U.S. patent application number 09/265989 was filed with the patent office on 2001-08-02 for poly (ethylene glycol) derivatives with proximal reactive groups.
Invention is credited to HARRIS, J. MILTON, KOZLOWSKI, ANTONI.
Application Number | 20010011115 09/265989 |
Document ID | / |
Family ID | 22139572 |
Filed Date | 2001-08-02 |
United States Patent
Application |
20010011115 |
Kind Code |
A1 |
HARRIS, J. MILTON ; et
al. |
August 2, 2001 |
POLY (ETHYLENE GLYCOL) DERIVATIVES WITH PROXIMAL REACTIVE
GROUPS
Abstract
An activated, substantially water soluble poly(ethylene glycol)
is provided having of a linear or branched poly(ethylene glycol)
backbone and at least one terminus linked to the backbone through a
hydrolytically stable linkage, wherein the terminus is branched and
has proximal reactive groups. The free reactive groups are capable
of reacting with active moieties in a biologically active agent
such as a protein or peptide thus forming conjugates between the
activated poly(ethylene glycol) and the biologically active
agent.
Inventors: |
HARRIS, J. MILTON;
(HUNTSVILLE, AL) ; KOZLOWSKI, ANTONI; (HUNTSVILLE,
AL) |
Correspondence
Address: |
ALSTON & BIRD LLP
BANK OF AMERICA PLAZA
101 SOUTH TRYON STREET, SUITE 4000
CHARLOTTE
NC
28280-4000
US
|
Family ID: |
22139572 |
Appl. No.: |
09/265989 |
Filed: |
March 11, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60077700 |
Mar 12, 1998 |
|
|
|
Current U.S.
Class: |
525/56 ; 523/406;
568/303; 568/377 |
Current CPC
Class: |
Y10S 530/816 20130101;
A61K 47/60 20170801; C08G 65/33313 20130101; C08G 65/33337
20130101; C08G 65/33396 20130101; C08G 65/329 20130101 |
Class at
Publication: |
525/56 ; 523/406;
568/303; 568/377 |
International
Class: |
C07C 049/00; C08F
016/06 |
Claims
What is claimed is:
1. An activated, water soluble polymer comprising a polymer
backbone and at least one terminus linked to said polymer backbone
through a stable linkage, wherein said at least one terminus
comprises a branching moiety and proximal reactive groups linked to
said branching moiety that are capable of forming covalent linkages
with another molecule.
2. The polymer of claim 1, wherein at least one of said proximal
reactive groups further comprises a tethering group linking said
reactive moiety to said branching moieity.
3. The polymer of claim 2, wherein said tethering group is an alkyl
chain.
4. The polymer of claim 1, wherein said polymer backbone is
selected from the group consisting of linear or branched
poly(ethylene glycol), linear or branched poly(alkylene oxide),
linear or branched poly(vinyl pyrrolidone), linear or branched
poly(vinyl alcohol), linear or branched polyoxazoline, and linear
or branched poly(acryloylmorpholine).
5. The polymer of claim 1, wherein said polymer backbone is
poly(ethylene glycol).
6. The polymer of claim 1, wherein said branching moiety is 6
7. An activated water soluble polymer having the formula:
POLY-Y--CHXX' wherein POLY is a polymer backbone, Y is a
hydrolytically stable linkage, and X and X' are reactive groups
having a reactive moiety, which may be the same or different, and
are selected from the group consisting of active esters, active
carbonates, aldehydes, isocyanates, isothiocyanates, epoxides,
alcohols, maleimides, vinylsulfones, hydrazides, dithiopyridines,
and iodoacetamides.
8. The polymer of claim 7, wherein POLY has a capping group on the
opposite terminus selected from the group consisting of --OH,
alkyl, and --Y--CHXX'.
9. The polymer of claim 8, wherein said polymer backbone is
selected from the group consisting of linear or branched
poly(ethylene glycol), linear or branched poly(alkylene oxide),
linear or branched poly(vinyl pyrrolidone), linear or branched
poly(vinyl alcohol), linear or branched polyoxazoline, and linear
or branched poly(acryloylmorpholine).
10. The polymer of claim 8, wherein POLY is poly(ethylene glycol)
or derivatives thereof.
11. The polymer of claim 8, wherein Y is selected from the group
consisting of --O--, --S-- and --CO--NH--.
12. The polymer of claim 8, wherein X and X' are represented by
-W-Z and -W'-Z' respectively wherein Z and Z' represent said
reactive moieties, and W and W' represent a tethering group
comprising a substantially linear chain of atoms.
13. The polymer of claim 12, wherein W and W' are selected from the
group consisting of alkyl chains, ether chains, ester chains, amide
chains, and combinations thereof.
14. The polymer of claim 12, wherein POLY is methoxy poly(ethylene
glycol), Y is --CO--NH--, W and W' are --(CH.sub.2).sub.m--O--
where m is 1-10, and Z and Z' are --CO.sub.2Q where Q is
N-succinimidyl, sulfo-N-succinimidyl, or the 1-benzotriazolyl
group:
CH.sub.3O-PEG-CO--NH--CH[(CH.sub.2).sub.m--OCO.sub.2Q].sub.2
15. The polymer of claim 12, wherein POLY is methoxy-poly(ethylene
glycol), Y is --CO--NH--, W and W' are
--(CH.sub.2).sub.m--O.sub.2C--CH.s- ub.2CH.sub.2-- where m is 1-10,
and Z and Z'-=--CO.sub.2Q where Q is N-succinimidyl,
sulfo-N-succinimidyl, or 1-benzotriazolyl group:
CH.sub.3O-PEG-CO--NH--CH[(CH.sub.2).sub.m--O.sub.2C--CH.sub.2CH.sub.2--CO-
.sub.2-Q].sub.2
16. The polymer of claim 12, wherein POLY is methoxy poly(ethylene
glycol), Y is --CO--NH--, W and W' are --(CH.sub.2).sub.m-- where m
is 1-10, and Z and Z'- are --CO.sub.2-Q where Q is N-succinimidyl,
sulfo-N-succinimidyl, or 1-benzotriazolyl group:
CH.sub.3O-PEG-CO--NH--CH- [(CH.sub.2).sub.m--CO.sub.2-Q].sub.2
17. The polymer of claim 12, wherein POLY is methoxy poly(ethylene
glycol), Y is --O--, W and W' are --(CH.sub.2).sub.m--O-- where m
is 1-10, and Z and Z'- are --CO.sub.2-Q where Q is N-succinimidyl,
sulfo-N-succinimidyl, or 1-benzotriazolyl group:
CH.sub.3O-PEG-O--CH[(CH.- sub.2).sub.m--O--CO.sub.2-Q].sub.2
18. The polymer of claim 12, wherein POLY is methoxy poly(ethylene
glycol), Y is --O--, W and W' are
--(CH.sub.2).sub.m--O.sub.2C--CH.sub.2C- H.sub.2-- where m is 1-10,
and Z and Z'- are --CO.sub.2-Q where Q is N-succinimidyl,
sulfo-N-succinimidyl, or 1-benzotriazolyl group:
CH.sub.3O-PEG-O--CH[(CH.sub.2).sub.m--O.sub.2C--CH.sub.2CH.sub.2--CO.sub.-
2-Q].sub.2
19. The polymer of claim 12, wherein POLY is methoxy poly(ethylene
glycol), Y is --CO--NH--, and W is a covalent bond directly to
--CH--, W' is (CH.sub.2).sub.m--, where m is 1-10, and Z and Z' are
--CO.sub.2-Q where Q is an N-succinimidyl, sulfo-N-succinimidyl, or
1-benzotriazolyl group:
CH.sub.3O-PEG-CO--NH--CH(CO.sub.2-Q)[(CH.sub.2).sub.mCO.sub.2-Q]
20. The polymer of claim 12, wherein POLY is methoxy poly(ethylene
glycol), Y is --O--, W and W' are --(CH.sub.2).sub.m where m is
1-10, and Z and Z' are --SO.sub.2--CH.dbd.CH.sub.2:
CH.sub.3O-PEG-O--CH.sub.2[(CH.s-
ub.2).sub.m--SO.sub.2--CH.dbd.CH.sub.2).sub.2
21. The polymer of claim 12, wherein POLY is linear, difunctional
poly(ethylene glycol), Y is --CONH--, W and W' are
--(CH.sub.2).sub.rO(CH.sub.2).sub.m-- where r is 1-10 and m is
1-10, and Z and Z' are --CHO:
[OHC--(CH.sub.2).sub.r--O--(CH.sub.2).sub.m].sub.2--C-
H--NHCO-PEG-CONH--CH[(CH.sub.2).sub.m--O--(CH.sub.2).sub.r--CHO].sub.2
22. An activated water soluble polymer having the formula:
B-[POLY-Y--CHXX'].sub.n wherein B is a central core; POLY is a
polymer backbone; Y is a hydrolytically stable linkage; n is from 2
to 200; X and X' are reactive groups having a reactive moiety
selected from the group consisting of active esters, active
carbonates, aldehydes, isocyanates, isothiocyanates, epoxides,
alcohols, maleimides, vinylsulfones, hydrazides, dithiopyridines,
and iodoacetamides.
