Process for preparing polyvalent and physiologically degradable carbohydrate-containing polymers by enzymatic glycosylation reactions and the use thereof for preparing carbohydrate building blocks

• US 6,406,894 B1
• Filed: 12/11/1997
• Issued: 06/18/2002
• Est. Priority Date: 12/11/1992
• Status: Expired due to Fees

• Current Assignee: Glycorex Transplantation AB
• Original Assignee: Glycorex AB
• Inventors: Hoersch, Brigitte, Seiffge, Dick, Ahlers, Michael, Bartnik, Eckart, Kretzschmar, Gerhard
• Primary Examiner(s): PRATS, FRANCISCO CHANDLER
• Application Number:
  US08/989,136
• Time in Patent Office: 1,650 Days
• Field of Search: 435/97, 435/100, 435/101, 536/55, 536/55.1, 536/55.2, 536/55.3, 536/123.1, 536/126, 536/4.1, 536/20, 536/21, 536/17.2, 536/18.5, 536/18.7, 536/45, 536/102, 536/124, 525/32.2
• US Class Current: 435/97
• CPC Class Codes: A61K 47/595 Polyamides, e.g. nylon polyamino acids A61K47/62
• CPC Class Codes: A61K 47/61 the organic macromolecular compound being a polysaccharide or a derivative thereof
• CPC Class Codes: A61K 47/64 Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent peptidic linkers A61K47/65
• CPC Class Codes: C07H 15/04 attached to an oxygen atom of the saccharide radical
• CPC Class Codes: C07H 15/203 Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
• CPC Class Codes: C07H 3/06 Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
• CPC Class Codes: C12P 19/18 produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
• CPC Class Codes: C12P 19/26 Preparation of nitrogen-containing carbohydrates
• CPC Class Codes: C12P 19/44 Preparation of O-glycosides, e.g. glucosides polysaccharides and not substituted disaccharides C12P19/04, C12P19/12

Abstract

A process for preparing polyvalent, physiologically degradable carbohydrate-containing polymers by enzymatic glycosylation reactions is described. The carbohydrate-containing polymers thus prepared may be used for preparing carbohydrate building blocks. The polyvalent carbohydrate-containing polymers of the invention cause no intolerance reactions in vivo, either in their intact form or in the form of degradation products. The carbohydrate side chain of the carbohydrate-containing polymer is assembled by enzymatic glycosylation reactions in homogeneous aqueous buffer systems directly on a biodegradable polymer. The yields of the glycosylation reaction are significantly improved over known processes, and are often quantitative. This also provides a significant increase in the loading densities. A process for preparing free oligosaccharides by means of the carbohydrate-containing polymers of the invention is also described.

Claims

1. A process for preparing a carbohydrate-containing polymer that is physiologically degradable in vivo in a mammal, wherein said carbohydrate-containing polymer comprises a non-naturally occurring hydrophilic, biodegradable polymer unit, a disaccharide or oligosaccharide unit, and a bifunctional spacer linking said disaccharide or oligosaccharide units to said polymer unit, comprising the steps of:

(a) covalently linking a monosaccharide or oligosaccharide unit to a spacer to form a monosaccharide or oligosaccharide-spacer complex wherein said spacer has the formula (mono- or oligosaccharide)—

O—

[Q₁—

(CH²)_p—

Q²]_r-(polymer), wherein Q¹ is —

CH₂or —

CO—

, Q² is —

NH or —

CO—

NH—

, p is an integer from 1 to 6, and r is 1 or 2;

(b) covalently linking said monosaccharide or oligosaccharide-spacer complex to a non-naturally occurring hydrophilic, biodegradable polymer unit to form an acceptor unit; and

(c) coupling a monosaccharide donor unit to said acceptor unit by enzymatic glycosylation.

2. The process according to claim 1, wherein the enzymatic glycosylation of said acceptor unit takes place in homogeneous aqueous phase.

3. The process according to claim 2, wherein said enzymatic glycosylation of said acceptor is carried out using nucleotide-activated carbohydrates as said monosaccharide donor units and wherein said glycosylation reaction is catalyzed by at least one glycosyltransferase.

4. The process according to claim 1, wherein said glycosylation reaction is carried out at between about pH 6.0 and about pH 8.5.

5. The process according to claim 3, further comprising adding alkaline phosphatase to the reaction medium when said monosaccharide donor unit is added in about equimolar amount or in excess.

6. The process according to claim 3, wherein between about 0.01 to about 10 units of said glycosyltransferase are used.

7. The process according to claim 2, wherein said enzymatic glycosylation is carried out at a temperature between about 10°

C. and about 40°

C. for between about 1 to about 5 days.

8. The process according to claim 1, wherein polyamino-acid has a molecular weight up to about 70 kD, and is in polyamide or polyanhydride form.

9. The process according to claim 8, wherein said polyamino-acid is selected from the group consisting of poly-α

,β

-(2-hydroxyethyl)-D,L-aspartamide, poly-D,L-succinimide, polyglutamate, poly-L-lysine methyl ester fumaramide and a copolymer of these polyamino-acids.

