U.S. patent application number 11/724009 was filed with the patent office on 2007-07-19 for polyethylene glycol aldehydes.
Invention is credited to Chee-Youb Won.
Application Number | 20070167606 11/724009 |
Document ID | / |
Family ID | 31495721 |
Filed Date | 2007-07-19 |
United States Patent
Application |
20070167606 |
Kind Code |
A1 |
Won; Chee-Youb |
July 19, 2007 |
Polyethylene glycol aldehydes
Abstract
Polyethylene glycol aldehyde compounds are provided. Methods of
making and using such compounds, as well as chemical intermediates
are also provided.
Inventors: |
Won; Chee-Youb; (Livingston,
NJ) |
Correspondence
Address: |
HOFFMANN-LA ROCHE INC.;PATENT LAW DEPARTMENT
340 KINGSLAND STREET
NUTLEY
NJ
07110
US
|
Family ID: |
31495721 |
Appl. No.: |
11/724009 |
Filed: |
March 14, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10623978 |
Jul 21, 2003 |
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11724009 |
Mar 14, 2007 |
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60398196 |
Jul 24, 2002 |
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Current U.S.
Class: |
528/425 ;
560/159; 564/152; 564/59 |
Current CPC
Class: |
C08G 65/331 20130101;
C08G 65/329 20130101; C08G 65/324 20130101 |
Class at
Publication: |
528/425 ;
560/159; 564/152; 564/059 |
International
Class: |
C08G 65/34 20060101
C08G065/34; C07C 271/20 20060101 C07C271/20; C07C 275/14 20060101
C07C275/14 |
Claims
1. A compound of formula (l):
R.sub.1--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--X--Y--NH--(CH.sub.2-
).sub.p--CHO (I) wherein R.sub.1 is a capping group, X is O or NH,
Y is selected from the group consisting of ##STR32## Z is a side
chain of an amino acid, m is from 1 to 17, n is from 10 to 10,000,
and p is from 1 to 3.
2. A compound according to claim 1, wherein R.sub.1 is selected
from the group consisting of halogen, epoxide, maleimide,
orthopyridyl disulfide, tosylate, isocyanate, hydrazine hydrate,
cyanuric halide, N-succinimidyloxy, sulfo-N-succinimidyloxy,
1-benzotriazolyloxy, 1-imidazolyloxy, p-nitrophenyloxy, and
##STR33##
3. A compound according to claim 1, wherein R.sub.1 is selected
from the group consisting of hydrogen, hydroxy, lower alkyl, lower
alkoxy, lower cycloalkyl, lower alkenyl, aryl, and heteroaryl.
4. A compound according to claim 1, wherein R.sub.1 is selected
from the group consisting of methoxy, hydroxy, and benzyloxy.
5. A compound according to claim 2, wherein R.sub.1 is
##STR34##
6. A compound according to claim 1 having the formula (III):
##STR35##
7. A compound according to claim 1 having the formula (IV):
##STR36##
8. A compound according to claim 1 having the formula (V):
##STR37##
9. A compound according to claim 1 having the formula (VI):
##STR38##
10. A compound of formula (Il): ##STR39## wherein R.sub.1 is a
capping group, m is from 1 to 17, n is from 10 to 10,000, and p is
from 1 to 3.
11. A compound according to claim 10, wherein p is 3.
12. A compound according to claim 11, wherein R.sub.1 is selected
from the group consisting of methoxy, hydroxy, and benzyloxy.
13. A compound according to claim 11, wherein m is from 1 to
14.
14. A compound according to claim 13, wherein m is from 1 to 7.
15. A compound according to claim 14, wherein m is from 1 to 4.
16. A compound according to claim 11, wherein n is from 20 to
5,000.
17. A compound according to claim 16, wherein n is from 50 to
2,500.
18. A compound according to claim 17, wherein n is from 75 to
1,000.
19. A compound according to claim 10, wherein p is 3, R.sub.1 is
methoxy, m is 1, and n is from 100 to 750.
20. A compound according to claim 10, wherein p is 2.
21. A compound according to claim 20, wherein R.sub.1 is selected
from the group consisting of methoxy, hydroxy, or benzyloxy.
22. A compound according to claim 20, wherein m is from 1 to
14.
23. A compound according to claim 22, wherein m is from 1 to 7.
24. A compound according to claim 23, wherein m is from 1 to 4.
25. A compound according to claim 20, wherein n is from 20 to
5,000.
26. A compound according to claim 25, wherein n is from 50 to
2,500.
27. A compound according to claim 26, wherein n is from 75 to
1,000.
28. A compound according to claim 10, wherein p is 2, R.sub.1 is
methoxy, m is 1, and n is from 100 to 750.
29. A compound according to claim 10, wherein p is 1.
30. A compound according to claim 29, wherein R.sub.1 is selected
from the group consisting of methoxy, hydroxy, or benzyloxy.
31. A compound according to claim 29, wherein m is from 1 to
14.
32. A compound according to claim 31, wherein m is from 1 to 7.
33. A compound according to claim 32, wherein m is from 1 to 4.
34. A compound according to claim 29, wherein n is from 20 to
5,000.
35. A compound according to claim 34, wherein n is from 50 to
2,500.
36. A compound according to claim 35, wherein n is from 75 to
1,000.
37. A compound according to claim 10, wherein p is 1, R.sub.1 is
methoxy, m is 1, and n is from 100 to 750.
38. A compound of formula (VIII): ##STR40## wherein m is from 1 to
17, n is from 10 to 10,000, and p is from 1 to 3.
39. A compound according to claim 38, wherein m is from 1 to
14.
40. A compound according to claim 39, wherein m is from 1 to 7.
41. A compound according to claim 40, wherein m is from 1 to 4.
42. A compound according to claim 38, wherein n is from 20 to
5,000.
43. A compound according to claim 42, wherein n is from 50 to
2,500.
44. A compound according to claim 43, wherein n is from 75 to
1,000.
45. A compound according to claim 38, wherein p is 3, m is 1 and n
is from 100 to 750.
46. A compound of formula (IX):
R.sub.1--(CH.sub.2--CH.sub.2--O).sub.n--CH.sub.213
CH.sub.2--X--Y--NH--(CH.sub.2).sub.p--CH(OCH.sub.2--CH.sub.3).sub.2
(IX) wherein R.sub.1 is a capping group, X is O or NH, Y is
selected from the group consisting of ##STR41## Z is a side chain
of an amino acid, m is from 1 to 17, n is from 10 to 10,000, and p
is from 1 to 3.
47. A compound according to claim 46, wherein R.sub.1is selected
from the group consisting of halogen, epoxide, maleimide,
orthopyridyl disulfide, tosylate, isocyanate, hydrazine hydrate,
cyanuric halide, N-succinimidyloxy, sulfo-N-succinimidyloxy,
1-benzotriazolyloxy, 1-imidazolyloxy, p-nitrophenyloxy, and
##STR42##
48. A compound according to claim 46, wherein R.sub.1 is selected
from the group consisting of hydrogen, hydroxy, lower alkyl, lower
alkoxy, lower cycloalkyl, lower alkenyl, aryl, and heteroaryl.
49. A compound according to claim 46, wherein R.sub.1 is selected
from the group consisting of methoxy, hydroxy, and benzyloxy.