23. The polymer of claim 22, wherein POLY is poly(ethylene glycol)
or derivatives thereof.
24. The polymer of claim 22, wherein B is moiety selected from the
group consisting of lysine, glycerol, pentaerythritol, and
sorbitol.
25. The polymer of claim 22, wherein Y is selected from the group
consisting of --O--, --S-- and --CO--NH--.
26. The polymer of claim 22, wherein X and X' are represented by
-W-Z and -W'-Z' respectively where Z and Z' represents said
reactive moieties, W and W' represent a tethering group comprising
a substantially linear chain of atoms.
27. The polymer of claim 22, wherein W and W' are selected from the
group consisting of alkyl chains, ether chains, ester chains, amide
chains, and combinations thereof.
28. The polymer of claim 26, wherein POLY is a poly(ethylene
glycol), n is 4, Y is --CONH--, W and W' are
--(CH.sub.2).sub.r--O(CH.sub.2).sub.m-- where r is 1-10 and m is
1-10, and Z and Z' are --CHO:
B{PEG-CONH--CH[(CH.sub.2).sub.m--O--(CH.sub.2).sub.r--CHO].sub.2}.sub.4
29. The polymer of claim 26, the polymer has the formula:
B{PEG-CO--NH--CH(CO.sub.2-Q)[(CH.sub.2).sub.mCO.sub.2-Q']}.sub.n
wherein PEG represents poly(ethylene glycol), Y is --CO--NH--, m is
1-10, Q and Q' are N-succinimidyl, sulfo-N-succinimidyl, or
1-benzotriazolyl group, and B and n are as described.
30. A method for preparing an activated water soluble polymer, said
polymer comprising of a polymer backbone and at least one branched
terminus having proximal reactive groups thereon, said terminus
linked to said polymer backbone through a stable linkage, said
method comprising: providing a first intermediate polymer
comprising said polymer backbone linked to a reactive end group;
providing a reactive compound having three free reactive groups
linked to a branching moiety, one of said three free groups being
capable of reacting with said reactive end group of the first
intermediate polymer to form said stable linkage; and reacting the
reactive end group of said intermediate polymer with said one of
said three free groups of the compound to form said stable
linkage.
31. The method of claim 30, wherein at least one of said two free
reactive moieties further comprises a tethering group linking said
reactive group to said branching moieity.
32. The method of claim 30, wherein said tethering group is an
alkyl chain.
33. The method of claim 30, wherein said polymer backbone is
selected from the group consisting of linear or branched
poly(ethylene glycol), linear or branched poly(alkylene oxide),
linear or branched poly(vinyl pyrrolidone), linear or branched
poly(vinyl alcohol), linear or branched polyoxazoline, and linear
or branched poly(acryloylmorpholine).
34. The method of claim 30, wherein said polymer backbone is
poly(ethylene glycol).
35. The method of claim 30, wherein said branching moiety is
--CH--.
36. The method of claim 30, wherein said compound is
H.sub.2NCH(CH.sub.2--OH).sub.2.
37. The method of claim 30, wherein said compound is
NaO--CH(CH.sub.2--O-Bz).sub.2.
38. The method of claim 30, wherein said compound is
H.sub.2N--CH(CH.sub.2CO.sub.2H).sub.2.
39. A method for preparing an activated water soluble polymer
represented by mPEGO--CH(CH.sub.2SO.sub.2CH.dbd.CH.sub.2).sub.2,
where mPEG represents methyl poly(ethylene glycol), comprising the
steps of: providing mPEG-O-Ms, wherein Ms represents mesylate;
providing NaO--CH(CH.sub.2--O-Bz).sub.2, wherein Bz represents a
benzyl moiety; reacting mPEG-O-Ms with
NaO--CH(CH.sub.2--O-Bz).sub.2 to form mPEGO--CH(CH.sub.2OBz).sub.2;
converting mPEGO--CH(CH.sub.2OBz).sub.2 to
mPEGO--CH(CH.sub.2OH).sub.2; substituting a mesylate group for both
hydroxyl groups in mPEGO--CH(CH.sub.2OH).sub.2 to form
mPEGO--CH(CH.sub.2OMs).sub.2; converting
mPEGO--CH(CH.sub.2OMs).sub.2 into
mPEGO--CH(CH.sub.2SCH.sub.2CH.sub.2OH).sub.2; oxidizing the sulfur
atom into a sulfone group; and converting the sulfone alcohol group
into a vinyl sulfone group, thus forming
mPEGO--CH(CH.sub.2SO.sub.2CH.dbd.CH.s- ub.2).sub.2.
40. A conjugate comprising: a biologically active agent; and an
activated water soluble polymer comprising a polymer backbone
having at least one terminus linked to said polymer backbone
through a stable linkage, wherein said at least one terminus
comprises a branching moiety having proximal reactive groups linked
to said branching moiety, said biologically active agent being
covalently linked to at least one of said proximal reactive
groups.
41. The conjugate of claim 40, wherein said reactive groups
comprise a reactive moiety capable of reacting with amino groups
and said biologically active agent is selected from the group
consisting of proteins, peptides, aminolipids, polysaccharides
having an amino group, amino-oligonucleotides, and pharmaceutical
agents having an amino group.
42. The conjugate of claim 40, wherein said reactive groups
comprise vinyl sulfone and said biologically active agent has a
thiol moiety.
43. The conjugate of claim 40, wherein at least one of said two
reactive groups comprises a tethering group linking the reactive
moiety to said branching moieity.
44. The conjugate of claim 43, wherein said tethering group is an
alkyl chain.
45. The conjugate of claim 40, wherein said polymer backbone is
selected from the group consisting of linear or branched
poly(ethylene glycol), linear or branched poly(alkylene oxide),
linear or branched poly(vinyl pyrrolidone), linear or branched
poly(vinyl alcohol), linear or branched polyoxazoline, and linear
or branched poly(acryloylmorpholine).
46. The conjugate of claim 40, wherein said polymer backbone is
poly(ethylene glycol).
