U.S. patent application number 10/586166 was filed with the patent office on 2008-12-25 for branched polymeric sugars and nucleotides thereof.
This patent application is currently assigned to NEOSE TECHNOLOGIES, INC.. Invention is credited to Caryn Bowe, Shawn DeFrees.
Application Number | 20080319183 10/586166 |
Document ID | / |
Family ID | 34637500 |
Filed Date | 2008-12-25 |
United States Patent
Application |
20080319183 |
Kind Code |
A1 |
DeFrees; Shawn ; et
al. |
December 25, 2008 |
Branched Polymeric Sugars and Nucleotides Thereof
Abstract
The present invention provides sugars, nucleotide sugars,
activated sugars that include one or more polymeric modifying
moiety within their structure. The invention is exemplified by
reference to linear and branched polymers, such as the
water-soluble polymer poly(ethylene glycol).
Inventors: |
DeFrees; Shawn; (North
Wales, PA) ; Bowe; Caryn; (Doylestown, PA) |
Correspondence
Address: |
MORGAN, LEWIS & BOCKIUS LLP (SF)
One Market, Spear Street Tower, Suite 2800
San Francisco
CA
94105
US
|
Assignee: |
NEOSE TECHNOLOGIES, INC.
Horsham
PA
|
Family ID: |
34637500 |
Appl. No.: |
10/586166 |
Filed: |
January 26, 2005 |
PCT Filed: |
January 26, 2005 |
PCT NO: |
PCT/US2005/002522 |
371 Date: |
July 23, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10997405 |
Nov 24, 2004 |
7405198 |
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10586166 |
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PCT/US04/03971 |
Feb 10, 2004 |
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10997405 |
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10366500 |
Feb 14, 2003 |
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PCT/US04/03971 |
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60539387 |
Jan 26, 2004 |
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60544411 |
Feb 12, 2004 |
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60546631 |
Feb 20, 2004 |
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60555504 |
Mar 22, 2004 |
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60570891 |
May 12, 2004 |
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60590573 |
Jul 23, 2004 |
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60592744 |
Jul 29, 2004 |
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60611790 |
Sep 20, 2004 |
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60614518 |
Sep 29, 2004 |
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60614518 |
Sep 29, 2004 |
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60623387 |
Oct 29, 2004 |
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60626678 |
Nov 9, 2004 |
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60641956 |
Jan 6, 2005 |
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60643347 |
Jan 13, 2005 |
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60524989 |
Nov 24, 2003 |
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60539387 |
Jan 26, 2004 |
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60555504 |
Mar 22, 2004 |
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60590573 |
Jul 23, 2004 |
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60592744 |
Jul 29, 2004 |
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60614518 |
Sep 29, 2004 |
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60623387 |
Oct 29, 2004 |
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Current U.S.
Class: |
536/26.8 |
Current CPC
Class: |
A61K 38/00 20130101;
A61K 47/60 20170801; A61P 7/00 20180101; A61P 17/02 20180101; C07H
19/10 20130101; C07K 14/505 20130101; A61P 7/06 20180101 |
Class at
Publication: |
536/26.8 |
International
Class: |
C07H 19/10 20060101
C07H019/10 |
Claims
1. A compound having a formula that is a member selected from:
##STR00059## wherein R.sup.1 is H, CH.sub.2OR.sup.7, COOR.sup.7 or
OR.sup.7 in which R.sup.7 represents H, substituted or
unsubstituted alkyl or substituted or unsubstituted heteroalkyl;
R.sup.2 is a member selected from H, OH, an activating group and a
moiety that includes a nucleotide; R.sup.3, R.sup.4, R.sup.5,
R.sup.6 and R.sup.6' are independently selected from H, substituted
or unsubstituted alkyl, OR.sup.9, and NHC(O)R.sup.10 wherein
R.sup.9 and R.sup.10 are independently selected from H, substituted
or unsubstituted alkyl or substituted or unsubstituted heteroalkyl,
and at least one of R.sup.3, R.sup.4, R.sup.5, R.sup.6 and R.sup.6'
includes a polymeric modifying moiety.
2. The compound according to claim 1 wherein R.sup.2 has the
formula: ##STR00060## in which R.sup.8 is a nucleoside.
3. The compound according to claim 2 wherein R.sup.8 is a member
selected from cytosine, uridine, guanosine, adenosine and
thymidine.
4. The compound according to claim 1 wherein at least one of
R.sup.3, R.sup.4, R.sup.5 and R.sup.6 includes the moiety:
##STR00061## wherein R.sup.11 is a polymeric modifying moiety; L is
a member selected from a bond and a linking group; and w is
selected from the integers from 1 to 6.
5. The compound according to claim 4 wherein said linking group is
a member selected from substituted or unsubstituted alkyl and
substituted or unsubstituted heteroalkyl moieties.
6. The compound according to claim 5 wherein the moiety:
##STR00062## has the formula: ##STR00063## wherein X.sup.2 and
X.sup.4 are independently selected from linkage fragments; X.sup.a
is a linkage fragment; R.sup.12 and R.sup.13 are independently
selected polymeric arms; and c is an integer from 1 to 20.
7. The compound according to claim 5 wherein said linking group has
the formula: ##STR00064## in which X.sup.a and X.sup.b are
independently selected linkage fragments; and L.sup.1 is a member
selected from a bond, substituted or unsubstituted alkyl or
substituted or unsubstituted heteroalkyl.
8. The compound according to claim 7 wherein X.sup.a and X.sup.b
are linkage fragments independently selected from S, SC(O)NH,
HNC(O)S, SC(O)O, O, NH, NHC(O), (O)CNH and NHC(O)O, and
OC(O)NH.
9. The compound according to claim 5 wherein said linker comprises
an acyl moiety.
10. The compound according to claim 9 wherein L-R.sup.11 has the
formula: ##STR00065## in which s is an integer from 0 to 20; and
R.sup.11 is said polymeric modifying moiety.
11. The compound according to claim 1, wherein said polymeric
modifying moiety has the formula: ##STR00066## wherein X.sup.2 and
X.sup.4 are independently selected from linkage fragments; X.sup.5
is a non-reactive group; and R.sup.12 and R.sup.13 are
independently selected polymeric arms.
12. The compound according to claim 11 wherein X.sup.2 and X.sup.4
are linkage fragments independently selected from S, SC(O)NH,
HNC(O)S, SC(O)O, O, NH, NHC(O), (O)CNH and NHC(O)O, OC(O)NH and
(CH.sub.2).sub.gY'' wherein g is an integer from 1 to 50; and Y''
is a member selected from O, S and NH.
13. The compound according to claim 11 wherein X.sup.4 is a peptide
bond; and R.sup.13 is an amino acid residue.
14. The compound according to claim 1 having the formula:
##STR00067## in which D is a member selected from --OH and
(R.sup.11).sub.w'-L-; G represents is a member selected from H,
(R.sup.11).sub.w'-L- and --C(O)(C.sub.1-C.sub.6)alkyl; w' is an
integer from 2 to 6, and at least one of D and G is
(R.sup.11).sub.w'-L-.
15. The compound according to claim 14 having the formula:
##STR00068## wherein L.sup.a is a member selected from substituted
or unsubstituted alkyl and substituted or unsubstituted
heteroalkyl.
16. The compound according to claim 1 having the formula:
##STR00069## wherein L.sup.a is a member selected from an amino
acid residue and a peptidyl residue having from 2 to 4 amino acid
residues; X.sup.2 and X.sup.4 are independently selected from
linkage fragments; X.sup.5 is a non-reactive group; and R.sup.12
and R.sup.13 are independently selected polymeric arms
17. The compound according to claim 16 having the formula:
##STR00070## wherein X.sup.2 and X.sup.4 are independently selected
from linkage fragments; X.sup.a is a linkage fragment; R.sup.12 and
R.sup.13 are independently selected polymeric arms; and c is an
integer from 1 to 20.
18. The compound according to claim 1, having the formula:
##STR00071## wherein AA-NH is an amino acid residue; and P is a
polymeric modifying group.
19. The compound according to claim 18 wherein -AA-NH is
--CH.sub.2NH.
20. The compound according to claim 1 wherein said compound is a
substrate for an enzyme that transfers a sugar moiety from a member
selected from an activated sugar, a nucleotide sugar and
combinations thereof onto an acceptor moiety of a substrate.
21. The compound according to claim 20 wherein said acceptor moiety
is a member selected from a glycosyl residue, an amino acid residue
and an aglycone.
22. A method of preparing cytidine monophosphate sialic
acid-poly(ethylene glycol), said method comprising: (a) contacting
mannosamine with an activated, N-protected amino acid under
conditions appropriate to form an amide conjugate between said
mannosamine and the N-protected amino acid; (b) contacting said
amide conjugate with pyruvate and sialic acid aldolase under
conditions appropriate to convert said amide conjugate to a sialic
acid amide conjugate; (c) contacting said sialic acid amide
conjugate with cytidine triphosphates, and a synthetase under
conditions appropriate to form a cytidine monophosphate sialic acid
amide conjugate; (d) removing the N-protecting group from said
cytidine monophosphate sialic acid amide conjugate, thereby
producing a free amine; and (e) contacting said free amine with an
activated PEG, thereby forming said cytidine monophosphate sialic
acid-poly(ethylene glycol).
23. The method according to claim 21, wherein said activated
N-protected amino acid has the formula: ##STR00072##
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application No. 60/539,387, filed Jan. 26, 2004; U.S. Provisional
Patent Application No. 60/555,504, filed Mar. 22, 2004; U.S.
Provisional Patent Application No. 60/590,573, filed Jul. 23, 2004;
U.S. Provisional Patent Application No. 60/555,504, filed Mar. 22,
2004; U.S. patent application Ser. No. 10/997,405, filed Nov. 24,
2004; U.S. Provisional Patent Application 60/544,411, filed Feb.
12, 2004; U.S. Provisional Patent Application 60/546,631, filed
Feb. 20, 2004; U.S. Provisional Patent Application May 12, 2004;
U.S. patent application ((Unassigned), Attorney Docket No.
40853-01-5138US), filed Jan. 10, 2005; PCT Application No.
((Unassigned), Attorney Docket No. 40853-01-5146WO), filed Dec. 3,
2004; U.S. Provisional Patent Application No. 60/590,649, filed
Jul. 23, 2004; U.S. Provisional Patent Application No. 60/611,790,
filed Sep. 20, 2004; U.S. Provisional Patent Application No.
60/592,744, filed Jul. 29, 2004; U.S. Provisional Patent
Application No. 60/614,518, filed Sep. 29, 2004; U.S. Provisional
Patent Application No. 60/623,387, filed Oct. 29, 2004; U.S.
Provisional Patent Application No. 60/626,678, filed Nov. 9, 2004;
and U.S. Provisional Patent Application No. ((Unassigned), Attorney
Docket No. 040853-01-5150), filed Jan. 6, 2005, the disclosure of
each of which is incorporated herein by reference in its entirety
for all purposes.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention resides in the field of modified
sugars and nucleotides thereof.
[0004] 2. Background
[0005] Post-expression in vitro modification of peptides is an
attractive strategy to remedy the deficiencies of methods that rely
on controlling glycosylation by engineering expression systems;
including both modification of glycan structures or introduction of
glycans at novel sites. A comprehensive toolbox of recombinant
eukaryotic glycosyltransferases is becoming available, malting in
vitro enzymatic synthesis of mammalian glycoconjugates with custom
designed glycosylation patterns and glycosyl structures possible.
See, for example, U.S. Pat. Nos. 5,876,980; 6,030,815; 5,728,554;
5,922,577; and WO/9831826; US2003180835; and WO 03/031464.
[0006] Enzyme-based syntheses have the advantages of
regioselectivity and stereoselectivity. Moreover, enzymatic
syntheses are performed using unprotected substrates. Three
principal classes of enzymes are used in the synthesis of
carbohydrates, glycosyltransferases (e.g., sialyltransferases,
oligosaccharyltransferases, N-acetylglucosaminyltransferases), and
glycosidases. The glycosidases are further classified as
exoglycosidases (e.g., .beta.-mannosidase, .beta.-glucosidase), and
endoglycosidases (e.g., Endo-A, Endo-M). Each of these classes of
enzymes has been successfully used synthetically to prepare
carbohydrates. For a general review, see, Crout et al., Curr. Opin.
Chem. Biol. 2: 98-111 (1998).
[0007] Glycosyltransferases modify the oligosaccharide structures
on glycopeptides. Glycosyltransferases are effective for producing
specific products with good stereochemical and regiochemical
control. Glycosyltransferases have been used to prepare
oligosaccharides and to modify terminal N- and O-linked
carbohydrate structures, particularly on glycopeptides produced in
mammalian cells. For example, the terminal oligosaccharides of
glycopeptides have been completely sialylated and/or fucosylated to
provide more consistent sugar structures, which improves
glycopeptide pharmacodynamics and a variety of other biological
properties. For example, .beta.-1,4-galactosyltransferase was used
to synthesize lactosamine, an illustration of the utility of
glycosyltransferases in the synthesis of carbohydrates (see, e.g.,
Wong et al., J. Org. Chem. 47: 5416-5418 (1982)). Moreover,
numerous synthetic procedures have made use of
.alpha.-sialyltransferases to transfer sialic acid from
cytidine-5'-monophospho-N-acetylneuraminic acid to the 3-OH or 6-OH
of galactose (see, e.g., Kevin et al., Chem. Eur. J. 2: 1359-1362
(1996)). Fucosyltransferases are used in synthetic pathways to
transfer a fucose unit from guanosine-5'-diphosphofucose to a
specific hydroxyl of a saccharide acceptor. For example, Ichikawa
prepared sialyl Lewis-X by a method that involves the fucosylation
of sialylated lactosamine with a cloned fucosyltransferase
(Ichikawa et al., J. Am. Chem. Soc. 114: 9283-9298 (1992)). For a
discussion of recent advances in glycoconjugate synthesis for
therapeutic use see, Koeller et al., Nature Biotechnology 18:
835-841 (2000). See also, U.S. Pat. Nos. 5,876,980; 6,030,815;
5,728,554; 5,922,577; and WO/9831826.
[0008] In addition to manipulating the structure of glycosyl groups
on polypeptides, interest has developed in preparing glycopeptides
that are modified with one or more non-saccharide modifying group,
such as water soluble polymers. Poly(ethyleneglycol) ("PEG") is an
exemplary polymer that has been conjugated to polypeptides. The use
of PEG to derivatize peptide therapeutics has been demonstrated to
reduce the immunogenicity of the peptides. For example, U.S. Pat.
No. 4,179,337 (Davis et al.) discloses non-immunogenic
polypeptides, such as enzymes and peptide hormones coupled to
polyethylene glycol (PEG) or polypropylene glycol. Between 10 and
100 moles of polymer are used per mole polypeptide. Although the in
vivo clearance time of the conjugate is prolonged relative to that
of the polypeptide, only about 15% of the physiological activity is
maintained. Thus, the prolonged circulation half-life is
counterbalanced by the dramatic reduction in peptide potency.
[0009] The loss of peptide activity is directly attributable to the
non-selective nature of the chemistries utilized to conjugate the
water-soluble polymer. The principal mode of attachment of PEG, and
its derivatives, to peptides is a non-specific bonding through a
peptide amino acid residue. For example, U.S. Pat. No. 4,088,538
discloses an enzymatically active polymer-enzyme conjugate of an
enzyme covalently bound to PEG. Similarly, U.S. Pat. No. 4,496,689
discloses a covalently attached complex of .alpha.-1 proteinase
inhibitor with a polymer such as PEG. Abuchowski et al. (J. Biol.
Chem. 252: 3578 (1977) discloses the covalent attachment of MPEG to
an amine group of bovine serum albumin. U.S. Pat. No. 4,414,147
discloses a method of rendering interferon less hydrophobic by
conjugating it to an anhydride of a dicarboxylic acid, such as
poly(ethylene succinic anhydride). PCT WO 87/00056 discloses
conjugation of PEG and poly(oxyethylated) polyols to such proteins
as interferon-.beta., interleukin-2 and immunotoxins. EP 154,316
discloses and claims chemically modified lymphokines, such as IL-2
containing PEG bonded directly to at least one primary amino group
of the lymphokine. U.S. Pat. No. 4,055,635 discloses pharmaceutical
compositions of a water-soluble complex of a proteolytic enzyme
linked covalently to a polymeric substance such as a
polysaccharide.
[0010] Another mode of attaching PEG to peptides is through the
non-specific oxidation of glycosyl residues on a glycopeptide. The
oxidized sugar is utilized as a locus for attaching a PEG moiety to
the peptide. For example M'Timkulu (WO 94/05332) discloses the use
of an amino-PEG to add PEG to a glycoprotein. The glycosyl moieties
are randomly oxidized to the corresponding aldehydes, which are
subsequently coupled to the amino-PEG.
[0011] In each of the methods described above, poly(ethyleneglycol)
is added in a random, non-specific manner to reactive residues on a
peptide backbone. For the production of therapeutic peptides, it is
clearly desirable to utilize a derivitization strategy that results
in the formation of a specifically labeled, readily
characterizable, essentially homogeneous product. A promising route
to preparing specifically labeled peptides is through the use of
enzymes, such as glycosyltransferases to append a modified sugar
moiety onto a peptide. The modified sugar moiety must function as a
substrate for the glycosyltransferase and be appropriately
activated. Hence, synthetic routes that provide facile access to
activated modified sugars are desirable. The present invention
provides such a route.
