التفاصيل البيبلوغرافية
| العنوان: |
CHARACTERIZATION OF N-GLYCAN MIXTURES BY NUCLEAR MAGNETIC RESONANCE |
| Document Number: |
20100279269 |
| تاريخ النشر: |
November 4, 2010 |
| Appl. No: |
12/595940 |
| Application Filed: |
April 15, 2008 |
| مستخلص: |
The present disclosure provides nuclear magnetic resonance (NMR) methods for characterizing mixtures of N-linked glycans. Without limitation, methods of the present disclosure may be useful in characterizing monosaccharide composition, branching, fucosylation, sulfation, phosphorylation, sialylation linkages, presence of impurities and/or efficiency of a labeling procedure (e.g., labeling with a fluorophore such as 2-AB). In certain embodiments, the methods can be used quantitatively. In certain embodiments, the methods can be combined with enzymatic digestion to further characterize glycan mixtures. |
| Inventors: |
Parsons, Ian Christopher (Belmont, MA, US); Lansing, Jonathan (Reading, MA, US); Beccati, Daniela (Boston, MA, US); Bailey, Scott (Someville, MA, US); Bosques, Carlos J. (Arlington, MA, US) |
| Assignees: |
Momenta Pharmaceuticals, Inc. (Cambridge, US) |
| Claim: |
1. A method for characterizing a mixture of N-glycans by NMR comprising steps of: providing a sample that includes a mixture of N-glycans; identifying whether the sample produces an NMR signal that is associated with a structural characteristic of an N-glycan, which structural characteristic is selected from the group consisting of: an oligomannose structure, a GlcNAc residue, a sialic acid residue, a sialic acid residue with an α2-3 linkage, a sialic acid residue with an α2-6 linkage, di- or tri-acetylated NeuAc, a fucose residue, a Man4 residue, a mono-antennary Man4 residue, a bi-antennary Man4 residue, a galactose residue in a lactosamine extension, a sulfated GlcNac residue, a phosphorylated mannose residue, a core fucose residue, an acetylated sialic acid residue, a Man4′ residue, a mono- or bi-antennary Man4′ residue, a GlcNAc1 residue, a GlcNAc2 residue, a Man3 residue, a GlcNac residue with a β(1-2) linkage to mannose, a GlcNAc residue with a β(1-4) or β(1-6) linkage to mannose, a GlcNAc residue in a lactosamine extension, an unsubstitued galactose residue, a galactose residue with an α(2-3) sialic acid attached, a galactose residue with an α(2-6) sialic acid attached, an antennary fucose residue, a label attached to GlcNAc1; and determining whether the sample does or does not include an N-glycan with the structural characteristic in light of the step of identifying. |
| Claim: |
2. The method of claim 1, wherein the step of identifying comprises at least one of the following: (i) determining the chemical shift of a 1H signal from the sample; (ii) determining the chemical shifts of a 1H-1H scalar correlation from the sample; (iii) determining the chemical shifts of a 1H-13C scalar correlation from the sample; (iv) obtaining a 1D 1H NMR spectrum of the sample; (v) obtaining a 2D NMR spectrum of the sample; (vi) obtaining a 2D 1H-1H TOCSY NMR spectrum of the sample; (vii) obtaining a 1D selective 1H TOCSY NMR spectrum of the sample; and (viii) obtaining a 2D 1H-13C HSQC NMR spectrum of the sample. |
| Claim: |
3-9. (canceled) |
| Claim: |
10. The method of claim 2, wherein the structural characteristic is an oligomannose structure and the step of identifying comprises at least one of the following: (i) determining whether the sample produces a 1H signal with a chemical shift in the range of ca. 4.5 ppm to ca. 5.5 ppm; and (ii) determining whether the sample produces a 1H signal with a chemical shift in the range of ca. 5.00 ppm to ca 5.10 ppm and/or ca. 5.35 ppm to ca. 5.45 ppm. |
| Claim: |
11. (canceled) |
| Claim: |
12. The method of claim 2, wherein the structural characteristic is a GlcNAc or sialic acid residue and the step of identifying comprises at least one of the following: (i) determining whether the sample produces a 1H signal corresponding to an acetyl methyl nucleus of a GlcNAc or sialic acid residue; (ii) determining whether the sample produces a 1H signal with a chemical shift in the range of ca. 0.7 ppm to ca. 2.8 ppm; and (iii) determining whether the sample produces a 1H signal with a chemical shift at ca. 2.0 ppm. |
| Claim: |
13-14. (canceled) |
| Claim: |
15. The method of claim 2, wherein the structural characteristic is a sialic acid residue and the step of identifying comprises at least one of the following: (i) determining whether the sample produces a 1H signal corresponding to an axial or equatorial H3 nucleus of a sialic acid residue; and (ii) determining whether the sample produces a 1H signal with a chemical shift in the range of ca. 1.6 ppm to ca. 1.9 ppm and/or in the range of ca. 2.6 ppm to ca. 2.8 ppm. |