47. The conjugate of claim 40, wherein said branching moiety is
7
48. The conjugate of claim 40, wherein said conjugate comprises two
separate biologically active agents, which can be the same or
different, each being linked to one of said two reactive groups.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is related to copending Provisional
Application Serial No. 60/077,700, filed Mar. 12, 1998,
incorporated by reference in its entirety, and claims the benefit
of its filing date under 35 U.S.C. .sctn.119(e).
FIELD OF THE INVENTION
[0002] This invention relates to derivatives of polyethylene glycol
and related hydrophilic polymers suitable for chemical coupling to
another molecule, including, for example, proteins, enzymes, small
drugs, and the like.
BACKGROUND OF THE INVENTION
[0003] Chemical attachment of the hydrophilic polymer poly(ethylene
glycol) ("PEG") to molecules and surfaces is of great utility in
biotechnology. In its most common form PEG is a linear polymer
terminated at each end with hydroxyl groups:
HO--CH.sub.2CH.sub.2O--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--OH
[0004] This polymer can be represented in brief form as HO-PEG-OH
where it is understood that the -PEG- symbol represents the
following structural unit:
--CH.sub.2CH.sub.2O--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--
[0005] In typical form n ranges from about 10 to about 2000.
[0006] PEG is commonly used as methoxy PEG-OH, or mPEG in brief, in
which one terminus is the relatively inert methoxy group, while the
other terminus is a hydroxyl group that is subject to ready
chemical modification.
CH.sub.3O--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--OH mPEG
[0007] PEG is also commonly used in branched forms that can be
prepared by addition of ethylene oxide to various polyols, such as
glycerol, pentaerythritol and sorbitol. For example, the four-arm,
branched PEG prepared from pentaerythritol is shown below:
C(CH.sub.2--OH).sub.4+n
C.sub.2H.sub.4O.fwdarw.C[CH.sub.2--O--(CH.sub.2CH.-
sub.2O).sub.n--CH.sub.2CH.sub.2--OH].sub.4
[0008] The branched PEGs can be represented in general form as
R(-PEG-OH).sub.n in which R represents the central "core" molecule,
such as glycerol or pentaerythritol, and n represents the number of
arms.
[0009] Branched PEGs can also be prepared in which two PEG "arms"
are attached to a central linking moiety having a single functional
group capable of joining to other molecules; e.g., Matsushima et
al., (Chem. Lett., 773, 1980) have coupled two PEGs to a central
cyanuric chloride moiety.
[0010] PEG is a well known polymer having the properties of
solubility in water and in many organic solvents, lack of toxicity,
and lack of immunogenicity. One use of PEG is to covalently attach
the polymer to insoluble molecules to make the resulting
PEG-molecule "conjugate" soluble. For example, it has been shown
that the water-insoluble drug paclitaxel, when coupled to PEG,
becomes water-soluble. Greenwald, et al., J. Org. Chem., 60:331-336
(1995).
[0011] In related work, U.S. Pat. No. 4,179,337 to Davis et al.
discloses that proteins coupled to PEG have enhanced blood
circulation lifetime because of reduced rate of kidney clearance
and reduced immunogenicity. These and other applications are also
described in Biomedical and Biotechnical Applications of
Polyethylene Glycol Chemistry, J. M. Harris, Ed., Plenum, New York
(1992), and Poly(ethylene glycol) Chemistry and Biological
Applications, J. M. Harris and S. Zalipsky, Eds., ACS, Washington
D.C. (1997).
[0012] To couple PEG to a molecule such as a protein, it is often
necessary to "activate" the PEG to prepare a derivative of the PEG
having a functional group at the terminus. The functional group can
react with certain moieties on the protein such as an amino group,
thus forming a PEG-protein conjugate. Many activated derivatives of
PEG have been described. An example of such an activated derivative
is the succinimidyl succinate "active ester":
CH.sub.3O-PEG-O.sub.2C--CH.sub.2CH.sub.2--CO.sub.2--NS
[0013] where NS= 1
[0014] Hereinafter, the succinimidyl active ester moiety will be
represented as --CO.sub.2--NS in chemical drawings.
[0015] The succinimidyl active ester is a useful compound because
it reacts rapidly with amino groups on proteins and other molecules
to form an amide linkage (--CO--NH--). For example, U.S. Pat. No.
4,179,337 to Davis et al. describes coupling of this derivative to
proteins (represented as PRO-NH.sub.2):
mPEG-O.sub.2CCH.sub.2CH.sub.2CO.sub.2NS+PRO-NH.sub.2.fwdarw.mPEG-O.sub.2C--
-CH.sub.2CH.sub.2--CONH-PRO
[0016] Bifunctional PEGs with active groups at both ends of the
linear polymer chain are also useful compounds when formation of a
crosslinked insoluble network is desired. Many such bifunctional
PEGs are known in the art. For example, U.S. Pat. No. 5,162,430 to
Rhee, et al. discloses using such bifunctional PEGs to crosslink
collagen.
[0017] Reactive PEGs have also been synthesized in which several
active functional groups are placed along the backbone of the
polymer. For example, lysine-PEG conjugates have been prepared in
the art in which a number of activated groups are placed along the
backbone of the polymer. Zalipsky et al. Bioconjugate Chemistry,
4:54-62 (1993).
[0018] U.S. Pat. No. 5,283,339 to Arnold et al. discloses PEG
compounds capable of chelating metals. The PEG compounds have a
terminal metal chelating group which has two free carboxylic acid
or amino groups, typcally linked to a nitrogen atom. The PEG
compounds are used to extract and precipitate proteins from
solutions with the carboxylic acid or amino groups together with
the nitrogen atom capable of forming ionic complexes with metal
ions. However, the metal chelating groups disclosed in the patent
generally are not useful in covalently coupling the PEG compounds
to proteins, peptides, or small drugs bearing functional groups
such as amines. The patent does not teach forming an activated PEG
derivative for covalently coupling to another molecule to form a
conjugate.
SUMMARY OF THE INVENTION
[0019] The invention described herein provides a water soluble
polymer such as poly(ethylene glycol) or related polymers that have
a branched moiety at one end of the polymer chain and two free
reactive groups linked to the branched moiety for covalent
attachment to another molecule. Each reactive moiety can have a
tethering group, such as an alkyl chain, linking a reactive group
to the branched moiety. Thus, the branched terminus allows the
activated water soluble polymer of this invention to react with two
molecules to form conjugates.
[0020] Because a tethering group having a desirable length can be
selected in preparing an activated polymer, the two reactive groups
can be held at a predetermined distance apart from each other. The
two molecules conjugated to the activated polymer through the two
reactive groups can also be held at a predetermined distance apart.
Accordingly, an activated PEG is provided in accordance with the
invention having two free reactive moieties branching out from one
PEG chain at a branched moiety. The two free reactive moieties are
capable of reacting with biologically active agents such as
proteins, thereby linking the activated polymer to the biologically
active agents.
[0021] In accordance with one embodiment of this invention, an
activated water soluble polymer is provided having the formula:
2
[0022] wherein POLY is a water soluble, substantially
non-immunogenic polymer backbone, Y is a hydrolytically stable
linkage, X and X' are reactive groups capable of reacting with a
moiety in another molecule such as a protein. Typically, the
polymer backbone is selected from the group consisting of linear
and branched poly(ethylene glycol), linear and branched
poly(alkylene oxide), linear and branched poly(vinyl pyrrolidone),
linear and branched poly(vinyl alcohol), linear and branched
polyoxazoline, linear and branched poly(acryloylmorpholine), and
derivatives thereof. Preferably, the polymer backbone is
poly(ethylene glycol) or a derivative thereof. The polymer backbone
POLY can have a capping group selected from the group consisting of
--OH, alkyls, and --Y--CHXX' wherein Y, X and X' are as described
above and can be the same or different on each terminus of the
PEG.
