb><SEP> NaOCl <SEP> 1.47M
<tb> (1.12 <SEP> mL,
<tb> 1.65mmol)
<tb> be <SEP> one
<tb> report <SEP> eq
<tb> NaOCl / <SEP> eq
<tb> anhydroglucose
<tb> = 0.52
<Tb>

27 mg of soluble fraction (OD = 1) is recovered; 21 mg of gel fraction and 452 mg of insoluble fraction (OD = 0.14).

<Desc / Clms Page number 32>

<Tb>
<Tb>

Example <SEP> 19
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> Temperature <SEP> NaOCl <SEP> pH
<tb> departure <SEP> NaBr
<tb> microfibrils <SEP> Same <SEP> 20 C <SEP> Same <SEP> ex. <SEP> 16, <SEP> Same as <SEP> ex. <SEP> 1
<tb> of <SEP> cellulose <SEP> ex. <SEP> is <SEP> a <SEP> report <SEP>
<tb> obtained <SEP> at <SEP> eq <SEP> NaOCl /
<tb> from <SEP> of <SEP> pulp <SEP> anhydroglucose
<tb> of <SEP> beets <SEP> = 0,75
<tb> sugar
<tb> processed <SEP> with
<tb> 50 <SEP> mg <SEP> approximately
<tb> of <SEP> NaBH4,
<tb> weak
<tb> agitation
<tb> during <SEP> 12h <SEP>: <SEP>
<tb> 510 <SEP> mg
<tb> (3.15mmol)
<Tb>

46 mg of soluble fraction (OD = 1) are recovered; 4 mg gel fraction and 444 mg insoluble fraction (OD = 0.20).

Example 20

<Tb>
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> Temperature <SEP> NaOCl <SEP> pH
<tb> departure <SEP> NaBr
<tb> microfibri <SEP> Same as <SEP> ex. <SEP> 1 <SEP> 20 C <SEP> Addition <SEP> with <SEP> Same as <SEP> ex. <SEP> 1
<tb> Lies <SEP> of <SEP> small
<tb> cellulose <SEP> volumes, <SEP> for
<tb> obtained <SEP> to <SEP> maintain <SEP> the
<tb> from <SEP> of <SEP> pH <SEP> to <SEP> 10, <SEP> of a <SEP>
<tb> pulp <SEP> of <SEP> solution <SEP> of
<tb> beets <SEP> NaOCl <SEP> 1.47
<tb> sugar <SEP> (0.38 <SEP>mL;<SEP> 0.56
<tb> crystallize <SEP> mmol, <SEP> or <SEP> a <SEP>
<tb> es <SEP> under <SEP> report <SEP> eq
<tb> form <SEP> of <SEP> NaOC1 / eq
<tb> cellulose <SEP> anhydroglucose
<tb> III <SEP>: <SEP> 510 <SEP> = <SEP> 0.52)
<tb> mg
<Tb>

(3,15mmol) ~~~~~~~~~~~~~~~~~~~~##-#####################
On récupère 40 mg de fraction soluble (DO= 1) ; 66 mg de fraction gel et 430 mg de fraction insoluble (DO= 0,18).

(3,15mmol) ~~~~~~~~~~~~~~~~~~~~ ## - ########################
40 mg of soluble fraction (OD = 1) are recovered; 66 mg of gel fraction and 430 mg of insoluble fraction (OD = 0.18).

<Desc / Clms Page number 33>

<Tb>
<Tb>

Exemple21
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> Temperature <SEP> NaOCl <SEP> pH <SEP> ~~~~~
<Tb>

départ NaBr Idem ex. Idem ex. 1 0-4 C Idem ex. 20, Idem ex. soit

departure NaBr Idem ex. Same as ex. 1 0-4 C Idem ex. 20, Idem ex. is

<Tb>
<tb> report <SEP> eq
<tb> NaOCl / <SEP> eq
<tb> anhydroglucose
<tb> = 0.52
<Tb>
34 mg of soluble fraction (OD = 1) are recovered; 9 mg of gel fraction and 458 mg of insoluble fraction (OD = 0.13).

Example 22

<Tb>
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> Temperature <SEP> NaOCl
<tb> departure <SEP> NaBr
<tb> Cellulose <SEP> Same as <SEP> ex. <SEP> 1 <SEP> 20 C <SEP> Addition <SEP> with <SEP> Same as <SEP> ex.1
<tb> microcrystal <SEP> small <SEP> for
<tb> line <SEP> volumes, <SEP> for
<tb> (Avicel) <SEP>. <SEP> maintain <SEP> the
<tb> 510 <SEP> mg <SEP> pH <SEP> to <SEP> 10 <SEP>, <SEP> of one
<tb> (3,15mmol) <SEP> solution <SEP> of
<tb>(3'15mol)<SEP> NaOCl <SEP> 1.42 <SEP> M,
<tb> (0.58 <SEP>mL;
<tb> 0.83mmol, <SEP> either
<tb> a <SEP> report <SEP> eq
<tb> NaOCl / eq
<tb> anhydroglucose
<tb> = <SEP> 0.26
<Tb>

28 mg of soluble fraction (OD = 1) are recovered; 35 mg of gel fraction and 453 mg of insoluble fraction (OD = 0.12).

Example 23

<Tb>
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> Temperature <SEP> NaOCl <SEP> pH
<Tb>

départ NaBr Idem ex. 22 Idem ex. 1 20 C Addition par Idem ex. petits

departure NaBr Idem ex. 22 Idem ex. 1 20 C Addition by Idem ex. small

<Tb>
<tb> volumes, <SEP> for
<tb> maintain <SEP> the
<tb> pH <SEP> to <SEP> 10, <SEP> of a
<tb> solution <SEP> of
<tb> NaOCl <SEP> 1.42 <SEP> M,
<tb>(1.16mL;
<tb> 1.65mmol, <SEP> either
<tb> a <SEP> report <SEP> eq
<tb> NaOC1 / eq
<tb> anhydroglucose
<tb> = <SEP> 0.52)
<Tb>

<Desc / Clms Page number 34>

63 mg of soluble fraction (OD = 1) are recovered; 25 mg of gel fraction and 443 mg of insoluble fraction (OD = 0.19)
Example 24

<Tb>
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> Temperature <SEP> NaOCl <SEP> pH
<tb> departure <SEP> NaBr
<tb> Cellulose <SEP> Same as <SEP> ex. <SEP> 1 <SEP> 20 C <SEP> Addition <SEP> with <SEP> Same as <SEP> ex. <SEP> 1
<tb> bacterial <SEP> small
<tb>: <SEP> 510 <SEP> mg <SEP> volumes, <SEP> for
<tb> (3,15mmol) <SEP> maintain <SEP> the
<tb> pH <SEP> to <SEP> 10, <SEP> of a
<tb> solution <SEP> of
<tb> NaOCl <SEP> 1.47M
<tb>(1.26mL;<SEP> 1.86
<tb> mmol, <SEP> (either <SEP> one
<tb> report <SEP> eq
<tb> NaOCl / eq
<tb> anhydroglucose
<tb> = <SEP> 0.6)
<Tb>

60 mg of soluble fraction (OD = 1) are recovered; 35 mg of gel fraction and 433 mg of insoluble fraction (OD = 0.17%).

Example 25 a. Preparation of tunicate whiskers by acid hydrolysis
A 20 g sample of cellulose pulp prepared from tunicate according to the procedure already described (Favier V., Chanzy H. and Cavaillé JY., Macromolecules, 28 (1995) 6365-6367), is hydrolyzed either with 2.5 N hydrochloric acid at reflux for 1 hour, or with a 22.5 N solution of 280 ml of sulfuric acid (65%) for 45 minutes at 45 ° C. The suspension is then filtered on a sinter of porosity N 4 and washed thoroughly with water until the washing water is neutral. The residue is resuspended, diluted and homogenized in small amounts with ultratrope, and then rapidly filtered on a sintered porosity N 1. The whisker suspension thus recovered is either freeze-dried or concentrated to bring it to the rate of dry matter desired and kept cool.

<Desc / Clms Page number 35>

b. Oxidation
The conditions of Example 1 are reproduced by using tunicate whiskers and a NaOCl / eq anhydroglucose eq = 0.06 ratio.

