Use of trans-sialidase and sialyltransferase for synthesis of sialyl.alpha.2.fwdarw.3.beta.galactosides

• US 5,409,817 A
• Filed: 05/04/1993
• Issued: 04/25/1995
• Est. Priority Date: 05/04/1993
• Status: Expired due to Fees

• Current Assignee: Neose Technologies, Inc.
• Original Assignee: Cytyc Corporation (Hologic Incorporated)
• Inventors: Ito, Yukishige, Paulson, James C.
• Primary Examiner(s): Lilling, Herbert J.
• Application Number:
  US08/057,528
• Time in Patent Office: 721 Days
• Field of Search: 435/72, 435/74, 435/97, 435/101, 435/193, 435/194, 435/175
• US Class Current: 435/74
• CPC Class Codes: C12N 9/1081 transferring other glycosyl groups (2.4.99)
• CPC Class Codes: C12N 9/1241 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: C12N 9/2402 hydrolysing O- and S- glycosyl compounds (3.2.1)
• CPC Class Codes: C12P 19/26 Preparation of nitrogen-containing carbohydrates
• CPC Class Codes: C12Y 302/01018 Exo-alpha-sialidase (3.2.1.18), i.e. trans-sialidase

Abstract

A single vessel cyclic synthesis process for preparation of a sialylα2→3βgalactoside is disclosed. In accordance with this process, a sialyltransferase acceptor is sialylated in an aqueous reaction medium by an α(2,3)sialyl transferase and CMP-sialic acid to form a sialyl donor substrate and CMP. In the presence of the trans-sialidase of Trypanosoma crusi, that sialyl donor substrate provides a sialyl group for a trans-sialidase acceptor, thereby preparing the sialylα2→3βgalactoside. The α(2,3)sialyltransferase acceptor is reformed upon trans-sialidation of the latter acceptor, and the sialyl donor substrate is reformed using the α(2,3)sialyltransferase and a CMP-sialic acid recycling system that combines CMP with sialic acid that is also present in the vessel. The K_m /V_max value for the α(2,3)sialyltransferase acceptor is less than one-tenth the value of K_m /V_max of the trans-sialidase acceptor for the α(2,3)sialyltransferase.

Claims

1. A process for forming a sialylα

2→

3β

galactoside that comprises the steps of;

combining the following components in a single vessel to form a reaction mixture;

(i) a catalytic amount of an α

(2,3)sialyltransferase;

(ii) a catalytic amount of a CMP-sialic acid synthetase;

(iii) a catalytic amount of Trypanosoma crusi trans-sialidase;

(iv) a sialic acid;

(v) an oligosaccharide acceptor for said α

(2,3)sialyltransferase having a β

-linked galactosyl unit at the oligosaccharide non-reducing terminus;

(vi) an oligosaccharide acceptor for said trans-sialidase having a β

-linked galactosyl unit at the oligosaccharide non-reducing terminus, said acceptor being free of fucosylation within two joined oligosaccharide units of said non-reducing terminal galactosyl unit;

said oligosaccharide acceptor of (v) having a K_m /V_max value for said α

(2,3)sialyltransferase that is less than one-tenth said K_m /V_max of said oligosaccharide acceptor of (vi) for that same enzyme;

(vii) a CMP-sialic acid recycling system that comprises at least 2 moles of phosphoenolpyruvate per each mole of sialic acid, and catalytic amounts of ATP, myokinase, pyruvate kinase and inorganic pyrophosphatase; and

(viii) a buffered aqueous reaction medium containing enzymatically sufficient amounts of metal ion cofactors for said enzymes and having a pH value of about 6 to about 8;

maintaining said reaction mixture at a temperature of about zero degrees C. to about 45°

C. for a time period sufficient for said acceptor (vi) to be sialylated and form said sialylα

2→

3β

galactoside.

2. The process according to claim 1 including the further step of recovering said formed sialylα

2→

3β

galactoside.

3. The process according to claim 1 wherein said acceptor of (vi) is 2-(trimethylsilyl)ethyl-β

-galactoside.

4. The process according to claim 1 wherein said acceptor of (v) is lacto-N-tetraose.

5. The process according to claim 1 wherein said acceptor of (vi) is Galβ

1→

3GalNAc1--OCH₂ CH₂ SiMe₃.

6. The process according to claim 1 wherein said oligosaccharide acceptor of (v) has a K_m /V_max that is less than one-tenth of the K_m /V_max value of said oligosaccharide acceptor of (vi) for said α

(2,3) sialyltransferase.

7. The process according to claim 1 wherein said sialic acid is 5-N-acetylneuraminic acid.

8. A process for forming a sialylα

2→

3β

galactoside that comprises the steps of;

combining the following compoents in a single vessel to form a reaction mixture;

(i) a catalytic amount of an α

(2,3)sialyltransferase;

(ii) a catalytic amount of a CMP-sialic acid synthetase;

(iii) a catalytic amount of Trypanosoma crusi trans-sialidase;

(iv) sialic acid;

(v) lacto-N-tetraose;

(vi) an oligosaccharide acceptor for said trans-sialidase having a β

-linked galactosyl unit at the oligosaccharide non-reducing terminus, said acceptor being free of fucosylation within two joined oligosaccharide units of said non-reducing terminal galactosyl unit;

said oligosaccharide acceptor of (v) having a K_m /V_max value for said α

(2,3)sialyltransferase that is less than one-tenth said K_m /V_max of said oligosaccharide acceptor of (vi) for that same enzyme;

(vii) a CMP-sialylic acid recycling system that comprises at least 2 moles of phosphoenolpyruvate per each mole of sialylic acid, and catalytic amounts of ATP, myokinase, pyruvate kinase and inorganic pyrophosphatase; and

(viii) a buffered aqueous reaction medium containing enzymatically sufficient amounts of metal ion cofactors for said enzymes and having a pH value of about 6 to about 8;

maintaining said reaction mixture at a temperature of about zero degrees C. to about 45°

C. for a time period sufficient for said acceptor (vi) to be sialylated and form said sialylα

2→

3β

galactoside.

9. The process according to claim 8 including the further step of recovering said formed sialylα

2→

3β

galactoside.