التفاصيل البيبلوغرافية
| العنوان: |
METHODS FOR LABELING GLYCANS |
| Document Number: |
20120264927 |
| تاريخ النشر: |
October 18, 2012 |
| Appl. No: |
12/595934 |
| Application Filed: |
April 15, 2008 |
| مستخلص: |
Methods for labeling glycans that include a step of freeze-drying a labeled glycan preparation. The labeled glycan preparation is maintained in a substantially frozen state for the duration of the freeze-drying process. |
| Inventors: |
Parsons, Ian Christopher (Belmont, MA, US); Thiruneelakantapillai, Lakshmanan (Boston, MA, US); Bosques, Carlos J. (Arlington, MA, US); Collins, Brian Edward (Arlington, MA, US) |
| Claim: |
1. A method comprising steps of: providing a labeled glycan preparation; and then freeze-drying the labeled glycan preparation, wherein the labeled glycan preparation is maintained in a substantially frozen state for the duration of the freeze-drying step. |
| Claim: |
2. The method of claim 1, wherein the step of providing a labeled glycan preparation comprises: providing a glycan preparation; and then reacting the glycan preparation with an aminated label in the presence of a reducing agent so that the aminated label reacts with the glycan by reductive amination and becomes covalently linked to the glycan. |
| Claim: |
3. The method of claim 2, wherein the aminated label is a compound of formula I: [chemical expression included] wherein R1′ and R1″ are each independently H, NH2, NHR2, CONH2, COOH, COR3, COOR4, SO3, SOnR5 where n is 1 or 2, or an alkyl, alkoxyalkyl, alkenyl, alkynyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl group, or when attached to adjacent carbon atoms R1′ and R141 may be taken together with the atoms to which they are attached to form a 5- to 7-membered ring optionally containing a heteroatom selected from O, N or S; and R2, R3, R4 and R5 are each independently H or an alkyl, alkoxyalkyl, alkenyl, alkynyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl group. |
| Claim: |
4. The method of claim 2, wherein the aminated label is a compound of formula II: [chemical expression included] wherein R6 is NH2, NHR2, CONH2, COOH, COR3, COOR4, SO3 or SO1R5 where n is 1 or 2; and R7′ and R7″ are each independently H, NH2, NHR2, CONH2, COOH, COR3, COOR4, SO3, SOnR5 where n is 1 or 2, or an alkyl, alkoxyalkyl, alkenyl, alkynyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl group, or when attached to adjacent carbon atoms R1 and R1′ may be taken together with the atoms to which they are attached to form a 5- to 7-membered ring optionally containing a heteroatom selected from O, N or S; and R2, R3, R4 and R5 are each independently H or an alkyl, alkoxyalkyl, alkenyl, alkynyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl group. |
| Claim: |
5. The method of claim 2, wherein the aminated label is a compound of formula III: [chemical expression included] wherein R8 is NH2, NHR2, CONH2, COOH, COR3, COOR4, SO3 or SO1R5 where n is 1 or 2; A is a fused 5- to 15-membered cycloheteroalkyl, aryl or heteroaryl ring system which is optionally substituted at 1 to 5 carbon positions with NH2, NHR2, CONH2, COOH, COR3, COOR4, SO3 or SOmR5 where m is 1 or 2, or an alkyl, alkoxyalkyl, alkenyl, alkynyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl group; and R2, R3, R4 and R5 are each independently H or an alkyl, alkoxyalkyl, alkenyl, alkynyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl group. |
| Claim: |
6. The method of claim 5, wherein A is a fused 5- to 7-membered cycloheteroalkyl, aryl or heteroaryl ring system which is optionally substituted at 1 to 5 carbon positions with NH2, NHR2, CONH2, COOH, COR3, COOR4, SO3 or SOmR5 where m is 1 or 2. |
| Claim: |
7. The method of claim 5, wherein A is a fused 6-membered heteroaryl ring system which is optionally substituted at 1 to 5 carbon positions with NH2, NHR2, CONH2, COOH, COR3, COOR4, SO3 or SOmR5 where m is 1 or 2. |
| Claim: |
8. The method of claim 2, wherein the aminated label is selected from the group consisting of 2-aminopyridine, 2,6-diaminopyridine, 2-aminobenzoic acid, 2-aminobenzamide, ortho-phenylenediamine, 6-aminoquinoline, 8-aminonaphthalene-1,3,6-trisulfonic acid and 1,2-diamino-4,5-methylenedioxy-benzene. |
| Claim: |
9-15. (canceled) |
| Claim: |
16. The method of claim 2, wherein the reducing agent is selected from a borane dimethylamine complex and a sodium cyanoborohydride complex. |
| Claim: |
17. (canceled) |
| Claim: |
18. The method of claim 2, wherein the step of reacting is performed in a solution selected from the group consisting of: (i) a solution that includes a mixture of methanol and acetic or citric acid; (ii) a solution that includes a mixture of dimethylformamide and acetic or citric acid; and iii) a solution that includes a mixture of dimethylsulfoxide and acetic or citric acid. |