<Tb>
<Tb>

Product <SEP> of <SEP> TEMPO <SEP> + <SEP> Temperature <SEP> NaOCl <SEP> pH
<tb> departure <SEP> NaBr
<tb> whiskers <SEP> of <SEP> TEMPO <SEP>: <SEP> 20 C <SEP> Addition <SEP> by <SEP> Idem
<tb> tunicate <SEP> 14,75mg <SEP>;<SEP> small <SEP> volumes, <SEP> ex.1
<tb> Prehydrolysed <SEP>: <SEP> 5 <SEP> 0.094 <SEP> mmol <SEP> for <SEP> maintain
<tb> 10 <SEP> mg <SEP> (3.15mmol) <SEP> NaBr <SEP>: <SEP><SEP> pH <SEP> to <SEP> 10,
<tb> brought <SEP> to <SEP> 100 <SEP> ml <SEP> 162mg <SEP>;<SEP> of a <SEP> solution
<tb> by <SEP> add. <SEP> of water <SEP> 1.57 <SEP> mmol <SEP> of <SEP> NaOCl <SEP> 1.47
<tb> dist. <SEP> M, (0.13mL; <SEP> 0.19
<tb> Dispersion <SEP> with <SEP> mmol, <SEP> or <SEP> one
<tb> a <SEP> sonicator <SEP> report <SEP> eq
<tb> (5min) <SEP> in <SEP> NaOCl / eq
<tb> cooling <SEP> the <SEP> anhydroglucose =
<tb> container <SEP> in <SEP> 0,06)
<tb> of <SEP> the <SEP> ice cream
<Tb>

The suspension is filtered on sintered N 4, then washed abundantly with water. The cellulosic residue thus obtained is resuspended in water and kept cool.

 503 mg of insoluble fraction (OD = 0.03) are recovered.

 The starting sample and the oxidized sample are monitored by infrared spectroscopy: FIGS. 5a, 5b, respectively.

Example 26

<Tb>
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> NaBr <SEP> Temperature <SEP> NaOCl <SEP> pH
<tb> departure
<tb> Same <SEP> ex. <SEP> 25 <SEP> Same <SEP> ex.25 <SEP> 20 C <SEP> Addition <SEP> by <SEP> Same as <SEP> ex. <SEP> 1
<tb> small <SEP> volumes,
<tb> for <SEP> maintain
<tb> the <SEP> pH <SEP> to <SEP> 10,
<tb> of a <SEP> solution
<tb> of <SEP> NaOCl <SEP> 1.47
<tb> M,
<tb> (0.26 <SEP>mL;
<tb> 0.38mmol, <SEP> either
<tb> a <SEP> report <SEP> eq
<tb> NaOCl / eq
<tb> anhydroglucose =
<tb> 0.12)
<Tb>

<Desc / Clms Page number 36>

The suspension is filtered on sintered N 4, then washed abundantly with water.

 500 mg of insoluble fraction (OD = 0.07) are recovered.

 The sample is monitored by infrared spectroscopy (Figure 5c).

Example 27

<Tb>
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> NaBr <SEP> Temperature <SEP> NaOCl <SEP> pH
<tb> departure
<tb> Same <SEP> ex. <SEP> 25 <SEP> Same as <SEP> ex. <SEP> 25 <SEP> 20 C <SEP> Addition <SEP> with <SEP> Same as <SEP> ex. <SEP> 1
<tb> small <SEP> volumes,
<tb> for <SEP> maintain
<tb> the <SEP> pH <SEP> to <SEP> 10,
<tb> of a <SEP> solution
<tb> of <SEP> NaOCl <SEP> 1.47
<tb> M,
<tb> (0.43 <SEP>mL;<SEP> 0.63
<tb> mmol, <SEP> (either <SEP> one
<tb> report <SEP> eq
<tb> NaOCl / eq
<tb> anhydroglucose =
<tb> 0.20)
<Tb>

The suspension is filtered on sintered N 4, then washed abundantly with water.

 481 mg of insoluble fraction (OD = 0.08) are recovered.

 The sample is monitored by infrared spectroscopy (Figure 5d).

Example 28

<Tb>
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> NaBr <SEP> Temperature <SEP> NaOCl <SEP> pH
<tb> departure
<tb> Same <SEP> ex. <SEP> 25 <SEP> Same as <SEP> ex. <SEP> 25 <SEP> 20 C <SEP> Addition <SEP> with <SEP> Same as <SEP> ex. <SEP> 1
<tb> small <SEP> volumes,
<tb> for <SEP> maintain
<tb> the <SEP> pH <SEP> to <SEP> 10,
<tb> of a <SEP> solution
<tb> of <SEP> NaOCl <SEP> 1.47
<tb> M,
<tb> (1.07 <SEP>mL;<SEP> 1.57
<tb> mmol, <SEP> (either <SEP> one
<tb> report <SEP> eq
<tb> NaOCl / eq
<tb> anhydroglucose =
<tb> 0.5)
<Tb>

The suspension is filtered on sintered N 4, then washed abundantly with water.

<Desc / Clms Page number 37>

 458 mg of insoluble fraction (OD = 0.10) are recovered.

The sample is monitored by infrared spectroscopy (Figure 5e). On the infrared spectra of FIG.
O the existence of a C-OH band at 1730 cm-1 of increasing intensity, which makes it possible to verify the increase in the degree of oxidation (OD) with the amount of NaOCl used. Samples are also controlled by X-ray (Figure 6). In this figure, the X-ray image of the sample of starting tunicate whiskers (a) and totally oxidized on the surface (b) of Example 28 is reported. It can be seen that the two images are identical and therefore that the crystallinity of the two samples is the same. FIG. 7 represents a transmission electron microscopy photo of tunicate (a) whisker whiskers after acid hydrolysis and (b) totally surface oxidized. The individualization of the crystals after oxidation can be observed. The diluted sample suspended in water is observed between crossed polarizers (Figure 8); we can observe a liquid crystal behavior (nematic chiral organization).

Example 29

<Tb>
<tb> Product <SEP> of <SEP> starting <SEP> TEMPO <SEP> + <SEP> Temperature <SEP> NaOCl <SEP> pH
<tb> NaBr
<tb> Paste <SEP> Borregard <SEP> Same as <SEP> 20 C <SEP> Addition <SEP> by <SEP> Same as <SEP> ex.l
<tb> (crushed <SEP> and <SEP> eg <SEP> 25 <SEP> small <SEP> volumes,
<tb> sieved): <SEP> 510 <SEP> mg <SEP> to <SEP> maintain
<tb> (3.15mmol) <SEP><SEP> pH <SEP> to <SEP> 10,
<tb> of a <SEP> solution
<tb><SEP> NaOCl <SEP> 1.28 <SEP> M
<tb> (2.21 <SEP>mL;<SEP> 2.83
<tb> mmol, <SEP> (either <SEP> one
<tb> report <SEP> eq
<tb> NaOCl / eq
<tb> anhydroglucose =
<tb> 0.9)
<Tb>

56 mg of soluble fraction (OD = 1) and 451 mg of insoluble fraction (OD = 0.19) are recovered.

<Desc / Clms Page number 38>

<Tb>
<Tb>

Example <SEP> 30
<Tb>

Produit de TEMPO + Température Na0C1 pH départ NaBr kôc -kddîHTT Idem Rayonne: Idem ex. 20 C Addition par Idem ex.

Product of TEMPO + Temperature Na0C1 pH starting NaBr koc -kddîHTT Idem Rayon: Idem ex. 20 C Addition by Idem ex.

Rayon: Same as ex. small volumes,

<Tb>
<tb> 510 <SEP> mg <SEP> (3.15 <SEP> 25 <SEP> small <SEP> volumes, <SEP>
<tb> mmol) <SEP><SEP> pH <SEP> to <SEP> 10,
<tb> of a <SEP> solution
<tb><SEP> NaOCl <SEP> 1.28 <SEP> M
<tb> (0.25 <SEP>mL;<SEP> 0.32
<tb> mmol, <SEP> (either <SEP> one
<tb> report <SEP> eq
<tb> NaOC1 / eq
<tb> anhydroglucose <SEP> =
<tb> 0.1)
<Tb>
495 mg of insoluble fraction (OD = 0.03) are recovered.