50. A compound according to claim 46, wherein R.sub.1 is
##STR43##
51. A compound according to claim 46, wherein m is from 1 to
14.
52. A compound according to claim 51, wherein m is from 1 to 7.
53. A compound according to claim 52, wherein m is from 1 to 4.
54. A compound according to claim 46, wherein n is from 20 to
5,000.
55. A compound according to claim 54, wherein n is from 50 to
2,500.
56. A compound according to claim 55, wherein n is from 75 to
1,000.
57. A compound according to claim 46, wherein R.sub.1 is methoxy, p
is 3, m is 1, and n is from 100 to 750.
58. A compound of formula (X): ##STR44## wherein R.sub.1 is a
capping group, m is from 1 to 17, n is from 10 to 10,000, and p is
from 1 to 3.
59. A compound according to claim 58, wherein R.sub.1 is selected
from the group consisting of halogen, epoxide, maleimide,
orthopyridyl disulfide, tosylate, isocyanate, hydrazine hydrate,
cyanuric halide, N-succinimidyloxy, sulfo-N-succinimidyloxy,
1-benzotriazolyloxy, 1-imidazolyloxy, p-nitrophenyloxy, and
##STR45##
60. A compound according to claim 58, wherein R.sub.1 is selected
from the group consisting of hydrogen, hydroxy, lower alkyl, lower
alkoxy, lower cycloalkyl, lower alkenyl, aryl, and heteroaryl.
61. A compound according to claim 58, wherein R.sub.1 is selected
from the group consisting of methoxy, hydroxy, and benzyloxy.
62. A compound according to claim 58, wherein R.sub.1 is
##STR46##
63. A compound according to claim 62, wherein m is from 1 to
14.
64. A compound according to claim 63, wherein m is from 1 to 7.
65. A compound according to claim 64, wherein m is from 1 to 4.
66. A compound according to claim 58, wherein n is from 20 to
5,000.
67. A compound according to claim 66, wherein n is from 50 to
2,500.
68. A compound according to claim 67, wherein n is from 75 to
1,000.
69. A compound according to claim 58, wherein R.sub.1 is methoxy, p
is 3, m is 1, and n is from 100 to 750.
70. A compound of formula (XI): ##STR47## wherein m is from 1 to
17, n is from 10 to 10,000, and p is from 1 to 3.
71. A compound according to claim 70, wherein m is from 1 to
14.
72. A compound according to claim 71, wherein m is from 1 to 7.
73. A compound according to claim 72, wherein m is from 1 to 4.
74. A compound according to claim 70, wherein n is from 20 to
5,000.
75. A compound according to claim 74, wherein n is from 50 to
2,500.
76. A compound according to claim 75, wherein n is from 75 to
1,000.
77. A compound according to claim 70, wherein p is 3, m is 1 and n
is from 100 to 750.
78. A method of making a polyethylene glycol aldehyde comprising
hydrolyzing a compound of formula (IX):
R.sub.1--(CH.sub.2--CH.sub.2--O).sub.n--CH.sub.2CH.sub.2--X--Y--NH--(CH.s-
ub.2).sub.p--CH--(OCH.sub.2--CH.sub.3).sub.2 (IX) to produce a
polyethylene glycol aldehyde of formula (I):
R.sub.1--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--X--Y--NH--(CH.sub.2-
).sub.p--CHO (I) wherein R.sub.1 is a capping group, X is O or NH,
Y is selected from the group consisting of ##STR48## Z is a side
chain of an amino acid, m is from 1 to 17, n is from 10 to 10,000,
and p is from 1 to 3.
79. A method of making a polyethylene glycol aldehyde comprising
hydrolyzing a compound of formula (X): ##STR49## to produce a
polyethylene glycol aldehyde of formula (II): ##STR50## wherein
R.sub.1 is a capping group, m is from 1 to 17, n is from 10 to
10,000, and p is from 1 to 3.
80. A method according to claim 79 wherein the compound of formula
(X) is produced by reacting a compound of formula (XII):
R.sub.1--(CH.sub.2--CH.sub.2--O).sub.n--CH.sub.2CH.sub.2--O--(CH.sub.2).s-
ub.m--COOH (XII) with a compound of formula (XIII):
H.sub.2N--(CH.sub.2).sub.p--CH--(OCH.sub.2--CH.sub.3).sub.2
(XIII).
81. A method according to claim 80 wherein the compound of formula
(XII) is produced by hydrolyzing a compound of formula (XIV):
R.sub.1--O--(CH.sub.2--CH.sub.2--O).sub.n--CH.sub.2CH.sub.2--O--(CH.sub.2-
).sub.m--COOR.sub.3 (XIV) wherein R.sub.3 is a branched or
unbranched C.sub.1-C.sub.4 alkyl.
82. A method according to claim 81 wherein the compound of formula
(XIV) is produced by reacting a compound of formula (XV):
R.sub.1--(CH.sub.2--CH.sub.2--O).sub.n--CH.sub.2CH.sub.2--OH (XV)
with a compound of formula (XVI):
R.sub.2--(CH.sub.2).sub.m--COOR.sub.3 (XVI) wherein R.sub.2 is
halogen.
83. A method of making a polyethylene glycol aldehyde comprising
hydrolyzing a compound of formula (XVII): ##STR51## to produce a
polyethylene glycol of formula (Vil): ##STR52## wherein m is from 1
to 17, n is from 10 to 10,000, and p is from 1 to 3.
84. A method according to claim 83 wherein the compound of formula
(VIII) is produced by reacting a compound of formula (XVIII):
HOOC--(CH.sub.2).sub.m--O--CH.sub.2CH.sub.2--(CH.sub.2--CH.sub.2--O).sub.-
n--CH.sub.2CH.sub.2--O--(CH.sub.2).sub.m--COOH (XVIII) with a
compound of formula (XIX):
H.sub.2N--(CH.sub.2).sub.p--CH--(OCH.sub.2--CH.sub.3).sub.2
(XIX).
85. A method according to claim 84 wherein the compound of formula
(XVIII) is produced by hydrolyzing a compound of formula (XX):
R.sub.3OOC--(CH.sub.2).sub.m--CH.sub.2CH.sub.2--O--(CH.sub.2--CH.sub.2--O-
).sub.n--CH.sub.2CH.sub.2--O--(CH.sub.2).sub.m--COOR.sub.3 (XXI)
wherein R.sub.3 is a branched or unbranched C.sub.1-C.sub.4
alkyl.
86. A method according to claim 85 wherein the compound of formula
(XX) is produced by reacting a compound of formula (XXI):
HO--CH.sub.2CH.sub.2--(CH.sub.2--CH.sub.2--O).sub.n--CH.sub.2CH.sub.2--OH
(XXI) with a compound of formula (XVI):
R.sub.2--(CH.sub.2).sub.m--COOR.sub.3 (XVI) wherein R.sub.2 is
halogen.
Description
PRIORITY TO RELATED APPLICATIONS
[0001] This application is a division of U.S. application Ser.
No.10/623,978, filed Jul. 21, 2003, now pending; which claims the
benefit of the U.S. Provisional Application 60/398,196 filed on
Jul. 24, 2002.
