Derivatives of oligosides, their process of preparation and...

Details Derivatives of oligosides, their process of preparation and... Derivatives of oligosides, their process of preparation and...

1996-02-21

2001-06-26

Jones, Dwayne C. (Department: 1614)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S414000, C536S023100, C536S123000, C530S330000, C530S322000

Reexamination Certificate

active

06251858

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to oligoside derivatives, their process of preparation, and their applications.
2. Description of the Related Art
Natural oligosides are able to be prepared in free form from various physiologic liquids such as milk, or extracts from natural or transformed products (honey, beer, etc.). Natural oligosides are also able to be obtained by cutting a glycoside bond from one of the sugar moieties of glycoconjugates (glycolipids, glycoproteins, polyosides, proteoglycans, etc.), by hydrolysis with the aid of enzymes or by chemical catalysis from said glycoconjugates.
The natural oligosides are able to be used as substrates, as inhibitors, as recognition signals, etc. In the majority of cases, it is advisable to fix the oligoside on a molecule, matrix or particle, which can be chosen from:
a matrix as a support for affinity chromatography;
a bead of gold or latex, for histology and cytology;
a protein for visualilzation, purification, etc., in particular 1) specific receptors of osides, receptors which are called lectins, adhesins, agglutinins, etc., or 2) proteins with or without enzymatic activity, which have an affinity for the osides, in particular the glycosyltransferases, exoglycosidases or endoglycosidases
a lipid for the characterization of the preceding receptors;
oligonucleotides for selectively increasing their capture by targeted cells;
a protein or polymers for the targeting of drugs, oligonucleotides or genes, or for obtaining intramolecular inhibitors.
The synthesis of derivatives of oligosides able to be linked by covalent means to a protein, a matrix, an oligonucleotide, or by general means to an organic molecule or a particle, in all cases preserving the integrity of the structure and functions of each of its sugar components—which is necessary for preserving the functional capacity of the oligoside can be obtained essentially in two ways: the total synthesis de novo and the intermediary transformation into glycosylamine. A third way which leads to an equally useful derivative but which destroys the terminal reductorose is amination in a reducing medium.
Concerning total synthesis, this requires a selective protection of the hydroxyls non-engaged in a glycoside bond, steps of condensation, steps of selective deprotection, and a step of final deprotection. Even if over the last decades the yields of some of these steps were able to be improved, this is a long process and the overall yield remains modest, and this all the more as the oligoside to be synthesized is more complex.
The yields for each step are between 20 and 95% according to the steps considered.
For example, the synthesis of a para-nitrophenyl derivative of a pentasaccharide such as the determinant of Lewis x:
Gal&bgr;3(Fuc&agr;4)GlcNAc&bgr;3Gal&bgr;4Glc&bgr;-O-p-C
6
H
4
—NO
2
requires in total several tens of steps. Each step has a yield of between 50 and 95%, more generally 80%. In total the yield of the product sought is of some %.
The elevated number of steps arises from the fact that the alcohol functions of each ose must be protected in a different manner depending on whether the hydroxyl under consideration will or will not be implicated in a condensation reaction.
A sugar such as GlcNAc which will be substituted 2 times will receive momentarily 3 different substitutes in order to permit a selective substitution on the hydroxyl 3 by galactose, on the hydroxyl 4 by fucose, the hydroxyl 6 remaining protected until the final deprotection.
For each step of condensation, the yield is affected by the fact that the product formed is in general a mixture of the two (&agr; and &bgr;) anomeric forms.