10. The process according to claim 1, wherein the linkages connecting the spacer to the monosaccharide or oligosaccharide unit and to the biodegradable polymer unit are formed by reactions selected from the group consisting of alkylation, reductive alkylation, acylation and addition onto a double bond.

11. The process according to claim 1, wherein the disaccharide or oligosaccharide unit of said carbohydrate-containing polymer is selected from the group consisting of:

SAα

2-6-Galβ

1-4-GlcNAc-;

SAα

2-3-Galβ

1-4-GlcNAc-;

SAα

2-3-Galβ

1-3-GlcNAc-;

Galβ

1-4-(Fucα

1-3-GlcNAc-;

Galβ

1-3-(Fucα

1-3)GlcNAc-;

SAα

2-3-Galβ

1-3-(Fucα

1-4)GlcNAc-;

SAα

2-3-Galβ

1-4-(Fucα

1-3)GlcNAc-, sialyl-Lewis X, sialyl-Lewis A, VIM-2, Lewis A, Lewis B, Lewis X, Lewis Y, Lewis A type¹, Lewis A type², Lewis B type¹, Lewis B type², Lewis H type¹ and Lewis H type².

12. The process according to claim 3, wherein said nucleotide-activated carbohydrate is selected from the group consisting of:

UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetyl-galactosamine, UDP-glucuronic acid, CMP-neuraminic acid, GDP-fucose, GDP-mannose, dTDP-glucose and dUDP-galactose.

13. The process according to claim 3, wherein said glycosyltransferase is selected from the group consisting of:

β

-1,4-galactosyltransferase, Gal-β

-1-4-GlcNAc α

-2-6-sialyltransferase, Gal-β

-1-3-GalNAc α

-2-3-sialyltransferase, Gal-β

-1-3(4)-GlcNAc α

-2-3-sialyltransferase, GalNAc α

-2-6-sialyltransferase, N-acetylglucosaminytransferases, α

-1-3-fucosyltransferase, α

-1-2-fucosyltransferase, α

-3/4-fucosyltransferase, and α

-1-2-mannosyltransferase.

14. The process according to claim 1, wherein said disaccharide or oligosaccharide unit comprises 2-20 monosaccharide units.

15. A carbohydrate-containing polymer that is physiologically degradable in vivo in a mammal, comprising:

(a) a non-naturally occurring hydrophilic, biodegradable polymer unit,

(b) a disaccharide or oligosaccharide unit, and

(c) a bifunctional spacer linking said disaccharide or oligosaccharide units to said biodegradable polymer unit, wherein said spacer has the formula (mono- or oligosaccharide)—

O—

[Q¹—

(CH₂)_p—

Q²]_r-(polymer), and wherein Q¹ is —

CH₂, or —

CO—

, Q² is —

NH or —

CO—

NH—

, p is an integer from 1 to 6, and r is 1 or 2.

16. A process for preparing a carbohydrate-containing polymer that is physiologically degradable in vivo in a mammal, wherein said carbohydrate-containing polymer comprises a hydrophilic, biodegradable polymer unit, a disaccharide or oligosaccharide unit, and a bifunctional spacer linking said disaccharide or oligosaccharide units to said polymer unit, comprising the steps of:

(a) covalently linking a hydrophilic, biodegradable polymer unit to a spacer to form a biodegradable polymer-spacer complex wherein said spacer has the formula (mono- or oligosaccharide)—

O—

[Q¹—

(CH₂)_p—

Q²]_r-(polymer), wherein Q¹ is —

CH₂ or —

CO—

, Q² is —

NH or —

CO—

NH—

, p is an integer from 1 to 6, and r is 1 or 2;

(b) covalently linking said biodegradable polymer-spacer complex to a monosaccharide or oligosaccharide unit to form an acceptor unit; and

(c) coupling a monosaccharide donor unit to said acceptor unit by enzymatic glycosylation.

17. A process for preparing a carbohydrate-containing polymer that is physiologically degradable in vivo in a mammal, wherein said carbohydrate-containing polymer comprises a non-naturally occurring hydrophilic, biodegradable polyamino-acid, a disaccharide or oligosaccharide unit, and a bifunctional spacer linking said disaccharide or oligosaccharide units to said polymer unit, comprising the steps of:

(a) covalently linking a non-naturally occurring hydrophilic, biodegradable polyamino-acid to a spacer to form a biodegradable polymer-spacer complex, wherein said spacer has the formula (mono- or oligosaccharide)—

O—

[Q¹—

(CH₂)_p—

Q²]_r-(polymer), wherein Q¹ is —

CH₂or —

CO—

, Q² is —

NH or —

CO—

NH—

, p is an integer from 1 to 6, and r is 1 or 2;

(b) covalently linking said biodegradable polyamino-acid-spacer complex to a monosaccharide or oligosaccharide unit to form an acceptor unit; and

(c) coupling a monosaccharide donor unit to said acceptor unit by enzymatic glycosylation.