FR2716200A1 - Process for the preparation of branched cyclomaltooligosaccharides, in particular branched cyclodextrins. - Google Patents

Abstract

Method for the preparation of branched cyclomaltooligosaccharides, in particular, branched cyclodextrines. The method comprises the steps of reacting a cyclomaltooligosaccharide, for example, a cyclodextrine, with at least one reducing agent selected from monosaccharides, reducing oligosaccharides, reducing polysaccharides and derivatives thereof, in the presence of anhydrous hydrogen fluoride, in order to substitute the hydroxyl groups in the cyclomaltooligosaccharide with the reducing agent, for example, glucose, maltose, mannose and 2-acetoamido-2-deoxy-D-glucose. According to the invention, the solubility in water of the starting cyclooligossacharide is enhanced by this method, while conferring to said substance useful environment-recognizable properties, for conveying therapeutic agents to the required site.

Description

PROCESS FOR THE PREPARATION OF CYCLOMALTOOLIGOSACCHARIDES
BRANCHED, ESPECIALLY BRANCHED CYCLODEXTRINS.

 The present invention relates to a process for the preparation of branched cyclomaltooligossaccharides, in particular branched cyclodextrins.

 Cyclomaltooligosaccharides such as cyclodextrins are cyclic oligosaccharides comprising, in the case of cyclodextrins a, ss and y, 6, 7 or 8 D-glucopyranose units linked to a- (1- + 4). These oligosaccharide macrocycles have the property of including in their cavities various molecules, of size adapted to that of the host structure. The generally apolar nature of these associations leads to preferentially include structures of the hydrophobic type, allowing in particular the solubilization in water of compounds poorly or not soluble in these media, thanks to the solubility in water of the cyclodextrins. As a result, the use of cyclodextrins has recently been developed in the food, agrochemical, cosmetic and pharmaceutical fields to improve the solubility, stability and bioavailability of substances of interest such as only active ingredients.

 However, the relatively low solubility in water of cyclodextrins, and in particular of the most economically accessible of them, ss-cyclodextrin which has a water solubility of 18.5 g / l 25 C, limits the use cyclodextrins in these different fields, especially since the stability constants of the inclusion complexes formed with the substances of interest are generally low.

As a result, much research has been undertaken to modify cyclodextrins to improve their solubility in water. For this purpose, they were provided with ether substituents and it was thus possible to obtain, by chemical synthesis, 2,6-di-O-methylcyclomaltoheptaose, 2,3,6-tri-O-methylcyclomaltoheptaose and 2- and 3- hydroxypropylcyclomaltooligosaccharides. Among these hydrophilic cyclodextrin derivatives, the 2-hydroxypropyl cyclodextrins are of some interest as described by Szente and Strattan in New Trends in Cyclodextrins and Derivatives,
D. Duchêne Ed, Editions de Santé, Paris 1991, pp.

57-96.

To improve the solubility in water of cyclodextrins, it has also been envisaged to produce branched cyclodextrins comprising one or more substituents aD-glucopyranosyl, a-maltosyl and a -maltotriosyle on the primary hydroxyl positions of the cyclodextrin. However, the preparation of these branched cyclodextrins involves biotechnological processes, namely the action of cyclomaltodextrins glucanotransferases on starch or amylopectin, or even the action of pullulanase or an isoamylase on a mixture of maltose or maltooligossacharides and the corresponding cyclodextrin, as described by
H.Hashimoto in New Trends in Cyclodextrines and
Derivatives, D. Duchêne Ed, Editions de Santé, Paris 1991, pp. 99-156. These branched cyclodextrins have a high solubility in water ranging from 970 g / l (750 mmol / l) for 6-Oa -D-glucopyranosyl-cyclomaltoheptaose, to 1700 g / l (1165 mmol / l) for 6- O-maltosyl-cyclomaltoheptaose.

These branched cyclodextrins therefore have interesting properties, but unfortunately their preparation by biotechnological routes has many drawbacks. Indeed, yields are low; the process leads to complex mixtures of acyclic and cyclic derivatives including maltose, maltotriose, maltotetraose, a-, ss- and y-free cyclodextrins which accompany the corresponding derivatives mono and di-substituted with a - D-glucose as well as a-maltose.