Moreover, it is necessary to point out that the intermediary products have to be purified, either by crystallization, or by chromatography, which contributes significantly to the total duration of the synthesis. Finally, it should be noted that the yields may be very weak for certain steps because of steric hindrance, which is specifically the case at the level of the branches: 2 sugars substituting a single monosaccharide.
All in all, this global synthesis is very costly and takes a long time.
In the case of the formation of a glycosylamine followed by acylation, this way depends on a reaction described at the end of the last century: an oside possessing a reducing sugar, incubated in the presence of an elevated concentration of ammoniac, of an ammonium salt or an aromatic amine, is transformed in a reversible manner into glycosylamine.
For example, the lactose gives a lactosylamine, with ammoniac:
Gal&bgr;4Glc+NH
3
→Gal&bgr;4Glc&bgr;-NH
2
+H
2
O
or with the aniline:
Gal&bgr;4Glc+NH
2
—C
6
H
5
→Gal&bgr;4Glc&bgr;-NH—C
6
H
5
+H
2
O
This reaction is however reversible, which is to say the isolated product, dissolved in a buffer, becomes hydrolyzed leading to give back the original products.
The glycosylamine may however be stabilized by acylation, for example by selective N-acetylation:
Gal&bgr;4Glc&bgr;-NH
2
+(CH
3
—CO)
2
—O→Gal&bgr;4Glc&bgr;-NH—CO—CH
3
+CH
3
—CO
2
H
On these bases, it has recently been proposed to substitute the amine of oligosylamines by an organic compound possessing a finctional reactive group. Manger et al., 1992, Biochemistry 31, 10724 and 31, 10733.
This route comprises the following steps:
1) incubation of the oside possessing a terminal reducing sugar in the presence of a highly concentrated ammonium salt.
For example,
Gal&bgr;4Glc+NH
4
&rlarr2;Gal&bgr;4Glc&bgr;-NH
3
+
+H
2
O
C
12
H
22
O
11
+NH
4
&rlarr2;C
12
H
24
O
10
N+H
2
O
2) Purification of the glycosylamine by chromatography on a column to eliminate the ammonium salt excess.
3) Substitution of the amine of the glycosylamine by reaction with an activated derivative of monochloroacetic acid, in alkaline medium.
For example,
Gal&bgr;4Glc&bgr;NH
2
+(Cl—CH
2
—CO)
2
—O→Gal&bgr;4Glc—NH—CO—CH
2
—Cl
4) Transformation of the chloroacetyl residues into glycyl residue.
The chloroacetylglycosylamide is incubated in the presence of a highly concentrated ammonium salt, which allows the introduction of an amine function. For example:
Gal&bgr;4Glc&bgr;-NH—CO—CH
2
—Cl+NH
4
+
→Gal&bgr;4Glc&bgr;-NH—CO—CH
2
—NH
3
+
, Cl
−
H
+
5) Purification of glycyl-glycosylamide by chromatography on column to eliminate the ammonium salts.
6) Condensation of the glycyl-glycosylamide and of a compound able to selectively react with an amine group of the glycyl residue.
For example:
Gal&bgr;4Glc&bgr;-NH—CO—CH
2
—NH
2
+R—CO-G→Gal&bgr;4Glc&bgr;-NH—CO—CH
2
—NH—CO—R+HG
in which G is an activator of the carboxylic function.
This route in 6 steps implies two steps of intermediary purification and the use of a toxic product: an activated derivative of chloroacetic acid.
The yield of each step is variable and ranges between 50 and 95%; the global yield is less than approximately 60%.
Another pathway has also been suggested, but it requires the transformation of the reducing sugar into polyol; this route was proposed in 1974 by Gray (Arch. Biochem. Biophys., 163, 426-428).
The oligoside is condensed with a compound comprising one (or many) amine function(s) in the presence of sodium cyanoborohydride in alkaline medium; the imine formed between the reducing sugar and the amine is reduced by the sodium cyanoborohydride into a secondary amine.
For example:
Gal&bgr;4Glc+NH
2
—R→
Gal&bgr;4-O—CH(CHOH—CH
2
OH)—CHOH—CHOH—CH═N—R
+NaBH
3
CN→Gal&bgr;4-O—CH(CHOH—CH
2
OH)—CHOH—CHOH—CH
2
—NH—R
The yield of this reaction varies according to the size of the partners and is usually between 5 and 70%.
There is a destruction of the reducing sugar, which is not desirable.
French patent number 2 227 823 concerns the N-osides of L pyrrolidone-2-carboxylic-5 acid, derivatives of said acid and their procedure of preparation, consisting of condensing the L pyrrolidone-2-carboxylic-5 acid and/or the L-glutamic acid and/or a salt of these acids with an ose or an oside, with reducin

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