Example 31

<Tb>
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> Temperature <SEP> NaOCl <SEP> pH
<Tb>

départ NaBr Idem ex. 30 Idem ex. 20 C Addition par Idem ex.

departure NaBr Idem ex. 30 Idem ex. 20 C Addition by Idem ex.

25 small volumes,

<Tb>
<tb> for <SEP> maintain
<tb> the <SEP> pH <SEP> to <SEP> 10,
<tb> of a <SEP> solution
<tb><SEP> NaOCl <SEP> 1.28 <SEP> M
<tb> (1.48 <SEP>mL;<SEQ> 1.89
<tb> mmol, <SEP> (either <SEP> one
<tb> report <SEP> eq
<tb> NaOCl / eq
<tb> anhydroglucose =
<tb> 0, <SEP> 6) <SEP>
<Tb>

104 mg of soluble fraction (OD = 1) and 407 mg of insoluble fraction (OD = 0.20) are recovered.

Example 32

<Tb>
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> Temperature <SEP> NaOCl <SEP> pH
<Tb>

départ NaBr Idem ex. 30 Idem ex. 25 20 C Addition par Idem petits volumes,

departure NaBr Idem ex. 30 Idem ex. 25 20 C Addition by Ditto small volumes,

<Tb>
<tb> for <SEP> maintain
<tb> the <SEP> pH <SEP> to <SEP> 10,
<tb> of <SEP> a <SEP> solution
<tb><SEP> NaOCl <SEP> 1.28 <SEP> M
<tb> (2.21 <SEP>mL;<SEP> 2.83
<tb> mmol, <SEP> (either <SEP> one
<tb> report <SEP> eq
<tb> NaOCl / eq
<tb> anhydroglucose <SEP> =
<tb> L <SEP> ~ <SEP> 0.9)
<Tb>

<Desc / Clms Page number 39>

152 mg of soluble fraction (OD = 1) and 390 mg of insoluble fraction (OD = 0.27) are recovered.

Example 33 a. Preparation of aggregates of starch crystals (or starch liquors) by acid hydrolysis
A starch sample (Waxilys type) was hydrolysed with 2.5N hydrochloric acid at 35 C for one week. The suspension is centrifuged and the solid residue is resuspended in a volume of water (100 mL) and then centrifuged. This treatment is carried out several times until neutrality of the washing water. The insoluble material is resuspended in water and stored at 4 ° C. in a cold room for subsequent use.

<Tb>
<Tb>

Product <SEP> of <SEP> TEMPO <SEP> + <SEP> NaBr <SEP> Temperature <SEP> NaOCl <SEP> pH
<tb> departure
<tb> dispersion <SEP> of <SEP> Same <SEP> ex.1 <SEP> 20 C <SEP> Same as <SEP> ex. <SEP> 27, <SEP> Same as <SEP> ex.
<tb> grain <SEP> either <SEP> a <SEP> report
<tb> Starch <SEP>;<SEP> eq <SEP> NaOCl / <SEP> eq
<tb> volume <SEP> of <SEP> the <SEP> anhydroglucose
<tb> suspension <SEP> = 0.20
<tb> for <SEP> have
<tb> 510 <SEP> mg
<tb> (3, <SEP> 15mmol) <SEP>
<Tb>

110 mg of soluble fraction (OD = 0.19) and 362 mg of insoluble fraction (OD = 0.06) are recovered.
Example 34

<Tb>
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> NaBr <SEP> Temperature <SEP> NaOCl <SEP> pH
<tb> departure
<tb> Same <SEP> ex. <SEP> 33 <SEP> Same as <SEP> ex.1 <SEP> 20 C <SEP> Addition <SEP> by <SEP> Same as <SEP> ex.l
<tb> small <SEP> volumes,
<tb> for <SEP> maintain
<tb> the <SEP> pH <SEP> to <SEP> 10,
<tb> of a <SEP> solution
<tb> of <SEP> NaOCl <SEP> 1.47
<tb> M,
<tb> (1.29 <SEP> mL <SEP>;
<tb> l, 9mmol, <SEP> is
<tb> a <SEP> report <SEP> eq
<tb> NaOCl / eq
<tb> anhydroglucose =
<tb> 0.6)
<Tb>

<Desc / Clms Page number 40>

167 mg of soluble fraction (OD = 0.30) and 318 mg of insoluble fraction (OD = 0.11) are recovered. Figure 9 shows a transmission electron microscopy photo of starch crystals after oxidation (0.6 eq NaOCl / eq anhydroglucose) (Example 34). We can observe the platelet organization of these crystals.

Example 35

<Tb>
<tb> Product <SEP> of <SEP> TEMPO <SEP> + <SEP> Temperature <SEP> NaOCl <SEP> PH
<tb> departure <SEP> NaBr
<tb> Same <SEP> ex. <SEP> 33 <SEP> Same as <SEP> ex.l <SEP> 20 C <SEP> Addition <SEP> by <SEP> Same as <SEP> ex.l
<tb> small <SEP> volumes,
<tb> for <SEP> maintain <SEP> the
<tb> pH <SEP> to <SEP> 10, <SEP> of a
<tb> solution <SEP> of <SEP> NaOCl
<tb> 1.47M <SEP>(2.14mL;
<tb> 3,15mmol, <SEP> (ie <SEP> one
<tb> report <SEP> eq
<tb> NaOCl / eq
<tb> anhydroglucose = <SEP> 1)
<Tb>

293 mg of soluble fraction (OD = 0.39) and 187 mg of insoluble fraction (OD = 0.12) are recovered.

Example 36 a. Preparation of chitin whiskers by acid hydrolysis
A sample of chitin paste, prepared from squid feather according to the procedure already described (Paillet M. and Dufresne A., Macromolecules, 34 (2001) 6527-6530), is hydrolysed with a solution of hydrochloric acid 2 5N at reflux for 3h. The suspension is then filtered on a frit of porosity N 4 and washed abundantly with water until neutrality of the washing water. The residue is resuspended, diluted and homogenized in small amounts with ultratrope, then rapidly filtered through a sintered N 1 porosity. The chitin suspension thus recovered is either lyophilized or concentrated so as to bring it to the desired dry matter content and stored at 4 ° C. in a cold room.

<Desc / Clms Page number 41>

c. Oxidation
510 mg (2.51 mmol) of chitin whiskers are dispersed in 100 ml of distilled water using a sonicator (ultrasound) for 5 minutes while cooling the container in an ice bath. The TEMPO (11.8 mg, 0.075 mmol) and NaBr (129 mg, 1.26 mmol) are added. The solution is maintained at 20 ° C. and the 1.47M NaOCl commercial solution (0.85 ml, 1.25 mmol, ie an anhydroglucose / NaOCl eq = 1 / 0.5 ratio) is added in small volumes so that maintain the pH at 10. After the addition of all the hypochlorite (generally between 5 and 60 minutes, more particularly in less than 30 minutes), the pH is constantly maintained at 10 by adding a sodium hydroxide solution (NaOH 2 % w / v). We continue to add sodium hydroxide until the pH no longer varies. Finally, the reaction is stopped by adding MeOH and then neutralized (pH7) with 1M HCl.

<Tb>
<Tb>

Product <SEP> of <SEP> TEMPO <SEP> + <SEP> NaBr <SEP> Temperature <SEP> NaOCl <SEP> pH
<tb> departure
<tb> Suspension <SEP> of <SEP> TEMPO <SEP>: <SEP> 20 C <SEP> Addition <SEP> by <SEP> Same <SEP> ex.1
<tb> whiskers <SEP> of <SEP> ll, 8mg <SEP>;<SEP> small <SEP> volumes,
<tb> chitin <SEP> 0.075 <SEP> mmol <SEP> for <SEP> maintain
<tb> prehydrolysed <SEP> NaBr <SEP>: <SEP> 129 <SEP><SEP> pH <SEP> to <SEP> 10,
<tb>: <SEP> 510 <SEP> mg <SEP> mg <SEP>;<SEP> 1.26 <SEP> of a <SEP> solution
<tb> (2.51mmol) <SEP> mmol <SEP> from <SEP> NaOCl <SEP> 1.47 <SEP> M
<tb> brought <SEP> to <SEP> 100 <SEP>(0.85mL;<SEP> 1.25
<tb> ml <SEP> by <SEP> add. <SEP> mmol, <SEP> (ie <SEP> one
<tb> water <SEP> dist. <SEP> report <SEP> eq
<tb> Dispersion <SEP> NaOCl / eq
<tb> with <SEP> an <SEP> anhydroglucose
<tb> sonicator <SEP> = <SEP> 0.5) <SEP>
<tb> (5min) <SEP> in
<tb> cooling
<tb> the <SEP> container
<tb> in <SEP> of <SEP> la
<tb> ice cream
<Tb>

The suspension is filtered on sintered N 4, then washed abundantly with water. The chitin residue thus obtained is resuspended in water and kept cool.