FIELD OF THE INVENTION
[0002] The present invention relates to polyethylene glycol
aldehydes, and to related methods of making and using such
derivatives, such as in the pegylation of polypeptides and other
biomolecules.
BACKGROUND OF THE INVENTION
[0003] Polyethylene glycol ("PEG") is a linear or branched, neutral
polyether, available in a variety of molecular weights. The
structure of PEG is HO--(CH.sub.2--CH.sub.2--O).sub.n--H, where n
indicates the number of repeats of the ethylene oxide unit in the
PEG.
[0004] PEG and PEG derivatives have been employed to modify a
variety of biomolecules. When attached to such molecules, PEG
increases their solubility and increases their size, but has little
effect on desirable properties.
[0005] Advantageously, PEG conjugated biomolecules may exhibit
increased retention and delayed metabolism in the body.
[0006] A variety of PEG derivatives has been developed for such
applications. Such PEG derivatives are described, for example, in
U.S. Pat. Nos. 5,252,714; 5,672,662; 5,959,265; 5,990,237; and
6,340,742.
[0007] Two general approaches have been used for the
functionalization of PEG: (1) changing the terminal hydroxyl group,
through a series of reactions, to a more active functional group
and/or (2) reaction of the PEG under controlled conditions with
difunctional compounds so that one of its functional groups reacts
with the PEG polymer and the other remains active. In most cases,
several steps must be conducted to achieve the desired PEG
derivatives. The desired PEG derivatives are often produced in low
yields and require a complicated purification process to isolate.
In addition, PEG derivatives may show nonspecific binding to the
biomolecules of interest, which can result in multiple PEGs
attached to a single biomolecule and/or PEG attachment at the
active site. Multiple PEG attachments may cause difficulty in
purification of the pegylated biomolecule. Multiple PEG
attachments, and/or pegylation at the active site, can also lead to
decreased activity of the biomolecule.
[0008] It would, therefore, be advantageous to provide improved PEG
derivatives suitable for conjugation with a variety of other
molecules, including polypeptides and other biomolecules containing
an .alpha.-amino group. There remains a need to provide PEG
derivatives that can be produced in high yield and purity, and that
can be conjugated to provide biomolecules having improved
performance characteristics.
[0009] These and other objects of the present invention are
described in greater detail below.
SUMMARY OF THE INVENTION
[0010] The compounds of the invention are aldehyde derivatives of
polyethylene glycol, having the general formula (I):
R.sub.1--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--X--Y--NH--(CH.sub.2-
).sub.p--CHO (I) wherein R.sub.1 is a capping group, X is O or NH,
Y is selected from the group consisting of ##STR1## Z is a side
chain of an amino acid, m is from 1 to 17, n is from 10 to 10,000,
and p is from 1 to 3.
[0011] The present invention also provides a compound of formula
(II): ##STR2## wherein R.sub.1, m, n, and p are defined as
above.
[0012] Another preferred embodiment of the present invention
provides a bifunctional polyethylene glycol aldehyde compound of
formula (VIII): ##STR3## wherein m, n, and p are is defined as
above.
[0013] The present invention also provides intermediate compounds
of formula (IX): ##STR4## wherein R.sub.1, X, Y, Z, m, n, and p are
defined as above.
[0014] The present invention further provides intermediate
compounds of formula (X): ##STR5## wherein R.sub.1, m, n, and p are
defined as above.
[0015] Also provided is an intermediate compound of formula (XI):
##STR6## wherein each m, n, and p is the same or different and is
defined as above.
[0016] The present invention further provides a method of making a
polyethylene glycol aldehyde comprising hydrolyzing a compound of
formula (IX):
R.sub.1--(CH.sub.2--CH.sub.2--O).sub.n--CH.sub.2CH.sub.2--X--Y--NH-
--(CH.sub.2).sub.p--CH--(OCH.sub.2--CH.sub.3).sub.2 (IX) to produce
a polyethylene glycol aldehyde of formula (I):
R.sub.1--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--X--Y--NH--(CH.sub.2-
).sub.p--CHO (I) wherein R.sub.1, X, Y, Z, m, n, and p are defined
as above.
[0017] The present invention also provides a method of making a
polyethylene glycol aldehyde comprising hydrolyzing a compound of
formula (X): ##STR7## to produce a polyethylene glycol aldehyde of
formula (11): ##STR8## wherein R.sub.1, m, n, and p are defined as
above.
[0018] The present invention provides a method of making a
polyethylene glycol aldehyde comprising hydrolyzing a compound of
formula (XVII): ##STR9## to produce a polyethylene glycol aldehyde
of formula (VIII): ##STR10## wherein m, n, and p are defined as
above.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The present invention provides a variety of compounds and
chemical intermediates and methods which may be used in connection
with the pegylation of polypeptides and other biomolecules. The
present invention provides a new chemical structure for
polyethylene glycol aldehydes.
[0020] The compounds of the invention are aldehyde derivatives of
polyethylene glycol, having the general formula (I):
R.sub.1--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--X--Y--NH--(CH.sub.2-
).sub.p--CHO (I) wherein R.sub.1 is a capping group, X is O or NH,
Y is selected from the group consisting of ##STR11## Z is a side
chain of an amino acid, m is from 1 to 17, n is from 10 to 10,000,
and p is from 1 to 3.
[0021] As used herein the R.sub.1 "capping group" is any suitable
chemical group which, depending upon preference, is generally
unreactive or generally reactive with other chemical moieties. The
terminal aldehyde group of the above formula permits ready covalent
attachment to a chemical moiety of interest, for example, to the
a-amino group of a polypeptide. The R.sub.1 capping group is
selected to permit or prevent bifunctionality, e.g., covalent
attachment to a second chemical moiety of interest.
[0022] In the case that the capping group is generally unreactive
with other chemical moieties R.sub.1 is relatively inert. If
R.sub.1 is relatively inert, then the structure of the resulting
polyethylene glycol aldehyde is monofunctional and therefore
covalently bonds with only one chemical moiety of interest.
Suitable generally unreactive R.sub.1 capping groups include:
hydrogen, hydroxyl, lower alkyl, lower alkoxy, lower cycloalkyl,
lower alkenyl, lower cycloalkenyl, aryl, and heteroaryl.
[0023] As used herein, the term "lower alkyl", means a substituted
or unsubstituted, straight-chain or branched-chain alkyl group
containing from 1 to 7, preferably from 1 to 4, carbon atoms, such
as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec.butyl,
tert.butyl, n-pentyl, n-hexyl, n-heptyl and the like. The lower
alkyl is optionally substituted with one or more groups
independently selected from halogen, lower alkyl, lower alkoxy,
lower cycloalkyl, lower alkenyl, lower cycloalkenyl, aryl, and
heteroaryl.
[0024] The term "lower alkoxy" means a lower alkyl group as defined
earlier which is bonded via an oxygen atom, with examples of lower
alkoxy groups being methoxy, ethoxy, n-propoxy, isopropoxy,
n-butoxy, sec.butoxy, tert.butoxy, n-pentoxy and the like. The
lower alkoxy is optionally substituted with one or more groups
independently selected from halogen, lower alkyl, lower alkoxy,
lower cycloalkyl, lower alkenyl, lower cycloalkenyl, aryl, and
heteroaryl.
