FR2838430A1 - INSOLUBILIZED POLYSACCHARIDE DERIVATIVES BY BRIDGING THEIR CHAINS. THEIR USE AS STATIONARY CHIRAL PHASES AND ENANTIOSELECTIVE MEMBRANES FOR THE SEPARATION OF ENANTIOMERS - Google Patents

Abstract

Derivatives of polysaccharides insolubilized by bridging their chains. Their use as stationary chiral phases and enantioselective membranes for the separation of enantiomers. The stationary chiral phases, the selector of which is a polysaccharide derivative, are those which are most used for the resolution of racemic compounds by chromatography, both on an analytical and a preparative scale. The most used stationary phases of this type consist of a polysaccharide derivative deposited on silica. The solubility of the chiral selector in many solvents makes it difficult to resolve racemic compounds poorly soluble in solvents compatible with the stability of the stationary phase. The invention relates to obtaining stationary phases in which the polysaccharide derivative is insolubilized on the surface of the support by simple bridging reaction between its chains using for this the residual free -OH functions present in the polysaccharide derivative. The bridging reaction is carried out by deposition of the polysaccharide derivative and of a polyfunctional reagent such as an acid anhydride or a diisocyanate on a support, evaporation of the solvent and dry heating of the product obtained at around 120 ° C. for approximately 1.5 hours. Then simply wash the product with a solvent such as chloroform at reflux to obtain a stable stationary phase in all the usual solvents in chromatography. The polysaccharide derivatives made insoluble according to the invention can also be used pure in the form of microbeads or membranes for the separation of chiral compounds.

Description

the interior of the device within sight of the user.

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  The present invention relates to the preparation and use of chromatographic supports for the resolution of racemic compounds. The chiral selector is a polysaccharide derivative which is made insoluble in the usual solvents of liquid-solid chromatography by a bridging reaction. This result is obtained using a polytonctional reagent capable of reacting with the residual -OH groups of the polysaccharide derivative. The insolubilization results from the formation of a three - dimensional network of sugar chains. The bridging reaction is carried out in a single step and dry after deposition on a

  support for the polysaccharide derivative and the polytonctional reagent.

  In the field of drugs and products for agricultural use, the activity of the various stereoisomers of a chiral compound may be different. For example, it is known that the use of a drug in its racemic form can have consequences

  dramatic. Hence the need to separate the isomers from the racemic compounds.

  If the duplication of racemic compounds is often done by crystallization of diastereoisomers, chromatographic techniques on chiral support have experienced a great boom in recent years justified by their high efficiency and the possibility of separating

  the enantiomers directly without prior treatment.

  If there is currently a large choice of chiral supports for splitting a racemic compound by chromatography on the analytical scale, it is not the same on the preparative scale. In the latter case, these are essentially supports whose

  chiral selector is a derivative of polysaccharide that are used.

  Cellulose in 1967 and then other native polysaccharides were used for the resolution of racemic compounds. Their enantioselectivity is however poor and in 1973 Hesse and Hagel showed that a polysaccharide derivative, in this case cellulose triacetate, led to a chiral support of better performance than the polysaccharide

not derivatized.

  Derivatives of pure polysaccharides have a drawback when they are used as a chiral support in a chromatographic column. These are mainly their poor mechanical properties and their swelling by solvents. This drawback has become unacceptable with the generalization of high performance liquid chromatography which requires the use of columns under pressure. To overcome this drawback, in 1984 Okamoto et al. (J. Am. Chem. Soc. 106 (1984) 5347) have shown that excellent chiral chromatographic supports can be obtained by depositing derivatives of silica on silica.

  cellulose and amylose and in particular triphenylcarbamates.

  Okamoto's chiral stationary phases have been marketed and are still very successful. However, they have a drawback: the solubility of the polysaccharide derivative in many solvents customary in chromatography, which means that only solvents compatible with the stability of the stationary phases are used as eluents. In fact, hydrocarbons such as hexane and alcohols with a small number of carbon atoms. However, in the field of medicaments which is the widest field of application of chiral chromatography, many racemic compounds to be split are very poorly soluble in the solvents compatible with the stability of the stationary phases in which the polysaccharide derivative is simply deposited on a support. This makes it necessary to inject very dilute solutions of the racemic compound into the chromatographic column to be split in order to avoid its precipitation and therefore to use large amounts of solvent. To overcome this drawback, several routes, some very complex, have been proposed for immobilizing the polysaccharide derivative on the support:

  Okamoto et al. (J. Liq. Chromatogr. 10 (1987) 1613) first deposit on a y -

  trinylated cellulose aminopropylsilica. This is followed by the hydrolysis of the trityl groups, the purpose of which is to regenerate the cellulose on the surface of the silica. This done, the cellulose is fixed to the support using a diisocyanate which reacts with its alcohol functions and the amine function of the modified silica. The polysaccharide thus fixed to the support is finally treated with a phenylisocyanate which reacts with its -OH groups. This method is long and does not

  not allow an easy conhôle of the various stages and reactions implemented.