SUMMARY OF THE INVENTION
[0012] The present invention provides polymeric species, sugars and
activated sugars conjugated to these polymeric species and
nucleotide sugars that include these polymers. The polymeric
species include both water-soluble and water-insoluble species.
Moreover, the polymers are either branched- or straight-chain
polymers. Exemplary sugar moieties include straight-chain and
cyclic structures and aldoses and ketoses.
[0013] The polymeric modifying group can be attached at any
position of the sugar moiety. In the discussion below, the
invention is exemplified by reference to an embodiment in which the
polymeric modifying group is attached to C-5 of a furanose or C-6
of a pyranose. Those of skill will appreciate that the focus of the
discussion is for clarity of illustration, the polymeric moiety can
be attached to other positions of both pyranoses and furanoses
using the methods set forth herein and art-recognized methods.
[0014] In an exemplary embodiment, the invention provides a sugar
or a sugar nucleotide that is conjugated to a polymer:
##STR00001##
[0015] In Formulae I and II, R.sup.1 is H, CH.sub.2OR.sup.7,
COOR.sup.7 or OR.sup.7, in which R.sup.7 represents H, substituted
or unsubstituted alkyl or substituted or unsubstituted heteroalkyl.
R.sup.2 is H, OH or a moiety that includes a nucleotide. An
exemplary R.sup.2 species according to this embodiment has the
formula:
##STR00002##
in which R.sup.8 is a nucleoside.
[0016] The symbols R.sup.3, R.sup.4, R.sup.5, R.sup.6 and R.sup.6'
independently represent H, substituted or unsubstituted alkyl,
OR.sup.9, NHC(O)R.sup.10. The index d is 0 or 1. R.sup.9 and
R.sup.10 are independently selected from H, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl or
sialic acid. At least one of R.sup.3, R.sup.4, R.sup.5, R.sup.6 or
R.sup.6' includes the polymeric modifying moiety e.g., PEG. In an
exemplary embodiment, R.sup.6 and R.sup.6', together with the
carbon to which they are attached are components of the side chain
of sialic acid. In a further exemplary embodiment, this side chain
is functionalized with the polymeric modifying moiety.
[0017] In an exemplary embodiment, the polymeric moiety is bound to
the sugar core, generally through a heteroatom on the core, through
a linker, L, as shown below:
##STR00003##
R.sup.11 is the polymeric moiety and L is selected from a bond and
a linking group. The index w represents and integer selected from
1-6, preferably 1-3 and more preferably 1-2. Exemplary linking
groups include substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl moieties and sialic acid. An exemplary
component of the linker is an acyl moiety.
[0018] When L is a bond it is formed between a reactive functional
group on a precursor of R.sup.11 and a reactive functional group of
complementary reactivity on a precursor of L. L can be in place on
the saccharide core prior to reaction with R.sup.11. Alternatively,
R.sup.11 and L can be incorporated into a preformed cassette that
is subsequently attached to the saccharide core. As set forth
herein, the selection and preparation of precursors with
appropriate reactive functional groups is within the ability of
those skilled in the art. Moreover, coupling the precursors
proceeds by chemistries that are well understood in the art.
[0019] In an exemplary embodiment L is a linking group that is
formed from an amino acid, or small peptide (e.g., 1-4 amino acid
residues) providing a modified sugar in which the polymeric
modifying moiety is attached through a substituted alkyl linker. An
exemplary linker is glycine.
[0020] In an exemplary embodiment, R.sup.6 includes the polymeric
modifying moiety. In another exemplary embodiment, R.sup.6 includes
both the polymeric modifying moiety and a linker, L, joining the
modifying moiety to the remainder of the molecule.
[0021] In an exemplary embodiment, the polymeric modifying moiety
is a branched structure that includes two or more polymeric chains
attached to central moiety. An exemplary structure of a useful
polymeric modifying moiety precursor according to this embodiment
of the invention has the formula:
##STR00004##
The sugars and nucleotide sugars according to this formula are
essentially pure water-soluble polymers. X.sup.3' is a moiety that
includes an ionizable (e.g., COOH, etc.) or other reactive
functional group, see, e.g., infra. C is carbon. X.sup.5 is
preferably a non-reactive group (e.g., H, unsubstitited alkyl,
unsubstituted heteroalkyl). R.sup.12 and R.sup.13 are independently
selected polymeric arms, e.g., nonpeptidic, nonreactive polymeric
arms. X.sup.2 and X.sup.4 are linkage fragments that are preferably
essentially non-reactive under physiological conditions, which may
be the same or different. Alternatively, these linkages can include
one or more moiety that is designed to degrade under
physiologically relevant conditions, e.g., esters, disulfides, etc.
X.sup.2 and X.sup.4 join polymeric arms R.sup.12 and R.sup.13 to C.
When X.sup.3' is reacted with a reactive functional group of
complementary reactivity on a linker, sugar or linker-sugar
cassette, X.sup.3' is converted to a component of linkage fragment
X.sup.3.
[0022] By reaction of the precursor with a suitable sugar or sugar
linker species the invention provides sugars and nucleotide sugars
that have the formulae:
##STR00005##
in which the identity of the radicals represented by the various
symbols is the same as that discussed hereinabove. L.sup.a is a
substituted or unsubstituted alkyl or substituted or unsubstituted
heteroalkyl moiety. In an exemplary embodiment, L.sup.a is a moiety
of the side chain of sialic acid that is functionalized with the
polymeric modifying moiety as shown.
[0023] The polymeric modifying moiety comprises two or more
repeating units that can be water-soluble or essentially insoluble
in water. Exemplary water-soluble polymers of use in the compounds
of the invention include PEG, e.g., m-PEG, PPG, e.g., m-PPG,
polysialic acid, polyglutamate, polyaspartate, polylysine,
polyethyeleneimine, biodegradable polymers (e.g., polylactide,
polyglyceride), and functionalized PEG, e.g., terminal-functionized
PEG.
[0024] The sugar moiety of the polymeric conjugates of the
invention is selected from both natural and unnatural furanoses and
hexanoses. The unnatural saccharides optionally include an
alkylated or acylated hydroxyl and/or amine moiety, e.g., ethers,
esters and amide substituents on the ring. Other unnatural
saccharides include an H, hydroxyl, ether, ester or amide
substituent at a position on the ring at which such a substituent
is not present in the natural saccharide. Alternatively, the
carbohydrate is missing a substituent that would be found in the
carbohydrate from which its name is derived, e.g., deoxy sugars.
Still further exemplary unnatural sugars include both oxidized
(e.g., -onic and -uronic acids) and reduced (sugar alcohols)
carbohydrates. The sugar moiety can be a mono-, oligo- or
poly-saccharide.
[0025] Exemplary natural sugars of use in the present invention
include glucose, glucosamine, galactose, galactosamine, fucose,
mannose, mannosamine, xylanose, ribose, N-acetyl glucose, N-acetyl
glucosamine, N-acetyl galactose, N-acetyl galactosamine, and sialic
acid.
[0026] An exemplary sialic acid-based conjugate has the
formula:
##STR00006##
in which AA is that portion of an amino acid residue that does not
include the carboxyl moiety and NP is a nucleotide phosphate. ONP
can also be replaced by an activating moiety to form an activated
sugar. As will be appreciated by those of skill in the art, the
polymeric modifying moiety-linker can also be attached to the
sialic acid side chain at C-6, C-7 and/or C-9.
[0027] Also provided is a synthetic method for producing an
activated sialic acid-PEG conjugate that is an appropriate
substrate for an enzyme, e.g., a glycosyltransferase. The method
includes the steps: (a) contacting mannosamine with an activated,
N-protected amino acid (or an amino acid functionalized with a
polymeric modifying moiety, a linker precursor or a
linker-polymeric modifying moiety cassette) under conditions
appropriate to form an amide conjugate between the mannosamine and
the N-protected amino acid; (b) contacting the amide conjugate with
pyruvate and sialic acid aldolase under conditions appropriate to
convert the amide conjugate to a sialic acid amide conjugate; (c)
contacting the sialic acid amide conjugate with cytidine
triphosphates, and a synthetase under conditions appropriate to
form a cytidine monophosphate sialic acid amide conjugate; (d)
removing the N-protecting group from the cytidine monophosphate
sialic acid amide conjugate, thereby producing a free amine; and
(e) contacting the free amine with an activated PEG (straight-chain
or branched), thereby forming the cytidine monophosphate sialic
acid-poly(ethylene glycol).
[0028] The nucleoside can be selected from both natural and
unnatural nucleosides. Exemplary natural nucleosides of use in the
present invention include cytosine, thymine, guanine, adenine and
uracil. The art is replete with structures of unnatural nucleosides
and methods of making them.
[0029] Exemplary modified sugar nucleotides of the invention
include polymerically-modified GDP-Man, GDP-Fuc, UDP-Gal,
UDP-GalNAc, UDP-Glc, UDP-GlcNAc, UDP-Glc, UDP-GlcUA and CMP-SA and
the like. Examples include UDP-Gal-2'--NH-PEG, UDP-Glc-2'--NH-PEG,
CMP-5'-PEG-SA and the like. Compounds encompassed by the invention
include those in which the L-R.sup.11 moiety is conjugated to a
furanose or a pyranose, e.g., at C-5 of a furanose or at C-6 of a
pyranose, generally through a heteroatom attached to this carbon
atom.
[0030] When the compound of the invention is a nucleotide sugar, or
activated sugar, the polymeric conjugates of the nucleotide sugars
are generally substrates for an enzyme that transfers the sugar
moiety and its polymeric substituent onto an appropriate acceptor
moiety of a substrate. Accordingly, the invention also provides
substrates modified by glycoconjugation using a polymeric conjugate
of a nucleotide sugar, or activated sugar, and an appropriate
enzyme. Substrates that can be glycoconjugated using a compound of
the invention include peptides, e.g., glycopeptides, peptides,
lipids, e.g., glycolipids and aglycones (sphingosines,
ceramides).
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1 is a table of sialyltransferases for which selected
modified sialic acid nucleotides and activated sugars are
substrates.
[0032] FIG. 2 is a general synthetic scheme of the invention for
preparing a sialic acid-poly(ethylene glycol) conjugate.
[0033] FIG. 3 is a synthetic scheme of the invention for preparing
a sialic acid-glycyl-poly(ethylene glycol) conjugate.
DETAILED DESCRIPTION OF THE INVENTION AND THE EMBODIMENTS
Abbreviations
[0034] Branched and unbranched PEG, poly(ethyleneglycol), e.g.,
m-PEG, methoxy-poly(ethylene glycol); Branched and unbranched PPG,
poly(propyleneglycol), e.g., m-PPG, methoxy-poly(propylene glycol);
Fuc, fucosyl; Gal, galactosyl; GalNAc, N-acetylgalactosaminyl; Glc,
glucosyl; GlcNAc, N-acetylglucosaminyl; Man, mannosyl; ManAc,
mannosaminyl acetate; Sia, sialic acid; and NeuAc,
N-acetylneuraminyl.
Definitions
[0035] The term "sialic acid" refers to any member of a family of
nine-carbon carboxylated sugars. The most common member of the
sialic acid family is N-acetyl-neuraminic acid
(2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onic
acid (often abbreviated as Neu5Ac, NeuAc, or NANA). A second member
of the family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in
which the N-acetyl group of NeuAc is hydroxylated. A third sialic
acid family member is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano
et al. (1986) J. Biol. Chem. 261: 11550-11557; Kanamori et al. J.
Biol. Chem. 265: 21811-21819 (1990)). Also included are
9-substituted sialic acids such as a 9-O--C.sub.1-C.sub.6
acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac,
9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. For review of
the sialic acid family, see, e.g., Varki, Glycobiology 2: 25-40
(1992); Sialic Acids Chemistry, Metabolism and Function, R.
Schauer, Ed. (Springer-Verlag, New York (1992)). The synthesis and
use of sialic acid compounds in a sialylation procedure is
disclosed in international application WO 92/16640, published Oct.
1, 1992.
[0036] As used herein, the term "modified sugar," refers to a
naturally- or non-naturally-occurring carbohydrate that is
enzymatically added onto an amino acid or a glycosyl residue of a
peptide in a process of the invention. The modified sugar is
selected from a number of enzyme substrates including, but not
limited to sugar nucleotides (mono-, di-, and tri-phosphates),
activated sugars (e.g., glycosyl halides, glycosyl mesylates) and
sugars that are neither activated nor nucleotides. The "modified
sugar" is covalently functionalized with a "modifying group."
Useful modifying groups include, but are not limited to,
water-soluble polymers, therapeutic moieties, diagnostic moieties,
biomolecules and the like. The modifying group is preferably not a
naturally occurring, or an unmodified carbohydrate. The locus of
functionalization with the modifying group is selected such that it
does not prevent the "modified sugar" from being added
enzymatically to a peptide.
[0037] The term "water-soluble" refers to moieties that have some
detectable degree of solubility in water. Methods to detect and/or
quantify water solubility are well known in the art. Exemplary
water-soluble polymers include peptides, saccharides, poly(ethers),
poly(amines), poly(carboxylic acids) and the like. Peptides can
have mixed sequences or be composed of a single amino acid, e.g.,
poly(lysine). An exemplary polysaccharide is poly(sialic acid). An
exemplary poly(ether) is poly(ethylene glycol), e.g., m-PEG.
Poly(ethylene imine) is an exemplary polyamine, and poly(acrylic)
acid is a representative poly(carboxylic acid). Exemplary polymers
are typically comprised of 2-8 polymeric units.
[0038] The polymer backbone of the water-soluble polymer can be
poly(ethylene glycol) (i.e. 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. The term PEG includes poly(ethylene glycol) in any
of its forms, including alkoxy PEG, difunctional PEG, multiarmed
PEG, forked PEG, branched PEG, pendent PEG (i.e. PEG or related
polymers having one or more functional groups pendent to the
polymer backbone), or PEG with degradable linkages therein.
[0039] 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. The central branch moiety can also be
derived from several amino acids, such as lysine. The branched
poly(ethylene glycol) can be represented in general form as
R(--PEG-OH).sub.m in which R represents the core moiety, such as
glycerol or pentaerythritol, and m represents the number of arms.
Multi-armed PEG molecules, such as those described in U.S. Pat. No.
5,932,462, which is incorporated by reference herein in its
entirety, can also be used as the polymer backbone.
[0040] Many other polymers are also suitable for the invention.
Polymer backbones that are non-peptidic and water-soluble, with
from 2 to about 300 termini, are particularly useful in the
invention. Examples of suitable polymers include, but are not
limited to, other poly(alkylene glycols), such as poly(propylene
glycol) ("PPG"), copolymers of ethylene glycol and propylene glycol
and the like, poly(oxyethylated polyol), poly(olefinic alcohol),
poly(vinylpyrrolidone), poly(hydroxypropylmethacrylamide),
poly(.alpha.-hydroxy acid), poly(vinyl alcohol), polyphosphazene,
polyoxazoline, poly(N-acryloylmorpholine), such as described in
U.S. Pat. No. 5,629,384, which is incorporated by reference herein
in its entirety, and copolymers, terpolymers, and mixtures thereof.
Although the molecular weight of each chain of the polymer backbone
can vary, it is typically in the range of from about 100 Da to
about 100,000 Da, often from about 6,000 Da to about 80,000 Da.
[0041] The term "glycoconjugation," as used herein, refers to the
enzymatically mediated conjugation of a modified sugar species to
an amino acid or glycosyl residue of a polypeptide, e.g., a mutant
human growth hormone of the present invention. A subgenus of
"glycoconjugation" is "glycol-PEGylation," in which the modifying
group of the modified sugar is poly(ethylene glycol), and alkyl
derivative (e.g., m-PEG) or reactive derivative (e.g.,
H.sub.2N-PEG, HOOC-PEG) thereof.
[0042] The term, "glycosyl linking group," as used herein refers to
a glycosyl residue to which a modifying group (e.g., PEG moiety,
therapeutic moiety, biomolecule) is covalently attached; the
glycosyl linking group joins the modifying group to the remainder
of the conjugate. In the methods of the invention, the "glycosyl
linking group" becomes covalently attached to a glycosylated or
unglycosylated peptide, thereby linking the agent to an amino acid
and/or glycosyl residue on the peptide. A "glycosyl linking group"
is generally derived from a "modified sugar" by the enzymatic
attachment of the "modified sugar" to an amino acid and/or glycosyl
residue of the peptide. The glycosyl linking group can be a
saccharide-derived structure that is degraded during formation of
modifying group-modified sugar cassette (e.g.,
oxidation.fwdarw.Schiff base formation.fwdarw.reduction), or the
glycosyl linking group may be intact. An "intact glycosyl linking
group" refers to a linking group that is derived from a glycosyl
moiety in which the saccharide monomer that links the modifying
group and to the remainder of the conjugate is not degraded, e.g.,
oxidized, e.g., by sodium metaperiodate. "Intact glycosyl linking
groups" of the invention may be derived from a naturally occurring
oligosaccharide by addition of glycosyl unit(s) or removal of one
or more glycosyl unit from a parent saccharide structure.
[0043] Where substituent groups are specified by their conventional
chemical formulae, written from left to right, they equally
encompass the chemically identical substituents, which would result
from writing the structure from right to left, e.g., --CH.sub.2O--
is intended to also recite --OCH.sub.2--.