[0025] The term "lower cycloalkyl" means a substituted or
unsubstituted cycloalkyl group containing from 3 to 7, preferably
from 4 to 6, carbon atoms, i.e. cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl or cycloheptyl. The lower cycloalkyl is
optionally substituted with one or more groups independently
selected from halogen, lower alkyl, lower alkoxy, lower cycloalkyl,
lower alkenyl, lower cycloalkenyl, aryl, and heteroaryl.
[0026] As used herein, the term "lower alkenyl" means a substituted
or unsubstituted, straight-chain or branched-chain alkenyl group
containing from 2 to 7, preferably from 2 to 5, carbon atoms, e.g.,
ethenyl, butenyl, pentenyl, hexenyl and the like. The lower alkenyl
is optionally substituted with one or more groups independently
selected from halogen, lower alkyl, lower alkoxy, lower cycloalkyl,
lower alkenyl, lower cycloalkenyl, aryl, and heteroaryl.
[0027] The term "lower cycloalkenyl" means a substituted or
unsubstituted, cycloalkenyl group containing from 4 to 7 carbon
atoms, e.g., cyclobutenyl, cyclopentenyl, cyclohexenyl and the
like. The lower cycloalkenyl is optionally substituted with one or
more groups independently selected from halogen, lower alkyl, lower
alkoxy, lower cycloalkyl, lower alkenyl, lower cycloalkenyl, aryl,
and heteroaryl.
[0028] The term "aryl" means a phenyl or naphthyl group which is
unsubstituted or optionally mono- or multiply-substituted by
halogen, lower alkyl, lower alkoxy, trifluoromethyl, hydroxyl,
carboxylic acid, carboxylic ester, nitro, amino, or phenyl,
particularly by halogen, lower alkyl, lower alkoxy,
trifluoromethyl, hydroxyl, nitro, amino and phenyl.
[0029] The term "heteroaryl" means a 5- or 6-membered
heteroaromatic group which contains one or more hetero atoms
selected from N, S, and O.
[0030] Preferred generally unreactive R.sub.1 capping groups
include methoxy, hydroxyl, or benzyloxy. An especially preferred RI
capping group is methoxy. When R.sub.1 is methoxy the aldehydes and
related compounds are sometimes referred to herein as "mPEG"
compounds, wherein the "m" stands for methoxy.
[0031] If the R.sub.1 capping group is generally reactive with
other chemical moieties, then R.sub.1 is a functional group capable
of reacting with some other functional group, such as an amine
and/or sulfhydryl in a peptide and/or protein. In such a case,
R.sub.1 may be a functional group that is capable of reacting
readily with electrophilic or nucleophilic groups on other
molecules, in contrast to those groups that require strong
catalysts or highly impractical reaction conditions in order to
react. If R.sub.1 is relatively reactive, the polyethylene glycol
aldehyde is bifunctional and may therefore covalently bond with two
chemical moieties.
[0032] Examples of suitable generally reactive R. capping groups
include: halogen, epoxide, maleimide, orthopyridyl disulfide,
tosylate, isocyanate, hydrazine hydrate, cyanuric halide,
N-succinimidyloxy, sulfo-N-succinimidyloxy, 1-benzotriazolyloxy,
1-imidazolyloxy, p-nitrophenyloxy, and ##STR12##
[0033] The term "halogen" means fluorine, chlorine, bromine, or
iodine.
[0034] A preferred generally reactive RI capping group is
##STR13##
[0035] The use of this R.sub.1 group results in a polyethylene
glycol aldehyde with aldehyde groups on both ends of the
polyethylene glycol aldehyde. And accordingly, the resultant
polyethylene glycol aldehyde exhibits binding properties on both
ends. It will be appreciated, however, that these bifunctional
compounds need not be perfectly symmetrical, and that the first m,
n, and/or p may be the same or different from the second m, n,
and/or p in the formula. It is preferred, however, that the
compound be symmetrical, meaning that both depicted m's have the
same value, both n's have the same value, and both p's have the
same value.
[0036] In the compounds of the present invention X is O or NH.
Preferably, X is O. Further, Y is selected from the group
consisting of ##STR14## wherein Z is a side chain of an amino
acid.
[0037] In the present invention, m is from 1 to 17. In a preferred
embodiment, m is from 1 to 14. More preferably m is from 1 to 7,
and even more preferably, m is from 1 to 4. Most preferably, m is
1.
[0038] In the case of a Y group with the general structure:
##STR15## the Y group exhibits a linkage to the amino acid through
a peptide bond.
[0039] Accordingly, this general structure results in specific
structures as simple as: ##STR16## when a single glycine is used as
the amino acid. When Z is CH.sub.3, then the amino acid is alanine.
If Z is CH.sub.2OH, the amino acid is serine.
[0040] Obviously, more complex structures are possible when more
and different amino acids are utilized, as can be appreciated from
an examination of the various amino acid structures shown below.
Preferably, only one amino acid is used. ##STR17## ##STR18##
[0041] In the present invention, n is from 10 to 10,000. In a
preferred embodiment of the present invention n is from 20 to
5,000. Preferably, n is from 50 to 2,500, even more preferably n is
from 75 to 1,000. Most preferably, n is from 100 to 750.
[0042] In the present invention, p is from 1 to 3. Preferably, p is
3.
[0043] In preferred embodiments, p is 3, R.sub.1 is methoxy, m is
1, and n is from 100 to 750; or p is 2, R.sub.1 is methoxy, m is 1,
and n is from 100 to 750; or p is 1, R.sub.1 is methoxy, m is 1,
and n is from 100 to 750.
[0044] The present inventin includes, but is not limited to,
compounds of formula I which are compounds of formulas II-VI as
follows: ##STR19##
[0045] Preferred R.sub.1 capping moieties are relatively
unreactive, with methoxy, hydroxl, and benzyloxy preferred.
[0046] Preferred compounds of the present invention fall within
Group A above.
[0047] Accordingly, the present invention provides a compound of
formula (II): ##STR20## wherein R.sub.1, m, n, and p are defined as
above.
[0048] In a preferred embodiment, R.sub.1 is methoxy, m is 1, and n
is from 100 to 750. More preferably, p is 3, R.sub.1 is methoxy, m
is 1, and n is from 100 to 750.
[0049] Another preferred embodiment of the present invention
provides a bifunctional polyethylene glycol aldehyde compound of
formula (VII): ##STR21## wherein m, n, and p are defined as
above.
[0050] In a preferred embodiment, RI is methoxy, m is 1, and n is
from 100 to 750. More preferably, p is 3, R.sub.1 is methoxy, m is
1, and n is from 100 to 750.
[0051] The present invention also provides a variety of chemical
intermediates which may be converted into the polyethylene glycol
aldehyde compounds of the invention described above. These
intermediates include compounds of formula (IX):
R.sub.1--(CH.sub.2--CH.sub.2--O).sub.n--CH.sub.2--CH.sub.2--X--Y--NH--(CH-
.sub.2).sub.p--CH--(OCH.sub.2--CH.sub.3).sub.2 (IX) wherein
R.sub.1, X, Y, Z, m, n, and p are defined as above.
[0052] In a preferred embodiment, p is 3, R.sub.1 is methoxy, m is
1, and n is from 100 to 750; or p is 2, R.sub.1 is methoxy, m is 1,
and n is from 100 to 750; or p is 1, R.sub.1 is methoxy, m is 1,
and n is from 100 to 750.