  In 1994 Kimata et al. (US Pat. 5,302,633) copolymerize a cellulose derivative and

  a silica each carrying an activated double bond. They use a sort (4-

  vinylbenzoyl) cellulose and an N-acryloyl-aminopropylsilica. This method of fixing a polysaccharide derivative to a support has two drawbacks. First, the group which it is desired to fix on the polysaccharide may be difficult to access by virtue of the compulsory presence of an activated double bond in its molecule. Next, the large number of activated double bonds carried by the polysaccharide derivative means that the crosslinking and attachment points to the support will be numerous, which affects the secondary structure of the polysaccharide derivative. However, it is known that this affects the performance of the stationary phase. In 1994 Oliveros et al. (EP 0 738 284 B1, J. Liq. Chromatogr. 18 (1995) 1521) describe the preparation of stationary phases in which the chiral selector is a polysaccharide derivative which has the originality of being substituted by two different groups: one selected for the enantioselective qualities which it confers on the polysaccharide which carries it, the other, in small proportion and which contains a non-activated aliphatic carbon-carbon double bond, is intended for fixing the polysaccharide derivative on the support. This fixing can be done by copolymerization between said derivative and the support if the latter is functionalized.

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  such as for example allylsilice or by crosslinking of the same derivative on inert supports such as virgin silica, alumina, graphite, etc. The extent of the crosslinking can here be controlled by the number of groups intended for fixing which are

  introduced into the chain of the polysaccharide.

  In 1996 Enamoto et al. (Anal. Chem. 68 (1996) 2798) fix on silica and by two different methods amylose obtained by enzymatic polymerization of a determined number of units of α-D-glucose-1phosphate. This amylose, fixed to the support, is treated with 3,5dimethylphenylisocyanate which leads to the desired chromatographic support. The good performance of these stationary phases can be explained by the great conformational freedom of the chain of the amylose derivative which is fixed to the support only by one of its ends. One difficulty with this method is polymerization

  enzymatic glucose units and moreover can only lead to amyloidosis.

  In 1998 Duval et al. (EP 0 864 586 A2) describe the preparation of grafted silicas of polysaccharide derivatives but the examples given do not differ from those of the publications by Oliveros et al. cited above only by the presence of an oxygen bridge in the chain of the

  substituent intended for the fixation of said derivatives.

  In 1996 and 1997, Francotte et al. describe three methods for fixing polysaccharide derivatives on supports. In the first (WO 96/27615) the polysaccharide derivative carrying a photopolymerizable group is deposited on silica and then irradiated with a mercury vapor lamp for approximately 16 hours. In the second (WO 97/04011) the polysaccharide derivative no longer carries a photopolymerizable group. As before, the polysaccharide derivative is deposited on the support and subjected to ultraviolet irradiation for approximately 20 hours. In the third (WO 97/49733) the polysaccharide derivative and a free radical initiator (oc, oc'-azobisisobutyronitrile or AIBN) are deposited on the support and then heated to dryness at 120 ° C. for several hours. It is not said, in this last publication, how the polysaccharide derivative binds to the support. Probably this results from bridging reactions between polysaccharide chains via free radicals originating from radical ruptures of certain bonds under the action of heat and the free radical initiator used in very large quantities since its proportion relative to to the polysaccharide derivative is usually

in the 1/1 report.

  In these latter methods, it is noted, in addition to very long reaction times, that the quantities of solvent used relative to the quantity of stationary phase treated are very large. In addition, each of them requires a binding extraction with the soxhlet of

  several hours in order to remove the polysaccharide derivative not attached to the support.

