Polysialic acid derivatives
A polysialic acid compound is reacted with a hetero-bifunctional reagent to introduce a pendant functional group for site-specific conjugation to sulfhydryl groups, for instance side chains of cysteine units in drugs, drug delivery systems, proteins or peptides. The functional group is, for instance, an N-maleimide group.
1. A polysialylated protein, wherein said polysialylated protein is formed by reacting
a compound comprising a polysaccharide having at least two sialic acid units linked 2.8 and/or 2.9 to one another, and having reducing and non-reducing terminal units and said polysaccharides having a pendant moiety linked to the reducing terminal sialic acid unit which pendant moiety includes a functional group selected from N-maleimide, vinyl sulfone, N-iodoacetamide and orthopyridyl disulfide
with a protein comprising a thiol group,
whereby said functional group reacts with said thiol group to form a thioether linkage.
2. The polysialylated protein according to claim 1 , wherein said thiol group is that of a cysteine moiety.
3. The polysialylated protein according to claim 1 , wherein said thiol group was introduced through a preliminary thiolation step.
4. The polysialylated protein according to claim 3 , wherein said preliminary thiolation step comprises reacting an amino group with 2-iminothiolane.
5. The polysialylated protein according to claim 4 , wherein said amino group is of a lysine moiety.
6. The polysialylated protein according to claim 1 , wherein said pendant moiety further comprises alkylene and/or arylene and/or an oxalkylene and/or oligooxa-alkylene and/or oligopeptide.
7. The polysialylated protein according to claim 1 , wherein said functional group is N-maleimido.
8. The polysialylated protein according to claim 1 , wherein said polysaccharide is a polysialic acid.
9. The polysialylated protein according to claim 8 , wherein said polysaccharide consists essentially of sialic acid units and said pendant moiety.
10. The polysialylated protein according to claim 8 , wherein said polysaccharide has at least 10 saccharide units.
11. The polysialylated protein according to claim 1 , wherein said compound has the formula
wherein:
R 1 is H or —CHOHCH 2 OH,
R 3 is —CH 2 CHR 4 R 5 or —CH(CH 2 OH)CHR 4 R)
wherein R 4 and R 5 together represent ═N—NR 6 or R 4 is H and R 5 is —NR 6 R 7
in which R 6 is an organic group comprising the said pendant functional group and R 7 is H;
O-Gly is a glycosyl (saccharide) group;
n is 1-50; and
Ac is acetyl.
12. The polysialylated protein according to claim 11 , wherein each O-Gly is a sialic acid unit.
13. The polysialylated protein according to claim 12 , wherein said polysaccharide has at least 10 saccharide units.
14. The polysialylated protein according to claim 1 , wherein said polysaccharide has at least 10 saccharide units.
15. The polysialylated protein according to claim 14 , wherein said polysaccharide has at least 50 saccharide units.