 498 mg of insoluble fraction (OD = 0.17) are recovered.

<Desc / Clms Page number 42>

 Figure 10 shows a transmission electron micrograph of chitin crystals: (a) after acid hydrolysis; (b) after oxidation NaOCl (0.5 eq NaOCl / eq anhydroglucose) (Example 36).

Example 37 a. Coupling reaction of oxidized polysaccharides soluble in water
78 mg (0.39 mmol) of 100% oxidized cellulose are dissolved in 5 ml of water. 74 μL (0.85 mmol) of 2-methoxyethylamine are added. The pH is brought to 8 with 0.5 N hydrochloric acid solution. 110 mg (0.57 mmol) of EDAC and 66 mg (0.57 mmol) of NHS dissolved in 2 ml of water are added. The solution is kept at 50 ° C. with stirring for 24 hours and the pH is maintained around 8. b. Purification
The coupling product can be purified either by dialysis (for several days) against distilled water, or by ultrafiltration for 60 hours with a YM1 membrane (Amicon, Inc.) which has a cutoff of Mw 1000, or by precipitation in ethanol followed by filtration or centrifugation.

 73 mg of product is recovered. The carbon 13 NMR spectrum of a 100% oxidized cellulose sample is shown in FIG. 11a and in FIG. 11b the carbon spectrum 13 of the same cellulose sample after coupling with 2-methoxyethylamine. According to these quantitative NMR spectra, it is possible to deduce a coupling ratio (TC) of the order of 70%, which makes it possible to calculate the yield of the reaction which is of the order of 83%.

The yield of the reaction is obtained by the equation:
Yield = [m / (TC x M + (1-TC) x 198)] x 100 / n With: m = mass of the sample in mg, TC = coupling rate (ie the% of carboxyl functions coupled, value between 0 and 100%), M = molar mass of the unit

<Desc / Clms Page number 43>

 coupled anhydrofluorose, n = number of moles of anhydroglucose units oxdated starting.

In the tables below, the coupling conditions used in Example 37, as well as in Examples 37 to 45, which relate to coupling reactions conducted according to similar protocols, were summarized.

<Tb>
<Tb>

<SEP> derivative of <SEP><SEP> amine derivative <SEP> and <SEP><SEP><SEP><SEP> conditions of
<tb> polysaccharide <SEP> conc. <SEP> operating systems <SEP> coupling
<tb> and <SEP> conc.
<Tb>

Cellulose <SEP> 2-methoxy <SEP> pH <SEP>: <SEP> 8 <SEP> 73 <SEP> mg
<tb> 100 <SEP> oxidized <SEP>. <SEP> ethylamine <SEP> EDAC <SEP>: <SEP> 110 <SEP> mg <SEP>SEP>SEP> Coupling rate
<tb> 78 <SEP> mg <SEP> (0.39 <SEP> m <SEP> 74 <SEP> L <SEP> (0.85 <SEP> mmol) <SEP> (0.57 <SEP> mmol) ) <SEP> and <SEP> of <SEP> the <SEP> order of
<tb> mol) <SEP> in <SEP> 5 <SEP> ml <SEP> NHS <SEP>: <SEP> 66 <SEP> mg <SEP> 70 <SEP>% <SEP> yield
<tb> water <SEP> (0.57 <SEP> mmol) <SEP> from <SEP> the <SEP> reaction
<tb> in <SEP> 2 <SEP> ml <SEP> of <SEP> the <SEP> order of
<tb> water. <SEP> 83 <SEP>%
<tb> Temperature <SEP>: <SEP>
<tb> 50 C, <SEP> 24 <SEP> h.
<Tb>

Example 38

<Tb>
<tb> Derivative <SEP> of <SEP><SEP> Derivative of Amine <SEP> and <SEP><SEP> Conditions <SEP> Performance of
<tb> polysaccharide <SEP> conc. <SEP> operating systems <SEP> coupling
<tb> and <SEP> conc.
<Tb>

Amylose <SEP> 2-methoxy <SEP> Same <SEP> ex. <SEP> 37 <SEP> 67 <SEP> mg
<tb> 100 <SEP>% <SEP> oxidized <SEP>: <SEP> ethylamine <SEP>SEP>SEP> coupling rate
<tb> 78 <SEP> mg <SEP> (0.39 <SEP> 67 <SEP> L <SEP> (0.77 <SE> mmol) <SEP> from <SEP> the order <SEP> of
<tb> mmol) <SEP> in <SEP> 5 <SEP> ml <SEP> 50 <SEP>% <SEP> (NMR <SEP> 13C),
<tb> of water <SEP> yield <SEP> of <SEP> la
<tb> reaction <SEP> of
<tb> the order <SEP> of <SEP> 78 <SEP>%
<Tb>
Example 39

<Tb>
<tb> Derivative <SEP> of <SEP><SEP> Derivative of Amine <SEP> and <SEP><SEP> Conditions <SEP> Performance of
<tb> polysaccharide <SEP> conc. <SEP> operating systems <SEP> coupling
<tb> and <SEP> conc.
<Tb>

Amyloidosis <SEP> Idem <SEP> ex. <SEP> 35 <SEP> Same as <SEP> ex. <SEP> 37 <SEP> 58 <SEP> mg
<tb> 100 <SEP>% <SEP> Oxidized <SEP>: <SEP>#<SEP> 14 <SEP>% <SEP> of
<tb> 78 <SEP> mg <SEP> (0.39 <SEP> coupling,
<tb> mmol) <SEP> in <SEP> 6 <SEP> ml <SEP> yield <SEP> of <SEP>
<tb> of <SEP> buffer <SEP> reaction <SEP> of
<tb> phosphate, <SEP> the order <SEP> of <SEP> 73%
<tb> pH <SEP> 7
<Tb>

<Desc / Clms Page number 44>

Example 40

<Tb>
<tb> Derivative <SEP> of <SEP><SEP> Derivative of Amine <SEP> and <SEP><SEP> Conditions <SEP> Performance of
<tb> polysaccharide <SEP> conc. <SEP> operating <SEP> coupling <SEP>
<Tb>

et conc. U-ïi -Kdir-37 Idem ex. 37 0,0'-bis Idem ex. 37 66 mg Idem ex. U,U Dis

and conc. U-Kdir-37 Idem ex. 37 0,0'-bis Idem ex. 37 66 mg Same as ex. U, U Dis

<Tb>
<tb> (aminopropyl) <SEP> = <SEP> 20 <SEP>% <SEP> of <SEP>
<tb> polyethylene <SEP> coupling,
<tb> glycol <SEP> 500 <SEP>: <SEP> yield <SEP> of <SEP>
<tb> 248 <SEP> L <SEP> (0.43 <SEP> mmol) <SEP> reaction <SEP> of <SEP>
<tb> L ~~~ <SEP> the order <SEP> of
<Tb>
Example 41

<Tb>
<tb> Derivative <SEP> of <SEP><SEP> Derivative of Amine <SEP> and <SEP><SEP> Conditions <SEP> Performance of
<tb> polysaccharide <SEP> conc. <SEP> operating <SEP> coupling <SEP>
<Tb>

et conc. 0 0,~bis '#######Tdem ex. 3744 4 mg de produit' Idem O,O'-bis Idem de produit Idem ex. 0,U Gis insoluble (aminopropyl) insoluble et polyéthylène 44,5 mg de

and conc. 0 0, ~ bis'####### Tdem ex. 3744 4 mg of product 'Idem O, O'-bis Idem of product Idem ex. 0, U insoluble Gis (aminopropyl) insoluble and polyethylene 44.5 mg of

<Tb>
<tb> glycol <SEP> 300 <SEP>: <SEP> soluble SEP product
<tb> 195 <SEP> L <SEP> (0.47 <SE> mmol) <SEP><SEP> Rate of <SEP> Coupling
<tb> yield <SEP> of <SEP> la
<tb> reaction <SEP> of
<tb> the order <SEP> of <SEP> 80-
<tb> 90%
<Tb>
Example 42

<Tb>
<tb> Derivative <SEP> of <SEP><SEP> Derivative of Amine <SEP> and <SEP><SEP> Conditions <SEP> Performance of
<tb> polysaccharide <SEP> conc. <SEP> operating systems <SEP> coupling
<Tb>

polysaccharide cône. operaoir et conc.

polysaccharide cone. operaoir and conc.