[0053] The present invention further provides intermediate
compounds of formula (X): ##STR22## wherein R.sub.1, m, n, and p
are defined as above.
[0054] In a preferred embodiment, p is 3, R.sub.1 is methoxy, m is
1, and n is from 100 to 750; or p is 2, R.sub.1 is methoxy, m is 1,
and n is from 100 to 750; or p is 1, R.sub.1 is methoxy, m is 1,
and n is from 100 to 750.
[0055] Also provided are intermediate compounds of formula (XI):
##STR23## wherein each m, n, and p is the same or different and is
defined as above.
[0056] In preferred embodiments, p is 3, R.sub.1 is methoxy, m is
1, and n is from 100 to 750; or p is 2, R.sub.1 is methoxy, m is 1,
and n is from 100 to 750; or p is 1, R.sub.1 is methoxy, m is 1,
and n is from 100 to 750.
[0057] The compounds of the present invention may be produced by
any suitable method, using known reagents and methods. However, the
present invention provides a specific method of making a
polyethylene glycol aldehyde comprising hydrolyzing a compound of
formula (IX): R.sub.1--(CH.sub.213
CH.sub.2--O),--CH.sub.2CH.sub.2--X--Y--NH--(CH.sub.2).sub.p--CH--(OCH.sub-
.2--CH.sub.3).sub.2 (IX) to produce a polyethylene glycol aldehyde
of formula (I):
R.sub.1--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--X--Y--NH--(CH.sub.2-
).sub.p--CHO (I) wherein R.sub.1, X, Y, Z, m, n, and p are defined
as above. Preferably, the hydrolysis is acid catalyzed. Suitable
catalytic acids include: trifluoroacetic acid, hydrochloric acid,
phosphoric acid, sulfuric acid, and nitric acid. Preferably, the
acid is trifluoroacetic acid.
[0058] In preferred embodiments, p is 3, R.sub.1 is methoxy, m is
1, and n is from 100 to 750; or p is 2, R.sub.1 is methoxy, m is 1,
and n is from 100 to 750; or p is 1, R.sub.1 is methoxy, m is 1,
and n is from 100 to 750.
[0059] The polyethylene glycol aldehyde compounds of formula (II)
may also be produced by any suitable method. By way of example,
however, polyethylene glycol aldehydes of formula (II) may be
produced as follows: First, the polyethylene glycol is dried.
Second, the polyethylene glycol is reacted with a halogenated
derivative of acetic acid. Hydrolyzing the resulting reaction
mixture results in a PEG carboxylic acid. Alternatively, the
product PEG carboxylic acid may also be derived from direct
oxidation of the PEG, after the drying step. Next, the PEG
carboxylic acid is then treated with an amine derivative of diethyl
acetal to produce a PEG acetal amine, which is reacted with a
halogenated carboxylic acid to produce a polyethylene glycol
aldehyde of formula. The polyethylene glycol aldehyde product is
then collected and purified.
[0060] The polyethylene glycol aldehyde product may be collected
and purified in any suitable manner. By way of example, the
polyethylene glycol aldehyde product may be extracted with
dichloromethane. The organic layer is dried over sodium sulfate,
filtered, concentrated, and precipitated with diethyl ether. The
product, PEG aldehyde, is collected by filtration and dried under
vacuum.
[0061] The present invention thus provides a method of making a
polyethylene glycol aldehyde comprising hydrolyzing a compound of
formula (X): ##STR24## to produce a polyethylene glycol aldehyde of
formula (II): ##STR25## wherein R.sub.1, m, n, and p are defined as
above.
[0062] The compound of formula (X) may be produced by reacting a
compound of formula (XII):
R.sub.1--(CH.sub.2--CH.sub.2--O).sub.n--CH.sub.2CH.sub.2--O--(CH.sub.2).s-
ub.m--COOH (XII) with a compound of formula (XII):
H.sub.2N--(CH.sub.2).sub.p--CH--(OCH.sub.2--CH.sub.3).sub.2
(XIII).
[0063] Another method to make PEG acid or PEG carboxylic acid is
direct oxidation. In this case, oxidizers such as CrO.sub.3 or
K.sub.2Cr.sub.2O.sub.7/H.sub.2SO.sub.4, HNO.sub.3 in the presence
of ammonium vanadate or Jone's reagent (CrO.sub.3 and
H.sub.2SO.sub.4), may be used.
[0064] The compound of formula (XII) may be produced by hydrolyzing
a compound of formula (XIV):
R.sub.1--O--(CH.sub.2--CH.sub.2--O).sub.n--CH.sub.2CH.sub.2--O--(CH.sub.2-
).sub.m--COOR.sub.3 (XIV) wherein R.sub.3 is a branched or
unbranched C.sub.1-C.sub.4 alkyl.
[0065] The compound of formula (XIV) may be produced by reacting a
compound of formula (XV):
R.sub.1--(CH.sub.2--CH.sub.2--O).sub.n--CH.sub.213 CH.sub.2--OH
(XV) with a compound of formula (XVI):
R.sub.2--(CH.sub.2).sub.m--COOR.sub.3 (XVI) wherein R.sub.2 is
halogen. Preferably R.sub.2 is bromine or chlorine. Suitable
compounds of formula (XVI) include t-butyl bromoacetate, methyl
bromoacetate, ethyl bromoacetate, t-butyl chloroacetate, methyl
chloroacetate, and ethyl chloroacetate. Other reagents that can be
used for this reaction step, i.e., substitutes for formula (XVI)
are, e.g., t-butyl bromoacetate, methyl bromoacetate, ethyl
bromoacetate, t-butyl chloroacetate, methyl chloroacetate, or ethyl
chloroacetate in the presence of potassium t-butoxide, an alkali
metal hydride such as sodium hydride or potassium naphtalide.
Preferably, the compound of formula (XVI) is t-butyl
bromoacetate.
[0066] In preferred embodiments, p is 3, R.sub.1 is methoxy, m is
1, and n is from 100 to 750; or p is 2, R.sub.1 is methoxy, m is 1,
and n is from 100 to 750; or p is 1, R.sub.1 is methoxy, m is 1,
and n is from 100 to 750.
[0067] Compounds of formulas (III)-(VI) (also identified as Groups
B-E, respectively) may likewise be made by any suitable means. By
way of example, however, the following reaction schemes may be used
to produce compounds of formulas (III)-(VI) (Groups B-E).
##STR26##
[0068] As with the polyethylene glycol aldehydes discussed above,
bifunctional polyethylene glycol aldehydes may be produced by any
suitable means. The present invention provides a method of making a
polyethylene glycol aldehyde comprising hydrolyzing a compound of
formula (XVII): ##STR27## to produce a polyethylene glycol aldehyde
of formula (Vil): ##STR28## wherein m, n, and p are defined as
above.
[0069] The compound of formula (VI) may be produced by reacting a
compound of formula (XVIII):
HOOC--(CH.sub.2).sub.m--O--CH.sub.2CH.sub.2--(CH.sub.2--CH.sub.2--O).sub.-
n--CH.sub.2CH.sub.2--O--(CH.sub.2).sub.m--COOH (XVIII) with a
compound of formula (XIX):
H.sub.2N--(CH.sub.2).sub.p--CH--(OCH.sub.2--CH.sub.3).sub.2
(XIX).
