Selective Desulfations of Heparin. M. Berton, Dr. David Pye. School of Environment and Life Sciences, University of Salford. CURS 2009-10. Extraction of GAGs.
Extraction, Characterization and Selective Desulfations of Heparin M. Berton, Dr. David Pye School of Environment and Life Sciences, University of Salford Proteoglycans (PGs) are complex macromolecules present at the cell surface. They consist of protein core onto which sulfated glycosaminoglycan (SGAG) chains are covalently bound. Both the protein and Heparan Sulfate (HS) part are thought to play a functional role in their biological activity. Usually several HS chains are presented for potential ligand binding interactions at the cell suface.[1] Heparin and HS are members of a family of complex polysaccharides known as GAGs A. They are made up of repeating C1, C4- linked disaccharide units B. They can be composed of a hexuronic acid, which is one of either GlcA or IdoA, and an GlcNAc or GlcNS. In addition to N-sulfation, many of these constituent disaccharides carry one or several O-sulfate substituents. Commonly O-sulfation occurs at C2 of IdoA and/or at C6 of GlcNS or GlcNAc B.[2]
1. 2. 3. 4.
Different fibroblast growth factors interact with different heparan sulfate sulfation sequences. From this rationale has come a comprehensive screen of the HS binding structures for FGFs. 4 groups were assessed .[3][4] 1. FGF-2 2-O-sulfation but not 6-O-sulfation 2. FGF-10 6-O-sulfation but not 2-O-sulfation 3. FGF-18 2-O-sulfation or 6-O-sulfation 4. FGF-4 & FGF-7 2-O- and 6-O-sulfation In FGF-FGFR-Heparin complex C a number of electrostatic interactions may exist between various negatively charged (COO- and SO3-) and positively charged basic amino acid residues exposed at the surface of FGFs and FGFR. The Pelligrini model C puts HS/heparin oligosaccharides at the heart of complex formation and biological activity. It can be seen how without stabilisation by the centrally positioned oligosaccharide FGFR dimerisation may not occur and a biological response not initiated.[5][6]
A
B
C
GAG extraction from mussels, Mytilus edulis. Selective 2-O-desulfation procedures of Heparin. Selective 6-O-desulfation procedures of Heparin. Selective N-desulfation procedures of Heparin.
5. 6.
HPLC extraction of deca- and dodeca-saccharides from Enoxaparin. Selective 2-O-desulfation procedures of dodecasaccharides.
Extraction of GAGs
Selective Desulfations
In order to establish the procedure for desulfation, methods were first tested and modified using heparin.
Separation of heparinderived oligosaccharides
[8]
[9]
The GAG were extracted from Blue Mussels. The extraction from giant African Snail has been described in detail in previous studies[7]. Which one with a few changes was used.
A method for the N-desulfation of py+ salt and Na+ salt has been described in detail in previous study[10].
The 2-O-desulfation was repeated on highly sulfated dp12 derived of Clexane (Enoxaparin)[11] to confirm the selectivity. Samples were analysed by several NMR analysis to obtain structural information.
dp12 was contained in fractions 16 to 21, dp10 in fractions 23 to 27 and dp8 in fractions 31 to 39.
0.16g of GAGs were extracted from 10g mussel powder. We observed that when [Alcalase] was increased from 5mg/mL to 10mg/mL we obtained 68% more of GAGs. We can obtain from 1Kg of blue mussel around 80g of soft body fat-free powder which one can be convert into 1.28g of GAGs. The optimal [NaOH]=0.1-0.2M for a full 2-Odesulfation. The results disagree with the previous study (optimal [NaOH]=0.5-0.6M) [8]. We can not assure the high selectivity because our NMR analysis did not give us enough resolution to identify all anomeric protons. The optimal reaction time for a full 6-Odesulfation is 80 min. Although it was accompanied by about 20% of 2-Odesulfation as a consequence of side The solvolysis is a powerful method but we can reaction 5. The risk to a simultaneous 2-O- affirm that is more selective than does hydrolysis. desulfation increase with the reaction time. They can not be considered as selective methods. It seems that Na+ salt is more reactive than the py+ salt. My supervisor Dr. David Pye, Dr. James Wilkinson, Roman Lagoutte (PhD), Jon A. Deakin, Kirit Amin, Dr. Steve Rossington, Kathrin Scherer (PhD), Ray Odgen.
In the dp12 was observed that the doublet, the last eluated, was not two different sulfate degree but may be just the two possible isomers[12].
We can see a really intensive peak at 1.75ppm corresponding to the NAc group, this composition was not expected. Enoxaparin does not contain a high amount of IdoA (2S).
[1] Bernfield,
M.; Annu. Rev. Cell Biol. 1992, 365-393.[2] Gatti, G.; Am. Chem. Soc. 1979, 12 (5), 1001-1007. [3] AshikariHada, S; J. of Bio. Chem. 2004, 279 (13), 12346-12354.[4] Ostrovsky, O.; J. of Bio. Chem. 2002, 277 (4), 2444-2453. [5] Pellegrini, L.; Nat. 2000, 407, 1029-1034.[6] Schlessinger, J.; Mol. Cell 2000, 6, 743-750. [7] Kim, Y. S.; J. of Bio. Chem. 1996, 271 (20), 11750-11755.[8] Holme, K. R.; Pat. 5,696,100, Dec. 9, 1997.[9] Kariya, Y.; J. of Bio. Chem. 2000, 275 (34), 25949-25958.[10] Nagasawa; Carb. Research 1976, No. 46, 87-95.[11] Bisio, A.; Thromb. Haemost. 2009, 102, 865-873. [12] Deakin, J. A.; J. of Bio. Chem. 2009, 284 (10), 6311-6321.
CURS 2009-10