Same as ex. 1.4 diaminobutane Same as ex. 29 mg of product 43.8 (0.44 insoluble and

<Tb>
<tb> mmol) <SEP> (0.44 <SEP> mg <SEP> of <SEP> product
<tb> mmol) <SEP> soluble
<tb> (training <SEP> of a
<tb> gel)
<Tb>

<Desc / Clms Page number 45>

Example 43

<Tb>
<tb> Derivative <SEP> of <SEP><SEP> Derivative of Amine <SEP> and <SEP><SEP> Conditions <SEP> Performance of
<tb> polysaccharide <SEP> conc. <SEP> operating systems <SEP> coupling
<tb> and <SEP> conc.
<Tb>

Same <SEP> ex. <SEP> 37 <SEP> 0- (2-aminopropyl) <SEP> Same <SEP> ex. <SEP> 37 <SEP> 26.5 <SEP> mg <SEP> from
<tb> -0 '<SEP> (2- <SEP> product
<tb> methoxyethylamine) <SEP> insoluble <SEP> and <SEP> 45
<tb> polypropylene <SEP> mg <SEP> of <SEP> product
<tb> glycol <SEP> 500 <SEP>: <SEP> soluble <SEP> (= <SEP> 15 <SEP>%
<tb> 490 <SEP> L <SEP> (0.80 <SEP> mmol) <SEP> of <SEP> Rate <SEP> of
<tb> coupling)
<tb> yield <SEP> of <SEP> la
<tb> reaction <SEP> of
<tb> the order <SEP> of <SEP> 60%
<Tb>
Example 44

<Tb>
<tb> Derivative <SEP> of <SEP><SEP> Derivative of Amine <SEP> and <SEP><SEP> Conditions <SEP> Performance of
<tb> polysaccharide <SEP> conc. <SEP> operating systems <SEP> coupling
<tb> and <SEP> conc.
<Tb>

Acetate <SEP> of <SEP> 2-methoxy <SEP> Same <SEP> ex. <SEP> 37 <SEP> 103 <SEP> mg
<tb> oxidized <SEP> cellulose <SEP> ethylamine <SEP><SEP><SEP> coupling rate
<tb> (68 <SEP>% <SEP> COONa) <SEP>: <SEP> 68 <SEP> L <SEP> (0.78 <SEP>#<SEP> 17 <SEP>%
<tb> 115 <SEP> mg <SEP> (0.39 <SEP> mmol) <SEP> (16 <SEP>% <SEP> CH20H <SEP> and
<tb> mmol <SEP> of <SEP> COONa) <SEP> 17 <SEP>% <SEP> acetate)
<tb> in <SEP> 7 <SEP> ml <SEP> of water <SEP> yield <SEP> of <SEP> la
<tb> reaction <SEP> of
<tb> the order <SEP> of <SEP> 90%
<Tb>
Example 45

<Tb>
<tb> Derivative <SEP> of <SEP><SEP> Derivative of Amine <SEP> and <SEP><SEP> Conditions <SEP> Performance of
<tb> polysaccharide <SEP> conc. <SEP> operating systems <SEP> coupling
<tb> and <SEP> conc.
<Tb>

Same <SEP> ex. <SEP> 44 <SEP>0,0'-bis<SEP> Same <SEP> ex. <SEP> 37 <SEP> 67 <SEP> mg
<tb> (aminopropyl) <SEP> Rate <SEP> of <SEP> coupling
<tb> polyethylene <SEP>#<SEP> 10 <SEP>%
<tb> glycol <SEP> 500 <SEP>: <SEP> (14 <SEP>% <SEP>) of <SEP> CHzOH <SEP> and
<tb> 463 <SEP> L <SEP> (0.81 <SEP> mmol) <SEP> 19% <SEP> acetate)
<tb> Yield <SEP> of <SEP>
<tb> reaction <SEP> of
<tb> the order <SEP> of <SEP> 59 <SEP>%
<Tb>

Example 46 a. Preparation of the polyNIPAM-amino terminal (polyNIPAM = poly-N-isopropylacrylamide):
In 22.3 mL of anhydrous dimethylformamide (DMF), 5.79 g (51.2 mmol) of N-isopropylacrylamide, 550.5 mg (4.8 mmol) of 2-aminoethanethiol hydrochloride (AET) are solubilized.

<Desc / Clms Page number 46>

 and 8.87 mg (0.54 mmol) 2,2'-azobis (2-methyl-propionitrile) (AIBN). The solution is frozen in liquid nitrogen and degassed several times under vacuum. The container is then sealed under vacuum and then kept at 75 ° C. for 15 hours. At the end of the reaction, the DMF is partially removed under reduced pressure and the polymer formed is precipitated in ethyl ether. The polymer is then purified by several cycles dissolving in a limited amount of DMF / precipitation in an excess of ethyl ether. The polymer is finally solubilized in water, purified by ultrafiltration (cut-off membrane = 500), and finally lyophilized.

5.5 g of Mw polyNIPAM are obtained between 1000 and 1200 (NMR and mass spectrometry). b. Coupling reaction
The conditions of Example 37 are reproduced using polyNIPAM c. Purification
Dialyzed for 2 days, then lyophilized. In order to remove the unreacted polyNIPAM, the recovered product is extracted into a soxhlet with chloroform for 12 hours.

In the tables below, the coupling conditions used in Example 46, as well as in Examples 47 and 48, which relate to coupling reactions conducted according to similar protocols, were summarized.

<Tb>
<Tb>

<SEP> derivative of <SEP> Amine <SEP> derivative <SEP><SEP><SEP> Conditions of
<tb> polysaccharide <SEP> and <SEP> conc. <SEP> operating systems <SEP> coupling
<tb> and <SEP> conc.
<Tb>

Cellulose <SEP> polyNIPAM <SEP> (Mw <SEP> pH <SEP>: <SEP> 8 <SEP> 124 <SEP> mg
<tb> 100 <SEP>% <SEP> Oxidized <SEP>: <SEP> 1000-1200): <SEP> 78 <SEP> EDAC <SEP>: <SEP> 151 <SEP> mg <SEP> Rate <SEP > of <SEP> coupling <SEP> of
<tb> 78 <SEP> mg <SEP> (0.39 <SEP> mg <SEP> (= <SEP> 0.07 <SE> mmol) <SEP> (0.79 <SE> mmol) <SEP> the order <SEP> of <SEP> 18 <SEP>%
<tb> mmol) <SEP> in <SEP> 5 <SEP> ml <SEP> and <SEP> NHS <SEP>: <SEP> 91 <SEP> yield <SEP> of <SEP>
<tb> water <SEP> mg <SEP> (0.79 <SEP> reaction <SEP> from <SEP> order
<tb> mmol) <SEP> in <SEP> 2 <SEP> of <SEP> 80 <SEP>%
<tb> ml <SEP> of water.
<Tb>

Temperature
<tb> ambient, <SEP> 24
<tb> h.
<Tb>

<Desc / Clms Page number 47>

Example 47

<Tb>
<tb> Derivative <SEP> of <SEP><SEP> Derivative of Amine <SEP> and <SEP><SEP> Conditions <SEP> Performance of
<tb> polysaccharide <SEP> conc. <SEP> operating systems <SEP> coupling
<tb> and <SEP> conc.
<Tb>

Same <SEP> ex. <SEP> 46 <SEP> polyNIPAM <SEP> (Mw <SEP> Same <SEP> eg <SEP> 46 <SEP> 154 <SEP> mg
<tb> 1000-1200): <SEP> 151 <SEP> mg <SEP> Rate <SEP> of <SEP> coupling <SEP> of
<tb> (= <SEP> 0.18 <SEP> mmol) <SEP> the order <SEP> of <SEP> 23 <SEP>%
<tb> yield <SEP> of <SEP> la
<tb> reaction <SEP> of <SEP> order
<tb> of <SEP> 90 <SEP>%
<Tb>

Example 48 a. Coupling reaction of Oxidized Cellulose Microcrystals
80 mg of OD cellulose of 0.23 (0.11 mmol COONa) are dispersed in 5 ml of water. 22 L (0.20 mmol) of benzylamine are added. The pH is brought to 8 with 0.5 N hydrochloric acid. EDAC (30 mg, 0.16 mmol) and NHS (17.9 mg, 0.16 mmol) are added, dissolved in 2 ml of water. . The pH is adjusted to 8 with 0.5 N HCl and 0.5 N NaOH. The solution is stirred at room temperature for 14 hours and the pH is maintained at 8. b. Purification
The coupling product can be separated either by centrifugation or by filtration.