[0070] The compound of formula (XVIII) may be produced by
hydrolyzing a compound of formula (XX):
R.sub.3OOC--(CH.sub.2).sub.m--CH.sub.2CH.sub.2--O--(CH.sub.2-CH.sub.2--O)-
.sub.n--CH.sub.2CH.sub.2--O--(CH.sub.2).sub.m--COOR.sub.3 (XX)
wherein R.sub.3 is a branched or unbranched C.sub.1-C.sub.4
alkyl.
[0071] The compound of formula (XX) may be produced by reacting a
compound of formula (XXI):
HO--CH.sub.2CH.sub.2--(CH.sub.2--CH.sub.2--O).sub.n--CH.sub.2CH.sub.2--OH
(XXI) with a compound of formula (XVI):
R.sub.2--(CH.sub.2).sub.m--COOR.sub.3 (XVI) wherein R.sub.2 is
halogen.
[0072] The polyethylene glycol aldehyde compositions of the present
invention discussed above may be used to derivatize a variety of
molecules, including biomolecules, using any suitable methods.
[0073] The PEG aldehyde compounds of the present invention are
N-terminus site-specific for the pegylation of peptides and other
biomolecules. The PEG aldehydes of the present invention form a
conjugate with the N-terminus a-amino group of the biomolecule or
protein forming a stable secondary amine linkage between the PEG
and the biomolecule or protein.
[0074] Biomolecules pegylated with PEG aldehydes of the present
invention show reproducibility in the number and location of PEG
attachment, resulting in a purification strategy that is less
complicated. This site-specific pegylation can result in a
conjugate where the pegylation site is far from the site where the
biomolecule or the peptide binds to the cell's receptors, which
will allow pegylated biomolecules, proteins, or peptides to retain
much or all of their biological activity. The PEG-aldehydes of the
present invention can react with any biomolecules that contain an
alpha (.alpha.) amino group.
[0075] Depending on the polyethylene glycol aldehyde selected the
polyethylene glycol may be covalently bonded to a biomolecule at
one end (monofunctional polyethylene glycol aldehyde) or at both
ends (bifunctional polyethylene glycol aldehyde).
[0076] As stated, the polyethylene glycol aldehydes of the present
invention may be used for N-terminus site-specific pegylation. The
site-specific N-terminal linkage results in pegylated polypeptides
which avoid cross-linking and multiple derivatizations of a single
polypeptide. To produce this site-specific covalent linkage, any
suitable reaction conditions may be used. Generally, the pH of the
reaction mixture is sufficiently acidic to activate the a-amino
acid of the polypeptide to be pegylated. Typically, the pH is about
5.5 to about 7.4, preferably about 6.5.
[0077] Accordingly, a method for attaching a polyethylene glycol
molecule to a polypeptide comprising: [0078] reacting at least one
polypeptide of formula (XXII): NH.sub.2B (XXII); with a
polyethylene glycol aldehyde molecule of formula (I):
R.sub.1--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--X--Y--NH--(CH.sub.2-
).sub.p--CHO (I) wherein R.sub.1, X, Y, Z, m, n, and p are defined
as above; to produce a compound of formula (XXIII):
R.sub.1--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--X--Y--NH--(CH.sub.2-
).sub.p--NHB (XXIII) wherein the polyethylene glycol aldehyde
molecule is bonded to the N-terminal amino group of the polypeptide
is provided.
[0079] In preferred embodiments, p is 3, R.sub.1 is methoxy, m is
1, and n is from 100 to 750; or p is 2, R.sub.1 is methoxy, m is 1,
and n is from 100 to 750; or p is 1, R.sub.1 is methoxy, m is 1,
and n is from 100 to 750.
[0080] The compounds of formula (XXII) may be any polypeptide,
including interferon-alpha, interferon-beta, consensus interferon,
erythropoietin (EPO), granulocyte colony stimulating factor (GCSF),
granulocyte/macrophage colony stimulating factor (GM-CSF),
interleukins (including IL-2, IL-10, and IL-12), and colony
stimulating factor.
[0081] The compounds of formula (XXII) may also be immunoglobulins,
such as, IgG, IgE, IgM, IgA, IgD, and subclasses thereof, and
fragments thereof. The term "antibody" or "antibody fragments"
refer to polyclonal and monoclonal antibodies, an entire
immunoglobulin or antibody or any functional fragment of an
immunoglobin molecule which binds to the target antigen. Examples
of such antibody fragments include Fv (fragment variable), single
chain Fv, complementary determining regions (CDRs), VL (light chain
variable region), VH (heavy chain variable region), Fab (fragment
antigen binding), F(ab)2', and any combination of those or any
other functional group of an immunoglobin peptide capable of
binding to a target antigen.
[0082] As stated, the pegylated compound may be prepared in any
desired manner. Conditions, e.g., pH, should be selected which
favor the site-specific pegylation of a-amino groups.
[0083] Generally, polypeptides may be pegylated with polyethylene
glycol compounds of the invention by adding the compound of formula
(XXII), and the PEG reagent in a molar ratio range of 1:1 to 1:100.
The reaction concentration may then placed in a borate, phosphate,
or tri buffer at room temperature or 4 degrees Celsius for about
0.5 to 24 hours at a pH range of 5.5 to 9.0. The molar ratio of PEG
reagent to peptide/proteins is generally from 1:1 to 100:1. The
concentration of peptide/proteins is generally from 1 to 10 mg/ml.
The concentration of buffer is usually 10 to 500 mM.
[0084] The pegylated compound may be isolated or purified in any
desired manner. By way of example, the resultant reaction mixture
may be diluted with an equilibration buffer (20 mM Tris, pH 7.5)
and the resulting mixture is then applied on a Q-Sepharose column.
After the mixture is applied on the QA column, it is washed with
the equilibration buffer eluted with 75 M NaCl; eluted with 200 mM
NaCl; eluted with 1M NaCl; and regenerated with 1M HOAC+1M NaCl and
0.5 NaOH. By using reverse phase HPLC, it is possible to separate
and isolate the N-terminal, monopegylated product from other
byproducts in the mixture. Each collected product can then be
confirmed by Matrix Assisted Laser Desorption/lonization-Time of
Flight Mass Spectrometry (MALbI-TOF).
[0085] In a preferred embodiment of the pegylation method of the
invention, a polypeptide of formula (XXII): NH.sub.2B (XXII); is
reacted with a polyethylene glycol aldehyde molecule of formula
(II):
R.sub.1--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--O--(CH.sub.2).sub.m-
--CO--NH--(CH.sub.2).sub.p--CHO (II) wherein R., m, n, and p are
defined as above; to produce a compound of formula (XXIV):
R.sub.1--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--O--(CH.sub.2).sub.m-
--CO--NH--(CH.sub.2).sub.p--NHB (XXIV) wherein the polyethylene
glycol aldehyde molecule is bonded to the N-terminal amino group of
the polypeptide.
[0086] In preferred embodiments, p is 3, R.sub.1 is methoxy, m is
1, and n is from 100 to 750; or p is 2, R.sub.1 is methoxy, m is 1,
and n is from 100 to 750; or p is 1, R.sub.1 is methoxy, m is 1,
and n is from 100 to 750.