| Claim: |
16. (canceled) |
| Claim: |
17. The method of claim 2, wherein the structural characteristic is a sialic acid residue with an α2-3 linkage and the step of identifying comprises at least one of the following: (i) determining whether the sample produces a 1H signal corresponding to an axial H3 nucleus of a sialic acid residue; and (ii) determining whether the sample produces a 1H signal with a chemical shift at ca. 1.7 ppm. |
| Claim: |
18. (canceled) |
| Claim: |
19. The method of claim 2, wherein the structural characteristic is a sialic acid residue with an α2-6 linkage and the step of identifying comprises at least one of the following: (i) determining whether the sample produces a 1H signal corresponding to an axial H3 nucleus of a sialic acid residue; and (ii) determining whether the sample produces a 1H signal with a chemical shift at ca. 1.8 ppm. |
| Claim: |
20. (canceled) |
| Claim: |
21. The method of claim 2, wherein the structural characteristic is di- or tri-acetylated NeuAc and the step of identifying comprises determining whether the sample produces a 1H signal with a chemical shift at ca. 2.15 ppm. |
| Claim: |
22. (canceled) |
| Claim: |
23. The method of claim 2, wherein the structural characteristic is a fucose residue and the step of identifying comprises at least one of the following: (i) determining whether the sample produces a 1H signal corresponding to a methyl nucleus of a fucose residue; and (ii) determining whether the sample produces a 1H signal with a chemical shift at ca. 1.2 ppm. |
| Claim: |
24-26. (canceled) |
| Claim: |
27. The method of claim 3, wherein the structural characteristic is a mono-antennary or bi-antennary Man4 residue and the step of identifying comprises determining whether the sample produces a 1H-1H scalar correlation between the H2 and H3 nuclei of a Man4 residue. |
| Claim: |
28-30. (canceled) |
| Claim: |
31. The method of claim 3, wherein the structural characteristic is a galactose residue in a lactosamine extension and the step of identifying comprises determining the chemical shifts of a 1H-1H scalar correlation between the H1 nucleus and another nucleus of a galactose residue. |
| Claim: |
32. The method of claim 3, wherein the structural characteristic is a sulfated GlcNac residue and the step of identifying comprises determining the chemical shifts of a 1H-1H scalar correlation between the H6 nucleus and another nucleus of a GlcNac residue. |
| Claim: |
33. The method of claim 3, wherein the structural characteristic is a phosphorylated mannose residue and the step of identifying comprises determining the chemical shifts of a 1H-1H scalar correlation between the H6 nucleus and another nucleus of a mannose residue. |
| Claim: |
34. The method of claim 4, wherein the structural characteristic is a core fucose residue and the step of identifying comprises determining the chemical shifts of a 1H-13C scalar correlation for the anomeric nucleus of a GlcNAc2 residue. |
| Claim: |
35. The method of claim 4, wherein the structural characteristic is a sialic acid residue and the step of identifying comprises determining whether the sample produces a 1H-13C scalar correlation with chemical shifts corresponding to an axial or an equatorial H3 nucleus of a sialic acid residue. |
| Claim: |
36-38. (canceled) |
| Claim: |
39. The method of claim 4, wherein the structural characteristic is an acetylated sialic acid residue and the step of identifying comprises determining the 1H chemical shift of a 1H-13C scalar correlation of the H7, H8 and/or H9 nuclei of a sialic acid residue. |
| Claim: |
40. (canceled) |
| Claim: |
41. The method of claim 4, wherein the structural characteristic is a Man4 residue and the step of identifying comprises determining the chemical shifts of a 1H-13C scalar correlation corresponding to an anomeric nucleus of a Man4 residue. |
| Claim: |
42-46. (canceled) |
| Claim: |
47. The method of claim 4, wherein the structural characteristic is a GlcNAc1 residue and the step of identifying comprises determining whether the sample produces a 1H-13C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a GlcNAc1 residue |
| Claim: |
48-51. (canceled) |
| Claim: |
52. The method of claim 4, wherein the structural characteristic is a Man3 residue and the step of identifying comprises determining whether the sample produces a 1H-13C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a Man3 residue. |