[0023] In a preferred embodiment, X and X' are represented by -W-Z
and -W'-Z' respectively, in which Z and Z' represent reactive
moieties for conjugating the polymer to another molecule. W and W'
represent tethering groups comprising a substantially linear chain
of atoms, e.g., alkyl chains, ether chains, ester chains, amide
chains, and combinations thereof. Examples of the reactive moieties
include, but are not limited to, active esters, active carbonates,
aldehydes, isocyanates, isothiocyanates, epoxides, alcohols,
maleimides, vinylsulfones, hydrazides, dithiopyridines, and
iodoacetamides.
[0024] In another embodiment of the activated polymer of this
invention, the activated water soluble polymer has the formula:
3
[0025] wherein
[0026] R is a central branch core;
[0027] POLY is a water soluble substantially non-immunogenic
polymer;
[0028] Y is a hydrolytically stable linkage;
[0029] n is from 2 to 200;
[0030] X and X' are reactive groups capable of reacting with a
moiety in another molecule such as a protein.
[0031] Many central branch core molecules for preparing branched or
dendritic PEGs are known and can all be used for R. Typically, R
can be a moiety derived from lysine, glycerol, pentaerythritol, or
sorbitol. Suitable polymer backbones include, but are not limited
to, linear and branched poly(ethylene glycol), linear and branched
poly(alkylene oxide), linear and branched poly(vinyl pyrrolidone),
linear and branched poly(vinyl alcohol), linear and branched
polyoxazoline, linear and branched poly(acryloylmorpholine), and
derivatives thereof. Preferably, is poly(ethylene glycol) or a
derivative thereof is used as the polymer backbone.
[0032] The reactive groups X and X' can be reactive moieties
directly linked to the branching moiety --CH. Preferably, X and X'
have a tethering group and are represented by -W-Z and -W'-Z'
respectively, in which Z and Z' represent reactive groups for
conjugating the polymer to another molecule. W and W' represent
tethering groups comprising a substantially linear chain of atoms,
e.g., alkyl chains, ether chains, ester chains, amide chains, and
combination thereof. Examples of the reactive groups include, but
are not limited to, active esters, active carbonates, aldehydes,
isocyanates, isothiocyanates, epoxides, alcohols, maleimides,
vinylsulfones, hydrazides, dithiopyridines, and iodoacetamides.
[0033] The activated water soluble polymer can be covalently linked
to a biologically active agent to form a conjugate. A suitable
biologically active agent can be any of those having a moiety
capable of reacting with at least one of the two reactive groups in
the terminus of the activated polymer. The biologically active
agent can have two such moieties and each of them can be linked to
one of the two reactive groups. Alternatively, the conjugate can
have two biologically active agents each being linked to one of the
two reactive moieties of the activated polymer. Because activated
polymers having different tethering groups can be prepared in
accordance with this invention, an activated polymer can be
provided in which the two reactive groups in a terminus of the
activated polymer are a desirable distance from each other. When
such an activated polymer is conjugated to two biologically active
agent molecules, the two molecules can be held at a desired
distance apart.
[0034] Accordingly, the activated PEG can be used with greater
versatility as compared to other PEG derivatives heretofore known
in the art to form various conjugates with molecules such as
proteins or peptides. Since PEG molecules conjugated to another
molecule can impart water solubility and reduced immunogenicity to
the other molecule, , the activated PEG derivatives of this
invention allows greater control and precision in modifying such
characteristics in a conjugate.
[0035] Thus, an activated water soluble polymer having proximal
reactive groups is provided. The polymer backbone has at least one
terminus having two reactive groups. The terminus has a branching
moiety and two free reactive moieties linked to the branching
moiety. The branching moiety is in turn linked to the polymer
backbone through a stable linkage.
DETAILED DESCRIPTION OF THE INVENTION
[0036] The terms "group," "functional group," "moiety," "active
moiety," "reactive site," "reactive group" and "reactive moiety"
are used in the art and herein to refer to distinct, definable
portions or units of a molecule. The terms are somewhat synonymous
in the chemical arts and are used herein to indicate that the
portions of molecules that perform some function or activity and
are reactive with other portions of molecules.
[0037] The term "linkage" is used herein to refer to groups or
bonds that normally are formed as the result of a chemical reaction
and typically are covalent linkages. Hydrolytically stable linkages
means that the linkages are substantially stable in water and do
not react with water at useful pHs, e.g., under physiological
conditions for an extended period of time, preferably
indefinitely.
[0038] The term "biologically active agent" when used herein means
any substance which can affect any physical or biochemical
properties of a biological organism, including but not limited to
viruses, bacteria, fungi, plants, animals, and humans. In
particular, as used herein, biologically active agent includes any
substance intended for the diagnosis, cure, mitigation, treatment,
or prevention of disease in humans or other animals, or to
otherwise enhance physical or mental well being of humans or
animals. Examples of biologically active agents include, but are
not limited to, organic and inorganic compounds, proteins,
peptides, lipids, polysaccharides, nucleotides, DNAs, RNAs, other
polymers, and derivatives thereof. Examples of biologically active
agents include antibiotics, fungicides, anti-viral agents,
anti-inflammatory agents, anti-tumor agents, cardiovascular agents,
anti-anxiety agents, hormones, growth factors, steroidal agents,
and the like. Other examples include, microorganisms such as
bacteria and yeast cells, viral particles, plant or animal or human
cells, and the like.
[0039] The polymer backbone is a water soluble substantially
non-immunogenic polymer, and is preferably poly(ethylene glycol)
(PEG). However, it should be understood that other related polymers
are also suitable for use in the practice of this invention and
that the use of the term PEG or poly(ethylene glycol) is intended
to be inclusive and not exclusive in this respect.
[0040] Poly(ethylene glycol) or PEG is useful in biological
applications because it has properties that are highly desirable
and is generally approved for biological or biotechnical
applications. PEG typically is clear, colorless, odorless, soluble
in water, stable to heat, inert to many chemical agents, does not
hydrolyze or deteriorate, and is generally nontoxic. Poly(ethylene
glycol) is considered to be biocompatible, which is to say that PEG
is capable of coexistence with living tissues or organisms without
causing harm. More specifically, PEG is non-immunogenic, which is
to say that PEG does not tend to produce an immune response in the
body. When attached to a molecule having some desirable function in
the body, such as a biologically active agent, to form a conjugate,
the PEG tends to mask the agent and can reduce or eliminate any
immune response so that an organism can tolerate the presence of
the agent. Accordingly, the conjugate is substantially non-toxic.
PEG conjugates tend not to produce a substantial immune response or
cause clotting or other undesirable effects. PEG having the formula
--CH.sub.2CH.sub.2--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2---
, where n is from about 8 to about 4000, is one useful polymer in
the practice of the invention. Preferably PEG having a molecular
weight of from about 200 to about 100,000 Da is used as polymer
backbone.
[0041] The polymer backbone can be linear or branched. Branched
polymer backbones are generally known in the art. Typically, a
branched polymer has a central branch core moiety and a plurality
of linear polymer chains linked to the central branch core. PEG is
commonly used in branched forms that can be prepared by addition of
ethylene oxide to various polyols, such as glycerol,
pentaerythritol and sorbitol. For example, the four-arm, branched
PEG prepared from pentaerythritol is shown below:
C(CH.sub.2--OH).sub.4+nC.sub.2H.sub.4O.fwdarw.C[CH.sub.2O--(CH.sub.2CH.sub-
.2O).sub.n--CH.sub.2CH.sub.2--OH].sub.4
[0042] The central branch moiety can also be derived from several
amino acids, e.g., lysine.