[0044] The term "alkyl," by itself or as part of another
substituent means, unless otherwise stated, a straight or branched
chain, or cyclic hydrocarbon radical, or combination thereof, which
may be fully saturated, mono- or polyunsaturated and can include
di- and multivalent radicals, having the number of carbon atoms
designated (i.e. C.sub.1-C.sub.10 means one to ten carbons).
Examples of saturated hydrocarbon radicals include, but are not
limited to, groups such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,
(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for
example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An
unsaturated alkyl group is one having one or more double bonds or
triple bonds. Examples of unsaturated alkyl groups include, but are
not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,
2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-
and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The
term "alkyl," unless otherwise noted, is also meant to include
those derivatives of alkyl defined in more detail below, such as
"heteroalkyl." Alkyl groups that are limited to hydrocarbon groups
are termed "homoalkyl".
[0045] The term "alkylene" by itself or as part of another
substituent means a divalent radical derived from an alkane, as
exemplified, but not limited, by
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--, and further includes those
groups described below as "heteroalkylene." Typically, an alkyl (or
alkylene) group will have from 1 to 24 carbon atoms, with those
groups having 10 or fewer carbon atoms being preferred in the
present invention. A "lower alkyl" or "lower alkylene" is a shorter
chain alkyl or alkylene group, generally having eight or fewer
carbon atoms.
[0046] The terms "alkoxy," "alkylamino" and "alkylthio" (or
thioalkoxy) are used in their conventional sense, and refer to
those alkyl groups attached to the remainder of the molecule via an
oxygen atom, an amino group, or a sulfur atom, respectively.
[0047] The term "heteroalkyl," by itself or in combination with
another term, means, unless otherwise stated, a stable straight or
branched chain, or cyclic hydrocarbon radical, or combinations
thereof, consisting of the stated number of carbon atoms and at
least one heteroatom selected from the group consisting of O, N, Si
and S, and wherein the nitrogen and sulfur atoms may optionally be
oxidized and the nitrogen heteroatom may optionally be quaternized.
The heteroatom(s) O, N and S and Si may be placed at any interior
position of the heteroalkyl group or at the position at which the
alkyl group is attached to the remainder of the molecule. Examples
include, but are not limited to, --CH.sub.2--CH.sub.2--O--CH.sub.3,
--CH.sub.2--CH.sub.2--NH--CH.sub.3,
--CH.sub.2--CH.sub.2--N(CH.sub.3)--CH.sub.3,
--CH.sub.2--S--CH.sub.2--CH.sub.3, --CH.sub.2--CH.sub.2,
--S(O)--CH.sub.3, --CH.sub.2--CH.sub.2--S(O).sub.2--CH.sub.3,
--CH.dbd.CH--O--CH.sub.3, --Si(CH.sub.3).sub.3,
--CH.sub.2--CH.dbd.N--OCH.sub.3, and
--CH.dbd.CH--N(CH.sub.3)--CH.sub.3. Up to two heteroatoms may be
consecutive, such as, for example, --CH.sub.2--NH--OCH.sub.3 and
--CH.sub.2--O--Si(CH.sub.3).sub.3. Similarly, the term
"heteroalkylene" by itself or as part of another substituent means
a divalent radical derived from heteroalkyl, as exemplified, but
not limited by, --CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2-- and
--CH.sub.2--S--CH.sub.2--CH.sub.2--NH--CH.sub.2--. For
heteroalkylene groups, heteroatoms can also occupy either or both
of the chain termini (e.g., alkyleneoxy, alkylenedioxy,
alkyleneamino, alkylenediamino, and the like). Still further, for
alkylene and heteroalkylene linking groups, no orientation of the
linking group is implied by the direction in which the formula of
the linking group is written. For example, the formula
--C(O).sub.2R'-- represents both --C(O).sub.2R'-- and
--R'C(O).sub.2--.
[0048] The terms "cycloalkyl" and "heterocycloalkyl", by themselves
or in combination with other terms, represent, unless otherwise
stated, cyclic versions of "alkyl" and "heteroalkyl", respectively.
Additionally, for heterocycloalkyl, a heteroatom can occupy the
position at which the heterocycle is attached to the remainder of
the molecule. Examples of cycloalkyl include, but are not limited
to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl,
cycloheptyl, and the like. Examples of heterocycloalkyl include,
but are not limited to, 1-(1,2,5,6-tetrahydropyridyl),
1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl,
3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,
tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,
2-piperazinyl, and the like.
[0049] The terms "halo" or "halogen," by themselves or as part of
another substituent, mean, unless otherwise stated, a fluorine,
chlorine, bromine, or iodine atom. Additionally, terms such as
"haloalkyl;" are meant to include monohaloalkyl and polyhaloalkyl.
For example, the term "halo(C.sub.1-C.sub.4)alkyl" is mean to
include, but not be limited to, trifluoromethyl,
2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the
like.
[0050] The term "aryl" means, unless otherwise stated, a
polyunsaturated, aromatic, substituent that can be a single ring or
multiple rings (preferably from 1 to 3 rings), which are fused
together or linked covalently. The term "heteroaryl" refers to aryl
groups (or rings) that contain from one to four heteroatoms
selected from N, O, and S, wherein the nitrogen and sulfur atoms
are optionally oxidized, and the nitrogen atom(s) are optionally
quaternized. A heteroaryl group can be attached to the remainder of
the molecule through a heteroatom. Non-limiting examples of aryl
and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl,
4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,
2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,
2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,
5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl,
3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl,
2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,
2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, tetrazolyl,
benzo[b]furanyl, benzo[b]thienyl, 2,3-dihydrobenzo[1,4]dioxin-6-yl,
benzo[1,3]dioxol-5-yl and 6-quinolyl. Substituents for each of the
above noted aryl and heteroaryl ring systems are selected from the
group of acceptable substituents described below.
[0051] For brevity, the term "aryl" when used in combination with
other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both
aryl and heteroaryl rings as defined above. Thus, the term
"arylalkyl" is meant to include those radicals in which an aryl
group is attached to an alkyl group (e.g., benzyl, phenethyl,
pyridylmethyl and the like) including those alkyl groups in which a
carbon atom (e.g., a methylene group) has been replaced by, for
example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl,
3-(1-naphthyloxy)propyl, and the like).
[0052] Each of the above terms (e.g., "alkyl," "heteroalkyl,"
"aryl" and "heteroaryl") is meant to include both substituted and
unsubstituted forms of the indicated radical. Preferred
substituents for each type of radical are provided below.
[0053] Substituents for the alkyl and heteroalkyl radicals
(including those groups often referred to as alkylene, alkenyl,
heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) are
generically referred to as "alkyl group substituents," and they can
be one or more of a variety of groups selected from, but not
limited to: --OR', .dbd.O, .dbd.NR', .dbd.N--OR', --NR'R'', --SR',
-halogen, --SiR'R''R''', --OC(O)R', --C(O)R', --CO.sub.2R',
--CONR'R'', --OC(O)NR'R'', --NR''C(O)R', --NR'--C(O)NR''R''',
--NR''C(O).sub.2R', --NR--C(NR'R''R''').dbd.NR''',
--NR--C(NR'R'').dbd.NR''', --S(O)R', --S(O).sub.2R',
--S(O).sub.2NR'R'', --NRSO.sub.2R', --CN and --NO.sub.2 in a number
ranging from zero to (2m'+1), where m' is the total number of
carbon atoms in such radical. R', R'', R''' and R'''' each
preferably independently refer to hydrogen, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g.,
aryl substituted with 1-3 halogens, substituted or unsubstituted
alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a
compound of the invention includes more than one R group, for
example, each of the R groups is independently selected as are each
R', R'', R''' and R'''' groups when more than one of these groups
is present. When R' and R'' are attached to the same nitrogen atom,
they can be combined with the nitrogen atom to form a 5-, 6-, or
7-membered ring. For example, --NR'R'' is meant to include, but not
be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above
discussion of substituents, one of skill in the art will understand
that the term "alkyl" is meant to include groups including carbon
atoms bound to groups other than hydrogen groups, such as haloalkyl
(e.g., --CF.sub.3 and --CH.sub.2CF.sub.3) and acyl (e.g.,
--C(O)CH.sub.3, --C(O)CF.sub.3, --C(O)CH.sub.2OCH.sub.3, and the
like).
[0054] Similar to the substituents described for the alkyl radical,
substituents for the aryl and heteroaryl groups are generically
referred to as "aryl group substituents." The substituents are
selected from, for example: halogen, --OR', .dbd.O, .dbd.NR',
.dbd.N--OR', --NR'R'', --SR', -halogen, --SiR'R''R''', --OC(O)R',
--C(O)R', --CO.sub.2R', --CONR'R'', --OC(O)NR'R'', --NR''C(O)R',
--NR'--C(O)NR''R''', --NR''C(O).sub.2R',
--NR--C(NR'R''R''').dbd.NR'''', --NR--C(NR'R'').dbd.NR''',
--S(O)R', --S(O).sub.2R', --S(O).sub.2NR'R'', --NRSO.sub.2R', --CN
and --NO.sub.2, --R', --N.sub.3, --CH(Ph).sub.2,
fluoro(C.sub.1-C.sub.4)alkoxy, and fluoro(C.sub.1-C.sub.4)alkyl, in
a number ranging from zero to the total number of open valences on
the aromatic ring system; and where R', R'', R''' and R'''' are
preferably independently selected from hydrogen, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted aryl and substituted or unsubstituted
heteroaryl. When a compound of the invention includes more than one
R group, for example, each of the R groups is independently
selected as are each R', R'', R''' and R'''' groups when more than
one of these groups is present. In the schemes that follow, the
symbol X represents "R" as described above.
[0055] Two of the substituents on adjacent atoms of the aryl or
heteroaryl ring may optionally be replaced with a substituent of
the formula -T-C(O)--(CRR').sub.q-U-, wherein T and U are
independently --NR--, --O--, --CRR'-- or a single bond, and q is an
integer of from 0 to 3. Alternatively, two of the substituents on
adjacent atoms of the aryl or heteroaryl ring may optionally be
replaced with a substituent of the formula
-A-(CH.sub.2).sub.r--B--, wherein A and B are independently
--CRR'--, --O--, --NR--, --S--, --S(O)--, --S(O).sub.2--,
--S(O).sub.2NR'-- or a single bond, and r is an integer of from 1
to 4. One of the single bonds of the new ring so formed may
optionally be replaced with a double bond. Alternatively, two of
the substituents on adjacent atoms of the aryl or heteroaryl ring
may optionally be replaced with a substituent of the formula
--(CRR').sub.s--X--(CR''R''').sub.d--, where s and d are
independently integers of from 0 to 3, and X is --O--, --NR'--,
--S--, --S(O)--, --S(O).sub.2--, or --S(O).sub.2NR'--. The
substituents R, R', R'' and R''' are preferably independently
selected from hydrogen or substituted or unsubstituted
(C.sub.1-C.sub.6)alkyl.
[0056] As used herein, the term "heteroatom" is meant to include
oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).
[0057] The use of reactive derivatives of PEG (or other linkers) to
attach one or more peptide moieties to the linker is within the
scope of the present invention. The invention is not limited by the
identity of the reactive PEG analogue. Many activated derivatives
of poly(ethyleneglycol) are available commercially and in the
literature. It is well within the abilities of one of skill to
choose, and synthesize if necessary, an appropriate activated PEG
derivative with which to prepare a substrate useful in the present
invention. See, Abuchowski et al. Cancer Biochem. Biophys., 7:
175-186 (1984); Abuchowski et al., J. Biol. Chem., 252: 3582-3586
(1977); Jackson et al., Anal. Biochem., 165: 114-127 (1987); Koide
et al., Biochem Biophys. Res. Commun., 111: 659-667 (1983)),
tresylate (Nilsson et al., Methods Enzymol., 104: 56-69 (1984);
Delgado et al., Biotechnol. Appl. Biochem., 12: 119-128 (1990));
N-hydroxysuccinimide derived active esters (Buckmann et al.,
Makromol. Chem., 182: 1379-1384 (1981); Joppich et al., Makromol.
Chem., 180: 1381-1384 (1979); Abuchowski et al., Cancer Biochem.
Biophys., 7: 175-186 (1984); Katre et al. Proc. Natl. Acad. Sci.
U.S.A., 84: 1487-1491 (1987); Kitamura et al., Cancer Res., 51:
4310-4315 (1991); Boccu et al., Z. Naturforsch., 38C: 94-99 (1983),
carbonates (Zalipsky et al., POLY(ETHYLENE GLYCOL) CHEMISTRY:
BIOTECHNICAL AND BIOMEDICAL APPLICATIONS, Harris, Ed., Plenum
Press, New York, 1992, pp. 347-370; Zalipsky et al., Biotechnol.
Appl. Biochem., 15: 100-114 (1992); Veronese et al., Appl. Biochem.
Biotech., 11: 141-152 (1985)), imidazolyl formates (Beauchamp et
al., Anal. Biochem., 131: 25-33 (1983); Berger et al., Blood, 71:
1641-1647 (1988)), 4-dithiopyridines (Woghiren et al., Bioconjugate
Chem., 4: 314-318 (1993)), isocyanates (Byun et al., ASAIO Journal,
M649-M-653 (1992)) and epoxides (U.S. Pat. No. 4,806,595, issued to
Noishiki et al., (1989). Other linking groups include the urethane
linkage between amino groups and activated PEG. See, Veronese, et
al., Appl. Biochem. Biotechnol., 11: 141-152 (1985).
[0058] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics. Naturally occurring amino acids are those encoded by the
genetic code, as well as those amino acids that are later modified,
e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an .alpha. carbon that is bound to a hydrogen, a
carboxyl group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. "Amino acid mimetics" refers to
chemical compounds having a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid.
[0059] "Peptide" refers to a polymer in which the monomers are
amino acids, amino acid analogues and/or amino acid mimetics and
are joined together through amide bonds, alternatively referred to
as a polypeptide. Additionally, unnatural amino acids, for example,
.beta.-alanine, phenylglycine and homoarginine are also included.
Amino acids that are not gene-encoded may also be used in the
present invention. Furthermore, amino acids that have been modified
to include reactive groups, glycosylation sites, polymers,
therapeutic moieties, biomolecules and the like may also be used in
the invention. All of the amino acids used in the present invention
may be either the D- or L-isomer. The L-isomer is generally
preferred. In addition, other peptidomimetics are also useful in
the present invention. As used herein, "peptide" refers to both
glycosylated and unglycosylated peptides. Also included are
peptides that are incompletely glycosylated by a system that
expresses the peptide. For a general review, see, Spatola, A. F.,
in CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS, PEPTIDES AND
PROTEINS, B. Weinstein, eds., Marcel Dekker, New York, p. 267
(1983).
[0060] The term "nucleoside" refers to a glycosylamine that is a
component of a nucleic acid and that comprises a nitrogenous base
linked either to .beta.-D-ribofuranose to form a ribonucleoside, or
to 2-deoxy-.beta.-D-ribofuranose to form a deoxyribonucleoside. The
base may be a purine e.g., adenine or guanosine, or a pyrimidine
e.g., thymidine, cytidine, uridine or pseudouridine. Nucleoside
also includes the unusual nucleoside used by microorganisms.
[0061] The term "targeting moiety," as used herein, refers to
species that will selectively localize in a particular tissue or
region of the body. The localization is mediated by specific
recognition of molecular determinants, molecular size of the
targeting agent or conjugate, ionic interactions, hydrophobic
interactions and the like. Other mechanisms of targeting an agent
to a particular tissue or region are known to those of skill in the
art. Exemplary targeting moieties include antibodies, antibody
fragments, transferrin, HS-glycoprotein, coagulation factors, serum
proteins, .beta.-glycoprotein, G-CSF, GM-CSF, M-CSF, EPO and the
like.
[0062] As used herein, "therapeutic moiety" means any agent useful
for therapy including, but not limited to, antibiotics,
anti-inflammatory agents, anti-tumor drugs, cytotoxins, and
radioactive agents. "Therapeutic moiety" includes prodrugs of
bioactive agents, constructs in which more than one therapeutic
moiety is bound to a carrier, e.g, multivalent agents. Therapeutic
moiety also includes proteins and constructs that include proteins.
Exemplary proteins include, but are not limited to, Erythropoietin
(EPO), Granulocyte Colony Stimulating Factor (GCSF), Granulocyte
Macrophage Colony Stimulating Factor (GMCSF), Interferon (e.g.,
Interferon-.alpha., -.beta., -.gamma.), Interleukin (e.g.,
Interleukin II), serum proteins (e.g., Factors VII, VIIa, VIII, IX,
and X), Human Chorionic Gonadotropin (HCG), Follicle Stimulating
Hormone (FSH) and Lutenizing Hormone (LH) and antibody fusion
proteins (e.g. Tumor Necrosis Factor Receptor ((TNFR)/Fc domain
fusion protein)).
[0063] As used herein, "anti-tumor drug" means any agent useful to
combat cancer including, but not limited to, cytotoxins and agents
such as antimetabolites, alkylating agents, anthracyclines,
antibiotics, antimitotic agents, procarbazine, hydroxyurea,
asparaginase, corticosteroids, interferons and radioactive agents.
Also encompassed within the scope of the term "anti-tumor drug,"
are conjugates of peptides with anti-tumor activity, e.g.
TNF-.alpha.. Conjugates include, but are not limited to those
formed between a therapeutic protein and a glycoprotein of the
invention. A representative conjugate is that formed between PSGL-1
and TNF-.alpha..
