Process for preparing glycopeptides having asparagine-linked oligosaccharides, and the glycopeptides

1. A process for preparing glycopeptide having at least two asparagine-linked oligosaccharides at a desired position of the peptide chain thereof, the process comprising:

(a) esterifying a hydroxyl group of a resin having the hydroxyl group and a carboxyl group of an amino acid having amino group nitrogen protected with a fat-soluble protective group,

(b) removing the fat-soluble protective group to form a free amino group,

(c) amidating the free amino group and a carboxyl group of an amino acid having amino group nitrogen protected with a fat-soluble protective group,

(d) removing the fat-soluble protective group to form a free amino group,

(e) repeating the steps (c) and (d) at least once,

(f) amidating the free amino group and a carboxyl group of the asparagine portion of an asparagine-linked oligosaccharide having amino group nitrogen protected with a fat-soluble protective group,

(g) removing the fat-soluble protective group to form a free amino group,

(h) amidating the free amino group and a carboxyl group of an amino acid having amino group nitrogen protected with a fat-soluble protective group,

(i) repeating the steps (g) and (h) at least once,

(j) removing the fat-soluble protective group to form a free amino group, and

(k) cutting off the resin with an acid;

wherein the steps (f) of amidating the free amino group and a carboxyl group of the asparagine portion of an asparagine-linked oligosaccharide having amino group nitrogen protected with a fat-soluble protective group and (g) of removing the fat-soluble protective group to form a free amino group are repeated after the step (g) or (j), and

wherein at least one of the asparagine-linked oligosaccharides in the step (f) has a residue of sialic acid or a derivative thereof at a terminal end thereof.

2. A process for preparing a glycopeptide according to claim 1 , wherein the asparagine-linked oligosaccharide of the step (f) of claim 1 has at least 6 sugar residues.

3. A process for preparing a glycopeptide according to claim 1 , wherein the asparagine-linked oligosaccharide of the step (f) of claim 1 has 9 to 11 sugar residues.

4. A process for preparing a glycopeptide according to claim 1 , wherein the asparagine-linked oligosaccharide of the step (f) of claim 1 has at least 6 sugar residues, and has a bifurcated oligosaccharide attached thereto.

5. A process for preparing a glycopeptide according to claim 1 , wherein at least one of the asparagine-linked oligosaccharides is an asparagine-linked asialooligosaccharide.

6. A process for preparing a glycopeptide according to claim 1 , wherein the fat-soluble protective group is 9-fluorenylmethoxycarbonyl (Fmoc) group.

7. A process for preparing a glycopeptides according to claim 1 ,

wherein at least one of the asparagine-linked oligosaccharides is an asparagine-linked disialooligosacharide or an asparagine-linked monosialooligosaccharide having amino group nitrogen protected with a fat-soluble protective group and the carboxyl group of the sialic acid protected with a benzyl or allyl group; and

the process further comprises the following step;

(l) preparing the asparagine-linked disialooligosaccharide or the asparagine-linked monosialooligosaccharide, wherein the benzyl or allyl group is introduced into the carboxyl group of the sialic acid under a condition of pH 5 to 6.

8. A process for preparing a glycopeptide according to claim 1 ,

wherein the process further comprises the following step after step (k),

(m) transferring fluoro-sialic acid to the resulting glycopeptides using a sialic acid transferase and CMP-7-fluoro-sialic acid, CMP-8-fuluoro-sialic acid, or CMP-9-fluoro-sialic acid.

9. A process for preparing a glycopeptide having at least two asparagine-linked oligosaccharides at a desired position of the peptide chain thereof, the process comprising:

(a) esterifying a hydroxyl group of a resin having the hydroxyl group and a carboxyl group of an amino acid having amino group nitrogen protected with a fat-soluble protective group,

(b) removing the fat-soluble protective group to form a free amino group,

(c) amidating the free amino group and a carboxyl group of an amino acid having amino group nitrogen protected with a fat-soluble protective group,

(d) removing the fat-soluble protective group to form a free amino group,

(e) repeating the steps (c) and (d) at least once,

(f) amidating the free amino group and a carboxyl group of the asparagine portion of an asparagine-linked oligosaccharide having amino group nitrogen protected with a fat-soluble protective group,

(g) removing the fat-soluble protective group to form a free amino group, and

(h) cutting off the resin with an acid;

wherein the steps (f) of amidating the free amino group and a carboxyl group of the asparagine portion of an asparagine-linked oligosaccharide having amino group nitrogen protected with a fat-soluble protective group, and (g) of removing the fat-soluble protective group to form a free amino group are repeated after the step (g), and

wherein at least one of the asparagine-linked oligosaccharides in the step (f) has a residue of sialic acid or a derivative thereof at a terminal end thereof.

