CN101087801B - Branched polymeric sugars and nucleotides thereof - Google Patents

Abstract

The present invention provides sugars, nucleotide sugars, activated sugars comprising one or more polymer modification moieties in their structure. The invention has been illustrated with reference to linear and branched polymers, such as the water-soluble polymer poly(ethylene glycol).

Description

Branched Polymeric Sugars and Their Nucleotides

Cross References to Related Applications

This application claims priority to the following documents: U.S. Provisional Patent Application No. 60/539,387, filed January 26, 2004; U.S. Provisional Patent Application No. 60/555,504, filed March 22, 2004; U.S. Provisional Patent Application No. .60/590,573, filed July 23, 2004; U.S. Provisional Patent Application No. 60/555,504, filed March 22, 2004; U.S. Patent Application No. 10/997,405, filed November 24, 2004; U.S. Provisional Patent Application 60/544,411, filed February 12, 2004; U.S. Provisional Patent Application 60/546,631, filed February 20, 2004; U.S. Provisional Patent Application, May 12, 2004; U.S. Patent Application ((not issued), Attorney Docket No. 40853-01-5138US), filed January 10, 2005; PCT Application No. ((Unauthorized), Attorney Docket No. 40853-01-5146WO), filed December 3, 2004; U.S. Provisional Patent Application No. 60/590,649, filed July 23, 2004; U.S. Provisional Patent Application No. 60/611,790, filed September 20, 2004; U.S. Provisional Patent Application No. 60/592,744, filed July 29, 2004; U.S. Provisional Patent Application No. 60/614,518, filed September 29, 2004; U.S. Provisional Patent Application No. 60/623,387, filed October 29, 2004; U.S. Provisional Patent Application No. 60/626,678 , filed Nov. 9, 2004; and U.S. Provisional Patent Application No. ((Unlicensed), Attorney Docket No. 040853-01-5150), filed Jan. 6, 2005, the disclosure of each document at It is hereby incorporated by reference in its entirety for all purposes.

technical field

The present invention belongs to the field of modified sugars and nucleotides thereof.

Background technique

Post-expression in vitro modification of peptides is an attractive strategy to remedy the deficiencies of methods that rely on the control of glycosylation by engineered expression systems; including both modification of glycan structure or introduction of glycan at novel sites . A comprehensive toolbox of recombinant eukaryotic glycosyltransferases is available, enabling the in vitro enzymatic synthesis of mammalian complex carbohydrates with commonly designed glycosylation patterns and glycosyl structures. See, eg, US Patent Nos. 5,876,980; 6,030,815; 5,728,554; 5,922,577; and WO/9831826; US2003180835; and WO 03/031464.

The advantages of enzyme-based synthesis are regioselectivity and stereoselectivity. Additionally, enzymatic synthesis was performed using unprotected substrates. Three main types of enzymes are used in the synthesis of carbohydrates, glycosyltransferases (eg, sialyltransferases, oligosaccharyltransferases, N-acetylglucosaminyltransferases), and glycosidases. Glycosidases are further classified into exoglycosidases (eg, β-mannosidase, β-glucosidase), and endoglycosidases (eg, Endo-A, Endo-M). Each of these classes of enzymes has been successfully used synthetically to make carbohydrates. For a general review, see Crout et al., Curr. Opin. Chem. Biol. 2:98-111 (1998).

Glycosyltransferases modify oligosaccharide structures on glycopeptides. Glycosyltransferases are efficient at generating specific products with good stereochemical and regiochemical control. Glycosyltransferases have been used to prepare oligosaccharides and modify terminal N- and O-linked carbohydrate structures, especially on glycopeptides produced in mammalian cells. For example, the terminal oligosaccharides of the glycopeptide are fully sialylated and/or fucosylated to provide a more consistent carbohydrate structure, which improves glycopeptide pharmacokinetics and various other biological properties. For example, β-1,4-galactosyltransferase is used for the synthesis of lactosamine, and the function of glycosyltransferase in the synthesis of carbohydrates is illustrated (see, e.g., Wong et al., J; Org Chem. 47:5416-5418( 1982)). In addition, many synthetic processes use sialyltransferases to transfer sialic acid from cytidine-5'-monophosphate-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 the synthetic pathway for the transfer of fucose units from guanosine-5'-diphosphate fucose to specific hydroxyl groups of sugar acceptors. For example, Ichikawa prepared sialyl Lewis-X by a method involving 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 complex carbohydrate synthesis for therapeutic use, see Koeller et al., Nature Biotechnology 18:835-841 (2000). See also US Patent Nos. 5,876,980; 6,030,815; 5,728,554; 5,922,577; and WO/9831826.

In addition to modulating the structure of glycosyl groups on polypeptides, interest has been directed towards preparing glycopeptides that are modified with one or more non-glycomodifying groups such as water-soluble polymers. Poly(ethylene glycol) ("PEG") is an exemplary polymer that is conjugated to a polypeptide. The use of PEG for derivatizing peptide therapeutics has been shown to reduce the immunogenicity of peptides. For example, US Patent No. 4,179,337 (Davis et al.) discloses non-immunogenic polypeptides, such as enzymes and peptide hormones, conjugated to polyethylene glycol (PEG) or polypropylene glycol. 10-100 moles of polymer are used per mole of polypeptide. Although the in vivo clearance time of the conjugate was prolonged relative to that of the polypeptide, only about 15% of its physiological activity was retained. Thus, the prolonged circulating half-life was offset by a drastic reduction in peptide potency.