[0064] As used herein, "a cytotoxin or cytotoxic agent" means any
agent that is detrimental to cells. Examples include taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracinedione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof. Other toxins include, for example,
ricin, CC-1065 and analogues, the duocarmycins. Still other toxins
include diptheria toxin, and snake venom (e.g., cobra venom).
[0065] As used herein, "a radioactive agent" includes any
radioisotope that is effective in diagnosing or destroying a tumor.
Examples include, but are not limited to, indium-111, cobalt-60.
Additionally, naturally occurring radioactive elements such as
uranium, radium, and thorium, which typically represent mixtures of
radioisotopes, are suitable examples of a radioactive agent. The
metal ions are typically chelated with an organic chelating
moiety.
[0066] Many useful chelating groups, crown ethers, cryptands and
the like are known in the art and can be incorporated into the
compounds of the invention (e.g., EDTA, DTPA, DOTA, NTA, HDTA, etc.
and their phosphonate analogs such as DTPP, EDTP, HDTP, NTP, etc).
See, for example, Pitt et al., "The Design of Chelating Agents for
the Treatment of Iron Overload," In, INORGANIC CHEMISTRY IN BIOLOGY
AND MEDICINE; Martell, Ed.; American Chemical Society, Washington,
D.C., 1980, pp. 279-312; Lindoy, THE CHEMISTRY OF MACROCYCLIC
LIGAND COMPLEXES; Cambridge University Press, Cambridge, 1989;
Dugas, BIOORGANIC CHEMISTRY; Springer-Verlag, New York, 1989, and
references contained therein.
[0067] Additionally, a manifold of routes allowing the attachment
of chelating agents, crown ethers and cyclodextrins to other
molecules is available to those of skill in the art. See, for
example, Meares et al., "Properties of In Vivo Chelate-Tagged
Proteins and Polypeptides." In, MODIFICATION OF PROTEINS: FOOD,
NUTRITIONAL, AND PHARMACOLOGICAL ASPECTS;" Feeney, et al., Eds.,
American Chemical Society, Washington, D.C., 1982, pp. 370-387;
Kasina et al., Bioconjugate Chem., 9: 108-117 (1998); Song et al.,
Bioconjugate Chem., 8: 249-255 (1997).
INTRODUCTION
[0068] The present invention provides polymeric species, and
sugars, activated sugars, and nucleotide sugars that are conjugated
to these polymers. The polymeric conjugates of the nucleotide
sugars are generally substrates for an enzyme that transfers the
sugar moiety and its polymeric substituent onto an appropriate
acceptor moiety of a substrate. Accordingly, the invention also
provides substrates modified by glycoconjugation using a polymeric
conjugate of a nucleotide sugar and an appropriate enzyme.
Substrates that can be glycoconjugated using a compound of the
invention include peptides, e.g., glycopeptides, lipids, e.g.,
glycolipids and aglycones (sphingosines, ceramides).
[0069] As discussed in the preceding sections, art-recognized
chemical methods of covalent PEGylation rely on chemical
conjugation through reactive groups on amino acids or
carbohydrates. Through careful design of the conjugate and the
reaction conditions, useful conjugates have been prepared using
chemically-mediated conjugation strategies. A major shortcoming of
chemical conjugation of polymers to proteins or glycoproteins is
the lack of selectivity of the activated polymers, which often
results in attachment of polymers at sites implicated in protein or
glycoprotein bioactivity. Several strategies have been developed to
address site selective conjugation chemistries, however, only one
universal method suitable for a variety of recombinant proteins has
been developed.
[0070] In contrast to art-recognized methods, the present invention
provides compounds that are of use in a novel strategy for highly
selective, site-directed glycoconjugation of branched water-soluble
polymers, e.g., glyco-PEGylation. In an exemplary embodiment of the
invention, site directed attachment of branched water-soluble
polymers is accomplished by in vitro enzymatic glycosylation of
specific peptide sequences using a nucleotide sugar or activated
sugar of the invention. Glyco-conjugation can be performed
enzymatically utilizing a glycosyltransferase, e.g., a
sialyltransferase, capable of transferring the species branched
water-soluble polymer-sugar, e.g., PEG-sialic acid, to a
glycosylation site ("glyco-PEGylation").
[0071] As discussed above, the present invention provides a
conjugate between a sugar having any desired carbohydrate
structure, modified with a polymeric moiety. Sugar nucleotides and
activated sugars based on these sugar structures are also a
component of the invention. The polymeric modifying moiety is
attached to the sugar moiety by enzymatic means, chemical means or
a combination thereof, thereby producing a modified nucleotide
sugar. The sugars are substituted with the polymeric modifying
moiety at any desired position. In an exemplary embodiment, the
sugar is a furanose that is substituted at one or more of C-1, C-2,
C-3, C-4 or C-5. In another embodiment, the invention provides a
pyranose that is substituted with the polymeric modifying moiety at
one or more of C-1, C-2, C-3, C-4, C-5 or C-6. Preferably, the
polymeric modifying moiety is attached directly to an oxygen,
nitrogen or sulfur pendent from the carbon. Alternatively, the
polymeric modifying moiety is attached to a linker that is
interposed between the sugar and the modifying moiety. The linker
is attached to an oxygen, nitrogen or sulfur pendent from the
selected carbon.
[0072] In a presently preferred embodiment, the polymeric
modifying-moiety is appended to a position, that is selected such
that the resulting conjugate functions as a substrate for an enzyme
used to ligate the modified sugar moiety to another species, e.g.,
peptide, glycopeptide, lipid, glycolipid, etc. Exemplary enzymes
are discussed in greater detail herein and include glycosyl
transferases (sialyl transferases, glucosyl transferases,
galactosyl transferases, N-acetylglucosyl transferases,
N-acetylgalactosyl transferases, mannosyl transferases, fucosyl
transferases, etc.). Exemplary sugar nucleotide and activated sugar
conjugates of the invention also include substrates for mutant
glycosidases and mutant glycoceramidases that are modified to have
synthetic, rather than hydrolytic activity.
[0073] In an exemplary embodiment, the conjugate of the invention
includes a sugar, activated sugar or nucleotide sugar that is
conjugated to one or more polymer, e.g. a branched polymer.
Exemplary polymers include both water-soluble and water-insoluble
species.
[0074] In an exemplary embodiment, the polymeric modifying group is
directly or indirectly attached to a pyranose or a furanose. For
example:
##STR00007##
In Formulae I and II, R.sup.1 is H, CH.sub.2OR.sup.7, COOR.sup.7 or
OR.sup.7, in which R.sup.7 represents H, substituted or
unsubstituted alkyl or substituted or unsubstituted heteroalkyl.
R.sup.2 is H, OH, NH or a moiety that includes a nucleotide. An
exemplary R.sup.2 species according to this embodiment has the
formula:
##STR00008##
in which X.sup.1 represents O or NH and R.sup.8 is a
nucleoside.
[0075] The symbols R.sup.3, R.sup.4, R.sup.5, R.sup.6 and R.sup.6'
independently represent H, substituted or unsubstituted-alkyl,
OR.sup.9, NHC(O)R.sup.10. The index d is 0 or 1. R.sup.9 and
R.sup.10 are independently selected from H, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl or
sialic acid. At least one of R.sup.3, R.sup.4, R.sup.5, R.sup.6,
and R.sup.6' includes the polymeric modifying moiety, e.g., PEG. In
an exemplary embodiment, R.sup.6 and R.sup.6', together with the
carbon to which they are attached are components of the side chain
of sialic acid. In still a further exemplary embodiment, this side
chain is modified with the polymeric modifying moiety (or a
linker-polymeric modifying moiety) at one or more of C-6, C-7 or
C-9.
[0076] The symbols R.sup.3, R.sup.4, R.sup.5 and R.sup.6
independently represent H, OR.sup.9, NHC(O)R.sup.10. R.sup.9 and
R.sup.10 are independently selected from H, substituted or
unsubstituted alkyl or substituted or unsubstituted heteroalkyl. At
least one of R.sup.3, R.sup.4, R.sup.5, R.sup.6, or R.sup.6'
include the polymeric modifying moiety.
[0077] In another exemplary embodiment, the sugar moiety is a
sialic acid moiety that has been oxidized and conjugated to a
polymeric modifying moiety, such as is described in commonly
assigned U.S. Provisional Patent Application No. ______ (Attorney
Docket No. 040853-01-5150), filed Jan. 6, 2005.
[0078] In an exemplary embodiment, the polymeric modifying moiety
is joined to the sugar core through a linker:
##STR00009##
in which R.sup.11 is the polymeric moiety and L is selected from a
bond and a linking group, and w is an integer from 1-6, preferably
1-3 and more preferably, 1-2.
[0079] When L is a bond it is formed between a reactive functional
group on a precursor of R.sup.11 and a reactive functional group of
complementary reactivity on a precursor of L. As set forth herein,
the selection and preparation of precursors with appropriate
reactive functional groups is within the ability of those skilled
in the art. Moreover, combining the precursors proceed by
chemistries that are well-understood in the art.
[0080] In an exemplary embodiment L is a linking group that is
formed from an amino acid, an amino acid mimetic, or small peptide
(e.g., 1-4 amino acid residues) providing a modified sugar in which
the polymeric modifying moiety is attached through a substituted
alkyl linker. The linker is formed through reaction of the amine
moiety and carboxylic acid (or a reactive derivative, e.g., active
ester, acid halide, etc.) of the amino acid with groups of
complementary reactivity on the precursors to L and R.sup.11. The
elements of the conjugate can be conjugated in essentially any
convenient order. For example the precursor to L can be in place on
the saccharide core prior to conjugating the precursors of R.sup.11
and L. Alternatively, an R.sup.11-L cassette, bearing a reactive
functionality on L can be prepared and subsequently linked to the
saccharide through a reactive functional group of complementary
reactivity on this species.
[0081] In an exemplary embodiments, the polymeric modifying moiety
is R.sup.3 and/or R.sup.6. In another exemplary embodiment, R.sup.3
and/or R.sup.6 includes both the polymeric modifying moiety and a
linker, L, joining the polymeric moiety to the remainder of the
molecule. In another exemplary embodiment, the polymeric modifying
moiety is R.sup.3. And, in a further exemplary embodiment, R.sup.3
includes both the polymeric modifying moiety and a linker, L,
joining the polymeric moiety to the remainder of the molecule. In
yet another exemplary embodiment in which the sugar is a sialic
acid, the polymeric modifying moiety is at R.sup.5 or attached at a
position of the sialic acid side chain, e.g., C-9.
Linear Polymer Conjugates
[0082] In an exemplary embodiment, the present invention provides a
sugar or activated sugar conjugate or nucleotide sugar conjugate
that is formed between a linear polymer, such as a water-soluble or
water-insoluble polymer. In the conjugates of the invention, the
polymer is attached to a sugar, activated sugar or sugar
nucleotide. As discussed herein, the polymer is linked to the sugar
moiety, either directly or through a linker.
[0083] An exemplary compound according to this embodiment has a
structure according to Formulae I or II, in which at least one of
R.sup.1, R.sup.3, R.sup.4, R.sup.5 or R.sup.6 has the formula:
##STR00010##
[0084] Another example according to this embodiment has the
formula:
##STR00011##
in which s is an integer from 0 to 20 and R.sup.11 is a linear
polymeric modifying moiety.
[0085] PEG moieties of any molecular weight, e.g., 2 Kda, 5 Kda, 10
Kda, 20 Kda, 30 Kda and 40 Kda are of use in the present
invention.
Branched Polymer Conjugates
[0086] In an exemplary embodiment, the polymeric modifying moiety
is a branched structure that includes two or more polymeric chains
attached to central moiety, having the formula:
##STR00012##
in which R.sup.11 and L are as discussed above and w' is an integer
from 2 to 6, preferably from 2 to 4 and more preferably from 2 to
3.
[0087] An exemplary precursor of use to form the conjugates
according to this embodiment of the invention has the formula:
##STR00013##
[0088] The branched polymer species according to this formula are
essentially pure water-soluble polymers. X.sup.3'is a moiety that
includes an ionizable, e.g., COOH, H.sub.2PO.sub.4, HSO.sub.3,
HPO.sub.3, etc.) or other reactive functional group, e.g., infra. C
is carbon. X.sup.5 is preferably a non-reactive group (e.g., H,
unsubstituted alkyl, unsubstituted heteroalkyl), and can be a
polymeric arm. R.sup.12 and R.sup.13 are independently selected
polymeric arms, e.g., nonpeptidic, nonreactive polymeric arms.
X.sup.2 and X.sup.4 are linkage fragments that are preferably
essentially non-reactive under physiological conditions, which may
be the same or different. Alternatively, these linkages can include
one or more moiety that is designed to degrade under
physiologically relevant conditions, e.g., esters, disulfides, etc.
X.sup.2 and X.sup.4 join polymeric arms R.sup.12 and R.sup.13 to C.
When X.sup.3' is reacted with a reactive functional group of
complementary reactivity on a linker, sugar or linker-sugar
cassette, X.sup.3' is converted to a component of linkage fragment
X.sup.3.
[0089] Exemplary linkage fragments for X.sup.2 and X.sup.4 include
S, SC(O)NH, HNC(O)S, SC(O)O, O, NH, NHC(O), (O)CNH and NHC(O)O, and
OC(O)NH, CH.sub.2S, CH.sub.2O, CH.sub.2CH.sub.2O,
CH.sub.2CH.sub.2S, (CH.sub.2).sub.aO, (CH.sub.2).sub.aS or
(CH.sub.2).sub.aY'-PEG or (CH.sub.2).sub.aY'-PEG wherein Y' is S or
O and a is an integer from 1 to 50.
[0090] In an exemplary embodiment, the precursor (III), or
activated derivative thereof, is bound to the sugar, activated
sugar or sugar nucleotide through a reaction between X.sup.3' and a
group of complementary reactivity on the sugar moiety.
Alternatively, X.sup.3' reacts with a reactive functional group on
a precursor to linker, L. One or more of R.sup.1, R.sup.3, R.sup.4,
R.sup.5 or R.sup.6 of Formulae I and II can include the branched
polymeric modifying moiety.
[0091] In an exemplary embodiment, the moiety:
##STR00014##
is the linker arm, L. In this embodiment, an exemplary linker is
derived from a natural or unnatural amino acid, amino acid analogue
or amino acid mimetic, or a small peptide formed from one or more
such species. For example, certain branched polymers found in the
compounds of the invention have the formula:
##STR00015##
[0092] X.sup.a is a linking moiety that is formed by the reaction
of a reactive functional group on a precursor of the branched
polymeric modifying moiety and the sugar moiety, or a precursor to
a linker. For example, when X.sup.3' is a carboxylic acid, it can
be activated and bound directly to an amine group pendent from an
amino-saccharide (e.g., GalNH.sub.2, GlcNH.sub.2, ManNH.sub.2,
etc.), forming an X.sup.a that is an amide. Additional exemplary
reactive functional groups and activated precursors are described
hereinbelow. The index c represents an integer from 1 to 10. The
other symbols have the same identity as those discussed above.
[0093] In another exemplary embodiment, X.sup.a is a linking moiety
formed with another linker:
##STR00016##
in which X.sup.b is a linking moiety and is independently selected
from those groups set forth for X.sup.a, and L.sup.1 is a bond,
substituted or unsubstituted alkyl or substituted or unsubstituted
heteroalkyl.
[0094] Exemplary species for X.sup.a and X.sup.b include S,
SC(O)NH, HNC(O)S, SC(O)O, O, NH, NHC(O), (O)CNH and NHC(O)O, and
OC(O)NH.
[0095] For example,
##STR00017##
in which s is an integer from 0 to 20 and R.sup.11 is a linear
polymeric modifying moiety.
[0096] In another exemplary embodiment, X.sup.4 is a peptide bond
to R.sup.3, which is an amino acid, di-peptide or tri-peptide in
which the alpha-amine moiety and/or side chain heteroatom is
modified with a polymer.
[0097] In a further exemplary embodiment, R.sup.6 includes the
branched polymeric modifying group and the modified sugar or
nucleotide sugar has a formula that is selected from:
##STR00018##
in which the identity of the radicals represented by the various
symbols is the same as that discussed hereinabove. L.sup.a is a
substituted or unsubstituted alkyl or substituted or unsubstituted
heteroalkyl moiety. In an exemplary embodiment, L.sup.a is a moiety
of the side chain of sialic acid that is functionalized with the
polymeric modifying moiety as shown.
[0098] In yet another exemplary embodiment, the invention provides
sugars and nucleotide sugars that have the formula:
##STR00019##
The identity of the radicals represented by the various symbols is
the same as that discussed hereinabove. As those of skill will
appreciate, the linker arm in Formulae VI and VIII is equally
applicable to other modified sugars set forth herein.
[0099] The embodiments of the invention set forth above are further
exemplified by reference to species in which the polymer is a
water-soluble polymer, particularly poly(ethylene glycol) ("PEG"),
e.g., methoxy-poly(ethylene glycol) ("m-PEG"). Those of skill will
appreciate that the focus in the sections that follow is for
clarity of illustration and the various motifs set forth using PEG
as an exemplary polymer are equally applicable to species in which
a polymer other than PEG is utilized.