 Chemical synthesis methods have however been recently published for the preparation of a-D-glucopyranosyl-cyclomaltohexaose and of a -D-glucopyranosyl-cyclomaltoheptaose as described by Takeo et al in J. Carbohydr. Chem. 7 (1988) 293-308 and by Ftigedi et al. in Carboydr.

Res., 175 (1988) 173-181. However, these chemical synthesis methods involve at least five stages of fine chemistry, result in overall yields of the order of 13%, and are not conveniently transposable to a preparatory level.

We also know from the document
EP-A-0-403 366, a process for the chemical synthesis of branched derivatives of cyclodextrins, 6-S-glycosyl-6-thiocyclodextrins starting from an l-thioglucose and a-, ss- and y-cyclodextrins activated, which makes it possible to obtain these branched derivatives in one step with good yields.

 The present invention specifically relates to a process for the preparation of other branched derivatives of cyclomaltooligosaccharides, which allows these derivatives to be easily obtained by chemical synthesis.

 According to the invention, the process for the preparation of branched cyclomaltooligosaccharides, is characterized in that a cyclomaltooligosaccharide is reacted with at least one reducing compound chosen from monosaccharides, reducing oligosaccharides, reducing polysaccharides and their derivatives, in the presence of anhydrous hydrogen fluoride, operating at least at the start of the reaction under conditions such that the anhydrous hydrogen fluoride is liquid, and using an amount of anhydrous hydrogen fluoride sufficient to obtain a pasty reaction mixture of cyclomaltooligosaccharide and of the reducing compound, and in that the reaction products of hydrogen fluoride are then separated.

We have known since the works of Fis Cher in 1895, as it is reported on page 281 of the book: The Carbohydrate, Chemistry and Biochemistry W. PIGMAN and D. HORTON (Ed.) Academic
Press, 1972, vol. 1A, p. 279-353, that the dares have the possibility of adding an alcohol to the cation formed in the anomeric position by the action of a protonating agent, but that this reaction was reversible and led in the case of disaccharides to generate the monosaccharide constituents by hydrolysis . The possibility of adding an alcohol is taken advantage of in the process of the invention by using cyclodextrin as alcohol to be added to the cation formed by the action of a protonating agent on the reducing compound chosen from monosaccharides, oligosaccharides reducing agents and reducing polysaccharides.

This reaction corresponds to the following reaction scheme when the reducing compound is D-glucose

<tb> HF <SEP> an <SEP> hydra <SEP> OH
<tb><SEP> 011
<tb><SEP> HO
<tb><SEP> n
<tb>
This reaction can take place on different hydroxyls, preferably in the primary hydroxyl position of the cyclomaltooligosaccharide which, as we know, is the most reactive. Also, at the end of the reaction, several branched cyclomaltooligosaccharides can be obtained having different substitution rates in
C-6.

 The solubilization in the primary hydroxyl position of the cyclomaltooligosaccharide is confirmed by the 13C NMR spectrum of the branched cyclodextrins obtained by the method of the invention, which shows the low-field displacement expected for the substituted C-6s.

 The use in the process of the invention of a particular protonating agent, constituted by anhydrous hydrogen fluoride makes it possible to avoid the usual drawbacks of the process described by Fischer, that is to say the hydrolysis of disaccharides and polysaccharides in their monosaccharide constituents and obtaining very poor yields, which was the case when using as protonating agent hydrochloric and sulfuric acids or organic acids. On the other hand, by using according to the invention anhydrous hydrogen fluoride as solvent and as glycosidation catalyst, it is possible to obtain branched cyclomaltooligosaccharides, that is to say substituted by monosaccharides, oligosaccharides or polysaccharides, in one single step with good yields.

 To implement the process of the invention, it is possible to intimately mix, preferably by kneading, the cyclomaltooligosaccharide considered and the monosaccharide, the reducing oligosaccharide or the reducing polysaccharide which it is desired to graft onto the cyclodextrin, in a suitable container. , for example Teflon, polyethylene or steel. One can then add to the mixture, a temperature such that the anhydrous hydrogen fluoride is liquid, for example by cooling, in an acetone-dry ice bath, an amount of hydrogen fluoride sufficient to obtain a homogeneous paste, then leave then the mixture return to room temperature while maintaining good stirring. After an appropriate reaction time, for example from 1 to 15 h, the reaction mixture is again cooled, for example in a dry ice acetone bath, and the reaction is stopped by addition of ether. The anhydrous hydrogen fluoride and the ether are then separated by decantation. The insoluble part which corresponds to the reaction products can be washed several times with ether, then dried and used as it is or separated into its various constituents by conventional methods. The products obtained can also be purified before separating them, for example to eliminate the presence of traces of residual fluorine, hydrogen fluoride or glycosyl fluoride. This purification can be carried out by dissolving the reaction products in water followed by the addition of calcium carbonate to the solution, to neutralize the residual acidity and cause the hydrolysis of any traces of glycosyl fluoride.

