Anti liver disease drug R-YEEE and method of synthesizing branched galactose-terminal glycoproteins

• US 6,383,812 B1
• Filed: 01/25/2000
• Issued: 05/07/2002
• Est. Priority Date: 05/28/1999
• Status: Expired due to Term

• Current Assignee: Academia Sinica (Government of The Republic of China)
• Original Assignee: Academia Sinica (Government of The Republic of China)
• Inventors: Huang, Chun-Ming, Tseng, Ping-Hui, Jiaang, Weir-Torn, Chen, Shui-Tein
• Primary Examiner(s): McGarry, Sean
• Assistant Examiner(s): EPPS -SMITH, JANET L
• Application Number:
  US09/490,734
• Time in Patent Office: 833 Days
• Field of Search: 530/333, 530/395, 530/391.9, 530/391.7, 435/375, 435/325, 435/455, 514/2-21
• US Class Current: 435/455
• CPC Class Codes: C07H 15/18 Acyclic radicals, substituted by carbocyclic rings

Abstract

The present invention provides a novel method of synthesizing branched galactose-terminal glycoproteins. A number of these glycoproteins have binding affinity to the asialoglycoprotein receptor. The present invention also provides novel conjugates having branched galactose-terminal glycoproteins that is complexed to a therapeutically effective agent, such as an isolated protein, polysacharides, lipids and radioactive isotope. These conjugates may be used to deliver the therapeutically effective agent to mammalian cells generally, and to hepatocytes specifically.

Claims

1. A method of synthesizing branched galactose-terminal glycoprotein comprising the steps of:

coupling an amino alcohol with a chemically protected ester of a dicarboxylic acid, which dicarboxylic acid is selected from the group consisting of glutamic and aspartic acids to produce A;

reacting A with the oxazoline of N-acetyl galactosamine to produce the amino acid derivative B;

reacting a 1,3 diacetate of N-acetyl galatosamine with a monobenzyl ester of a dicarboxylic acid selected from the group consisting of succinic, glutaric, adipic, maleic and fumaric acids to produce C;

reacting C with a chemically protected amino alkyl alcohol to produce D;

attaching D to an amide resin;

reacting the attached D with B to form E that is attached to the amide resin;

reacting the attached E with an amino acid having a chemically protected amino group;

deprotecting and cleaving from the resin to form the branched galactose-terminal glycoprotein.

2. The method of claim 1 wherein the amino alcohol is 6-amino hexanol.

3. The method of claim 1 wherein the chemically protected ester of a dicarboxylic acid is γ

-Fmoc-Glu(tBu).

4. The method of claim 1 wherein the chemically protected ester of a dicarboxylic acid is γ

-Fmoc-Asp(tBu).

5. The method of claim 1 wherein the monobenzyl ester of a dicarboxylic acid is succinic acid monobenzyl ester.

6. The method of claim 1 wherein the attached E is reacted with tyrosine having a chemically protected amino group.

7. The method of claim 1 wherein the chemically protected ester of a dicarboxylic acid is γ

-Fmoc-Glu(tBu) and the monobenzyl ester of a dicarboxylic acid is the monobenzyl ester of succinic acid.

8. The method of claim 7 wherein E has at least two galactose terminal groups.

9. The method of claim 7 wherein E has at least three galactose terminal groups.

10. The method of claim 9 wherein the branched galactose-terminal glycoprotein is YEE(GalNAcAH)₃.

11. A method of synthesizing branched galactose-terminal glycoprotein comprising the steps of:

coupling an amino alcohol with a chemically protected ester of a dicarboxylic acid, which dicarboxylic acid is selected from the group consisting of glutamic and aspartic acids, to produce A;

reacting A with the oxazoline of the N-acetyl galactosamine to produce the amino acid derivative B;

attaching B to an amide resin;

reacting the attached B with unattached B to form C that is attached to the amide resin;

reacting the attached C with an amino acid having a chemically protected amino group;

deprotecting and cleaving from the resin to form the branched galactose-terminal glycoprotein.

12. The method of claim 11 wherein the amino alcohol is 6-amino hexanol.

13. The method of claim 11 wherein the chemically protected ester of a dicarboxylic acid is γ

-Fmoc-Glu(tBu).

14. The method of claim 11 wherein the chemically protected ester of a dicarboxylic acid is γ

-Fmoc-Asp(tBu).

15. The method of claim 11 wherein the attached C is reacted with tyrosine having a chemically protected amino group.

16. The method of claim 11 wherein C has at least two galactose terminal groups.

17. The method of claim 11 wherein C has at least three galactose terminal groups.

18. The method of claim 17 wherein the branched galactose-terminal glycoprotein is YEEE(GalNAcAH)₃.

19. A branched galactose-terminal glycoprotein having binding affinity to the asialoglycoprotein receptor produced by the process of claim 11.

20. The branched galactose-terminal glycoprotein of claim 19, having three galactose terminal groups.

21. The branched galactose terminal glycoprotein having the following chemical structure:

22. A conjugate having a branched galatose-terminal glycoprotein having the chemical structure according to claim 21, which is complexed to a therapeutically effective agent.

23. The conjugate of claim 22, wherein the therapeutically effective agent is selected from the group consisting of a nucleic acid, protein, polysaccharide, lipid and radioactive isotope.

24. The conjugate of claim 23, wherein the nucleic acid is DNA.

25. The conjugate of claim 23, wherein the nucleic acid is RNA.

26. The conjugate of claim 23, wherein the protein is interferon.

27. A method of delivering a therapeutically effective agent to a mammalian cell comprising administering to a mammal a conjugate having a branched galatose-terminal glycoprotein having the chemical structure according to claim 21, which is complexed to a therapeutically effective agent.

28. A method of transfecting hepatocytes comprising adding to the hepatocytes a composition comprising a complex comprising an isolated nucleic acid and a branched galatose-terminal glycoprotein having the chemical structure according to claim 21.

29. The method of claim 28, wherein the isolated nucleic acid is DNA.

30. The method of claim 29, wherein the isolated nucleic acid comprises sequences of antisense oligonucleotides of PKC (protein kinase C) α

;

having a sequence of (5′

-3′

) CAGCCATGGTTCCCCCCAAC (SEQ ID NO;

     2).