Water-Soluble Polymers
[0100] Many water-soluble polymers are known to those of skill in
the art and are useful in practicing the present invention. The
term water-soluble polymer encompasses species such as saccharides
(e.g., dextran, amylose, hyalouronic acid, poly(sialic acid),
heparans, heparins, etc.); poly(amino acids), e.g., poly(aspartic
acid) and poly(glutamic acid); nucleic acids; synthetic polymers
(e.g., poly(acrylic acid), poly(ethers), e.g., poly(ethylene
glycol); peptides, proteins, and the like. A polymer typically
comprises at least two polymeric units. In an exemplary embodiment
the polymer is from 2-25 units. In another exemplary embodiment the
polymer comprises 2-8 polymeric units. The present invention may be
practiced with any water-soluble polymer with the sole limitation
that the polymer must include a point at which the remainder of the
conjugate can be attached.
[0101] Methods for activation of polymers can also be found in WO
94/17039, U.S. Pat. No. 5,324,844, WO 94/18247, WO 94/04193, U.S.
Pat. No. 5,219,564, U.S. Pat. No. 5,122,614, WO 90/13540, U.S. Pat.
No. 5,281,698, and more WO 93/15189, and for conjugation between
activated polymers and peptides, e.g. Coagulation Factor VIII (WO
94/15625), hemoglobin (WO 94/09027), oxygen carrying molecule (U.S.
Pat. No. 4,412,989), ribonuclease and superoxide dismutase
(Veronese at al., App. Biochem. Biotech. 11: 141-45 (1985)).
[0102] Preferred water-soluble polymers are those in which a
substantial proportion of the polymer molecules in a sample of the
polymer are of approximately the same molecular weight; such
polymers are "homodisperse."
[0103] The present invention is further illustrated by reference to
a poly(ethylene glycol) conjugate. Several reviews and monographs
on the functionalization and conjugation of PEG are available. See,
for example, Harris, Macronol. Chem. Phys. C25: 325-373 (1985);
Scouten, Methods in Enzymology 135: 30-65 (1987); Wong et al.,
Enzyme Microb. Technol. 14: 866-874 (1992); Delgado et al.,
Critical Reviews in Therapeutic Drug Carrier Systems 9: 249-304
(1992); Zalipsky, Bioconjugate Chem. 6: 150-165 (1995); and Bhadra,
et al., Pharmazie, 57:5-29 (2002). Routes for preparing reactive
PEG molecules and forming conjugates using the reactive molecules
are known in the art. For example, U.S. Pat. No. 5,672,662
discloses a water soluble and isolatable conjugate of an active
ester of a polymer acid selected from linear or branched
poly(alkylene oxides), poly(oxyethylated polyols), poly(olefinic
alcohols), and poly(acrylomorpholine).
[0104] U.S. Pat. No. 6,376,604 sets forth a method for preparing a
water-soluble 1-benzotriazolylcarbonate ester of a water-soluble
and non-peptidic polymer by reacting a terminal hydroxyl of the
polymer with di(1-benzotriazoyl)carbonate in an organic solvent.
The active ester is used to form conjugates with a biologically
active agent such as a protein or peptide.
[0105] WO 99/45964 describes a conjugate comprising a biologically
active agent and an activated water soluble polymer comprising a
polymer backbone having at least one terminus linked to the polymer
backbone through a stable linkage, wherein at least one terminus
comprises a branching moiety having proximal reactive groups linked
to the branching moiety, in which the biologically active agent is
linked to at least one of the proximal reactive groups. Other
branched poly(ethylene glycols) are described in WO 96/21469, U.S.
Pat. No. 5,932,462 describes a conjugate formed with a branched PEG
molecule that includes a branched terminus that includes reactive
functional groups. The free reactive groups are available to react
with a biologically active species, such as a protein or peptide,
forming conjugates between the poly(ethylene glycol) and the
biologically active species. U.S. Pat. No. 5,446,090 describes a
bifunctional PEG linker and its use in forming conjugates having a
peptide at each of the PEG linker termini.
[0106] Conjugates that include degradable PEG linkages are
described in WO 99/34833; and WO 99/14259, as well as in U.S. Pat.
No. 6,348,558. Such degradable linkages are applicable in the
present invention.
[0107] The art-recognized methods of polymer activation set forth
above are of use in the context of the present invention in the
formation of the branched polymers set forth herein and also for
the conjugation of these branched polymers to other species, e.g.,
sugars, sugar nucleotides and the like.
[0108] Exemplary modifying groups are discussed below. The
modifying groups can be selected for their ability to impart to a
peptide one or more desirable property. Exemplary properties
include, but are not limited to, enhanced pharmacokinetics,
enhanced pharmacodynamics, improved biodistribution, providing a
polyvalent species, improved water solubility, enhanced or
diminished lipophilicity, and tissue targeting.
[0109] Exemplary poly(ethylene glycol) molecules of use in the
invention include, but are not limited to, those having the
formula:
##STR00020##
in which A.sup.2 is H, OH, NH.sub.2, substituted or unsubstituted
alkyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted heteroalkyl, e.g.,
acetal, OHC--, H.sub.2N--(CH.sub.2).sub.q--, HS--(CH.sub.2).sub.q,
or (CH.sub.2).sub.qC(Y.sup.b)Z.sup.b. The index "e" represents an
integer from 1 to 2500. The indices b, d, and q independently
represent integers from 0 to 20. The symbols Z.sup.a and Z.sup.b
independently represent OH, NH.sub.2, leaving groups, e.g.,
imidazole, p-nitrophenyl, HOBT, tetrazole, halide, S--R.sup.a, the
alcohol portion of activated esters; --(CH.sub.2).sub.pC(Y.sup.b)V,
or --(CH.sub.2).sub.pU(CH.sub.2).sub.sC(Y.sup.b).sub.v. The symbol
Y.sup.a represents H(2), .dbd.O, .dbd.S, .dbd.N--R.sup.b. The
symbols X.sup.a, Y.sup.a, A.sup.1, and U independently represent
the moieties O, S, N--R.sup.c. The symbol V represents OH,
NH.sub.2, halogen, S--R.sup.a, the alcohol component of activated
esters, the amine component of activated amides, sugar-nucleotides,
and proteins. The indices p, q, s and v are members independently
selected from the integers from 0 to 20. The symbols R.sup.a,
R.sup.b, and R.sup.c independently represent H, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heterocycloalkyl and substituted or unsubstituted heteroaryl.
[0110] Specific embodiments of linear and branched polymers, e.g.,
PEGs, of use in the invention include:
##STR00021##
and carbonates and active esters of these species, such as:
##STR00022##
can be used to form the linear and branched polymeric species,
linker arm conjugates of these species and conjugates between these
compounds and sugars and nucleotide sugars. The indices e and f are
independently selected from 1 to 2500.
[0111] Other exemplary activating, or leaving groups, appropriate
for activating linear PEGs of use in preparing the compounds set
forth herein include, but are not limited to the species:
##STR00023##
It is well within the abilities of those of skill in the art to
select an appropriate activating group for a selected moiety on the
precursor to the polymeric modifying moiety.
[0112] PEG molecules that are activated with these and other
species and methods of making the activated PEGs are set forth in
WO 04/083259.
[0113] In exemplary embodiments, the branched polymer is a PEG
based upon a cysteine, serine, lysine, di- or tri-lysine core.
Thus, further exemplary branched PEGs include:
##STR00024##
The indices e and f are independently selected from 1 to 2500.
[0114] In yet another embodiment, the branched PEG moiety is based
upon a tri-lysine peptide. The tri-lysine can be mono-, di-, tri-,
or tetra-PEG-ylated. Exemplary species according to this embodiment
have the formulae:
##STR00025##
in which e, f and f' are independently selected integers from 1 to
2500; and q, q' and q'' are independently selected integers from 0
to 20.
[0115] In exemplary embodiments of the invention, the PEG is m-PEG
(5 kD, 10 kD, 20 kD, 30 kD or 40 kD). An exemplary branched PEG
species is a lysine, serine- or cysteine-(m-PEG).sub.2 in which the
m-PEG is a 20 kD m-PEG.
[0116] As will be apparent to those of skill, the branched polymers
of use in the invention include variations on the themes set forth
above. For example the di-lysine-PEG conjugate shown above can
include three polymeric subunits, the third bonded to the
.alpha.-amine shown as unmodified in the structure above.
Similarly, the use of a tri-lysine functionalized with three or
four polymeric subunits is within the scope of the invention.
[0117] Those of skill in the art will appreciate that one or more
of the m-PEG arms of the branched polymer can be replaced by a PEG
moiety with a different terminus, e.g., OH, COOH, NH.sub.2,
C.sub.2-C.sub.10-alkyl, etc. Moreover, the structures above are
readily modified by inserting alkyl linkers (or removing carbon
atoms) between the carbon atom and the functional group of the side
chain. Thus, "homo" derivatives and higher homologues, as well as
lower homologies are within the scope of cores for branched PEGs of
use in the present invention.
[0118] The branched PEG species set forth herein are readily
prepared by methods such as that set forth in the scheme below:
##STR00026##
in which X.sup.b is O, NH or S and r is an integer from 1 to 10.
The indices e and f are independently selected integers from 1 to
2500. Exemplary branched PEG species are 10,000, 15,000, 20,000,
30,000 and 40,000 daltons.
[0119] Thus, according to this scheme, a natural or unnatural amino
acid is contacted with an activated m-PEG derivative, in this case
the tosylate, forming 1 by alkylating the side-chain heteroatom
X.sup.b. The mono-functionalized m-PEG amino acid is submitted to
N-acylation conditions with a reactive m-PEG derivative, thereby
assembling branched m-PEG 2. As one of skill will appreciate, the
tosylate leaving group can be instead any suitable leaving group,
e.g., halogen, mesylate, triflate, etc. Similarly, the reactive
carbonate utilized to acylate the amine can be instead an active
ester, e.g., N-hydroxysuccinimide, etc., or the acid can be
activated in situ using a dehydrating agent such as
dicyclohexylcarbodiimide, carbonyldiimidazole, etc.
[0120] In the exemplary scheme set forth above, the modifying group
is a linear PEG moiety, however, any modifying group, e.g.,
water-soluble polymer, water-insoluble polymer, branched polymer,
therapeutic moiety, etc., can be incorporated in a glycosyl moiety
through
[0121] Further branched polymeric species of use in the compounds
of the invention are exemplified by branched cores functionalized
with PEG, such as the examples set forth below:
##STR00027##
in which R.sup.14 is OH or another reactive functional group. An
exemplary reactive functional group is C(O)Q', in which Q' is
selected such that C(O)Q' is a reactive functional group. Exemplary
species for Q' include halogen, NHS, pentafluorophenyl, HOBT, HOAt,
and p-nitrophenyl. The index "e" and the index "f" are integers
independently selected from 1 to 2500.
[0122] The branched compounds set forth above, and additional
branched compounds of use in the compounds of the invention are
readily prepared from such starting materials as:
##STR00028##
Polymer Modified Sugar Species
[0123] The sugar moiety of the nucleotide sugars of the invention
can be selected from both natural and unnatural furanoses and
hexanoses. The unnatural saccharides optionally include an
alkylated or acylated hydroxyl and/or amine moiety, e.g., ethers,
esters and amide substituents on the ring. Other unnatural
saccharides include an H, hydroxyl, ether, ester or amide
substituent at a position on the ring at which such a substituent
is not present in the natural saccharide. The sugar moiety can be a
mono-, oligo- or poly-saccharide.
[0124] Exemplary natural sugars of use in the present invention
include glucose, galactose, fucose, mannose, xylanose, ribose,
N-acetyl glucose, sialic acid and N-acetyl galactose.
[0125] Similarly, the nucleoside can be selected from both natural
and unnatural or unusual nucleosides. Exemplary natural nucleosides
of use in the present invention include cytosine, thymine, guanine,
adenine and uracil. Unusual nucleosides may include but are not
limited to such molecules as spongouridin and spongothymidin. The
art is replete with structures of unnatural and unusual nucleosides
and methods of making them.
[0126] Exemplary modified sugar nucleotides of the invention
include GDP-Man, GDP-Fuc, UDP-Gal, UDP-Gal-NH.sub.2, UDP-GalNAc,
UDP-Glc, UDP-Glc-NH.sub.2, UDP-GlcNAc, UDP-Glc, UDP-GlcUA and
CMP-Sia. As with the sugars of the invention discussed above, the
sugar nucleotides of the invention can be substituted with a
polymeric modifying moiety (or linker-modifying moiety) at any
position of the saccharide. For example, compounds encompassed by
the invention include those in which the L-R.sup.11 moiety is
conjugated to C-5 of a furanose-based nucleotide sugar or C-6 of a
pyranose-based nucleotide sugar.
[0127] Exemplary moieties attached to the conjugates disclosed
herein include, but are not limited to, PEG derivatives (e.g.,
alkyl-PEG, acyl-PEG, acyl-alkyl-PEG, alkyl-acyl-PEG carbamoyl-PEG,
aryl-PEG), PPG derivatives (e.g., alkyl-PPG, acyl-PPG,
acyl-alkyl-PPG, alkyl-acyl-PPG carbamoyl-PPG, aryl-PPG),
therapeutic moieties, diagnostic moieties, mannose-6-phosphate,
heparin, heparan, SLe.sub.x, mannose, mannose-6-phosphate, Sialyl
Lewis X, FGF, VFGF, proteins, chondroitin, keratan, dermatan,
albumin, integrins, antennary oligosaccharides, peptides and the
like. Methods of conjugating the various modifying groups to a
saccharide moiety are readily accessible to those of skill in the
art (POLY (ETHYLENE GLYCOL CHEMISTRY: BIOTECHNICAL AND BIOMEDICAL
APPLICATIONS, J. Milton Harris, Ed., Plenum Pub. Corp., 1992; POLY
(ETHYLENE GLYCOL) CHEMICAL AND BIOLOGICAL APPLICATIONS, J. Milton
Harris, Ed., ACS Symposium Series No. 680, American Chemical
Society, 1997; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press,
San Diego, 1996; and Dunn et al., Eds. POLYMERIC DRUGS AND DRUG
DELIVERY SYSTEMS, ACS Symposium Series Vol. 469, American Chemical
Society, Washington, D.C. 1991).
[0128] Exemplary sugar nucleotides that of the present invention,
in their modified form, include nucleotide mono-, di- or
triphosphates or analogs thereof of a UDP-glycoside, CMP-glycoside,
or a GDP-glycoside. Even more preferably, the modified sugar
nucleotide is selected from an UDP-galactose, UDP-galactosamine,
UDP-glucose, UDP-glucosamine, GDP-mannose, GDP-fucose, CMP-sialic
acid, or CMP-NeuAc. N-acetylamine derivatives of the sugar
nucleotides are also of use in the method of the invention.
[0129] In other embodiments, the modified sugar is an activated
sugar. Activated modified sugars, which are useful in the present
invention are typically glycosides which have been synthetically
altered to include an activated leaving group. As used herein, the
term "activated leaving group" refers to those moieties, which are
easily displaced in enzyme-regulated nucleophilic substitution
reactions. Many activated sugars are known in the art. See, for
example, Vocadlo et al., In CARBOHYDRATE CHEMISTRY AND BIOLOGY,
Vol. 2, Ernst et al. Ed., Wiley-VCH Verlag: Weinheim, Germany,
2000; Kodama et al., Tetrahedron Lett. 34: 6419 (1993); Lougheed,
et al., J. Biol. Chem. 274: 37717 (1999)).
[0130] Examples of activating groups (leaving groups) include
fluoro, chloro, bromo, tosylate ester, mesylate ester, triflate
ester and the like. Preferred activated leaving groups, for use in
the present invention, are those that do not significantly
sterically encumber the enzymatic transfer of the glycoside to the
acceptor. Accordingly, preferred embodiments of activated glycoside
derivatives include glycosyl fluorides and glycosyl mesylates, with
glycosyl fluorides being particularly preferred. Among the glycosyl
fluorides, .alpha.-galactosyl fluoride, .alpha.-mannosyl fluoride,
.alpha.-glucosyl fluoride, .alpha.-fucosyl fluoride,
.alpha.-xylosyl fluoride, .alpha.-sialyl fluoride,
.alpha.-N-acetylglucosaminyl fluoride,
.alpha.-N-acetylgalactosaminyl fluoride, .beta.-galactosyl
fluoride, .beta.-mannosyl fluoride, .beta.-glucosyl fluoride,
.beta.-fucosyl fluoride, .beta.-xylosyl fluoride, .beta.-sialyl
fluoride, .beta.-N-acetylglucosaminyl fluoride and
.beta.-N-acetylgalactosaminyl fluoride are most preferred.
[0131] By way of illustration, glycosyl fluorides can be prepared
from the free sugar by first acetylating the sugar and then
treating it with HF/pyridine. This generates the thermodynamically
most stable anomer of the protected (acetylated) glycosyl fluoride
(i.e., the .alpha.-glycosyl fluoride). If the less stable anomer
(i.e., the .beta.-glycosyl fluoride) is desired, it can be prepared
by converting the peracetylated sugar with HBr/HOAc or with HCl to
generate the anomeric bromide or chloride. This intermediate is
reacted with a fluoride salt such as silver fluoride to generate
the glycosyl fluoride. Acetylated glycosyl fluorides may be
deprotected by reaction with mild (catalytic) base in methanol
(e.g. NaOMe/MeOH). In addition, many glycosyl fluorides are
commercially available.
[0132] Other activated glycosyl derivatives can be prepared using
conventional methods known to those of skill in the art. For
example, glycosyl mesylates can be prepared by treatment of the
fully benzylated hemiacetal form of the sugar with mesyl chloride,
followed by catalytic hydrogenation to remove the benzyl
groups.