| Claim: |
19-20. (canceled) |
| Claim: |
21. The method of claim 1, wherein the step of providing a labeled glycan preparation comprises: providing a glycan preparation that includes a glycan with a sialic acid group; and then reacting the glycan preparation with an aminated label so that the aminated label reacts with the sialic acid group via a condensation mechanism and becomes covalently linked to the glycan, wherein the aminated label is a compound of formula IIA: [chemical expression included] wherein R7′ and R7″ are as defined in claim 4. |
| Claim: |
22. The method of claim 2, wherein in the step of providing a glycan preparation, the glycan preparation is a freeze-dried glycan preparation. |
| Claim: |
23. The method of claim 2, wherein in the step of providing a glycan preparation, the glycan preparation is a freeze-dried glycan preparation and the step of reacting comprises steps of: re-suspending the freeze-dried glycan preparation by adding a solution that includes the aminated label; and then adding a solution that includes the reducing agent. |
| Claim: |
24. (canceled) |
| Claim: |
25. The method of claim 2, wherein in the step of providing a glycan preparation, the glycan preparation is a freeze-dried glycan preparation and the step of reacting comprises steps of: re-suspending the freeze-dried glycan preparation by adding a solution that includes the aminated label and the reducing agent. |
| Claim: |
26. (canceled) |
| Claim: |
27. The method of claim 2 further comprising a step of: removing excess aminated label from the labeled glycan preparation before the freeze-drying step, wherein excess aminated label may be removed by paper chromatography or by dialysis. |
| Claim: |
28-29. (canceled) |
| Claim: |
30. The method of claim 27 further comprising a step of: drying the labeled glycan preparation by evaporation before the step of removing. |
| Claim: |
31. (canceled) |
| Claim: |
32. The method of claim 1, wherein the step of freeze-drying comprises steps of: placing the labeled glycan preparation in a container; freezing the labeled glycan preparation by reducing the temperature within the container to below the eutectic point of the labeled glycan preparation; and drying the labeled glycan preparation by reducing the pressure within the container. |
| Claim: |
33-35. (canceled) |
| Claim: |
36. The method of claim 32, wherein in the step of freezing, the temperature is reduced to a temperature in the range of about −240 to 0° C. |
| Claim: |
37. (canceled) |
| Claim: |
38. The method of claim 32, wherein in the step of freezing, the temperature is gradually reduced over a period of about 5 to 20 minutes. |
| Claim: |
39. The method of claim 32, wherein in the step of freezing, the temperature is cycled up and down within a temperature range. |
| Claim: |
40. The method of claim 39, wherein in the step of freezing, the temperature is cycled around a gradually decreasing temperature. |
| Claim: |
41. (canceled) |
| Claim: |
42. The method of claim 39, wherein in the step of freezing, the temperature is cycled anywhere within the range of about −240 to 25° C. |
| Claim: |
43. (canceled) |
| Claim: |
44. The method of claim 32, wherein in the step of drying, the pressure is reduced to a point where a solvent in the labeled glycan preparation can sublimate. |
| Claim: |
45-46. (canceled) |
| Claim: |
47. The method of claim 32, wherein in the step of drying, the temperature within the container is increased. |
| Claim: |
48-49. (canceled) |
| Claim: |
50. The method of claim 47, wherein in the step of drying, the temperature within the container remains at least 25° C. below the melting point of the labeled glycan preparation. |
| Claim: |
51. (canceled) |
| Claim: |
52. The method of any one of claims 3-5, wherein one or more of the following conditions is met: (i) one or more of the hydrogen atoms is optionally isotopically labeled as 2H or 3H; (ii) one or more of the carbon atoms is optionally isotopically labeled as 13C; (iii) one or more of the oxygen atoms is optionally isotopically labeled as 18O; (iv) one or more of the nitrogen atoms is optionally isotopically labeled as 15N; and (v) one or more of the sulfur atoms is optionally isotopically labeled as 33S or 34S. |
| Claim: |
53-56. (canceled) |
| Current U.S. Class: |
536/53 |
| Current International Class: |
07 |
| رقم الانضمام: |
edspap.20120264927 |
| قاعدة البيانات: |
USPTO Patent Applications |