10. A process for preparing a glycopeptides according to claim 9 ,

wherein at least one of the asparagines-linked oligosaccharides is an asparagine-linked disialooligosacharide or an asparagine-linked monosialooligosaccharide having amino group nitrogen protected with a fat-soluble protective group and the carboxyl group of the sialic acid protected with a benzyl or allyl group; and

the process further comprises the following step;

(i) preparing the asparagine-linked disialooligosaccharide or the asparagine-linked monosialooligosaccharide, wherein the benzyl or allyl group is introduced into the carboxyl group of the sialic acid under a condition of pH 5 to 6.

11. A process for preparing a glycopeptide according to claim 9 ,

Wherein the process further comprises the following step after step (h),

transferring fluoro-sialic acid to the resulting glycopeptides using a sialic acid transferase and CMP-7-fluoro-sialic acid, CMP-8-fuluoro-sialic acid, or CMP-9-fluoro-sialic acid.

12. A process for preparing a glycopeptide having at least two asparagine-linked oligosaccharides at a desired position of the peptide chain thereof, the process comprising:

(a) esterifying a hydroxyl group of a resin having the hydroxyl group and a carboxyl group of the asparagine portion of an asparagine-linked oligosaccharide having an amino group nitrogen protected with a fat-soluble protective group,

(b) removing the fat-soluble protective group to form a free amino group,

(c) amidating the free amino group and a carboxyl group of an amino acid having amino group nitrogen protected with a fat-soluble protective group,

(d) removing the fat-soluble protective group to form a free amino group,

(e) repeating the steps (c) and (d) at least once, and

(f) cutting off the resin with an acid; and

wherein the following steps are performed after the step (b), (d), or (e): (c′) amidating the free amino group and a carboxyl group of the asparagine portion of an asparagine-linked oligosaccharide having amino group nitrogen protected with a fat-soluble protective group, and (d′) of removing the fat-soluble protective group to form a free amino group, and

wherein at least one of the asparagine-linked oligosaccharides in the step (a) or step (c′) has a residue of sialic acid or a derivative thereof at a terminal end thereof.

13. A process for preparing a glycopeptides according to claim 12 ,

wherein at least one of the asparagines-linked oligosaccharides is an asparagine-linked disialooligosacharide or an asparagine-linked monosialooligosaccharide having amino group nitrogen protected with a fat-soluble protective group and the carboxyl group of the sialic acid protected with a benzyl or allyl group; and

the process further comprises the following step;

(g) preparing the asparagine-linked disialooligosaccharide or the asparagine-linked monosialooligosaccharide, wherein the benzyl or allyl group is introduced into the carboxyl group of the sialic acid under a condition of pH 5 to 6.

14. A process for preparing a glycopeptides according to claim 12 ,

wherein the process further comprises the following step after step (f),

(h) transferring fluoro-sialic acid to the resulting glycopeptides using a sialic acid transferase and CMP-7-fluoro-sialic acid, CMP-8-fuluoro-sialic acid, or CMP-9-fluoro-sialic acid.

15. A process for preparing a glycopeptide having at least two asparagine-linked oligosaccharides at a desired position of the peptide chain thereof, the process comprising:

(a) esterifying a hydroxyl group of a resin having the hydroxyl group and a carboxyl group of an amino acid having amino group nitrogen protected with a fat-soluble protective group,

(b) removing the fat-soluble protective group to form a free amino group,

(c) amidating the free amino group and a carboxyl group of the asparagine portion of an asparagine-linked oligosaccharide having amino group nitrogen protected with a fat-soluble protective group,

(d) removing the fat-soluble protective group to form a free amino group,

(e) amidating the free amino group and a carboxyl group of an amino acid having amino group nitrogen protected with a fat-soluble protective group,

(f) removing the fat-soluble protective group to form a free amino group,

(g) repeating the steps (e) and (f) at least once, and

(h) cutting off the resin with an acid; and

wherein the steps (c) of amidating the free amino group and a carboxyl group of the asparagine portion of an asparagine-linked oligosaccharide having amino group nitrogen protected with a fat-soluble protective group, and (d) of removing the fat-soluble protective group to form a free amino group are repeated after the step (d), (f), or (g), and

wherein at least one of the asparagine-linked oligosaccharides in the step (c) has a residue of sialic acid or a derivative thereof at a terminal end thereof.

16. A process for preparing a glycopeptide according to claim 15 ,

wherein at least one of the asparagines-linked oligosaccharides is an asparagine-linked disialooligosacharide or an asparagine-linked monosialooligosaccharide having amino group nitorogen protected with a fat-soluble protective group and the carboxyl group of the sialic acid protected with a benzyl or allyl group; and

the process further comprises the following step;

(i) preparing the asparagine-linked disialooligosaccharide or the asparagine-linked monosialooligosaccharide, wherein the benzyl or allyl group is introduced into the carboxyl group of the sialic acid under a condition of pH 5 to 6.

17. A process for preparing a glycopeptide according to claim 15 ,

wherein the process further comprises the following step after step (h),

(j) transferring fluoro-sialic acid to the resulting glycopeptides using a sialic acid transferase and CMP-7-fluoro-sialic acid, CMP-8-fuluoro-sialic acid, or CMP-9-fluoro-sialic acid.