To separate the reaction products, the methods described in particular by
Hashimoto in New Trends in Cyclodextrines and
Derivatives, D. Duchêne, Editions de Santé, Paris, 1991, p. 99-156 as well as purification techniques by gel exclusion.

 To carry out the process of the invention, one can use anhydrous hydrogen fluoride alone or dilute it in a nonaqueous solvent.

As examples of such solvents, mention may be made of sulfur dioxide, dioxane, pyridine, etc.

 According to the invention, the amounts of cyclomaltooligosaccharides and of reducing compounds used are chosen according to the result which it is desired to obtain. Preferably, the stoichiometry corresponding to the number of hydroxyls of the cyclomaltooligossacharide capable of being substituted by the reducing compound is not exceeded. Generally, the molar ratio of the reducing compound chosen from monosaccharides, oligosaccharides and polysaccharides to the cyclomaltooligosaccharide is from 0.5 to 50 and preferably from 1 to 10.

 The cyclomaltooligosaccharides used in the process of the invention can in particular be the cyclodextrins a, ss and y.

 The reducing compound used can be a monosaccharide such as glucose, galactose, mannose, a disaccharide such as maltose, lactose, as well as oligosaccharides and small reducing polysaccharides.

 It is also possible to use derivatives of these monosaccharides, oligosaccharides and polysaccharides, that is to say the substituted derivatives of these compounds which retain the property of forming a cation by the action of a protonating agent and thus being able to react with the cyclomaltooligosaccharide .

 By way of example of such derivatives, mention may be made of 2-acetamido-2-deoxy-D-glucose.

 According to the invention, the quantity of hydrogen fluoride added must be sufficient so that a homogeneous paste is obtained in order to obtain a substitution rate for the regular cyclomaltooligosaccharide.

Generally, an amount of anhydrous hydrogen fluoride ranging from 0.1 & 1 ml per gram of mixture of cyclomaltooligosaccharide and reducing compound (s) is used for this purpose. However, good results can be obtained when 0.4 ml of anhydrous hydrogen fluoride is used per gram of mixture.

The process of the invention leads to a mixture of branched cyclomaltooligosaccharides, but quite interestingly and unexpectedly, the presence of linear oligosaccharides has not been demonstrated in the reaction products, which confirms the absence of hydrolysis of cyclomaltooligosaccharide, and of course not to exceed the stoichiometry corresponding to the number of hydroxyls which can be substituted. Furthermore, it has been observed that this reaction leads to the introduction almost exclusively of anomerism substituents a when the
D-glucose is the monosaccharide used. Indeed, the 13C NMR technique, applied to the crude product of the reaction between ss-cyclodextrin and D-glucose, indicates that this reaction leads to almost exclusively introducing substituents of anomerism a, since the predominant signal is at 96.5 ppm, which corresponds to the C-1 of the glucopyranoside substituent and that a diffuse signal is obtained around 103 ppm attributable to the presence of glucopyranoside of anomerism ss, which does not exceed 5% of the value of the integral of the previous signal. In addition, the absence of glucofuranoside substituent is confirmed by the absence of lower field anomeric signals.

 The mixtures of branched cyclomaltooligosaccharides obtained by the process of the invention are very advantageous, because they have a high solubility in water. Indeed, in the case of the reaction involving an equimolecular proportion of cyclomaltoheptaose and D-glucose, the solubility in water of the products obtained reaches 91 g / l at 250C (69 mmol / liter) while the solubility of cyclomaltoheptaose departure is only 19 g / l.

This solubility increases very quickly with the substitution rate of cyclomaltooligosaccharide, since, for 3 molecular equivalents of D-glucose compared to cyclomaltoheptaose, a solubility of 1320 g / l is obtained, which is very high compared to 19 g / l of the starting cyclomaltoheptaose.