[0087] Additional illustrations of the use of the compounds of the
present invention are disclosed in the U.S. Provisional Patent
Applications entitled "Pegylated T20 Polypeptide," U.S. Ser. No.
60/398,195, filed Jul. 24, 2002, and "Pegylated T1249 Polypeptide,
U.S. Ser. Nos. 60/439,213 filed Jan. 10, 2003, and 60/398,190 filed
Jul. 24, 2002, all of which are incorporated herein by reference as
if recited in full.
[0088] Further provided, is a method for attaching a polyethylene
glycol molecule to a polypeptide comprising: [0089] reacting a
polypeptide of formula (XXII): NH.sub.2B (XXII); with a
polyethylene glycol aldehyde molecule of formula (VII): ##STR29##
wherein each m, n, and p is the same or different and is defined as
above; to produce a compound of formula (XXV): ##STR30##
[0090] wherein the polyethylene glycol aldehyde molecule is bonded
to the N-terminal amino group of the polypeptides.
[0091] In preferred embodiments, p is 3, m is 1, and n is from 100
to 750; or p is 2, m is 1, and n is from 100 to 750; or p is 1, m
is 1, and n is from 100 to 750.
[0092] The pegylated polypeptides may be used in any desired
manner. Suitably, however, they are used to prepare pharmaceutical
compositions, by admixture with a pharmaceutically acceptable
excipient. Such pharmaceutical compositions may be in unit dosage
form. They may be injectable solutions or suspensions, transdermal
delivery devices, or any other desired form.
[0093] The following examples are provided to further illustrate
the present invention. These examples are illustrative only and are
not intended to limit the scope of the invention in any way.
EXAMPLES
Example 1
Preparation of PEG Aldehyde Compounds
[0094] Five grams of PEG (molecular weight of 1,000 to 60,000
daltons) in 50 to 100 ml of toluene is azeotropically dried by
refluxing for 1 to 3 hours, followed by the removal of 20 to 30 mL
of toluene. The resulting solution is cooled to room temperature
then potassium tert-butoxide (1 to 10 molar excess) in 20-50 ml of
absolute tert-butanol and 20-50 ml of toluene is added to the PEG
solution. The resulting mixture is then stirred for two hours at
room temperature under argon.
[0095] Tert-butyl bromoacetate (1 to 10 molar excess) is added to
the reaction via syringe and the reaction mixture stirred overnight
at room temperature under argon gas. Depending on the desired size
of the "m" group defined in formula (XVI), tert-butyl bromoacetate
can be replaced with another halogenated derivative of acetic acid,
e.g., propionic acid, butyric acid, etc.
[0096] The reaction solution is then condensed by rotary
evaporation and the residue precipitated by the addition of diethyl
ether. The precipitated product, PEG t-butyl carboxy ester, is
filtered off and dried in vacuo.
[0097] PEG t-butyl carboxy ester (4 g) is then dissolved in 50 to
100 ml of 1 N sodium hydroxide and the solution stirred at room
temperature overnight. The pH of the mixture is adjusted to 2.5 to
3.0 by addition of 1 to 6N hydrochloric acid, and the mixture
extracted with dichloromethane. The organic layer is then dried
over sodium sulfate, filtered, concentrated, and precipitated into
diethyl ether. The product, PEG-carboxylic acid, is collected by
filtration and dried under vacuum.
[0098] The PEG-carboxylic acid (3 g) is then dissolved in anhydrous
dichloromethane (20-30 ml) followed by the addition of
4-aminobutylraldehyde diethyl acetal (1-5 molar excess),
1-hydroxybenzotriazole (1-5 molar excess), and
dicyclohexylcarbodiimide (1-5 molar excess). Depending on the
desired size of the "p" group defined in formula (XIII),
4-aminobutylraldehyde diethyl acetal can be replaced with another
amine derivative of diethyl acetal, e.g., 3-aminopropionaldehyde
diethyl acetal or 2-aminoacetalaldehyde diethyl acetal.
[0099] The resulting mixture is stirred overnight at room
temperature under argon gas. The reaction mixture is filtered,
concentrated, and precipitated with a mixture of 2-propanol and
diethyl ether (1:1). The PEG acetal product is dried in vacuo
overnight.
[0100] The PEG acetal product is then dissolved in 10-200 ml of
1-90% CF.sub.3COOH, and the solution is stirred at room temperature
overnight. The pH of the mixture is adjusted to 6.0 by addition of
1 N NaOH solution, and sodium chloride (10 wt %) is then added and
the pH of the solution is adjusted to 7.0 by addition of 1 N NaOH.
The mixture is then extracted with dichloromethane. The organic
layer is dried over sodium sulfate, filtered, concentrated, and
precipitated with diethyl ether. The product, PEG aldehyde, is
collected by filtration and dried under vacuum.
Example 2
Preparation of mPEG.sub.10K-butanoaldehyde
[0101] The following represents a general reaction scheme for
preparing mPEG10k-butanoaldehyde of the invention: ##STR31##
[0102] First, Carboxymethyl PEG (mPEG) of molecular weight 10,000
daltons (30.0 g, 3 mmol) in 300 mL of toluene was azeotropically
dried by refluxing for 2 hours, followed by the removal of 100 ml
of toluene. The resulting solution was cooled to room temperature
then potassium tert-butoxide (0.68 g, 6 mmol) in 20 ml of absolute
tert-butanol and 20 ml of toluene was added to the PEG solution
(1). The resulting mixture was stirred for two hours at room
temperature under argon.
[0103] Tert-butyl bromoacetate (1.00 mL, 6.75 mmol) was added to
the reaction via syringe and the reaction was stirred overnight at
room temperature under argon. The reaction solution was then
condensed by rotary evaporation. The residue was precipitated by
addition of diethyl ether. The precipitated product was filtered
off and dried in vacuo. Yield: 28 g. NMR (d.sub.6-DMSO): 1.40 ppm
(t, 9H, --CH3); 3.21 ppm (s, --OCH.sub.3); 3.50 ppm (s,
--O--CH.sub.2CH.sub.2--O--); 3.96 ppm (s, 2H,
--O--CH.sub.2--COO--).
[0104] Next, mPEG.sub.10k t-butyl carboxymethyl ester (20 g) was
dissolved in 200 mL of 1N sodium hydroxide and the solution was
stirred at room temperature overnight (2). The pH of the mixture
was adjusted to 2.5 by addition of 6 N hydrochloric acid, and the
mixture was extracted with dichloromethane (50 mL, 40 mL, and 30
mL). The organic layer was dried over sodium sulfate, filtered,
concentrated, and precipitated with diethyl ether. The product,
m-PEG.sub.10k-carboxymethyl acid, was collected by filtration and
dried under vacuum. Yield: 18 g. NMR (d.sub.6-DMSO): 3.21 ppm (s,
--OCH.sub.3); 3.5 ppm (s, --O--CH.sub.2CH.sub.2--O--); 3.99 ppm (s,
2H, --O--CH.sub.2--COOH).