 Centrifugation: The suspension (20,000 g) is centrifuged for 30 minutes and the insoluble material is collected, washed with water several times and lyophilized.

 Filtration: Sartorius SM 162 49 stainless steel pressure filter filter is filtered on a cellulose nitrate membrane with a porosity of 5 μm to recover the insoluble part, washed with water several times and freeze-dried.

 70.4 mg of the coupling product is recovered.

 In the tables below, the coupling conditions used in Example 48, as well as in Examples 49 to 52 which relate to coupling reactions conducted according to similar protocols, were summarized.

<Desc / Clms Page number 48>

<Tb>
<Tb>

<SEP> derivative of <SEP><SEP> amine derivative <SEP> and <SEP><SEP><SEP><SEP> conditions of
<tb> polysaccharid <SEP> conc. <SEP> operating systems <SEP> coupling
<tb> e <SEP> and <SEP> conc.
<Tb>

Liners <SEP> of <SEP> Benzylamine <SEP>: <SEP> 22 <SEP> pL <SEP> pH <SEP>: <SEP> 8 <SEP> 70 <SEP> mg
<tb> cotton <SEP> oxidized <SEP> (0.20 <SEP> mmol) <SEP> EDAC <SEP>: <SEP> mg <SEP> (OD <SEP> = <SEP> 0.16)
<tb> (OD <SEP> = <SEP> 0.23): <SEP> (0.16 <SEP> mmol) <SEP> and <SEP> Rate <SEP> of <SEP> coupling
<tb> 80 <SEP> mg <SEP> NHS <SEP>: <SEP> 17.9 <SEP> mg <SEP> of <SEP> the order <SEP> of
<tb> (0.11 <SEP> mmol <SEP> of <SEP> (0.16 <SEP> mmol) <SEP> 30 <SEP>%
<tb> COONa) <SEP> in <SEP> 5 <SEP> in <SEP> 1 <SEP> ml
<tb> ml <SEP> of water <SEP> of water.
<Tb>

Temp. <SEP> ambient,
<tb> 14 <SEP> h
<Tb>
Example 49

<Tb>
<tb> Derivative <SEP> of <SEP><SEP> Derivative of Amine <SEP> and <SEP><SEP> Conditions <SEP> Performance of
<tb> polysaccharid <SEP> conc. <SEP> operating systems <SEP> coupling
<tb> e <SEP> and <SEP> conc.
<Tb>

Liners <SEP> of <SEP>0.0'-bis<SEP> pH: <SEP> 8 <SEP> 65 <SEP> mg
<tb> cotton <SEP> oxidized <SEP> (aminopropyl) <SEP> EDAC <SEP>: <SEP> 24.3 <SEP> mg <SEP> (OD <SEP> = <SEP> 0.13)
<tb> (OD <SEP> = <SEP> 0.17): <SEP> polyethylene <SEP> glycol <SEP> (0.13 <SEP> mmol) <SEP> and <SEP><SEP> rate of <SEP>SEP> coupling
<tb> 80 <SEP> mg <SEP> (0.08 <SEP> 500 <SEP>: <SEP> 83 <SEP> uL <SEP> (0.14 <SEP> NHS <SEP>: <SEP> 16 <SEP> mg <SEP> of <SEP> the order <SEP> of <SEP> 24
<tb> mmol <SEP> of <SEP> mmol) <SEP> (0.14 <SEP> mmol) <SEP>%
<tb> COONa) <SEP> in <SEP> in <SEP> 1 <SEP> ml
<tb> 5 <SEP> ml <SEP> of water <SEP> of water.
<Tb>

Temp. <SEP>: <SEP>
<tb> ambient, <SEP> 18 <SEP> h
<Tb>
Example 50

<Tb>
<tb> Derivative <SEP> of <SEP><SEP> Derivative of Amine <SEP> and <SEP><SEP> Conditions <SEP> Performance of
<tb> polysaccharid <SEP> conc. <SEP> operating systems <SEP> coupling
<tb> e <SEP> and <SEP> conc.
<Tb>

Liners <SEP> of <SEP> 2-methoxyethylamine <SEP> pH <SEP>: <SEP> 66 <SEP> mg
<tb> cotton <SEP> oxidized <SEP> 27 <SEP> uL <SEP> (0.32 <SEP> mmol) <SEP> EDAC <SEP>: <SEP> 24.8 <SEP> mg <SEP> ( OD <SEP> = <SEP> 0.19)
<tb> (OD <SEP> = <SEP> 0.25): <SEP> (0.13 <SEP> mmol) <SEP> and <SEP> Rate <SEP> of <SEP> coupling
<tb> 80 <SEP> mg <SEP> (0.14 <SEP> NHS <SEP>: <SEP> 16 <SEP> mg <SEP> of <SEP> the order <SEP> of
<tb> mmol <SEP> of <SEP> (0.14 <SEP> mmol) <SEP> 24 <SEP>%
<tb> COONa) <SEP> in <SEP> in <SEP> 1 <SEP> ml
<tb> 5 <SEP> ml <SEP> of water <SEP> of water.
<Tb>

Temp .: <SEP> 50 <SEP> C,
<tb> 20 <SEP> h.
<Tb>

* The samples of oxidized cotton linters (D0 = 0.25) before and after coupling are checked in infrared spectroscopy, figure 12 a and 12 b, respectively. We can observe the decrease of the O = C-OH band at 1730 cm-1 and the appearance of 2 low intensity bands at 1657 cm-1 (# C = O) and 1544 cm-1 (# NH) which correspond to at amide I and amide II absorptions.

<Desc / Clms Page number 49>

Example 51 a. Preparation of the polyNIPAM-amino terminal
In this example, the conditions of Example 46 are reproduced. However, to increase the DP of the polyNIPAM, the amount of AET (0.2173 g, 1.9 mmol) is decreased.

<Tb>
<Tb>

<SEP> derivative of <SEP> Amine <SEP> derivative <SEP><SEP><SEP> Conditions of
<tb> polysaccharide <SEP> and <SEP> conc. <SEP> operating systems <SEP> coupling
<tb> and <SEP> conc.
<Tb>

Tunicate <SEP> oxidized <SEP> polyNIPAM <SEP> (Mw <SEP> pH: <SEP> 8 <SEP> 297 <SEP> mg
<tb> (OD <SEP> = <SEP> 0.1): <SEP> 200 <SEP> 2000-2500): <SEP> 400 <SEP> EDAC <SEP>: <SEP> 67 <SEP> mg <SEP> (OD <SEP> = <SEP> 0.07)
<tb> mg <SEP> (0.12 <SEP> mmol <SEP> of <SEP> mg <SEP> (= <SEP> 0.18 <SEP> mmol) <SEP> (0.35 <SEP> mmol) ) <SEP> and <SEP> Rate <SEP> of <SEP> coupling
<tb> COONa) <SEP> in <SEP> 15 <SEP> NHS <SEP>: <SEP> 40 <SEP> mg <SEP> of <SEP> the order <SEP> of
<tb> ml <SEP> of water <SEP> (0.35 <SEP> mmol) <SEP> 30 <SEP>%
<tb> in <SEP> 2 <SEP> ml
<tb> water.
<Tb>