 The chain length of the substituents corresponding to the reducing compounds used also plays an important role in the solubility of the branched products obtained. Indeed, when D-glucose is replaced by maltose and equimolecular proportions of cyclomaltoheptaose and maltose are used, the solubility in water of the mixture of products obtained reaches 1,200 g / l (803 mmol / l ), whereas it is 91 g / l in the case of D-glucose.

 The products obtained by the process of the invention can therefore have many applications because of their improved solubility in water. The properties of the substituents grafted onto the cyclomaltooligosaccharide can also be used for other applications, for example its possibility of recognition by its environment. Thus, oligosaccharide recognition signals can be grafted onto a cyclodextrin allowing the transport and vectorization of products for therapeutic use. By way of example of such possibilities, mention may be made of the grafting of 2-acetamido-2-deoxy-D-glucose and of D-mannose which are components frequently associated with the membrane glycoproteins of cells and thus make it possible to preferentially transport products for therapeutic use towards these membrane glycoproteins.

 Other characteristics and advantages of the invention will appear better on reading the following examples, which are of course given for illustrative and non-limiting purposes.

Exerrle 1:
To a carefully crushed mixture of cyclomaltoheptaose (3.15 g, 2.8 mmol) and D-glucose (2.8 mmol) in a polyethylene container placed in an acetone-dry ice bath, the anhydrous hydrogen fluoride (1.8 ml). The pasty mixture is homogenized using a steel spatula until a syrup is obtained. The container is then closed and stored at room temperature while maintaining magnetic stirring for 1 hour. After this time, the container is again cooled in liquid nitrogen and added with ether. The resulting white precipitate is separated from the supernatant by decantation, washed again with ether (3x50 ml), filtered and dried, which leads to a white pulverulent substance (3.6 g).

 The mixture obtained is analyzed by mass spectrometry by the ionization technique using the bombardment of accelerated atoms in a glycerol / thioglycerol matrix with sodium iodide as cationization agent. With this technique, the cationized or protonated molecular ions of the glycosylcyclodextrin constituents of the mixture obtained represent the majority, if not exclusive, signals of this spectrum, which allows an excellent characterization of the mass distribution of the oligosaccharide constituents of the crude mixture (as described in article by C. Bosso, J. Defaye, A.

Heyraud and J. Ulrich. "Fast atom bombardment-mass spectrometry for the characterization of cello- and malto-oligosaccharides" published in Carbohydr Res. 125 (1984) 309-317).

This analysis by mass spectrometry of the product obtained (FAB +; glycerol-thioglycerol matrix added with INa) gives the following series of signals at m / z 1157 (100, [CD + Na +]), 1319 (10, [CDGlc + Na ] +), 1481 (10, [CDGlc2 + Na] +), 1643 (4, [CDGlc3 + Na] +), 1805 (1, [CDGlc4 + Na] +)
Thus, for a 1: 1 proportion of D-glucose and of cyclomaltoheptaose, the presence on the spectrum is noted, independently of an ion at m / z 1157 ([M + Na] + of unreacted cyclomaltoheptaose), of ions at m / z 1319, 1481, 1643 and 1805 corresponding respectively to the cationized molecular ions of a monoglucosylcyclomaltoheptaose as well as of di-, triet tetraglucosylcyclomaltoheptaoses.

 The solubility of this substance in water is 91 g / l (69 mmol / l).

Example 2
The same procedure is followed as in Example 1, starting from a mixture of cyclomaltoheptaose (3.15 g, 2.8 mmol) and D-glucose (1.5 g, 8.4 mmol) which is supplemented with anhydrous hydrogen fluoride (2.3 ml). After the series of operations in Example 1, a white pulverulent substance (4.5 g) is obtained which shows in mass spectrometry (FAB +; glycerol-thioglycerol;
INa) the following series of signals at m / z 1157 (100), 1319 (90), 1481 (60), 1643 (50), 1805 (45), 1967 (35, [CdGlcs + Na] +), 2129 ( 30, [CDGlc6 + Na] +), 2291 (25, [CDGlc7 + Na] +), 2453 (25, [CDGlc8 + Na] +), 2615 (20, [CDGlcg + Na] +), 2777 (10, [CDGlclo + Na] +), 2939 (5, [CDGlcll + Na] +), 3101 (1, [CDGlc12 + Na] +), 3263 (1, [CDGlc13 + Na] +), 3425 (1, [CDGlcl4 + Na] +), 3587 (> 1, [CDGlcls + Na] +).