[0043] The branched polyethylene glycols can be represented in
general form as R(-PEG-OH).sub.n in which R represents the core
moiety, such as glycerol or pentaerythritol, and n represents the
number of arms. Suitable branched PEGs can be prepared in
accordance with International Publication No. WO 96/21469, entitled
Multi-Armed, Monofunctional, and Hydrolytically Stable Derivatives
of Poly(Ethylene Glycol) and Related Polymers For Modification of
Surfaces and Molecules, which was filed Jan. 11, 1996, the contents
of which are incorporated herein in their entirety by reference.
These branched PEGs can then be modified in accordance with the
teachings herein.
[0044] Many other water soluble substantially non-immunogenic
polymers than PEG are also suitable for the present invention.
These other polymers can be either in linear form or branched form,
and include, but are not limited to, other poly(alkylene oxides)
such as poly(propylene glycol) ("PPG"), copolymers of ethylene
glycol and propylene glycol and the like; poly(vinyl alcohol)
("PVA") and the like. The polymers can be homopolymers or random or
block copolymers and terpolymers based on the monomers of the above
polymers, straight chain or branched.
[0045] Specific examples of suitable additional polymers include,
but are not limited to, difunctional poly(acryloylmorpholine)
("PAcM"), and poly(vinylpyrrolidone)("PVP"). PVP and
poly(oxazoline) are well known polymers in the art and their
preparation should be readily apparent to the skilled artisan. PAcM
and its synthesis and use are described in U.S. Pat. Nos. 5,629,384
and 5,631,322, the contents of which are incorporated herein by
reference in their entirety.
[0046] Although the molecular weight of each chain of the polymer
backbone can vary, it is typically in the range of from about 100
to about 100,000, preferably from about 6,000 to about 80,000.
[0047] Those of ordinary skill in the art will recognize that the
foregoing list for substantially water soluble non-immunogenic
polymer backbone is by no means exhaustive and is merely
illustrative, and that all polymeric materials having the qualities
described above are contemplated.
[0048] The activated polymer of this invention also has proximal
reactive groups linked to at least one arm of the polymer backbone.
As will be apparent, the term "proximal" is used herein to mean
that the terminus has two free reactive moieties capable of
reacting with two other moieties in another molecule or two other
molecules, which can be the same or different.
[0049] The terminus typically has a branching moiety covalently
linked to a polymer chain of the polymer backbone through a
hydrolytically stable linkage. Typically, there are two free
reactive groups branching out from the branching moiety. The term
"free" as used herein means that each of the two free reactive
groups has two ends, one of which is covalently linked to the
branching moiety and the other end is not linked to any other
moiety or group through covalent linkage, and (available for
reaction with another moiety or group, e.g., of another
molecule).
[0050] Typically the branching moiety is a stable, non-reactive,
and inert moiety that is covalently linked to a polymer chain and
to the two reactive groups. The branching moiety should not form a
hydrogen bond or ionic bond with metal ions or moieties or
molecules. It is believed that the ability to form strong hydrogen
bonds or ionic bonds would interfere with the branching moiety's
function. The branching atom, i.e., the atom the two free reactive
groups are linked to is not a nitrogen atom (N), but is typically a
carbon atom (C).
[0051] At least one of the two free reactive groups may comprise
two portions: a reactive moiety at the free end and a tethering
group linking the reactive moiety to the branching moiety. The
reactive moiety is a moiety capable of reacting with a moiety in
another molecule, e.g., a biologically active agent such as
proteins, peptides, etc. Examples of suitable reactive moieties
include, but are not limited to, active esters, active carbonates,
aldehydes, isocyanates, isothiocyanates, epoxides, alcohols,
maleimides, vinylsulfones, hydrazides, dithiopyridines,
N-succinimidyl, and iodoacetamides. The selection of a free
reactive moiety is determined by the moiety in another molecule to
which the free reactive moiety is to react. For example, when the
moiety in another molecule is a thiol moiety, then a vinyl sulfone
moiety is preferred for the free reactive moiety of the activated
polymer. On the other hand, an N-succinimidyl moiety is preferred
to react to an amino moiety in a biologically active agent.
[0052] The tethering group can have a predetermined length such
that the reactive moiety linked to it is at a predetermined
distance away from the branching moiety, and consequently, a
predetermined distance from the other reactive moiety of the
terminus. Typically, the tethering group is non-reactive and is a
substantially linear chain of atoms, e.g., alkyl chains, ether
chains, ester chains, amide chains, and combinations thereof.
[0053] Thus, in a preferred embodiment, the activated polymer of
this invention can be represented by formula I: 4
[0054] or formula II: 5
[0055] In the above formula, POLY is a linear polymer chain of a
water soluble substantially non-immunogenic polymer backbone,
preferably is poly(ethylene glycol) or a derivative thereof. In the
activated polymer represented by formula I, the polymer backbone
has only one polymer chain. Y is a hydrolytically stable linkage,
which can comprise an atom or a group such as --O--, --S-- and
--CO--NH--. It will be apparent to skilled artisan that many other
hydrolytically stable linkages can also be employed in this
embodiment.
[0056] X and X' are free reactive groups, which can be same or
different, each having a reactive moiety capable of reacting with a
moiety in another molecule such as a protein. In the activated
polymer as represented by formula I, the polymer backbone POLY can
have a capping group at the end opposite to the terminus having
proximal reactive groups. The capping group can be, for example,
--OH, various alkyl, and can also contain proximal reactive groups
--Y--CHXX' wherein Y, X and X' are as described above. Accordingly,
the activated polymer can have two terminals with proximal reactive
groups, one on each end of the polymer backbone.
[0057] In formula II, R is the central core as described above.
POLY is a polymer chain of the water soluble substantially
non-immunogenic polymer backbone. Y is a hydrolytically stable
linkage. n is from 2 to 200 representing the number of polymer
chains or arms in the polymer backbone, as described above.
[0058] As will be apparent, the branching moiety as described above
is CH in this embodiment of the activated polymer. Typically it
does not become charged in normal conditions, and does not form an
ionic bond with a metal ion.
[0059] In a preferred embodiment, X and X' can have a tethering
group in addition to a reactive moiety and can be represented by
-W-Z and -W'-Z' respectively, in which Z and Z' represent free
reactive moieties for conjugating the polymer to another molecule.
W and W' represent tethering groups. Z and Z' can be different or
same reactive moieties.
[0060] Some examples of preferred embodiments of the activated
polymers of this invention are provided as follows:
CH.sub.3O-PEG-CO--NH--CH(CH.sub.2--OCO.sub.2--NS).sub.2
CH.sub.3O-PEG-CO--NH--CH(CH.sub.2--O.sub.2C--CH.sub.2CH.sub.2--CO.sub.2--N-
S).sub.2
CH.sub.3O-PEG-CO--NH--CH(CH.sub.2--CO.sub.2--NS).sub.2
CH.sub.3O-PEG-O--CH(CH.sub.2--OCO.sub.2--NS).sub.2
CH.sub.3O-PEG-O-CH(CH.sub.2--O.sub.2C--CH.sub.2CH.sub.2--CO.sub.2--NS).sub-
.2
(OHC--CH.sub.2CH.sub.2--O--CH.sub.2).sub.2--CH--NHCO-PEG-CONH--CH(OCH.sub.-
2CH.sub.2--CHO).sub.2
PEG-[CONH--CH(OCH.sub.2CH.sub.2--CHO).sub.2].sub.4
CH.sub.3O-PEG-CO--NH--CH(CO.sub.2--NS)(CH.sub.2CH.sub.2CO.sub.2--NS)
[0061] In these examples, --NS represents the N-succinimidyl
moiety.