[0105] The mPEG.sub.10k-carboxymethyl acid (3 g, 0.3 mmol) was
dissolved in anhydrous dichloromethane (20 mL) followed by the
addition of 4-aminobutylraldehyde diethyl acetal (50 mg, 0.3 mmol),
1-hydroxybenzotriazole (40 mg, 0.3 mmol), and
dicyclohexylcarbodiimide (80 mg, 0.39 mmol) (3). The mixture was
stirred overnight at room temperature under argon. The reaction
mixture was filtered, concentrated, and precipitated with a mixture
of 2-propanol and diethyl ether (1:1). The product,
mPEG.sub.10k-butanoacetal, was dried in vacuo overnight. Yield: 2.7
g. NMR (d.sub.6-DMSO): 1.07-1.12 ppm (t, 6H,
(--O--CH.sub.2-CH.sub.3).sub.2); 1.46 ppm (m, 4H,
--NHCH.sub.2CH.sub.2CH.sub.2--CH--); 3.08-3.11 ppm (q, 2H,
--NHCH.sub.2CH.sub.2CH.sub.2--CH--); 3.21 ppm (s, --OCH.sub.3); 3.5
ppm (s, --O--CH.sub.2CH.sub.2--O--); 3.85 ppm (s, 2H,
--O--CH.sub.2--CO--NH--); 4.44 ppm (t, 1H,
--NHCH.sub.2CH.sub.2CH.sub.2--CH--); 7.67 ppm (--NH--).
[0106] Finally, the mPEG.sub.10k-butanoacetal (5 g, 0.5 mmol) was
dissolved in 50 mL of 10% CF.sub.3COOH and the solution was stirred
at room temperature overnight (4). The pH of the mixture was
adjusted to 6.0 by addition of 1 N NaOH solution, and sodium
chloride (10 wt %) was added and then the pH of the solution was
adjusted to 7.0 by addition of 1 N NaOH. The mixture was extracted
with dichloromethane. The organic layer was dried over sodium
sulfate, filtered, concentrated, and precipitated into diethyl
ether. The product, mPEG.sub.10k-butanoaldehyde (5), was collected
by filtration and dried under vacuum. Yield: 4.1 g (82%). NMR
(d.sub.6-DMSO): 3.21 ppm (s, --OCH.sub.3); 3.5 ppm (s,
--O--CH.sub.2CH.sub.2--O); 3.85 ppm (s, 2H,
--O--CH.sub.2--CO--NH--); 7.67 ppm (--NH--); 9.66 ppm
(--CHO--).
Example 3
Preparation of mPEG.sub.10k-acetal aldehyde
[0107] mPEG.sub.10k-acetal aldehyde was prepared by dissolving
mPEG.sub.10k-diethyl acetal (1 g, Mol. Wt. 10,000), which was
prepared according to the procedure in Example 1, in 10 ml of 80%
trifluoacetic acid (Aldrich, 99+%). The reaction solution was
stirred overnight at room temperature under argon gas. 1 N NaOH was
then added dropwise to the reaction solution until a pH of 6.0 was
obtained. Next, NaCl (10 wt %) was added to the above solution. The
pH was then adjusted to 6.95.+-.0.05 by adding 0.1 N NaOH. The
solution was then extracted with methylene chloride. The organic
layer was then dried over sodium sulfate, filtered, concentrated,
and precipitated with diethyl ether. The product, mPEG,Ok-acetal
aldehyde, was collected by filtration and dried under vacuum.
Yield: 0.85 g (85%).
Example 4
Preparation of mPEG.sub.10k-propionaldehyde
[0108] mPEG.sub.10k-propionaldehyde was prepared by dissolving
mPEG.sub.10k-propionacetal (2 g, Mol. Wt. 10,000), which was
prepared according to the procedure in example 1, in 20 ml of 80%
trifluoacetic acid (Aldrich, 99+%). The reaction solution was
stirred overnight at room temperature under argon gas. 1 N NaOH was
then added dropwise to the reaction solution until a pH of 6.0 was
obtained. Next, NaCI (10 wt %) was added to the above solution. The
pH was then adjusted to 6.95.+-.0.05 by adding 1 N NaOH. The
solution was then extracted with methylene chloride. The organic
layer was then dried over sodium sulfate, filtered, concentrated,
and precipitated with diethyl ether. The product,
mPEG.sub.10k-propionaldehyde, was collected by filtration and dried
under vacuum. Yield: 1.8 g (90%).
Example 5
Preparation of PEG.sub.20k-di-butanoaldehyde
[0109] PEG.sub.20k-di-butanoaldehyde was prepared by dissolving
PEG.sub.20k-di- butyraldehyde diethyl acetal (3.1 g, Mol. Wt.
20,000), which was prepared according to the procedure in example
1, in 20 ml of 80% trifluoacetic acid (Aldrich, 99+%). The reaction
solution was stirred overnight at room temperature under argon gas.
1 N NaOH was then added dropwise to the reaction solution until a
pH of 6.0 was obtained. Next, NaCI (10 wt %) was added to the above
solution. The pH was then adjusted to 6.95.+-.0.05 by adding 0.1 N
NaOH. The solution was then extracted with methylene chloride. The
organic layer was then dried over sodium sulfate, filtered,
concentrated, and precipitated with diethyl ether. The product,
PEG.sub.20k-di-butanoaldehyde, was collected by filtration and
dried under vacuum. Yield: 2.5 g (81%).
Example 6
Preparation of mPEG.sub.20k-butanoaldehyde
[0110] mPEG.sub.20k-butanoaldehyde was prepared by dissolving
mPEG.sub.20k-butyraldehyde diethyl acetal (3.0 g, Mol. Wt. 20,000),
which was prepared according to the procedure in Example 1, in 30
ml of 80% trifluoacetic acid (Aldrich, 99+%). The reaction solution
was stirred overnight at room temperature under argon gas. 1 N NaOH
was then added dropwise to the reaction solution until a pH of 6.0
was obtained. Next, NaCl (10 wt %) was added to the above solution.
The pH was then adjusted to 6.95.+-.0.05 by adding 1 N NaOH. The
solution was then extracted with methylene chloride. The organic
layer was then dried over sodium sulfate, filtered, concentrated,
and precipitated with diethyl ether. The product,
mPEG.sub.20k-butanoaldehyde, was collected by filtration and dried
under vacuum. Yield: 2.5 g (83.3%).
Example 7
Preparation of mPEG.sub.20k-butanoaldehyde
[0111] mPEG.sub.20k-butanoaldehyde was prepared by dissolving
mPEG.sub.20k-butyraldehyde diethyl acetal (14.7 g, Mol. Wt.
20,000), which was prepared according to the procedure in Example
1, in 200 ml of 10% trifluoacetic acid (Aldrich, 99+%). The
reaction solution was stirred overnight at room temperature under
argon gas. 1 N NaOH was then added dropwise to the reaction
solution until a pH of 6.0 was obtained. Next, NaCl (10 wt %) was
added to the above solution. The pH was then adjusted to
6.95.+-.0.05 by adding 0.1 N NaOH. The solution was then extracted
with methylene chloride. The organic layer was then dried over
sodium sulfate, filtered, concentrated, and precipitated with
diethyl ether. The product, mPEG.sub.20k- butanoaldehyde, was
collected by filtration and dried under vacuum. Yield: 13.1 g
(89%).
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