[0133] In a further exemplary embodiment, the modified sugar is an
oligosaccharide having an antennary structure. In another
embodiment, one or more of the termini of the antennae bear the
modifying moiety. When more than one modifying moiety is attached
to an oligosaccharide having an antennary structure, the
oligosaccharide is useful to "amplify" the modifying moiety; each
oligosaccharide unit conjugated to the peptide attaches multiple
copies of the modifying group to the peptide. The general structure
of a typical conjugate of the invention as set forth in the drawing
above, encompasses multivalent species resulting from preparing a
conjugate of the invention utilizing an antennary structure. Many
antennary saccharide structures are known in the art, and the
present method can be practiced with them without limitation.
[0134] In an exemplary embodiment, the activated, modified sugar is
a substrate for a mutant enzyme that transfers the sugar onto an
appropriate acceptor moiety of a substrate. Exemplary mutant
enzymes include, e.g., those set forth in commonly assigned PCT
publications WO03/046150 and WO03/045980
[0135] Water-soluble polymer modified sugar, activated sugar and
nucleotide sugar species in which the sugar moiety is modified with
a water-soluble polymer, e.g., a water-soluble polymer, are of use
in the present invention. An exemplary modified sugar nucleotide
bears a sugar group that is modified through an amine moiety on the
sugar. Modified sugar nucleotides, e.g., saccharyl-amine
derivatives of a sugar nucleotide, are also of use in the methods
of the invention. For example, a saccharyl amine (without the
modifying group) can be enzymatically conjugated to a peptide (or
other species) and the free saccharyl amine moiety subsequently
conjugated to a desired modifying group. Alternatively, the
modified sugar nucleotide can function as a substrate for an enzyme
that transfers the modified sugar to a saccharyl acceptor on a
substrate, e.g., a peptide, glycopeptide, lipid, aglycone,
glycolipid, etc.
[0136] In one embodiment, the sugar is conjugated to a branched
polymeric species, such as those set forth herein.
[0137] In another embodiment, the sugar moiety is a modified sialic
acid. When sialic acid is the sugar, the sialic acid is substituted
with the modifying group at either the 9-position on the pyruvyl
side chain or at the 5-position on the amine moiety that is
normally acetylated in sialic acid.
[0138] In another embodiment, in which the saccharide core is
galactose or glucose, R.sup.5 is NHC(O)Y.
[0139] In an exemplary embodiment, the modified sugar is based upon
a 6-amino-N-acetyl-glycosyl moiety. As shown below for
N-acetylgalactosamine, the 6-amino-sugar moiety is readily prepared
by standard methods:
##STR00029##
[0140] In the scheme above, the index n represents an integer from
1 to 2500, preferably from 10 to 1500, and more preferably from 10
to 1200. The symbol "A" represents an activating group, e.g., a
halo, a component of an activated ester (e.g., a
N-hydroxysuccinimide ester), a component of a carbonate (e.g.,
p-nitrophenyl carbonate) and the like. Those of skill in the art
will appreciate that other PEG-amide nucleotide sugars are readily
prepared by this and analogous methods. Moreover, a branched
polymer, as set forth herein, can be substituted for the linear
PEG.
[0141] Another exemplary polymerically modified nucleotide sugar of
the invention in which the C-6 position is modified has the
formula:
##STR00030##
in which X.sup.6 is a bond or O, J is S or O, and y is 0 or 1. The
indices e and f are independently selected from 1 to 2500.
[0142] In other exemplary embodiments, the amide moiety is replaced
by a group such as a urethane or a urea.
[0143] In the discussion that follows, a number of specific
examples of modified sugars that are useful in practicing the
present invention are set forth. In the exemplary embodiments, a
sialic acid derivative is utilized as the sugar nucleus to which
the modifying group is attached. The focus of the discussion on
sialic acid derivatives is for clarity of illustration only and
should not be construed to limit the scope of the invention. Those
of skill in the art will appreciate that a variety of other sugar
moieties can be activated and derivatized in a manner analogous to
that set forth using sialic acid as an example. For example,
numerous methods are available for modifying galactose, glucose,
N-acetylgalactosamine and fucose to name a few sugar substrates,
which are readily modified by art recognized methods. See, for
example, Elhalabi et al., Curr. Med. Chem. 6: 93 (1999); and
Schafer et al., J. Org. Chem. 65: 24 (2000)).
[0144] In FIG. 2, a general scheme according to the present
invention is set forth. Thus, according to FIG. 2, an amide
conjugate between mannosamine and a protected amino acid is formed
by contacting mannosamine with an N-protected amino acid under
conditions appropriate to form the conjugate. The carboxyl terminus
of the protected amino acid is activated in situ or it is
optionally converted to a reactive group that is stable to storage,
e.g., N-hydroxy-succinimide. The amino acid can be selected from
any natural or non-natural amino acid. Those of skill in the art
understand how to protect side-chain amino acids from undesirably
reacting in the method of the invention. The amide conjugate is
reacted with pyruvate and sialic acid aldolase under conditions
appropriate to convert the amide conjugate to a sialic acid amide
conjugate, which is subsequently converted to a nucleotide
phosphate sialic acid amide conjugate by reaction of the sialic
acid amide conjugate with a precursor of the nucleotide phosphate
and an appropriate enzyme. In an exemplary embodiment, the
precursor is cytidine triphosphate and the enzyme is a synthetase.
Following the formation of the nucleotide sugar, the amino acid
amine is deprotected, providing a free, reactive amine amine. The
amine serves as a locus for conjugating the modifying moiety to the
nucleotide sugar. In FIG. 2, the modifying moiety is exemplified by
a water-soluble polymer, i.e., poly(ethylene glycol), e.g., PEG,
m-PEG, etc.
[0145] The present invention is further exemplified in FIG. 3,
which sets forth a scheme for preparing sialic
acid-glycyl-PEG-cytidine monophosphate. Similar to the scheme set
forth in FIG. 2, that of FIG. 3 originates with mannosamine. The
sugar is conjugated with FMOC-glycine, using the
N-hydroxysuccinimide activated derivative of the protected amino
acid. The resulting amide conjugate is converted to the
corresponding sialic acid by the action of sialic acid aldolase on
the conjugate and pyruvate. The resulting sialic acid conjugate is
converted to the cytidine monophosphate analogue using cytidine
triphosphate and a synthetase. The CMP-analogue is deprotected by
removing the protecting group from the amino acid amine moiety,
converting this moiety to a reactive locus for conjugation. The
amine moiety is reacted with an activated PEG species
(m-PEG-O-nitrophenyl carbonate), thereby forming the sialic
acid-glycyl-PEG-cytidine monophosphate.
[0146] Exemplary sugar cores based upon sialic acid have the
formula:
##STR00031##
in which D is --OH or (R.sup.11).sub.w'-L-. The symbol G represents
H, (R.sup.11).sub.w'-L- or --C(O)(C.sub.1-C.sub.6)alkyl. R.sup.11
is as is as described above. At least one of D and G is
R.sup.11-L-.
[0147] In another embodiment, the invention provides a sugar,
activated sugar or sugar nucleotide that comprises the
structure:
##STR00032##
in which L.sup.2 is as described above in the context of L, e.g., a
bond, substituted or unsubstituted alkyl or substituted or
unsubstituted heteroalkyl group. The index e represents an integer
from 1 to about 2500.
[0148] In another embodiment, the sugar or sugar nucleotide
comprises the structure:
##STR00033##
in which s is selected from the integers from 0 to 20, and e is 1
to 2500.
[0149] Selected sialic acid-based nucleotide sugars functionalized
with a branched polymer have the formula:
##STR00034##
in which AA is an amino acid residue, PEG is poly(ethylene glycol)
or methoxy-poly(ethylene glycol) and NP is a nucleotide, which is
linked to the glycosyl moiety via a phosphodiester bond
("nucleotide phosphate"). Those of skill will appreciate that ONP
can be replaced by an activating moiety as discussed herein.
[0150] In still further embodiments, the sialic acid derivative has
a structure that is a member selected from:
##STR00035##
in which X.sup.6 is a bond or O, and J is S or O. The indices a, b
and c are independently selected from 0 to 20, and e and f are
independently selected from 1 to 2500.
[0151] Moreover, as discussed above, the present invention provides
nucleotide sugars that are modified with a water-soluble polymer,
which is either straight-chain or branched. For example, compounds
having the formula shown below are within the scope of the present
invention:
##STR00036##
in which X.sup.6 is O or a bond, and J is S or O. The indices e and
f are independently selected from 1 to 2500.
[0152] Also provided are conjugates of peptides and glycopeptides,
lipids and glycolipids that include the compositions of the
invention. The conjugates are formed by combining a nucleotide
sugar or activated sugar of the invention and a substrate with an
appropriate acceptor moiety for the sugar moiety and an enzyme for
which the modified nucleotide sugar is a substrate under conditions
appropriate to transfer the modified sugar from the nucleotide
sugar onto the acceptor moiety. For example, the invention provides
conjugates having the following formulae:
##STR00037##
wherein J and X.sup.6 are as discussed above. The indices a, b, c,
e and f are as discussed above.
[0153] Selected compounds of the invention are based on species
having the stereochemistry of mannose, galactose and glucose. The
general formulae of these compounds are:
##STR00038##
in which one of R.sup.3-R.sup.6 is the modifying moiety, e.g.,
polymeric modifying moiety or the polymeric modifying moiety-linker
construct.
[0154] As discussed above, certain compounds of the present
invention are polymeric modified sugar nucleotides. Exemplary sugar
nucleotides that are used in the present invention in their
modified form include nucleotide mono-, di- or triphosphates or
analogs thereof. In a preferred embodiment, the modified sugar
nucleotide is selected from a UDP-glycoside, CMP-glycoside, or a
GDP-glycoside. Even more preferably, the modified sugar nucleotide
is selected from an UDP-galactose, UDP-galactosamine, UDP-glucose,
UDP-glucosamine, GDP-mannose, GDP-fucose, CMP-sialic acid, or
CMP-Sia. In an exemplary embodiment, the nucleotide mono- di- or
tri-phosphate is attached to C-1.
[0155] The saccharyl-amine derivatives of the sugar nucleotides are
also of use in the method of the invention. For example, the
saccharyl amine (without the modifying group) can be enzymatically
conjugated to a peptide (or other species) and the free saccharyl
amine moiety subsequently conjugated to a desired modifying
group.
[0156] The sugar nucleotide conjugates of the invention are
described generically by the formula:
##STR00039##
in which the symbols represent groups as discussed above. When the
sugar core is mannose, the polymeric modifying moiety is preferably
at R.sup.3, R.sup.4 or R.sup.6. For glucose, the polymeric
modifying moiety is optionally at R.sup.5 or R.sup.6. The index "u"
is 0, 1 or 2.
[0157] A further exemplary nucleotide sugar of the invention, based
on GDP mannose has the structure:
##STR00040##
[0158] In a still further exemplary embodiment, the invention
provides a conjugate, based on UDP galactose having the
structure:
##STR00041##
[0159] In another exemplary embodiment, the nucleotide sugar is
based on glucose and has the formula:
##STR00042##
In each of the three preceding formulae, the identity of the
radicals and indices is as discussed above.
[0160] As is apparent to those of skill in the art, the linear PEG
moiety can be replaced by a branched polymeric or other linear
polymeric species as described herein.
[0161] In one embodiment in which the saccharide core is galactose
or glucose, R.sup.5 is NHC(O)Y.
Water-Insoluble Polymers
[0162] In another embodiment, analogous to those discussed above,
the modified sugars include a water-insoluble polymer, rather than
a water-soluble polymer. A water-insoluble polymer, like a water
soluble polymer is typically comprised of at least two polymeric
units. In one exemplary embodiment the polymer is comprised of from
2 to 25 polymeric units. In another exemplary embodiment the
polymer is comprised of 2 to 8 polymeric units. The conjugates of
the invention may also include one or more water-insoluble
polymers. This embodiment of the invention is illustrated by the
use of the conjugate as a vehicle with which to deliver a
therapeutic peptide in a controlled manner. Polymeric drug delivery
systems are known in the art. See, for example, Dunn et al., Eds.
POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium Series
Vol. 469, American Chemical Society, Washington, D.C. 1991. Those
of skill in the art will appreciate that substantially any known
drug delivery system is applicable to the conjugates of the present
invention.
[0163] Representative water-insoluble polymers include, but are not
limited to, polyphosphazines, poly(vinyl alcohols), polyamides,
polycarbonates, polyalkylenes, polyacrylamides, polyalkylene
glycols, polyalkylene oxides, polyalkylene terephthalates,
polyvinyl ethers, polyvinyl esters, polyvinyl halides,
polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes,
poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl
methacrylate), poly(isobutyl methacrylate), poly(hexyl
methacrylate), poly(isodecyl methacrylate), poly(lauryl
methacrylate), poly(phenyl methacrylate), poly(methyl acrylate),
poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl
acrylate) polyethylene, polypropylene, poly(ethylene glycol),
poly(ethylene oxide), poly (ethylene terephthalate), poly(vinyl
acetate), polyvinyl chloride, polystyrene, polyvinyl pyrrolidone,
pluronics and polyvinylphenol and copolymers thereof.
[0164] Synthetically modified natural polymers of use in conjugates
of the invention include, but are not limited to, alkyl celluloses,
hydroxyalkyl celluloses, cellulose ethers, cellulose esters, and
nitrocelluloses. Particularly preferred members of the broad
classes of synthetically modified natural polymers include, but are
not limited to, methyl cellulose, ethyl cellulose, hydroxypropyl
cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl
cellulose, cellulose acetate, cellulose propionate, cellulose
acetate butyrate, cellulose acetate phthalate, carboxymethyl
cellulose, cellulose triacetate, cellulose sulfate sodium salt, and
polymers of acrylic and methacrylic esters and alginic acid.
[0165] These and the other polymers discussed herein can be readily
obtained from commercial sources such as Sigma Chemical Co. (St.
Louis, Mo.), Polysciences (Warrenton, Pa.), Aldrich (Milwaukee,
Wis.), Fluka (Ronkonkoma, N.Y.), and BioRad (Richmond, Calif.), or
else synthesized from monomers obtained from these suppliers using
standard techniques.
[0166] Representative biodegradable polymers of use in the
conjugates of the invention include, but are not limited to,
polylactides, polyglycolides and copolymers thereof, poly(ethylene
terephthalate), poly(butyric acid), poly(valeric acid),
poly(lactide-co-caprolactone), poly(lactide-co-glycolide),
polyanhydrides, polyorthoesters, blends and copolymers thereof. Of
particular use are compositions that form gels, such as those
including collagen, pluronics and the like.
[0167] The polymers of use in the invention include "hybrid`
polymers that include water-insoluble materials having within at
least a portion of their structure, a bioresorbable molecule. An
example of such a polymer is one that includes a water-insoluble
copolymer, which has a bioresorbable region, a hydrophilic region
and a plurality of crosslinkable functional groups per polymer
chain.
[0168] For purposes of the present invention, "water-insoluble
materials" includes materials that are substantially insoluble in
water or water-containing environments. Thus, although certain
regions or segments of the copolymer may be hydrophilic or even
water-soluble, the polymer molecule, as a whole, does not to any
substantial measure dissolve in water.
[0169] For purposes of the present invention, the term
"bioresorbable molecule" includes a region that is capable of being
metabolized or broken down and resorbed and/or eliminated through
normal excretory routes by the body. Such metabolites or break down
products are preferably substantially non-toxic to the body.
[0170] The bioresorbable region may be either hydrophobic or
hydrophilic, so long as the copolymer composition as a whole is not
rendered water-soluble. Thus, the bioresorbable region is selected
based on the preference that the polymer, as a whole, remains
water-insoluble. Accordingly, the relative properties, i.e., the
kinds of functional groups contained by, and the relative
proportions of the bioresorbable region, and the hydrophilic region
are selected to ensure that useful bioresorbable compositions
remain water-insoluble.
[0171] Exemplary resorbable polymers include, for example,
synthetically produced resorbable block copolymers of
poly(.alpha.-hydroxy-carboxylic acid)/poly(oxyalkylene, (see, Cohn
et al., U.S. Pat. No. 4,826,945). These copolymers are not
crosslinked and are water-soluble so that the body can excrete the
degraded block copolymer compositions. See, Younes et al., J
Biomed. Mater. Res. 21: 1301-1316 (1987); and Cohn et al., J
Biomed. Mater. Res. 22: 993-1009 (1988).
[0172] Presently preferred bioresorbable polymers include one or
more components selected from poly(esters), poly(hydroxy acids),
poly(lactones), poly(amides), poly(ester-amides), poly(amino
acids), poly(anhydrides), poly(orthoesters), poly(carbonates),
poly(phosphazines), poly(phosphoesters), poly(thioesters),
polysaccharides and mixtures thereof. More preferably still, the
bioresorbable polymer includes a poly(hydroxy) acid component. Of
the poly(hydroxy) acids, polylactic acid, polyglycolic acid,
polycaproic acid, polybutyric acid, polyvaleric acid and copolymers
and mixtures thereof are preferred.
[0173] In addition to forming fragments that are absorbed in vivo
("bioresorbed"), preferred polymeric coatings for use in the
methods of the invention can also form an excretable and/or
metabolizable fragment.
[0174] Higher order copolymers can also be used in the present
invention. For example, Casey et al., U.S. Pat. No. 4,438,253,
which issued on Mar. 20, 1984, discloses tri-block copolymers
produced from the transesterification of poly(glycolic acid) and an
hydroxyl-ended poly(alkylene glycol). Such compositions are
disclosed for use as resorbable monofilament sutures. The
flexibility of such compositions is controlled by the incorporation
of an aromatic orthocarbonate, such as tetra-p-tolyl orthocarbonate
into the copolymer structure.