[0062] In accordance with another aspect of this invention, a
method for preparing the activated water soluble polymer of this
invention is also provided.
[0063] Typically, in the first step, there is a first intermediate
polymer provided having a polymer backbone and a reactive end group
covalently linked to of the polymer backbone.
[0064] In addition, a compound having three reactive groups linked
to a branching moiety is provided. This compound typically has a
branching moiety forming a central core and three free groups
branching out from the central core. When the three free groups are
linked to the same atom in the branching moiety, the atom is not a
nitrogen atom. One of the three free groups can react with the
reactive end group of the first intermediate polymer to form a
hydrolytically stable linkage. The other two free groups can be
ultimately converted into the two free reactive groups on the
terminus of the activated polymer of this invention. Examples of
these compounds include, H.sub.2NCH(CH.sub.2--OH).sub.2,
NaO--CH(CH.sub.2--O-Bz).sub.2,
H.sub.2N--CH(CH.sub.2CO.sub.2H).sub.2, and the like. As will be
apparent, in these examples, the branching moiety is CH. The
H.sub.2N-- and NaO-- moieties can be used to link the compound to
the first intermediate polymer to form a hydrolytically stable
linkage, while the hydroxyl groups, carboxylic acid groups, and
--CH.sub.2--O-Bz groups can be ultimately converted into free
reactive moieties of the activated polymer of this invention.
[0065] Thus, in the second step of the method, the compound having
three reactive groups is reacted with the first intermediate
polymer to form a second intermediate polymer which includes a
hydrolytically stable linkage linking the first intermediate
polymer and the compound having three reactive groups, thus leaving
only two free groups at the terminus of the polymer chain.
[0066] In the third step, the two free groups of the compound are
converted into two free reactive moieties linked to the branching
moiety. A number of methods known in the art can be employed in the
conversion. For example, the free groups can be reacted to a
compound which can impart a free reactive moiety. Alternatively,
the two free groups in the second intermediate polymer can be
oxidized or reduced or substituted to form two new free reactive
moieties. Such methods will be apparent to skilled artisan in view
of the Examples given below.
[0067] In accordance with yet another aspect of this invention, a
conjugate is provided formed by covalently linking the activated
water soluble polymer of this invention to another molecule, e.g.,
a biologically active agent. Typically, a suitable biologically
active agent can be any biologically active agent having a moiety
capable of reacting with at least one of the two proximal reactive
groups in the terminus of the activated polymer.
[0068] The biologically active agent can have two such moieties and
each of them can be linked to one of the two reactive groups.
Alternatively, the conjugate can have two biologically active
agents each being linked to one of the two reactive moieties of the
activated polymer. For example, the reactive moieties in the
activated polymer can be vinyl sulfone moieties, which can react
with a thiol moiety. If a protein has only one thiol moiety, then
two of such protein molecules can be linked to the activated
polymer through the two vinyl sulfone moieties. When a protein has
two thiol moieties, the reaction between the protein and the
activated polymer can be controlled such that each activated
polymer molecule is conjugated to two protein molecules.
Alternatively, the reaction can also be controlled such that the
two vinyl sulfone moieties of an activated polymer are reacted with
two thiol moieties on the same protein molecule.
[0069] Other moieties in biologically active agents useful for
reacting with the free reactive moieties of the bivalent terminus
of an activated polymer of this invention include, e.g., amino
groups, carboxylic acid groups, etc. It will be apparent for
skilled artisan once apprised of the present invention to select
appropriate free reactive moieties in an activated polymer for
reaction with a given moiety in a biologically active agent. For
example, if conjugation is through reaction with an amino group in
a biologically active agent, moieties such as --CO.sub.2--NS or
aldehyde is preferably used as a free reactive moiety in the
activated polymer for conjugation.
[0070] Because activated polymers having different tethering groups
can be prepared in accordance with this invention, an activated
polymer can be provided in which the two reactive groups in a
bivalent terminus of the activated polymer are in a desirable
distance from each other. When such an activated polymer is
conjugated to two biologically active agent molecules, the two
molecules can be held at a desired distance apart.
[0071] The following examples are given to illustrate the
invention, but should not be considered in limitation of the
invention:
[0072] Example 1. Synthesis of mPEG.sub.20K-OCH.sub.2CH.sub.2CONHCH
(CH.sub.2O.sub.2CCH.sub.2 CH.sub.2CO.sub.2NS).sub.2
[0073] (NS=N-succinimidyl)
[0074] Example 2. Synthesis of
mPEG.sub.20K-OCH(CH.sub.2--SO.sub.2CH.dbd.C- H.sub.2).sub.2
[0075] Example 3. Synthesis of
mPEG.sub.5K-O.sub.2CNH--CH(CH.sub.2CO.sub.2- NS).sub.2
[0076] Example 4. Synthesis of
mPEG.sub.5k-O--CH.sub.2CH.sub.2CH(CO.sub.2H- ).sub.2
EXAMPLE 1
[0077] Reactions:
[0078] 1.
mPEG.sub.20K-OCH.sub.2CH.sub.2CO.sub.2NS+H.sub.2NCH(CH.sub.2--OH-
).sub.2
[0079]
mPEG.sub.20K-OCH.sub.2CH.sub.2CONHCH(CH.sub.2OH).sub.2+NHS
[0080] NS=N-succinimidyl;
[0081] NHS=N-hydroxysuccinimide
[0082] 2.
mPEG.sub.20K-OCH.sub.2CH.sub.2CONHCH(CH.sub.2--OH).sub.2+2SA
[0083]
mPEG.sub.20K-OCH.sub.2CH.sub.2CONHCH(CH.sub.2--O.sub.2CCH.sub.2CH.-
sub.2CO.sub.2H).sub.2
[0084] SA=succinic anhydride
[0085] 3.
mPEG.sub.20K-OCH.sub.2CH.sub.2CONHCH(CH.sub.2--O.sub.2CCH.sub.2C-
H.sub.2CO.sub.2H).sub.2+NHS+DCC
[0086]
mPEG.sub.20K-OCH.sub.2CH.sub.2CONHCH(CH.sub.2--O.sub.2CCH.sub.2CH.s-
ub.2CONS).sub.2
[0087] DCC=dicyclohexylcarbodiimide
[0088] Preparations:
[0089] 1.
mPEG.sub.20K-OCH.sub.2CH.sub.2CONHCH(CH.sub.2OH).sub.2
[0090] A solution of mPEG.sub.20K-OCH.sub.2CH.sub.2CO.sub.2NS (mSPA
20K, 20 g, 0.001 moles)), H.sub.2NCH(CH.sub.2--OH).sub.2 (serinol,
0.14 g, 0.00154 moles), and triethylamine (0.3 ml) in acetonitrile
(100 ml) was stirred under nitrogen overnight and the solvent
removed by distillation. The product was chromatographed on DEAE
sepharose eluted with water and the eluate was saturated with NaCl
and extracted with chloroform. The resulting chloroform phase was
dried over magnesium sulfate, filtered, and the filtrate evaporated
to dryness under vacuum to yield 20 g of product as a white solid
showing a single peak with gel permeation chromatography
(Ultrahydrogel 250, pH 7.2 buffer).
[0091] 2.
mPEG.sub.20K-OCH.sub.2CH.sub.2CONHCH(CH.sub.2--O.sub.2CCH.sub.2C-
H.sub.2CO.sub.2H).sub.2
[0092] A solution of the product from (1) (20 g, 0.002 moles) and
butylated hydroxytoluene (BHT) (0.02 g) in 220 ml of chloroform was
subjected to distillation until about 150 ml of solvent had
distilled. Succinic anhydride (2.0 g, 0.02 moles), pyridine (1.62
ml, 0.02 moles), and 40 ml of toluene were added and the resulting
mixture heated at 84 C. for 20 h under nitrogen. The product was
precipitated with 850 ml of ether and collected by filtration.
