Compounds and methods for purification of serine proteases
View Patent ↗Disclosed herein are compounds, compositions, methods and kits for purifying a serine protease and serine proteases purified with the compounds, compositions and methods.
1. A compound of Formula I:
or a salt thereof,
wherein:
R 1 is —CF 3 , —SO 2 CH 3 , —X-L-R,
R 2 is —OCH 3 , or X-L-R;
R 3 is hydrogen or chloro;
X is O, S, SO 2 , C(O)NH, NHC(O) or NH;
L-R is -L 1 -R,
L 1 -R is
L 2 -R is H,
L 3 -R is
R is NH 2 ;
q is 2, 3, 4, 5, 6 or 7;
r is 1, 2, 3, 4, 5, 6 or 7;
s is 1, 2, 3, 4, 5, 6 or 7;
t is 1, 2, 3, 4, 5, 6 or 7;
n, m, and p are either 0 or 1, with the provisos that
(1) when R 1 is
—CF 3 or —SO 2 CH 3 , and R 2 is —OCH 3 , then one of n, m, and p must be 1, and the others of n, m, and p must be zero; and
(2) when R 1 is other than
—CF 3 or —SO 2 CH 3 , or R 2 is X-L-R, then all of n, m, and p must be zero.
2. The compound of claim 1 , which is of Formula I-A:
wherein the variables are as defined in claim 1 .
3. The compound of claim 1 , which is of Formula I-C:
4. The compound of claim 1 , wherein R 1 is
5. The compound of claim 1 , wherein R 2 is —OCH 3 .
6. The compound of claim 1 , wherein R 2 is X-L-R.
7. The compound of claim 1 , wherein R 3 is hydrogen.
8. The compound of claim 1 , wherein R 3 is chloro.
9. The compound of claim 1 , wherein each X is O.
10. The compound of claim 1 , wherein each X is S or SO 2 .
11. The compound of claim 1 , wherein each X is NH.
12. The compound of claim 1 , wherein L-R is -L 1 -R or
13. The compound of claim 1 , wherein L 1 -R is
14. The compound of claim 1 , wherein L-R is selected from the group consisting of:
—X-L-R is selected from the group consisting of
15. The compound of claim 1 , wherein each of q, r, s, and t is at least 3.
16. A compound selected from:
or a salt thereof.
17. An affinity solid support of Formula II:
or a salt thereof,
wherein:
R 21 is —CF 3 , —SO 2 CH 3 , —X-L-Y—Z,
R 22 is —OCH 3 , or X-L-Y—Z;
R 23 is hydrogen or chloro;
X is O, S, SO 2 , C(O)NH, NHC(O) or NH;
L-Y—Z is -L 1 -Y—Z,
L 1 -Y—Z is
L 2 -Y—Z is
L 3 -Y—Z is
Y—Z is
Z is a solid support;
q is 2, 3, 4, 5, 6 or 7;
r is 1, 2, 3, 4, 5, 6 or 7;
s is 1, 2, 3, 4, 5, 6 or 7;
t is 1, 2, 3, 4, 5, 6 or 7;
v is 1, 2, 3, 4, 5, 6 or 7;
u is 1, 2, 3, 4, 5, 6 or 7;
w is 1, 2 or 3;
n, m, and p are either 0 or 1, with the provisos that
(1) when R 21 is
—CF 3 or —SO 2 CH 3 , and R 22 is —OCH 3 , then one of n, m, and p must be 1, and the others of n, m, and p must be zero; and
(2) when R 21 is other than
—CF 3 or —SO 2 CH 3 , or R 22 is X-L-Y—Z, then all of n, m, and p must be zero.
18. The affinity solid support of claim 17 of Formula II-A:
wherein the variables are as defined in claim 17 .
19. The affinity solid support of claim 17 of Formula II-B:
wherein the variables are as defined in claim 17 .
20. The affinity solid support of claim 17 of Formula II-D:
21. The affinity solid support of claim 17 , wherein R 21 is
22. The affinity solid support of claim 17 , wherein R 22 is —OCH 3 .
23. The affinity solid support of claim 17 , wherein R 22 is X-L-Y—Z.
24. The affinity solid support of claim 17 , wherein R 23 is hydrogen.
25. The affinity solid support of claim 17 , wherein R 23 is chloro.
26. The affinity solid support of claim 17 , wherein each X is O.
27. The affinity solid support of claim 17 , wherein each X is S or SO 2 .
28. The affinity solid support of claim 17 , wherein each X is NH.
29. The affinity solid support of claim 17 , wherein L-Y—Z is -L 1 -Y—Z or
30. The affinity solid support of claim 17 , wherein L-Y—Z is selected from the group consisting of
31. The affinity solid support of claim 17 , wherein L 1 -Y—Z is selected from the group consisting of
32. The affinity solid support of claim 17 , wherein each of q, r, s, and t is at least 3.
33. The affinity solid support of claim 17 , wherein —X-L-Y—Z is selected from the group consisting of
34. The affinity solid support of claim 17 which is
or a salt thereof.
35. The affinity solid support of claim 17 , wherein Z is a cross-linked agarose resin.
36. A method for purifying a serine protease comprising
(1) adding a first composition comprising the serine protease to an affinity solid support of claim 17 or a salt thereof to form a second composition comprising the serine protease and the affinity solid support of claim 17 , and
(2) eluting the serine protease from the second composition with an elution buffer comprising a competitive agent.
37. The method of claim 36 , wherein the competitive agent is arginine and/or benzamidine, or a salt thereof.
38. The method of claim 36 , wherein the serine protease is a fXa derivative.
39. The method of claim 36 , wherein the serine protease is a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2, or a polypeptide having at least about 80% sequence identity to SEQ ID NO: 1 or 2.
40. A kit for purifying a serine protease comprising
(1) an affinity solid support of claim 17 , and
(2) an elution buffer comprising a competitive agent.
41. A kit for purifying a serine protease comprising a compound of claim 2 and a resin of the Formula NC—O—Z or
wherein Z is a solid support.