[0175] Other polymers based on lactic and/or glycolic acids can
also be utilized. For example, Spinu, U.S. Pat. No. 5,202,413,
which issued on Apr. 13, 1993, discloses biodegradable multi-block
copolymers having sequentially ordered blocks of polylactide and/or
polyglycolide produced by ring-opening polymerization of lactide
and/or glycolide onto either an oligomeric diol or a diamine
residue followed by chain extension with a di-functional compound,
such as, a diisocyanate, diacylchloride or dichlorosilane.
[0176] Bioresorbable regions of coatings useful in the present
invention can be designed to be hydrolytically and/or enzymatically
cleavable. For purposes of the present invention, "hydrolytically
cleavable" refers to the susceptibility of the copolymer,
especially the bioresorbable region, to hydrolysis in water or a
water-containing environment. Similarly, "enzymatically cleavable"
as used herein refers to the susceptibility of the copolymer,
especially the bioresorbable region, to cleavage by endogenous or
exogenous enzymes.
[0177] When placed within the body, the hydrophilic region can be
processed into excretable and/or metabolizable fragments. Thus, the
hydrophilic region can include, for example, polyethers,
polyalkylene oxides, polyols, poly(vinyl pyrrolidine), poly(vinyl
alcohol), poly(alkyl oxazolines), polysaccharides, carbohydrates,
peptides, proteins and copolymers and mixtures thereof.
Furthermore, the hydrophilic region can also be, for example, a
poly(alkylene) oxide. Such poly(alkylene) oxides can include, for
example, poly(ethylene) oxide, poly(propylene) oxide and mixtures
and copolymers thereof.
[0178] Polymers that are components of hydrogels are also useful in
the present invention. Hydrogels are polymeric materials that are
capable of absorbing relatively large quantities of water. Examples
of hydrogel forming compounds include, but are not limited to,
polyacrylic acids, sodium carboxymethylcellulose, polyvinyl
alcohol, polyvinyl pyrrolidine, gelatin, carrageenan and other
polysaccharides, hydroxyethylenemethacrylic acid (HEMA), as well as
derivatives thereof, and the like. Hydrogels can be produced that
are stable, biodegradable and bioresorbable. Moreover, hydrogel
compositions can include subunits that exhibit one or more of these
properties.
[0179] Bio-compatible hydrogel compositions whose integrity can be
controlled through crosslinking are known and are presently
preferred for use in the methods of the invention. For example,
Hubbell et al., U.S. Pat. Nos. 5,410,016, which issued on Apr. 25,
1995 and 5,529,914, which issued on Jun. 25, 1996, disclose
water-soluble systems, which are crosslinked block copolymers
having a water-soluble central block segment sandwiched between two
hydrolytically labile extensions. Such copolymers are further
end-capped with photopolymerizable acrylate functionalities. When
crosslinked, these systems become hydrogels. The water soluble
central block of such copolymers can include poly(ethylene glycol);
whereas, the hydrolytically labile extensions can be a
poly(.alpha.-hydroxy acid), such as polyglycolic acid or polylactic
acid. See, Sawhney et al., Macromolecules 26: 581-587 (1993).
[0180] In another embodiment, the gel is a thermoreversible gel.
Thermoreversible gels including components, such as pluronics,
collagen, gelatin, hyalouronic acid, polysaccharides, polyurethane
hydrogel, polyurethane-urea hydrogel and combinations thereof are
presently preferred.
[0181] In yet another exemplary embodiment, the conjugate of the
invention includes a component of a liposome. Liposomes can be
prepared according to methods known to those skilled in the art,
for example, as described in Eppstein et al., U.S. Pat. No.
4,522,811, which issued on Jun. 11, 1985. For example, liposome
formulations may be prepared by dissolving appropriate lipid(s)
(such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl
choline, arachadoyl phosphatidyl choline, and cholesterol) in an
inorganic solvent that is then evaporated, leaving behind a thin
film of dried lipid on the surface of the container. An aqueous
solution of the active compound or its pharmaceutically acceptable
salt is then introduced into the container. The container is then
swirled by hand to free lipid material from the sides of the
container and to disperse lipid aggregates, thereby forming the
liposomal suspension.
[0182] The above-recited microparticles and methods of preparing
the microparticles are offered by way of example and they are not
intended to define the scope of microparticles of use in the
present invention. It will be apparent to those of skill in the art
that an array of microparticles, fabricated by different methods,
is of use in the present invention.
[0183] The structural formats discussed above in the context of the
water-soluble polymers, both straight-chain and branched are
generally applicable with respect to the water-insoluble polymers
as well. Thus, for example, the cysteine, serine, dilysine, and
trilysine branching cores can be functionalized with two
water-insoluble polymer moieties. The methods used to produce these
species are generally closely analogous to those used to produce
the water-soluble polymers.
[0184] The in vivo half-life of therapeutic glycopeptides can also
be enhanced with PEG moieties such as polyethylene glycol (PEG).
For example, chemical modification of proteins with PEG
(PEGylation) increases their molecular size and decreases their
surface- and functional group-accessibility, each of which are
dependent on the size of the PEG attached to the protein. This
results in an improvement of plasma half-lives and in
proteolytic-stability, and a decrease in immunogenicity and hepatic
uptake (Chaffee et al. J. Clin. Invest. 89: 1643-1651 (1992);
Pyatak et al. Res. Commun. Chem. Pathol Pharmacol. 29: 113-127
(1980)). PEGylation of interleukin-2 has been reported to increase
its antitumor potency in vivo (Katre et al. Proc. Natl. Acad. Sci.
USA. 84: 1487-1491 (1987)) and PEGylation of a F(ab')2 derived from
the monoclonal antibody A7 has improved its tumor localization
(Kitamura et al. Biochem. Biophys. Res. Commun. 28: 1387-1394
(1990)). Thus, in another embodiment, the in vivo half-life of a
peptide derivatized with a PEG moiety by a method of the invention
is increased relevant to the in vivo half-life of the
non-derivatized peptide.
[0185] The increase in peptide in vivo half-life is best expressed
as a range of percent increase in this quantity. The lower end of
the range of percent increase is about 40%, about 60%, about 80%,
about 100%, about 150% or about 200%. The upper end of the range is
about 60%, about 80%, about 100%, about 150%, or more than about
250%.
Preparation of Modified Sugars
[0186] In general, the sugar moiety and the modifying group are
linked together through the use of reactive groups, which are
typically transformed by the linking process into a new organic
functional group or unreactive species. The sugar reactive
functional group(s), is located at any position on the sugar
moiety. Reactive groups and classes of reactions useful in
practicing the present invention are generally those that are well
known in the art of bioconjugate chemistry. Currently favored
classes of reactions available with reactive sugar moieties are
those, which proceed under relatively mild conditions. These
include, but are not limited to nucleophilic substitutions (e.g.,
reactions of amines and alcohols with acyl halides, active esters),
electrophilic substitutions (e.g., enamine reactions) and additions
to carbon-carbon and carbon-heteroatom multiple bonds (e.g.,
Michael reaction, Diels-Alder addition). These and other useful
reactions are discussed in, for example, March, ADVANCED ORGANIC
CHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985;
Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego,
1996; and Feeney et al., MODIFICATON OF PROTEINS; Advances in
Chemistry Series, Vol. 198, American Chemical Society, Washington,
D.C., 1982.
[0187] Useful reactive functional groups pendent from a sugar
nucleus, linker precursor or polymeric modifying moiety precursor
include, but are not limited to: [0188] (a) carboxyl groups and
various derivatives thereof including, but not limited to,
N-hydroxysuccinimide esters, N-hydroxybenztriazole esters, acid
halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl,
alkenyl, alkynyl and aromatic esters; [0189] (b) hydroxyl groups,
which can be converted to, e.g., esters, ethers, aldehydes, etc.
[0190] (c) haloalkyl groups, wherein the halide can be later
displaced with a nucleophilic group such as, for example, an amine,
a carboxylate anion, thiol anion, carbanion, or an alkoxide ion,
thereby resulting in the covalent attachment of a new group at the
functional group of the halogen atom; [0191] (d) dienophile groups,
which are capable of participating in Diels-Alder reactions such
as, for example, maleimido groups; [0192] (e) aldehyde or ketone
groups, such that subsequent derivatization is possible via
formation of carbonyl derivatives such as, for example, imines,
hydrazones, semicarbazones or oximes, or via such mechanisms as
Grignard addition or alkyllithium addition; [0193] (f) sulfonyl
halide groups for subsequent reaction with amines, for example, to
form sulfonamides; [0194] (g) thiol groups, which can be, for
example, converted to disulfides or reacted with acyl halides;
[0195] (h) amine or sulfhydryl groups, which can be, for example,
acylated, alkylated or oxidized; [0196] (i) alkenes, which can
undergo, for example, cycloadditions, acylation, Michael addition,
etc; and [0197] (j) epoxides, which can react with, for example,
amines and hydroxyl compounds.
[0198] The reactive functional groups can be chosen such that they
do not participate in, or interfere with, the reactions necessary
to assemble the reactive sugar nucleus or modifying group.
Alternatively, a reactive functional group can be protected from
participating in the reaction by the presence of a protecting
group. Those of skill in the art understand how to protect a
particular functional group such that it does not interfere with a
chosen set of reaction conditions. For examples of useful
protecting groups, see, for example, Greene et al., PROTECTIVE
GROUPS IN ORGANIC SYNTHESIS, John Wiley & Sons, New York,
1991.
[0199] In the discussion that follows, a number of specific
examples of modified sugars that are useful in practicing the
present invention are set forth. In the exemplary embodiments, a
sialic acid derivative is utilized as the sugar nucleus to which
the modifying group is attached. The focus of the discussion on
sialic acid derivatives is for clarity of illustration only and
should not be construed to limit the scope of the invention. Those
of skill in the art will appreciate that a variety of other sugar
moieties can be activated and derivatized in a manner analogous to
that set forth using sialic acid as an example. For example,
numerous methods are available for modifying galactose, glucose,
N-acetylgalactosamine and fucose to name a few sugar substrates,
which are readily modified by art recognized methods. See, for
example, Elhalabi et al., Curr. Med. Chem. 6: 93 (1999); and
Schafer et al., J. Org. Chem. 65: 24 (2000)).
[0200] In Scheme 1 below, the amino glycoside 1, is treated with
the active ester of a protected amino acid (e.g., glycine)
derivative, converting the sugar amine residue into the
corresponding protected amino acid amide adduct. The adduct is
treated with an aldolase to form .alpha.-hydroxy carboxylate 2.
Compound 2 is converted to the corresponding CMP derivative by the
action of CMP-SA synthetase, followed by catalytic hydrogenation of
the CMP derivative to produce compound 3. The amine introduced via
formation of the glycine adduct is utilized as a locus of PEG or
PPG attachment by reacting compound 3 with an activated (m-) PEG or
(m-) PPG derivative (e.g., PEG-C(O)NHS, PPG-C(O)NHS), producing 4
or 5, respectively.
##STR00043##
As those of skill will appreciate, the polymeric modifying moiety
can also be a branched moiety, such as those described herein.
[0201] An exemplary scheme for preparing the branched
polymerically-modified sugars of the invention is provided
below:
##STR00044## ##STR00045##
[0202] Another exemplary scheme for preparing the
polymerically-modified sugars of the invention is set forth
below:
##STR00046##
[0203] Table 1 sets forth representative examples of sugar
monophosphates that are derivatized with a polymeric modifying
moiety, e.g., a branched- or straight-chain PEG or PPG moiety.
Certain of the compounds of Table 1 are prepared by the method of
Scheme 1. Other derivatives are prepared by art-recognized methods.
See, for example, Keppler et al., Glycobiology 11: 11R (2001); and
Charter et al., Glycobiology 10: 1049 (2000)). Other amine reactive
polymeric modifying moiety precursors and components, e.g., PEG and
PPG analogues are commercially available, or they can be prepared
by methods readily accessible to those of skill in the art.
TABLE-US-00001 TABLE 1 ##STR00047## CMP-SA-5-NH--R ##STR00048##
CMP-NeuAc-9-O--R ##STR00049## CMP-KDN-5-O--R ##STR00050##
CMP-NeuAc-9-NH--R ##STR00051## CMP-NeuAc-8-O--R ##STR00052##
CMP-NeuAc-8-NH--R ##STR00053## CMP-NeuAc-7-O--R ##STR00054##
CMP-NeuAc-7-NH--R ##STR00055## CMP-NeuAc-4-O--R ##STR00056##
CMP-NeuAc-4-NH--R
in which R is the polymeric (branched or straight-chain) modifying
moiety.
[0204] The modified sugar phosphates of use in practicing the
present invention can be substituted in other positions as well as
those set forth above. Presently preferred substitutions of sialic
acid are set forth in the formula below:
##STR00057##
in which one or more of X.sup.c, Y.sup.a, Y.sup.b, Y.sup.c and Z is
a linking group, which is preferably selected from --O--, --N(H)--,
--S, CH.sub.2--, and N(R).sub.2. When X.sup.c, Y.sup.a, Y.sup.b,
Y.sup.c and Z is a linking group, it is attached to the polymeric
modifying moiety as represented by R.sup.c, R.sup.d, R.sup.e,
R.sup.f and R.sup.g. Alternatively, these symbols represent a
linker that is bound to a branched- or straight-chain water-soluble
or water-insoluble polymer, therapeutic moiety, biomolecule or
other moiety. When R.sup.c, R.sup.d, R.sup.e, R.sup.f or R.sup.g is
not a polymeric modifying moiety, the combination of
X.sup.cR.sup.c, Y.sup.aR.sup.d, Y.sup.bR.sup.e, Y.sup.cR.sup.f or
ZR.sup.g is H, OH or NC(O)CH.sub.3.
[0205] Also provided is a synthetic method for producing an
activated sialic acid-polymeric modifying group conjugate that is
an appropriate substrate for an enzyme that transfers the modified
sugar moiety onto an acceptor, e.g., a glycosyltransferase. The
method includes the steps: (a) contacting mannosamine with an
activated, N-protected amino acid (or an amino acid functionalized
with a polymeric modifying moiety, a linker precursor or a
linker-polymeric modifying moiety cassette) under conditions
appropriate to form an amide conjugate between the mannosamine and
the N-protected amino acid; (b) contacting the amide conjugate with
pyruvate and sialic acid aldolase under conditions appropriate to
convert the amide conjugate to a sialic acid amide conjugate; (c)
contacting the sialic acid amide conjugate with cytidine
triphosphates, and a synthetase under conditions appropriate to
form a cytidine monophosphate sialic acid amide conjugate; (d)
removing the N-protecting group from the cytidine monophosphate
sialic acid amide conjugate, thereby producing a free amine; and
(e) contacting the free amine with an activated PEG (straight-chain
or branched), thereby forming the cytidine monophosphate sialic
acid-poly(ethylene glycol).
Cross-Linking Groups
[0206] Preparation of the modified sugar for use in the methods of
the present invention includes attachment of a modifying group to a
sugar residue and forming a stable adduct, which is a substrate for
a glycosyltransferase. The sugar and modifying group can be coupled
by a zero- or higher-order cross-linking agent. Exemplary
bifunctional compounds which can be used for attaching modifying
groups to carbohydrate moieties include, but are not limited to,
bifunctional poly(ethyleneglycols), polyamides, polyethers,
polyesters and the like. General approaches for linking
carbohydrates to other molecules are known in the literature. See,
for example, Lee et al., Biochemistry 28: 1856 (1989); Bhatia et
al., Anal. Biochem. 178: 408 (1989); Janda et al., J. Am. Chem.
Soc. 112: 8886 (1990) and Bednarski et al., WO 92/18135. In the
discussion that follows, the reactive groups are treated as benign
on the sugar moiety of the nascent modified sugar. The focus of the
discussion is for clarity of illustration. Those of skill in the
art will appreciate that the discussion is relevant to reactive
groups on the modifying group as well.
[0207] An exemplary strategy involves incorporation of a protected
sulfhydryl onto the sugar using the heterobifunctional crosslinker
SPDP (n-succinimidyl-3-(2-pyridyldithio)propionate and then
deprotecting the sulfhydryl for formation of a disulfide bond with
another sulfhydryl on the modifying group.
[0208] A variety of reagents are used to modify the components of
the modified sugar with intramolecular chemical crosslinks (for
reviews of crosslinking reagents and crosslinking procedures see:
Wold, F., Meth. Enzymol. 25: 623-651, 1972; Weetall, H. H., and
Cooney, D. A., In: ENZYMES AS DRUGS. (Holcenberg, and Roberts,
eds.) pp. 395-442, Wiley, New York, 1981; Ji, T. H., Meth. Enzymol.
91: 580-609, 1983; Mattson et al., Mol. Biol. Rep. 17: 167-183,
1993, all of which are incorporated herein by reference). Preferred
crosslinking reagents are derived from various zero-length,
homo-bifunctional, and hetero-bifunctional crosslinking reagents.
Zero-length crosslinking reagents include direct conjugation of two
intrinsic chemical groups with no introduction of extrinsic
material.