If we compare these results with those of Example 1, we observe that when the proportion
D-glucose / cyclomaltoheptaose is 3, the mass distribution as it appears in the spectrum is modified since we note the presence, independently of that of the previous ions of Example 1 even more intense, of ions with m / z 1967, 2129, 2291, 2453, 2615, 2777, 2939, 3101, 3263, 3425 and 3587 indicative of the associated presence of tri-to pentadecaglucosylcyclomaltoheptaoses.

 Thus, the mass distribution of the mixture of branched cyclodextrins is directly proportional to the amount of glucose reacted.

 The water solubility of this substance is -1320 g / l (-894 mmol / l).

Example 3
The procedure of Example 1 is applied to a mixture of cyclomaltoheptaose (3.15 g, 2.8 mmol) and maltose (1H20, 1.0 g, 2.8 mmol), which is added with fluoride. anhydrous hydrogen (1.8 ml).

After the series of operations in Example 1, 4.0 g of a white pulverulent substance are obtained which shows in mass spectrometry (FAB +; glycerol-thioglycerol; INa), the following series of signals at m / z 1157 (100, [CD + Na] +) 1481 (40, [CDMalt + Na] +), 1805 (20, [CDMalt2 + Na] +), 2129 (10, [CDMalt3 + Na] +), 2453 (5 , [CDMalt4 + Na +), 2777 (2, [CDMalt5 + Na] +), 3101 (1, [CDMalt6 + Na]) +).

 The solubility in water of this substance is -1200 g / l (-803 mmol / l).

Example 4
The procedure of Example 1 is applied to a mixture of cyclomaltohexaose (2.7 g, 2.8 mmol) and maltose (1H2O, 1.0 g, 2.8 mmol) to which hydrogen fluoride is added. anhydrous (1.8 ml). Obtaining, by repeating the series of operations of Example 1, 3.6 g of a white pulverulent substance which shows in mass spectrometry (FAB +; glycerol-thioglycerol;
ICs), the following series of signals at m / z 1105 (100, [CD + Cs] +), 1429 (30, [CDMalt + Csi +), 1753 (15, [CDMalt2 + Cs] +), 2077 (10, [CDMalt3 + Cs] +), 2401 (7, [CDMalt4 + Cs] +), 2725 (5, [CDMalt5 + Cs] +), 3049 (3, [CDMalt6 + Cs] +), 3373 (2, [CDMalt7 + Cs] +), 3697 (1, [CDMalt8 + Cs] +).

 The solubility in water of this substance is -1450 g / l (-1088 mmol / l).

Example 5
The procedure of Example 1 is applied to a mixture of cyclomaltooctaose (3.6 g, 2.8 mmol) and maltose (1 H 2 O, 1.0 g, 2.8 mmol), to which the fluoride d anhydrous hydrogen (2.3 ml). After the series of operations in Example 1, 4.4 g of a white pulverulent substance are obtained which shows in mass spectrometry (FAB +, glycerol-thioglycerol,
ICs), the following signal series at m / z 1429 (100, [CD + Cs] +), 1753 (40, [CDMalt + Cs] +), 2077 (30, [CDMalt2 + Cs] +), 2401 ( 15, ICDMalt3 + Cs) +), 2725 (10, {CDMalt4 + Cs} +), 3049 (5, CDMalt5 + Cs} +), 3373 (3, [CDMalt6 + Cs] +), 3697 (2, [CDMalt7 + Cs] +), 4021 (1, [CDMaltg + Cs] +).

 The solubility of this substance in water is -1580 g / l (954 mmol / l).