After drying, the product was dissolved in 200 ml of water, 20 g of
NaCl added, and the pH adjusted to 3 with aqueous phosphoric acid.
The product was extracted with chloroform (200+150+100 ml) and the
combined extracts dried over magnesiun sulfate. Evaporation of the
dried solution yielded the product as a white solid (16 g). The
molecular weight was determined to be 20,940 Da by potentiometric
titration.
[0093] 3.
mPEG.sub.20K-OCH.sub.2CH.sub.2CONHCH(CH.sub.2--O.sub.2CCH.sub.2C-
H.sub.2CONS).sub.2
[0094] A solution of the product from (2) (15 g, 0.0015 moles),
N-hydroxysuccinimide (0.21 g, 0.00179 moles),
dicyclohexylcarbodiimide, 0.37 g, 0.00177 moles) in methylene
chloride (100 ml) was stirred at room temperature under nitrogen
overnight. The suspension was filtered, product precipitated twice
from methylene chloride by addition of ether (850 ml) and collected
by filtration to obtain a white solid (13.0 g) which had 97.7%
substitution by proton nmr. The proton nmr displayed a broad
multiplet at 3.50 ppm (PEG backbone methylene groups), a singlet at
3.23 ppm PEG methyl), a singlet at 2.80 ppm (NS methylenes), and
multiplets at 2.68 and 2.95 ppm (succinate methylenes).
EXAMPLE 2
[0095] Reactions:
[0096] 1. HO--CH(CH.sub.2--O-Bz).sub.2+NaH (toluene)
NaO--CH(CH.sub.2OBz).sub.2
[0097] Bz=Benzyl
[0098] 2. NaO--CH(CH.sub.2--O-Bz).sub.2+mPEG20K-O-Ms
mPEG.sub.20KO--CH(CH.sub.2OBz).sub.2
[0099] Ms=mesylate
[0100] 3.
mPEG.sub.20KO--CH(CH.sub.2OBz).sub.2+HCO.sub.2H/MeOH/H.sub.2O/Pd-
/C
[0101] mPEG.sub.20KO--CH(CH.sub.2OH).sub.2
[0102] 4. mPEG.sub.20KO--CH(CH.sub.2OH).sub.2+MsCl/Et.sub.3N
mPEG.sub.20KO--CH(CH.sub.2OMs).sub.2
[0103] 5.
mPEG.sub.20KO--CH(CH.sub.2OMs).sub.2+HSCH.sub.2CH.sub.2OH
[0104] mPEG.sub.20KO--CH(CH.sub.2SCH.sub.2CH.sub.2OH).sub.2
[0105] 6.
mPEG.sub.20KO--CH(CH.sub.2SCH.sub.2CH.sub.2OH).sub.2+H.sub.2WO.s-
ub.4
[0106]
mPEG.sub.20KO--CH(CH.sub.2SO.sub.2CH.sub.2CH.sub.2OH).sub.2
[0107] 7.
mPEG.sub.20KO--CH(CH.sub.2SO.sub.2CH.sub.2CH.sub.2OH).sub.2+MsCl-
/ET.sub.3N
[0108] mPEG.sub.20KO--CH(CH.sub.2SO.sub.2CH=CH.sub.2).sub.2
[0109] Preparations:
[0110] 1. mPEG.sub.20KO--CH(CH.sub.2OBz).sub.2
[0111] A solution of 18 g (0.0641 moles) of
1,3-dibenzyloxy-2-propanol in 80 ml of toluene was distilled until
15 ml of toluene was removed. The azeotropically dried solution was
then added to a suspension of 2.56 g (0.064 moles) of NaH in 80 ml
of toluene and the resulting mixture stirred while heating to 37-40
C. before filtering. The filtrate was then added to a solution of
azeotropically-dried mPEG.sub.20K mesylate in about 350 ml of
toluene and the resulting mixture was heated for 20 h at 125 C.
under N.sub.2. The product was precipitated with cold ether, wash
on the filter with hexane, and dried under vacuum to yield 70.4 g
of white solid shown to be pure by proton nmr.
[0112] 2. mPEG.sub.20KO--CH(CH.sub.2OH).sub.2
[0113] To a solution of 15 g (0.00075 moles) of the product from
(1) in 9.2 ml of formic acid and 0.8 ml of water was added 2.0 g of
Pd/C (10%) and the mixture was stirred for 2 h under nitrogen. The
mixture was then filtered and the pH of the filtrate adjusted to
7.2. The reuslting solution was extracted with CH.sub.2Cl.sub.2 and
the extract dried over MgSO.sub.4. Evaporation of the solution
yielded 12.9 g of product which displayed no benzyl groups in the
proton nmr.
[0114] 3. mPEG.sub.20KO--CH(CH.sub.2OMs).sub.2
[0115] To an azeotropically-dried solution of the product from (2)
(8.0 g, 0.000889 moles) in toluene (100 ml) containing 0.008 g of
BHT was added a solution of mesyl chloride (0.090 ml, 0.00116
moles) and triethylamine (0.210 ml, 0.0011 moles) in 10 ml of
ET.sub.3N and the resulting solution was stirred overnight at room
temperature under nitrogen. Ethanol (1 ml) was added and 50 ml of
the solvent was removed by distillation before adding 500 ml of
ether to precipitate the product. The product was collected by
filtration and dried under vacuum to yield 7.6 g of the mesylate
derivative shown by nmr to be 100% substituted.
[0116] 4. mPEG.sub.20KO--CH(CH.sub.2SCH.sub.2CH.sub.2OH).sub.2
[0117] A solution of the product of (3), (7.0 g, 0.00035 moles),
mercaptoethanol (0.56, 0.0080 moles) ml, NaOH (0.22 g), in toluene
(30 ml and ethanol (60 ml) was heated at 60 C. for 2 h under
N.sub.2. The pH was adjusted to 7 and the product extracted with
methylene chloride (3.times.100 ml). After drying the extract over
MgSO4, the solvent was removed and the product precipitated with
250 ml of ethyl ether. The product was collected by filtration and
dried under vacuum to get 6.6 g of white solid which was shown by
nmr to be 97.3% substituted.
[0118] 5.
mPEG.sub.20KO--CH(CH.sub.2SO.sub.2CH.sub.2CH.sub.2OH).sub.2
[0119] A solution containing the product from (4), 6.5 g (0.00065
moles), and tungstic acid (0.16 g) in water (14 ml) was prepared
and the pH adjusted to 6.6. Hydrogen peroxide (30%, 0.65 ml) was
added and the mixture stirred at room temperature overnight. The pH
was adjusted to 7.5 and the mixture stirred 1 h before extracting
with CH.sub.2Cl.sub.2 (3.times.30 ml). The mixture was dried over
MgSO4, filtered, and the filtrate concentrated to 25 ml. The
product was precipitated with 200 ml of ether and collected by
filtration to obtain 5.3 g of product after vacuum drying. The
product was shown by nmr to have 86% substitution.
[0120] 6.
mPEG.sub.20KO--CH(CH.sub.2SO.sub.2CH.dbd.CH.sub.2).sub.2
[0121] A solution of the product from (5), (5.2 g, 0.00052 moles),
Et.sub.3N (0.63 ml, 0.00452 moles), BHT (0.005 g), and MsCl (0.15
ml, 0.001938 moles) in CH.sub.2Cl.sub.2 (25 ml) was stirred at room
temperature for 42 h at room temperature. Ethanol (1 ml) was added
and the mixture was stirred 15 minutes. Methylene chloride (50 ml)
was added and the resulting solution was washed with aquous 1M HCl
followed by 5% aqueous Na.sub.2HPO.sub.4. After drying over MgSO4,
the solution was concentrated to 30 ml and the product precipitated
with 300 ml of ether. The product was collected by filtration and
dried under vacuum to yield the product (4.6 g) as a white solid.