Conjugation of Modified Sugars to Peptides
[0209] The modified sugars are conjugated to a glycosylated or
non-glycosylated peptide using an appropriate enzyme to mediate the
conjugation. Thus, the compounds of the invention, particularly the
nucleotide sugars are preferably substrates for enzymes that
transfer sugar moieties from a nucleotide sugar onto an amino acid,
glycosyl, or aglycone acceptor moiety. Nucleotide sugars that act
as sugar donors for acceptors, e.g., galactosyl acceptors, e.g.,
GalNAc, Gal.beta.1,4GlcNAc, Gal.beta.1,4GalNAc, Gal.beta.1,3GalNAc,
lacto-N-tetraose, Gal.beta.1,3GlcNAc, Gal.beta.1,3Ara,
Gal.beta.1,6GlcNAc, Gal.beta.1,4Glc (lactose), and other acceptors
well known to those of skill in the art (see, e.g., Paulson et al.,
J. Biol. Chem. 253: 5617-5624 (1978)).
[0210] Exemplary enzymes for which the modified nucleotide sugars
of the invention are substrates include glycosyltransferases. The
glycosyltransferase can be cloned, or isolated from any source.
Many cloned glycosyltransferases are known, as are their
polynucleotide sequences. See, e.g., "The WWW Guide To Cloned
Glycosyltransferases," (http://www.vei.co.uk/TGN/gt_guide.htm).
Glycosyltransferase amino acid sequences and nucleotide sequences
encoding glycosyltransferases from which the amino acid sequences
can be deduced are also found in various publicly available
databases, including GenBank, Swiss-Prot, EMBL, and others.
[0211] Glycosyltransferases for which the compounds of the
invention are substrates include, but are not limited to,
galactosyltransferases, fucosyltransferases, glucosyltransferases,
N-acetylgalactosaminyltransferases,
N-acetylglucosaminyltransferases, glucuronyltransferases,
sialyltransferases, mannosyltransferases, glucuronic acid
transferases, galacturonic acid transferases, and
oligosaccharyltransferases. Suitable glycosyltransferases include
those obtained from eukaryotes, as well as from prokaryotes.
[0212] In some embodiments, the compound of the invention is a
substrate for a fucosyltransferase. Fucosyltransferases are
generally known to those of skill in the art, and are exemplified
by enzymes that transfer L-fucose from GDP-fucose to a hydroxy
position of an acceptor sugar.
[0213] In another group of embodiments, the compound is a substrate
for a galactosyltransferase. Exemplary galactosyltransferases
include .alpha.(1,3) galactosyltransferases (E.C. No. 2.4.1.151,
see, e.g., Dabkowski et al., Transplant Proc. 25:2921 (1993) and
Yamamoto et al. Nature 345: 229-233 (1990), bovine (GenBank j04989,
Joziasse et al., J. Biol. Chem. 264: 14290-14297 (1989)), murine
(GenBank m26925; Larsen et al., Proc. Nat'l. Acad. Sci. USA 86:
8227-8231 (1989)), porcine (GenBank L36152; Strahan et al.,
Immunogenetics 41: 101-105 (1995)). Another suitable .alpha.1,3
galactosyltransferase is that which is involved in synthesis of the
blood group B antigen (EC 2.4.1.37, Yamamoto et al., J. Biol. Chem.
265: 1146-1151 (1990) human)). Yet a further exemplary
galactosyltransferase is core Gal-T1. Still further examples
include .beta.(1,4) galactosyltransferases, which include, for
example, EC 2.4.1.90 (LacNAc synthetase) and EC 2.4.1.22 (lactose
synthetase) (bovine (D'Agostaro et al., Eur. J. Biochem. 183:
211-217 (1989)), human (Masri et al., Biochem. Biophys. Res.
Commun. 157: 657-663 (1988)), murine (Nakazawa et al., J. Biochem.
104: 165-168 (1988)), as well as E.C. 2.4.1.38 and the ceramide
galactosyltransferase (EC 2.4.1.45, Stahl et al., J. Neurosci. Res.
38: 234-242 (1994)). Other suitable galactosyltransferases include,
for example, .alpha.1,2 galactosyltransferases (from e.g.,
Schizosaccharomyces pombe, Chapell et al., Mol. Biol. Cell 5:
519-528 (1994)). Also suitable in the practice of the invention are
soluble forms of .alpha.1,3-galactosyltransferase such as that
reported by Cho et al., J. Biol. Chem., 272: 13622-13628
(1997).
a) Sialyltransferases
[0214] Sialyltransferases are another type of glycosyltransferase
for which the compounds of the invention are substrates. Examples
include ST3Gal III (e.g., a rat or human ST3Gal III), ST3Gal IV,
ST3Gal I, ST6Gal I, ST3Gal V, ST6Gal II, ST6GalNAc I, ST6GalNAc II,
and ST6GalNAc III (the sialyltransferase nomenclature used herein
is as described in Tsuji et al., Glycobiology 6: v-xiv (1996)). An
exemplary .alpha.(2,3)sialyltransferase referred to as
.alpha.(2,3)sialyltransferase (EC 2.4.99.6) transfers sialic acid
to the non-reducing terminal Gal of a Gal.beta.1.fwdarw.3Glc
disaccharide or glycoside. See, Van den Eijnden et al., J. Biol.
Chem. 256: 3159 (1981), Weinstein et al., J. Biol. Chem. 257: 13845
(1982) and Wen et al., J. Biol. Chem. 267: 21011 (1992). Another
exemplary .alpha.2,3-sialyltransferase (EC 2.4.99.4) transfers
sialic acid to the non-reducing terminal Gal of the disaccharide or
glycoside. see, Rearick et al., J. Biol. Chem. 254: 4444 (1979) and
Gillespie et al., J. Biol. Chem. 267: 21004 (1992). Further
exemplary enzymes include Gal-.beta.-1,4-GlcNAc .alpha.-2,6
sialyltransferase (See, Kurosawa et al. Eur. J. Biochem. 219:
375-381 (1994)). Other sialyltransferases for which the compounds
of the invention are substrates include those that form polysialic
acids. Examples include the .alpha.-2,8-polysialyltransferases,
e.g., ST8SiaI, ST8SiaII, ST8SiaIII, ST8SiaIV and ST8SiaV. See for
example, Angata et al. J. Biol. Chem. 275: 18594-18601 (2000); Kono
et al., J. Biol. Chem. 271: 29366-29371 (1996); Greiner et al.,
Infect. Immun. 72: 4249-4260 (2004); and Jones et al., J. Biol.
Chem. 277: 14598-14611 (2002).
[0215] An example of a sialyltransferase that is useful in the
claimed methods is ST3Gal III, which is also referred to as
.alpha.(2,3)sialyltransferase (EC 2.4.99.6). This enzyme catalyzes
the transfer of sialic acid to the Gal of a Gal.beta.1,3GlcNAc or
Gal.beta.1,4GlcNAc glycoside (see, e.g., Wen et al., J. Biol. Chem.
267: 21011 (1992); Van den Eijnden et al., J. Biol. Chem. 256: 3159
(1991)). Still further sialyltransferases include those isolated
from Campylobacter jejuni, including the .alpha.(2,3). See, e.g,
WO99/49051.
[0216] Preferably, the compounds of the invention are substrates
for an enzyme that transfers the modifies sialic acid to the
sequence Gal.beta.1,4GlcNAc-, the most common penultimate sequence
underlying the terminal sialic acid on fully sialylated
carbohydrate structures.
b) GalNAc Transferases
[0217] Selected compounds of the invention are substrates for
N-acetylgalactosaminyltransferases. Exemplary
N-acetylgalactosaminyltransferases include, but are not limited to,
.alpha.(1,3) N-acetylgalactosaminyltransferase, .beta.(1,4)
N-acetylgalactosaminyltransferases (Nagata et al., J. Biol. Chem.
267: 12082-12089 (1992) and Smith et al., J. Biol Chem. 269: 15162
(1994)) and polypeptide N-acetylgalactosaminyltransferase (Homa et
al., J. Biol. Chem. 268: 12609 (1993)).
c) Glycosidases
[0218] This invention also encompasses substrates for wild-type and
mutant glycosidases. Mutant .beta.-galactosidase enzymes have been
demonstrated to catalyze the formation of disaccharides through the
coupling of .alpha.-glycosyl fluoride to a galactosyl acceptor
molecule. (Withers, U.S. Pat. No. 6,284,494; issued Sep. 4, 2001).
Other glycosidases of use in this invention include, for example,
.beta.-glucosidases, .beta.-galactosidases, .beta.-mannosidases,
.beta.-acetyl glucosaminidases, .beta.-N-acetyl galactosaminidases,
.beta.-xylosidases, .beta.-fucosidases, cellulases, xylanases,
galactanases, mannanases, hemicellulases, amylases, glucoamylases,
.alpha.-glucosidases, .alpha.-galactosidases, .alpha.-mannosidases,
.alpha.-N-acetyl glucosaminidases, .alpha.-N-acetyl
galactose-aminidases, .alpha.-xylosidases, .alpha.-fucosidases, and
neuraminidases/sialidases, endoglycoceramidases.
[0219] The following examples are provided to illustrate selected
embodiments of the invention and are not to be construed as
limiting its scope.
EXAMPLES
Example 1
Preparation of UDP-GalNAc-6'-CHO
[0220] UDP-GalNAc (200 mg, 0.30 mmoles) was dissolved in a 1 mM
CuSO.sub.4 solution (20 mL) and a 25 mM NaH.sub.2PO.sub.4 solution
(pH 6.0; 20 mL). Galactose oxidase (240 U; 240 .mu.L) and catalase
(13000 U; 130 .mu.L) were then added, the reaction system equipped
with a balloon filled with oxygen and stirred at room temperature
for seven days. The reaction mixture was then filtered (spin
cartridge; MWCO 5K) and the filtrate (.about.40 mL) was stored at
4.degree. C. until required. TLC (silica; EtOH/water (7/2);
R.sub.f=0.77; visualized with anisaldehyde stain).
Example 2
Preparation of UDP-GalNAc-6'--NH.sub.2)
[0221] Ammonium acetate (15 mg, 0.194 mmoles) and NaBH.sub.3CN (1M
THF solution; 0.17 mL, 0.17 mmoles) were added to the
UDP-GalNAc-6'-CHO solution from above (2 mL or .about.20 mg) at
0.degree. C. and allowed to warm to room temperature overnight. The
reaction was filtered through a G-10 column with water and the
product collected. The appropriate fractions were freeze-dried and
stored frozen. TLC (silica; ethanol/water (7/2); R.sub.f=0.72;
visualized with ninhydrin reagent).
Example 3
Preparation of UDP-GalNAc-6-NHCO(CH.sub.2).sub.2--O-PEG-OMe (1
KDa)
[0222] The
galactosaminyl-1-phosphate-2-NHCO(CH.sub.2).sub.2--O-PEG-OMe (1
KDa) (58 mg, 0.045 mmoles) was dissolved in DMF (6 mL) and pyridine
(1.2 mL). UMP-morpholidate (60 mg, 0.15 mmoles) was then added and
the resulting mixture stirred at 70.degree. C. for 48 h. The
solvent was removed by bubbling nitrogen through the reaction
mixture and the residue purified by reversed phase chromatography
(C-18 silica, step gradient between 10 to 80%, methanol/water). The
desired fractions were collected and dried at reduced pressure to
yield a white solid. TLC (silica, propanol/H.sub.2O/NH.sub.4OH,
(30/20/2), R.sub.f=0.54). MS (MALDI): Observed, 1485, 1529, 1618,
1706.
Example 4
Preparation of Cystein-PEG.sub.2 (2)
##STR00058##
[0224] 4.1 Synthesis of Compound 1
[0225] Potassium hydroxide (84.2 mg, 1.5 mmol, as a powder) was
added to a solution of L-cysteine (93.7 mg, 0.75 mmol) in anhydrous
methanol (20 L) under argon. The mixture was stirred at room
temperature for 30 min, and then mPEG-O-tosylate of molecular mass
20 kilodalton (Ts; 1.0 g, 0.05 mmol) was added in several portions
over 2 hours. The mixture was stirred at room temperature for 5
days, and concentrated by rotary evaporation. The residue was
diluted with water (30 mL), and stirred at room temperature for 2
hours to destroy any excess 20 kilodalton mPEG-O-tosylate. The
solution was then neutralized with acetic acid, the pH adjusted to
pH 5.0 and loaded onto a reversed phase chromatography (C-18
silica) column. The column was eluted with a gradient of
methanol/water (the product elutes at about 70% methanol), product
elution monitored by evaporative light scattering, and the
appropriate fractions collected and diluted with water (500 mL).
This solution was chromatographed (ion exchange, XK 50 Q, BIG
Beads, 300 ml, hydroxide form; gradient of water to water/acetic
acid-0.75N) and the pH of the appropriate fractions lowered to 6.0
with acetic acid. This solution was then captured on a reversed
phase column (C-18 silica) and eluted with a gradient of
methanol/water as described above. The product fractions were
pooled, concentrated, redissolved in water and freeze-dried to
afford a white solid (1). Structural data for the compound were as
follows: .sup.1H-NMR (500 MHz; D.sub.2O) .delta. 2.83 (t, 2H,
O--C--CH.sub.2--S), 3.05 (q, 1H, S--CHH--CHN), 3.18 (q, 1H, (q, 1H,
S--CHH--CHN), 3.38 (s, 3H, CH.sub.3O), 3.7 (t, OCH.sub.2CH.sub.2O),
3.95 (q, 1H, CHN). The purity of the product was confirmed by SDS
PAGE.
[0226] 4.2 Synthesis of Compound 2 (Cysteine-PEG.sub.2)
[0227] Triethylamine (.about.0.5 mL) was added dropwise to a
solution of compound 1 (440 mg, 22 .mu.mol) dissolved in anhydrous
CH.sub.2Cl.sub.2 (30 mL) until the solution was basic. A solution
of 20 kilodalton mPEG-O-p-nitrophenyl carbonate (660 mg, 33
.mu.mol) and N-hydroxysuccinimide (3.6 mg, 30.8 .mu.mol) in
CH.sub.2Cl.sub.2 (20 mL) was added in several portions over 1 hour
at room temperature. The reaction mixture was stirred at room
temperature for 24 hours. The solvent was then removed by rotary
evaporation, the residue was dissolved in water (100 mL), and the
pH adjusted to 9.5 with 1.0 N NaOH. The basic solution was stirred
at room temperature for 2 hours and was then neutralized with
acetic acid to a pH 7.0. The solution was then loaded onto a
reversed phase chromatography (C-18 silica) column. The column was
eluted with a gradient of methanol/water (the product elutes at
about 70% methanol), product elution monitored by evaporative light
scattering, and the appropriate fractions collected and diluted
with water (500 mL). This solution was chromatographed (ion
exchange, XK 50 Q, BIG Beads, 300 mL, hydroxide form; gradient of
water to water/acetic acid-0.75N) and the pH of the appropriate
fractions lowered to 6.0 with acetic acid. This solution was then
captured on a reversed phase column (C-18 silica) and eluted with a
gradient of methanol/water as described above. The product
fractions were pooled, concentrated, redissolved in water and
freeze-dried to afford a white solid (2). Structural data for the
compound were as follows: .sup.1H-NMR (500 MHz; D.sub.2O) .delta.
2.83 (t, 2H, O--C--CH.sub.2--S), 2.95 (t, 2H, O--C--CH.sub.2--S),
3.12 (q, 1H, S--CHH--CHN), 3.39 (s, 3H CH.sub.3O), 3.71 (t,
OCH.sub.2CH.sub.2O). The purity of the product was confirmed by SDS
PAGE.
Example 5
Preparation of UDP-GalNAc-6-NHCO(CH.sub.2).sub.2--O-PEG-OMe (1
KDa)
[0228] Galactosaminyl-1-phosphate-2-NHCO(CH.sub.2).sub.2--O-PEG-OMe
(1 kilodalton) (58 mg, 0.045 mmoles) was dissolved in DMF (6 mL)
and pyridine (1.2 mL). UMP-morpholidate (60 mg, 0.15 mmoles) was
then added and the resulting mixture stirred at 70.degree. C. for
48 h. The solvent was removed by bubbling nitrogen through the
reaction mixture and the residue purified by reversed phase
chromatography (C-18 silica, step gradient between 10 to 80%,
methanol/water). The desired fractions were collected and dried at
reduced pressure to yield a white solid. TLC (silica,
propanol/H.sub.2O/NH.sub.4OH, (30/20/2), R.sub.f=0.54). MS (MALDI):
Observed, 1485, 1529, 1618, 1706.
SDS PAGE Procedure
[0229] The purity of the products, 1 and 2, were confirmed by SDS
PAGE. A 4-20% Tris-Glycine SDS PAGE gel (Invitrogen) was used. The
sample was mixed 1:1 with SDS Sample Buffer, and was run in
Tris-Glycine Running Buffer (LC2675-5) at a constant voltage (125
V) for 1 hr 50 min. After electrophoresis, the gel was washed with
water (100 mL) for 10 min followed by a wash with a 5% barium
chloride aqueous solution (100 mL) for 10 min. Products 1 or 2 were
visualized by staining the gels with 0.1 N iodine solution (4.0 mL)
at room temperature and the staining process stopped by washing the
gels with water. The visualized product bands were scanned with an
HP Scanjet 7400C, and the image of the gel was optimized with the
HP Precision Scan Program.
[0230] While this invention has been disclosed with reference to
specific embodiments, it is apparent that other embodiments and
variations of this invention may be devised by others skilled in
the art without departing from the true spirit and scope of the
invention.
[0231] All patents, patent applications, and other publications
cited in this application are incorporated by reference herein in
their entirety for all purposes.
* * * * *
References