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  The importance of stationary chiral phases for the resolution of racemic compounds on the preparative scale is always great. Novel chromatographic methods or techniques such as the so-called simulated mobile bed (or SMB of simulating mobile bed) are capable of producing large quantities of optically pure enantiomers from a racemic compound. To do this, it is desirable that the stationary chiral phases used have a large capacity, are particularly stable and support all kinds of solvents in order to work with optimal conditions without stresses other than those specific to the separation itself. However, the capacity and the enantioselectivity of the stationary phases in which the chiral selector is a polysaccharide derivative are large. It follows that the search for stationary phases of this type which are stable and easy to obtain is still relevant. A particularly simple method of fixing polysaccharide derivatives is described here. The fixation is obtained by the insolubilization of the polysaccharide derivative on the surface of the support by bridging using a polytonctional reagent which reacts with the residual -OH functions generally always present in the polysaccharide derivatives. The present invention relates to the insolubilization by bridging of polysaccharide derivatives of which 1.5 to 2.95 H functions per osidic unit have been transformed into esters -O-CO-R or into carbamates - CO-NH-R or into esters and into carbamates. In these formulas, the group R generally represents an alkyl group or an aryl group or a

  arylalkyl group or an alkylaryl group.

  When R is an aryl group or an arylalkyl group or an alkylaryl group, the beuzenic rings may be substituted by one or more identical or different atoms or groups chosen from halogen atoms, linear or branched alkyl groups and linear alkoxy groups or branched. The group R is preferably phenyl or

  phenylethyl, substituted or unsubstituted on the benzene ring.

  When R is an alkyl group, it may be branched or not and substituted by one or more identical or different atoms or groups chosen from halogen atoms and

  alkoxy groups. Preferably the group R is then the methyl group.

  The polysaccharide derivatives according to the invention have a degree of polymerization

  between 5 and 2000 and preferably from 10 to 500.

  Generally, the polysaccharide derivatives according to the invention are derivatives of the

  cellulose, amylose, chitosan and amylopectin.

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  The bridging reagent carries or is capable of giving rise to at least two functions capable of reacting with the free -OH groups of the polysaccharide derivative. It can be represented by the general formulas Ia and Ib:

X / CO \

  Ia A Ib A / O Y CO In which: a) X and Y. identical or different, each represent a function capable of reacting with free OH groups of a polysaccharide derivative. Preferably -X and -Y represent the following functional groups: -N = C = 0 or -SO2Cl or COCI or -CO2H or epoxide b) A is an alkyl group or an aryl or polyaryl group or an arylalkyl group or an alkylaryl group may bear identical or different substituents chosen from halogen atoms, alkyl groups, alkoxy groups,

acyl groups, ester groups.

  The derivatization of the polysaccharides which it is desired to use according to the invention takes place

  by the conventional methods described in the literature.

  Preferably the polysaccharide derivative is fixed by bridging on an inorganic support such as silica, modified or not, alumina, titanium oxide, zirconium oxide, graphite

  or materials such as paper or other natural or synthetic macromolecules.

  The amount of polysaccharide derivative fixed by bridging on the support is between 5 and 40% by mass relative to the support and preferably between 5 and 25% According to the invention, the bridging of the polysaccharide derivative is carried out hot in the absence of solvent as follows: a) If a support is used, this is introduced into a solution of the polysaccharide derivative and of the bridging reagent. The solvent is then evaporated and the resulting product is heated to 120 ° C. for 1 to 2 hours. The product obtained

  is washed with chloroform at reflux.

  b) If the polysaccharide derivative is pure in the form of microbeads or sheets, it is introduced into a solution of the bridging reagent, the solvent of which does not dissolve the polysaccharide derivative. The rest of the treatment is the same as below

above in a).

  The polysaccharide derivatives according to the invention are characterized in that the bridging reaction makes them insoluble in organic solvents such as for example

  chloroform, toluene, ethyl acetate or acetone.

  The insolubilized polysaccharide derivatives according to the present invention are useful for the separation of a chemical from a mixture, the resolution of racemic compounds and more generally the separation of chiral compounds. The methods used to do this are essentially chromatographic: in the liquid, solid, super or subcritical phase, in a simulated moving bed or by enantioselective diffusion through a membrane.

Example 1:

  2.6 grams of silica with a particle diameter of 5 m and a porosity of 100 angstroms are introduced into a flask containing a solution of 0.5 grams of triacetylcellulose sold by the company Fluka with a molar mass of 37,000 and the substitution size of which is 2 , 48 and 0.4 grams of diacetyltartaric anhydride in 50 mL of pyridine. Leave to stir for a few minutes and slowly evaporate the solvent under reduced pressure at C. Heat dry in an oil bath at around 120 C for approximately 1.5 hours. Leave to cool and take up in 50 mL of chloroform with magnetic stirring at room temperature for about 2 hours and finish by bringing to reflux for 2 hours. Filter through sintered glass, wash on filter first with chloroform and then with acetone. To dry; we get about 3 grams of support. Centesimal analysis: C 10.19%. If this silica is then washed again with pyridine at room temperature for 2.5 hours with magnetic stirring, filtered and washed on a filter with chloroform and diethyl ether,

  The centesimal analysis then indicates 9.60% of carbon.