The degree of substitution was 92.5% by nmr. The .sup.1H nmr
spectrum (dmso-d6) displayed absorptions at 3.51 ppm (PEG backbone
CH.sub.2), 3.23 ppm, CH.sub.3O), 6.2 and 7.0 ppm, m, vinyl H. Note
in this example that Y=O, W= CH.sub.2, and
Z=SO.sub.2CH.dbd.CH.sub.2.
EXAMPLE 3
[0122] Reactions:
[0123] 1. mPEG.sub.5KBTC+H.sub.2N--CH(CH.sub.2CO.sub.2H).sub.2
mPEG.sub.5KO.sub.2CNHCH
[0124] (CH.sub.2CO.sub.2H).sub.2
[0125] BTC=1-benzotriazolyl carbonate
[0126] 2.
mPEG.sub.5KO.sub.2CNHCH(CH.sub.2CO.sub.2H).sub.2+NHS/DCC
[0127] mPEG.sub.5KO.sub.2CNHCH(CH.sub.2CO.sub.2NS).sub.2
[0128] Preparations:
[0129] 1. mPEG.sub.5KO.sub.2CNHCH(CH.sub.2CO.sub.2H).sub.2
[0130] To a solution of .beta.-glutamic acid (0.10 g, 0.00068
moles), boric acid (0.1 g) in 10 ml of water at pH 8 was added
mPEG.sub.5KBTC over 15 m while maintaining the pH at 8.15-8.25 by
addition of NaOH solution. NaCl (6 g) was added and the pH of the
solution was adjusted to 2 with 10% H.sub.3PO.sub.4. The product
was extracted into CH.sub.2Cl.sub.2 (100+80+50 ml) and the combined
extracts were dried over MgSO.sub.4. The mixture was filtered and
the filtrate evaporated under vacuum to yield 7.8 g of product. The
mixture was determined to be 75.5% of the mPEG glutamic acid
derivative and 24.5% mPEG. This mixture was purified by
chromatography on DEAE sepharose by first eluting with water and
then eluting the desired product with 0.5 M NaCl. Extraction of the
product from the NaCl solution (pH 2) with methylene chloride
followed by drying the extract over MgSO4 and evaporation of the
solvent yielded 6.1 g of material shown to be 100% pure by GPC.
[0131] 2. mPEG.sub.5KO.sub.2CNHCH(CH.sub.2CO.sub.2NS).sub.2
[0132] A solution of
mPEG.sub.5KO.sub.2CNHCH(CH.sub.2CO.sub.2H).sub.2 (6.0 g, 0.00116
moles), NHS (0.385 g, 0.001627 moles), DCC (0.676 g, 0.00162 moles)
in methylene chloride (50 ml) was stirred overnight at room
temperature under nitrogen. The resulting suspension was filtered
and the filtrate was added to 500 ml of cold ethyl ether. The
precipitate was collected by filtration and dried under vacuum to
obtain 5.5 g of product which was shown by nmr to have 100%
substitution. The .sup.1H nmr spectrum (dmso-d6) displayed
absorptions at 3.51 ppm (PEG backbone CH.sub.2), 3.23 ppm,
CH.sub.3O), 4.29 ppm (--NHC+E,uns H--), 4.05 ppm (--C+E,uns
H.sub.+E,uns 2--O--CONH--, 3.24 ppm (C+E,uns H.sub.+E,uns
2CO.sub.2NS), 2.81 (NS C+E,uns H.sub.+E,uns 2).
EXAMPLE 4
[0133] Reactions:
NaH
[0134]
CH.sub.3O-PEG-O--CH.sub.2CH.sub.2--OMs+CH.sub.2(CO.sub.2C.sub.2H.su-
b.5).sub.2.fwdarw.CH.sub.3--O-PEG-O--CH.sub.2CH.sub.2CH(CO.sub.2C.sub.2H.s-
ub.5).sub.2
1. NaOH
2. HCl
[0135]
CH.sub.3--O-PEG-O--CH.sub.2CH.sub.2CH(CO.sub.2C.sub.2H.sub.5).sub.2-
.fwdarw.CH.sub.3--O-PEG-O--CH.sub.2CH.sub.2CH(CO.sub.2H).sub.2
1. LiAlH.sub.4
[0136]
CH.sub.3--O-PEG-O--CH.sub.2CH.sub.2CH(CO.sub.2H).sub.2.fwdarw.CH.su-
b.3--O-PEG-O--CH.sub.2CH.sub.2CH(CH.sub.2OH).sub.2
2. HCl
[0137] Preparations:
[0138] 1. Preparation of
CH.sub.3--O-PEG-O--CH.sub.2CH.sub.2CH(CO.sub.2H).- sub.2 (Steps 1
and 2 above)
[0139] Diethyl malonate (8.8 ml) in 150 ml of dry dioxane was added
dropwise to NaH (2.4 g) in 60 ml of toluene under argon.
MPEG.sub.5000 mesylate (30 g) in 250 ml of toluene was
azeotropically distilled to remove 150 ml of toluene and the
residue was added to the above diethyl malonate solution. After
refluxing the mixture for 3-4 hours, it was evaporated under vacuum
to dryness and dried in vacuo overnight. The dried material was
then dissolved in 200 ml of IN NaOH, the solution was stirred for 2
days at room temperature, and the pH adjusted to 3 with 1N HCl.
NaCl was added to the solution to a concentration of about 15% and
the mixture was then extracted with 350 ml of CH.sub.2Cl.sub.2 in
several portions. The combined extracts were dried over
Na.sub.2SO.sub.4, concentrated under vacuum and the product
precipitated by addition of isopropanol/ether (1:1). The product
was collected by filtration and dried under vacuum overnight to
obtain 24.7 g of product as a white powder. GPC (Ultrahydrogel 250)
showed the product to be 98% pure.
[0140] .sup.1H NMR (dmso-d6, ppm): 1.96 (t, CH.sub.2C+E,uns
H.sub.+E,uns 2--C); 3.51 (br m, PEG --CH.sub.2CH.sub.2--O--).
[0141] 2. Preparation of
CH.sub.3--O-PEG.sub.5000-O--CH.sub.2CH.sub.2CH(CH-
.sub.2OH).sub.2
[0142]
CH.sub.3--O-PEG.sub.5000-O--CH.sub.2CH.sub.2CH(CO.sub.2H).sub.2 (5
g) was dissolved in 50 ml of toluene and 9.8 ml of LiAlH.sub.4 (1 M
in THF) was added. After stirring overnight at room temperature,
the mixture was evaporated to dryness under vacuum and 150 ml of
water and 22.5 g of NaCl were added. The pH was adjusted to 6.5
with aqueous HCl and the resulting solution was extracted with
3.times.50 ml of methylene chloride. The combined extracts were
dried over Na.sub.2SO.sub.4 and the solution was evaporated to
dryness. The product was precipitated with ethyl ether and
collected by filtration. After chromatography on DEAE sepharose,
the product was 90% pure by GPC (Ultrahydrogel 250).
[0143] .sup.1H NMR (dmso-d6, ppm): 3.51 (br m, PEG
--CH.sub.2CH.sub.2--O--- ); 1.5 (br mult, CH; 4.32 (t, OH).
* * * * *