Example 6
The procedure of Example 1 is applied to a mixture of cyclomaltoheptaose (3.15 g, 2.8 mmol) and D-mannose (1.5 g, 8.4 mmol) to which hydrogen fluoride is added. anhydrous (2.3 ml). After the series of operations of Example 1, 4.4 g of a white pulverulent substance are obtained which shows, in mass spectrometry (FAB +, glycerol-thioglycerol,
INa), the following series of signals at m / z 1267 (100, [CD + Cs] +), 1429 (80, [CDMan + Cs] +), 1591 (60, [CDMan2 + Cs] +), 1753 ( 50, [CDMan3 + Cs] +), 1915 (40, [CDMan4 + Cs] +), 2077 (30, [CDMans + Cs] +), 2239 (25, [CDMan6 + CsJ +), 2401 (20, [CDMan7 + Cs] +), 2563 (17, [CDMang + Cs] +), 2725 (15, [CDMang + Cs] +), 2887 (12, [CDMan10 + Cs] +), 3049 (10, [CDManll + Cs ] +), 3211 (7, [CDMan12 + Cs] +), 3373 (5, [CDMan13 + Cs] +), 3535 (3, [CDManl4 + Cs] +), 3697 (2, [CDMan15 + Cs] + ), 3859 (1, [CDMan16 + Csl +), 9021 (1, [CDManl7 + Cs] +).

 The solubility in water of this substance is -1400 g / l (-853 mmol / l).

Example 7
To a carefully ground mixture of cyclomaltoheptaose (2.56 g, 2.26 mmol) and 2-acetamido-2-deoxy-D-glucose (1.0 g, 4.52 mmol), in a container polyethylene placed in an acetone-dry ice bath, anhydrous hydrogen fluoride (2 ml) is added. The pasty mixture is homogenized using a steel spatula until a syrup is obtained. The non-closed container is then brought to ambient temperature while maintaining agitation until it stops by itself as a result of the evaporation of HF.

After 15 hours, the residue is dissolved in water (30 ml) and added until the powdered CaCo3 is neutral. The suspension is then freed of the insoluble salts by filtration and lyophilized, which leads to a white pulverulent substance (3.4 g) which shows in mass spectrometry (FAB +; glycerol-thioglycerol matrix; ICs) a series of signals at m / z 1267 (100, [CD + Cs] +), 1470 (60, [CDGleNAc + Cs) +), 1673 (15, [CD (GleNac) 2 + Cs] +), 1876 (1, [CD (GleNAc) 3 + Cs] +).

 The solubility of this substance in water is -1100 g / l.

Claims (9)

 1. Process for the preparation of branched cyclomaltooligosaccharides, characterized in that a cyclomaltooligosaccharide is reacted with at least one reducing compound chosen from monosaccharides, reducing oligosaccharides, reducing polysaccharides and their derivatives, in the presence of anhydrous hydrogen fluoride , operating at least at the start of the reaction under conditions such that the anhydrous hydrogen fluoride is liquid, and using an amount of anhydrous hydrogen fluoride sufficient to obtain a pasty reaction mixture of the cyclomaltooligosaccharide and the reducing compound, and in that the reaction products of hydrogen fluoride are then separated.  2. Method according to claim 1, characterized in that the reaction products are then purified by dissolution in water followed by addition of calcium carbonate to the solution to remove traces of residual fluorine, hydrofluoric acid and / or fluoride of the reducing compound.  3. Method according to any one of claims 1 and 2, characterized in that the anhydrous hydrogen fluoride is mixed with a non-aqueous solvent.  4. Method according to claim 3, characterized in that the non-aqueous solvent is chosen from sulfur dioxide, dioxane and pyridine.  5. Method according to any one of claims 1 to 4, characterized in that the molar ratio of the reducing compound to the cyclomaltooligosaccharide is from 0.5 to 50.  6. Method according to any one of claims 1 to 5, characterized in that the cyclomaltooligosaccharide is chosen from cyclodextrins a, ss and y.  7. Method according to any one of claims 1 to 6, characterized in that the reducing compound is chosen from D-glucose, maltose, D-mannose and 2-acetamido-2-deoxy-D-glucose.  8. Method according to any one of claims 1 to 7, characterized in that the amount of anhydrous hydrogen fluoride is 0.1 to 1 ml / g of mixture of cyclomaltooligosaccharide and compound (s) reducing (s) .  9. Process for improving the solubility in water of a cyclomaltooligosaccharide, characterized in that it consists in replacing the cyclomaltooligosaccharide with at least one reducing compound chosen from monosaccharides, reducing oligosaccharides, reducing polysaccharides and their derivatives implementing the method according to any one of claims 1 to 8.