| Claim: |
53. (canceled) |
| Claim: |
54. The method of claim 4, wherein the structural characteristic is a GlcNac residue with a β(1-2) linkage to mannose and the step of identifying comprises determining whether the sample produces a 1H-13C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a GlcNac residue with a β(1-2) linkage to mannose. |
| Claim: |
55. (canceled) |
| Claim: |
56. The method of claim 4, wherein the structural characteristic is a GlcNAc residue with a β(1-4) or β(1-6) linkage to mannose and the step of identifying comprises determining whether the sample produces a 1H-13C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a GlcNAc residue with a β(1-4) or β(1-6) linkage to mannose. |
| Claim: |
57. (canceled) |
| Claim: |
58. The method of claim 4, wherein the structural characteristic is a GlcNAc residue in a lactosamine extension and the step of identifying comprises determining whether the sample produces a 1H-13C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a GlcNac residue in a lactosamine extension. |
| Claim: |
59. (canceled) |
| Claim: |
60. The method of claim 4, wherein the structural characteristic is an unsubstitued galactose residue and the step of identifying comprises determining whether the sample produces a 1H-13C scalar correlation with chemical shifts corresponding to an anomeric nucleus of an unsubstitued galactose residue. |
| Claim: |
61. (canceled) |
| Claim: |
62. The method of claim 4, wherein the structural characteristic is a galactose residue with an α(2-3) sialic acid attached and the step of identifying comprises determining whether the sample produces a 1H-13C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a galactose residue with an α(2-3) sialic acid attached. |
| Claim: |
63. (canceled) |
| Claim: |
64. The method of claim 4, wherein the structural characteristic is a galactose residue with an α(2-6) sialic acid attached and the step of identifying comprises determining whether the sample produces a 1H-13C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a galactose residue with an α(2-6) sialic acid attached. |
| Claim: |
65. (canceled) |
| Claim: |
66. The method of claim 4, wherein the structural characteristic is a galactose residue in a lactosamine extension and the step of identifying comprises determining whether the sample produces a 1H-13C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a galactose residue in a lactosamine extension. |
| Claim: |
67. (canceled) |
| Claim: |
68. The method of claim 4, wherein the structural characteristic is an oligomannose structure and the step of identifying comprises determining whether the sample produces a 1H-13C scalar correlation with chemical shifts corresponding to an anomeric nucleus of an oligomannose structure. |
| Claim: |
69-70. (canceled) |
| Claim: |
71. The method of claim 4, wherein the structural characteristic is a core fucose residue and the step of identifying comprises determining whether the sample produces a 1H-13C scalar correlation with chemical shifts corresponding to an anomeric or methyl nucleus of a core fucose residue. |
| Claim: |
72-74. (canceled) |
| Claim: |
75. The method of claim 4, wherein the structural characteristic is an antennary fucose residue and the step of identifying comprises determining whether the sample produces a 1H-13C scalar correlation with chemical shifts corresponding to a methyl nucleus of an antennary fucose residue. |
| Claim: |
76. (canceled) |
| Claim: |
77. The method of claim 2, wherein the structural characteristic is a label attached to an N-glycan and the step of identifying comprises determining whether the sample produces a scalar correlation signal corresponding to said N-glycan. |
| Claim: |
78-82. (canceled) |
| Claim: |
83. The method of claim 1 further comprising steps of treating the sample with a digestive enzyme to produce a digested sample and repeating the step of identifying with the digested sample. |
| Claim: |
84-91. (canceled) |
| Claim: |
92. A method for characterizing a mixture of N-glycans by NMR comprising steps of: providing a sample that includes a mixture of N-glycans; detecting in the sample an NMR signal that is associated with a structural characteristic of an N-glycan; and quantifying the amount of the structural characteristic present in the sample. |
| Current U.S. Class: |
435/4 |
| Current International Class: |
12; 01 |
| رقم الانضمام: |
edspap.20100279269 |
| قاعدة البيانات: |
USPTO Patent Applications |