Temperature
<tb> ambient, <SEP> 24 <SEP> h.
<Tb>

Example 52

<Tb>
<tb> Derivative <SEP> of <SEP> Amino <SEP> Derivative <SEP> Conditions <SEP> Yield <SEP> of
<tb> polysaccharide <SEP> and <SEP> conc. <SEP> operating systems <SEP> coupling
<tb> and <SEP> conc.
<Tb>

Linter <SEP> of <SEP> cotton <SEP> polyNIPAM <SEP> (Mw <SEP> Ditto <SEP> eg <SEP> 51 <SEP> 318 <SEP> mg
<tb> oxidized <SEP> (OD <SEP> = <SEP> 1000-1200): <SEP> 400 <SEP> (OD <SEP> = <SEP> 0.08)
<tb> 0.16): <SEP> 200 <SEP> mg <SEP> mg <SEP>SEP>SEP> Coupling rate
<tb> (0.20 <SEP> mmol <SEP> of <SEP>(<SEP> 0.18 <SEP> mmol) <SEP> of <SEP> the order <SEP> of
<tb> COONa) <SEP> in <SEP> 15 <SEP> 50 <SEP>%
<tb> ml <SEP> of water
<Tb>
Example 53

<Tb>
<tb> Derivative <SEP> of <SEP> Amino <SEP> Derivative <SEP> Conditions <SEP> Yield <SEP> of
<tb> polysaccharide <SEP> and <SEP> conc. <SEP> operating systems <SEP> coupling
<tb> and <SEP> conc.
<Tb>

Aggregates <SEP> 2-methoxy <SEP> pH <SEP>: <SEP> 108 <SEP> mg
<tb> starch <SEP> oxidized <SEP> ethylamine <SEP> 27 <SEP> uL <SEP> EDAC <SEP>: <SEP> 24.8 <SEP> mg <SEP> (OD <SEP> = <SEP> 0.08)
<tb> (OD <SEP> = <SEP> 0.12) <SEP>: <SEP> (0.32 <SEP> mmol) <SEP> (0.13 <SEP> mmol) <SEP> and <SEP > Rate <SEP> of <SEP> coupling
<tb> 200 <SEP> mg <SEP> (0.14 <SEP> NHS <SEP>: <SEP> 16 <SEP> mg <SEP> of <SEP> the order <SEP> of
<tb> mmol <SEP> of <SEP> COONa) <SEP> (0.14 <SEP> mmol) <SEP> 33 <SEP>%
<tb> in <SEP> 15 <SEP> ml <SEP> of water <SEP> in <SEP> 1 <SEP> ml
<tb> water.
<Tb>

Temp .: <SEP> 50 <SEP> C,
<tb> 20 <SEP> h
<Tb>

<Desc / Clms Page number 50>

Example 54

<Tb>
<tb> Derivative <SEP> of <SEP> Amino <SEP> Derivative <SEP> Conditions <SEP> Yield <SEP> of
<tb> polysaccharide <SEP> and <SEP> conc. <SEP> operating systems <SEP> coupling
<tb> and <SEP> conc.
<Tb>

Chitin <SEP> oxidized <SEP> Same <SEP> ex. <SEP> 53 <SEP> Same as <SEP> ex. <SEP> 53 <SEP> 115mg
<tb> (OD <SEP> = <SEQ> 0.17) <SEP>: <SEP> (DO <SEP> = <SEP> 0.11)
<tb> 200 <SEP> mg <SEP> (0.10 <SEP> Rate <SEP> of <SEP> coupling
<tb> mmol <SEP> of <SEP> COONa) <SEP> of <SEP> the order <SEP> of <SEP> 35
<tb> in <SEP> 15 <SEP> ml <SEP> of water <SEP>%
<Tb>

Example 55
This example was carried out in order to study the influence of the order of introduction of the reagents (EDAC, NHS and amine) in the coupling yields. at. Method A
150 mg of cellulose of OD 0.24 (0.21 mmol of COONa) are dispersed in 10 ml of water. 161 L (1.85 mmol) of 2-methoxyethylamine are added. The pH is brought to 8 with 0.5 N hydrochloric acid. 177 mg (0.92 mmol) of EDAC and 106.7 mg (0.93 mmol) of NHS are dissolved in 2 ml of water, and this solution is added to the reaction medium. The pH is adjusted to 8 with 0.5N HCl and 0.5N NaOH. The solution is stirred at 50 ° C. for 24 hours and the pH is maintained at 8.b. Method B
150 mg of cellulose of OD 0.24 (0.21 mmol of COONa) are dispersed in 10 ml of water. 177 mg (0, 92 mmol) of EDAC and 106.7 mg (0.93 mmol) of NHS are dissolved in 2 ml of water, and this solution is added to the reaction medium. The pH is adjusted to 8 with 0.5N NaOH. The solution is stirred at room temperature for 24 hours. 161 L (1.85 mmol) of 2-methoxyethylamine is added. The pH is brought to 8 with 0.5N HCl. The solution is stirred at 50 ° C. for 24 hours and the pH is maintained at 8 ° C.

<Desc / Clms Page number 51>

c. Method C
150 mg of OD cellulose of 0.24 (0.21 mmol COONa) are dispersed in 10 ml of water. 106.7 mg (0.93 mmol) of NHS are dissolved in 1 ml of water, and this solution is added to the reaction medium. The pH is brought to 8 with 0.5 N NaOH. 161 μL (1.85 mmol) of 2-methoxyethylamine is added. 177 mg (0.92 mmol) of EDAC are dissolved in 1 ml of water, and this solution is added to the reaction medium. The pH is adjusted to 8 with 0.5 N HCl. The solution is stirred at 50 ° C. for 24 hours and the pH is maintained at 8 ° C.

In the tables below, the coupling conditions used in Example 55, as well as in Examples 56 to 58 which relate to coupling reactions conducted according to similar protocols, were summarized.

<Tb>
<Tb>

Product <SEP> of <SEP> Derivative <SEP> of amine, <SEP> Method <SEP> Yield <SEP> of
<tb> Start <SEP> EDAC <SEP> and <SEP> NHS <SEP> of <SEP> Coupling <SEP> Coupling
<tb> Microfibrils <SEP> 2-methoxyethylamine <SEP> 102 <SEP> mg <SEP> (OD <SEP> =
<tb> of <SEP> cellulose <SEP> 161 <SEP> L <SEP> (1.85 <SEP> mmol) <SEP> 0.13)
<tb> obtained <SEP> at <SEP> Rate <SEP> of
<tb> from <SEP> of <SEP> EDAC <SEP> 177 <SEP> mg <SEP> coupling <SEP> = <SEP> 46 <SEP>%
<tb> pulp <SEP> of <SEP> (0.92 <SEP> mmol) <SEP>% N <SEP> = <SEP> 0.86 <SEP> either
<tb> beets <SEP> a <SEP> rate <SEP> of
<tb> sugar <SEP> NHS <SEP> 106.7 <SEP> mg <SEP> coupling <SEP> of
<tb> oxidized <SEP> of <SEP> OD <SEP> (0.93 <SEQ> mmol) <SEP> 45%
<tb> = <SEP>0.24;<SEP> 116 <SEP> mg <SEP> (DO <SEP> =
<tb> 150 <SEP> mg <SEP> (0.21 <SEP> 0.16)
<tb> mmol <SEP> of <SEP> COONa) <SEP> Rate <SEP> of
<tb> coupling <SEP>;<SEP> 33 <SEP>%
<tb> 114 <SEP> mg <SEP> (DO <SEP> =
<tb> C <SEP> 0.15)
<tb> Rate <SEP> of
<tb> coupling <SEP> = <SEP> 37 <SEP>%
<tb>% N <SEP> = <SEP> 0.85 <SEP>
<tb> a <SEP> rate <SEP> of
<tb> coupling <SEP> of
<tb> 44%
<Tb>