Example 2:

  The procedure is as in Example 1 but starting from a yaminopropylsilica (centesimal analysis: C 5.35%, H 1.68% and N 1.88%) obtained from the same virgin silica as that used in 1 example 1. Centesimal analyzes: After treatment with chloroform at

  boiling:% C = 14.62; followed by washing with pyridine:% C = 12.55.

  We note from these two examples: - That the polysaccharide derivative has indeed been made insoluble in solvents

  energetic such as chloroform and acetone by a bridging reaction.

  - That if we take into account the percentage of carbon in the starting aminopropyl silica, the rate of fixation of the cellulose derivative on the support is substantially the same on aminopropylsilica as on virgin silica. This tends to show that the insolubilization of the polysaccharide derivative is due mainly to bridging

of its chains.

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Claims (10)

  1. Polysaccharide derivative of which 1.5 to 2.95 -OH functions per osidic unit have been transformed into ester-OCO-R or carbamate-ON = CO-R or into ester and carbamate characterized in that, initially soluble in organic solvents such as, for example, chloroform, toluene, ethyl acetate or acetone, it is made insoluble in these same solvents by an interchanging bridging reaction by the action of a polyfunctional reagent which reacts with - OH free present in said derivative.   2. A polysaccharide derivative according to claim 1 characterized in that the syrnbole R of the ester and carbamate groups represents an alkyl or aryl or arylalkyl or alkylaryl group in which the alkyl chains can be linear or branched and the aromatic rings substituted or not by a or several identical or different atoms or groups chosen from halogen atoms, alkyl groups   linear or branched and alkoxy groups.   3. Polysaccharide derivative according to the preceding claims, characterized in that the   bridging reaction is carried out using a polyfunctional reagent capable of reacting and establishing at least two bonds with free -OH groups of the derivative of   polysaccharide which one wishes to insolubilize.   4. Polysaccharide derivative according to the preceding claims, characterized in that the   or the functions carried by the polyfunctional reagent used for its insolubilization by bridging are carboxylic acid anhydride, carboxylic acid chloride, acid   carboxylic, sulfonic acid chloride, isocyanate or epoxide.   5. Polysaccharide derivative according to the preceding claims, characterized in that it   it is a derivative of cellulose, amylose, amylopectin or chitosan, the   degree of polymerization is between 5 and 2000.   6. Method for obtaining a polysaccharide derivative according to the claims   previous steps whose steps are: - Dissolution of a polysaccharide derivative of which 1.5 to 2.95 -OH functions per osidic unit have been transformed into ester-OCO-R or carbamate-ON = CO-R or into ester and carbamate with R as defined in claim 2, and a   polyfunctional reagent as defined in claims 3 and 4 in a   appropriate organic solvent and impregnation of a chromatographic support,   mineral or organic, natural or synthetic. 8 2838430   - Evaporation of the solvent, heating to 120 C of the resulting product for   about 1.5 hours and finish by washing with chloroform at reflux.   7. Method for obtaining a polysaccharide derivative according to claims 1 to 5 including   the steps are: - Microbeads or sheets obtained from a polysaccharide derivative of which 1.5 to 2.95 -OH functions per osidic unit have been transformed into ester -OCO-R or carbamate -ON = COR or into ester and carbamate with R as defined in claim 2, are impregnated with a solution of a reagent   polytonctional as defined in claims 3 and 4 in a solvent   organic in which the polysaccharide derivative is not soluble.   - Evaporation of the solvent, change around 120 C of the resulting product for   about 1.5 hours finish with a chloroform wash at reflux.   8. Stationary phase for the separation of chiral compounds by chromatography according to claims 1 to 6 characterized in that the support is chosen from silica, modified silica, alumina, zirconium oxide, graphite or materials such as   paper or other natural or synthetic macromolecules.   9. Stationary phase for the separation of chiral compounds by chromatography according to claims 1 to S and 7 characterized in that the polysaccharide derivative is used in the form of microbeads.   10. Membrane for the separation of chiral compounds according to claims 1 to 5 and 7