<Desc / Clms Page number 52>

<Tb>
<tb> Example56
<tb> Product <SEP> of <SEP> Derivative <SEP> of Amine, <SEP> Method <SEP> Yield <SEP> of
<tb> start <SEP> EDAC <SEP> and <SEP> NHS <SEP> from <SEP><SEP> coupling <SEP> coupling
<tb> Same <SEP> ex. <SEP> 55 <SEP> 0- (2-aminopropyl) <SEP> 90 <SEP> mg <SEP> (OD)
<tb> -0 '(2- <SEP> A <SEP> 0.15)
<tb> methoxyethylamine) <SEP><SEP> rate of <SEP>
<tb> polypropylene <SEP> coupling <SEP> = <SEP>
<tb> glycol <SEP> 500 <SEP>: <SEP> 37 <SEP>%
<tb> 1.13 <SEP> mL <SEP> (1.8 <SEP> mmol)
<tb> EDAC <SEP> 177 <SEP> mg
<tb> (0.92 <SEP> mmol)
<tb> NHS <SEP> 106.7 <SEP> mg
<tb> (0.93 <SEP> mmol)
<Tb>
Example 57

<Tb>
<tb> Product <SEP> of <SEP> Derivative <SEP> of amine, <SEP> Method <SEP> Yield <SEP> ae
<tb> start <SEP> EDAC <SEP> and <SEP> NHS <SEP> from <SEP><SEP> coupling <SEP> coupling
<tb> Linters <SEP> of <SEP> Same <SEP> ex. <SEP> 55 <SEP> 124 <SEP> n <SEP> (DO
<tb><SEP> Oxidized Cotton <SEP><SEP><SEP> Rate
<tb> (OD <SEP> = <SEP> (0.21. <SEP> coupling <SEP> - <SEP> 58 <SEP>%
<Tb>

150 mg (0,21 mmol de COONa) %N = 0,99 soit taux de

150 mg (0.21 mmol COONa)% N = 0.99 is the

<Tb>
<tb> coupling <SEP> of
<tb> 54%
<tb> 116 <SEP> mg <SEP> (OD
<tb> B <SEP> 0.15)
<tb> Rate <SEP> of
<tb> coupling <SEP> - <SEP> 37
<tb>% N <SEP> = <SEP> 0.81 <SEP> either
<tb> a <SEP> rate <SEP> of
<tb> coupling <SEP> of
<tb> 42%
<tb> 128 <SEP> mg <SEP> (DO <SEP> =
<tb> C <SEP> 0.10)
<tb> Rate <SEP> of
<tb> z | ~~~ <SEP> coupling <SEP> = <SEP> 58 <SEP>%
<Tb>
Example 58

<Tb>
<tb> Product <SEP> of <SEP> Derivative <SEP> of Amine, <SEP> Method <SEP> Yield <SEP> of
<tb> start <SEP> EDAC <SEP> and <SEP> NHS <SEP> from <SEP><SEP> coupling <SEP> coupling
<tb> Same <SEP> ex. <SEP> 57 <SEP> Same as <SEP> ex. <SEP> 56 <SEP> 95 <SEP> mg <SEP> (OD
<tb> Rate <SEP> of
<tb> coupling <SEP> - <SEP> 46 <SEP>%
<Tb>

Claims (17)

1. Crystalline polysaccharide derivatives in which at least a part of the -CH 2 OH groups are oxidized to -COOH groups, the latter groups being able to be partly or wholly in the form of salts or functionalized, characterized in that they are present in form of aggregates comprising microcrystals and / or individual microfibrils, with lateral sizes of microcrystals and microfibrils of 1 to 30 nm, their length being up to several tens of microns, or even about 100 m, the aggregates, microcrystals and microfibrils being insoluble in water.  2. Derivatives according to claim 1, characterized in that the polysaccharides are carboxylic derivatives, the -COOH groups being at the surface and, where appropriate, in the amorphous phases.  3. Derivatives according to claim 2, characterized in that the average number of carboxyl groups per saccharide unit varies from 0 to 0.4 depending on the polysaccharide.  4. Derivatives according to claim 1, characterized in that they are carboxylates, especially alkali metal carboxylates, in particular sodium, alkaline earth metals, heavy metals, radioactive isotopes or quaternary ammonium, acetates, propionates, acetopropionates, aceto-butyrates, sulfo-acetates.  5. Derivatives according to claim 1, characterized in that the polysaccharides comprise functional groups -COR1, and where appropriate -CH20R2, in which. RI represents OX or NX, X being a C1-C12 alkyl or alkylamino radical, the alkyl or alkylamino radical being optionally substituted by one or more -OH groups, or an aromatic group, in particular phenyl, or a chain; polymer in particular alkyl polyethylene or polypropylene glycol or alkoxy polyethylene or polypropylene glycol; <Desc / Clms Page number 54>  . R2 represents COX or X, X being as defined above.  6. Derivatives according to any one of the preceding claims, characterized in that the base polysaccharide is selected from the group consisting of cellulose, chitin, starch, amylose, nigerane ((a (l-> 3 a (1-> 4) glucan)), (1-> 3) glucan, (1-> 4) mannan, ss (1-> 3) xylan, inulin, agarose, carrageenans .  7. Process for obtaining polysaccharides according to any one of the preceding claims, this process comprising the radical oxidation of the -CH 2 OH groups of the polysaccharides in the presence of a catalytic amount of an aminoxide radical, and in the presence of an agent. oxidizer, characterized in that the oxidation step is carried out in a controlled manner by adding the oxidizing agent dropwise so as to maintain the pH at 10 by addition of sodium hydroxide until the end of the reaction.  8. Process according to claim 7, characterized in that the radical oxidation reaction is carried out with, as aminoxide radical, the 2,2,6,6-tetramethylpiperidine-1-oxide (TEMPO) radical, the 4-hydroxy TEMPO analogue. acylated derivatives such as 4-acetoxy, 4-phosphonooxy and 4-benzyloxy-TEMPO, amine derivatives such as 4-amino, 4-acetamido and 4-maleimido-TEMPO and, as an oxidizing agent, an agent capable of reoxidizing nitroxides reduced, such as ozone, and in particular hypochlorites, such as sodium hypochlorite, or hydrogen peroxide or organic hydroperoxides, or enzymes, such as peroxidases or oxidases, particularly polyphenol oxidases, laccases and lipases.  9. Process according to claim 8, characterized in that catalytic amounts of these compounds of the order of from 0.1 to 5 moles of reagent per 100 anhydroglucose units are used. <Desc / Clms Page number 55>  10. Process according to any one of claims 7 to 9, characterized in that the oxidation reaction is carried out in the presence of sodium bromide, this compound being advantageously used in a proportion of 1 to 70 moles per 100 anhydroglucose units.  11. Method according to any one of claims 7 to 10, characterized in that the oxidation reaction is carried out at a temperature of -5 C to 50 C, preferably 0 to 25 C.  12. Method according to any one of claims 7 to 11, characterized in that the oxidation is stopped by addition of an alcohol, such as methanol and the medium is neutralized to a pH of about 7, the oxidized derivatives being recovered for example by filtration and / or centrifugation and several washes, especially with water.  13. Process according to any one of Claims 7 to 12, characterized in that, to prepare the polysaccharide derivatives according to Claim 5 or 6, a coupling reaction of the carboxylic derivatives obtained with an amine of formula HN (X ), which X is as defined in claim 5.  14. Process according to claim 13, characterized in that the coupling step is carried out in solution in water and comprises the amidation of the carboxylic derivatives using a water-soluble amidation reagent, such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) and an esterifying reagent, such as N-hydroxysuccinimide sulfonate (NHS), at a temperature of 5 to 100 C, preferably from 20 to 60 [deg.] C., most preferably from 45 to 55 [deg.] C., at a basic pH of 7 to 10, preferably from 7.5 to 8, and the recovery of the amide-linked derivative formed.  15. The method of claim 13 or 14, characterized in that said carboxylic derivatives of polysaccharides are crystallites and or microfibrils of cellulose. <Desc / Clms Page number 56>  16. Process according to any one of Claims 13 to 15, characterized in that the quantities of reagents used correspond to. 1 to 10 moles of amine for 1 mol anhydroglucose oxidized or not. 1 to 10 moles of EDAC for 1 mol anhydroglucose oxidized or not 1 to 10 moles of NHS for 1 mol anhydroglucose oxidized or not.  17. Application of polysaccharide derivatives according to any one of claims 1 to 6, as viscosifying agents or stabilizers, or superabsorbent polymers, or as metal chelating agents.