The loss of peptide activity can be directly attributed to the non-selective nature of the chemistry used to bind the water soluble polymer. The main mode of attachment of PEG (and its derivatives) to peptides is through non-specific bonding of peptide amino acid residues. For example, US Patent No. 4,088,538 discloses an enzyme-active polymer-enzyme conjugate of an enzyme covalently bound to PEG. Similarly, US Patent No. 4,496,689 discloses a covalently linked complex of alpha-1 protease inhibitor with a polymer such as PEG. Abuchowski et al. (J.Biol.Chem. 252:3578 (1977) disclose the covalent attachment of MPEG to the amine groups of bovine serum albumin. U.S. Pat. (e.g. poly(ethylene succinic anhydride)) to reduce the hydrophobicity of interferon. PCT WO87/00056 discloses the effect of PEG and poly(oxyethylated) polyols on e.g. proteins (e.g. interferon-beta, interleukin- 2 and immunotoxins). EP 154,316 discloses and claims chemically modified lymphokines such as IL-2 comprising PEG directly bonded to at least one primary amino group of lymphin. U.S. Patent No. 4,055,635 discloses covalently linked to Pharmaceutical composition of a water-soluble complex of a proteolytic enzyme of a polymeric substance such as a polysaccharide.

Another mode of attachment of PEG to peptides is through non-specific oxidation of glycosyl residues on glycopeptides. Oxidized sugars are used as sites for linking PEG moieties to peptides. For example M'Timkulu (WO94/05332) discloses the use of amino-PEG for the addition of PEG to glycoproteins. The glycosyl moiety is randomly oxidized to the corresponding aldehyde, which is subsequently coupled to amino-PEG.

In each of the above methods, poly(ethylene glycol) is added in a random, non-specific manner to reactive residues of the peptide backbone. For the production of therapeutic peptides, it is clear that derivatization strategies leading to the formation of specifically labeled, easily characterized, substantially homogeneous products are required. A promising approach to making specifically labeled peptides is through the attachment of modified sugar moieties to the peptides using enzymes such as glycosyltransferases. The modified sugar moiety must function as a substrate for the glycosyltransferase and be properly activated. Therefore, there is a need for synthetic routes that provide facile access to activated modified sugars. The present invention provides such an approach.

Contents of the invention

The present invention provides polymeric substances, sugars and activated sugars bound to these polymeric substances and nucleotide sugars comprising these polymeric substances. Polymeric materials include both water-soluble and water-insoluble materials. Furthermore, the polymers are branched or linear polymers. Exemplary sugar moieties include linear and cyclic structures and aldoses and ketoses.

The polymer modifying group can be attached anywhere on the sugar moiety. In the following discussion, the invention is illustrated by reference to embodiments in which the polymer modifying group is attached to C-5 of the furanose or C-6 of the pyranose. Those skilled in the art will recognize that the focus of the discussion is for clarity, and that polymer moieties may be attached to other positions on both pyranose and furanose sugars using methods described herein and art-recognized methods.

In an exemplary embodiment, the invention provides a sugar or sugar nucleotide conjugated to a polymer:

In general formulas I and II, R ¹ is H, CH ₂ OR ⁷ , COOR ⁷ or OR ⁷ , wherein R ⁷ represents H, substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl. R ² is H, OH, or a group comprising nucleotides. Exemplary ^R according to this embodiment has the general formula:

wherein ^R is a nucleoside.

The symbols R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁶ ′ independently represent H, substituted or unsubstituted alkyl, OR ⁹ , NHC(O)R ¹⁰ . Flag d is 0 or 1. R ⁹ and R ¹⁰ are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, or sialic acid. At least one of ^R3 , ^R4 , ^R5 , ^R6 and ^R6 ' includes a polymer modifying moiety (eg, PEG). In an exemplary embodiment, ^R6 and ^R6 ' together with the carbon to which they are attached are components of the sialic acid side chain. In a further exemplary embodiment, the side chain is functionalized with a polymer modifying moiety.

In an exemplary embodiment, the polymer moiety is bound to the sugar core, typically via a heteroatom on the core, via a linker L, as follows:

R ¹¹ is a polymer moiety and L is selected from a bond and a linking group. The notation w represents an 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 a linker is an acyl group.

When L is a bond, it is formed between the reactive functional group on the precursor of R ¹¹ and the complementary reactive reactive functional group on the precursor of L. L can be in place on the sugar core prior to the reaction with ^R11 . Alternatively, R ¹¹ and L can be introduced into a pre-formed cassette that is subsequently attached to the sugar core. As illustrated herein, the selection and preparation of precursors with appropriate reactive functional groups is within the purview of those skilled in the art. In addition, the conjugation of precursors is performed using chemistries well known in the art.

In an exemplary embodiment, L is a linker formed from an amino acid or a small peptide (e.g., 1-4 amino acid residues), providing a modified sugar in which the polymer-modified moiety is linked by a substituted alkyl linker . An exemplary linker is glycine.

In an exemplary embodiment, R ⁶ includes a polymer modifying moiety. In another exemplary embodiment, ^R6 includes both a polymer modifying moiety and a linker L that bonds the modifying moiety to the remainder of the molecule.

In an exemplary embodiment, the polymer modifying portion is a branched structure comprising two or more polymer chains attached to a central portion. Exemplary structures of useful polymer modifying moiety precursors according to this embodiment of the invention have the general formula:

Sugars and nucleotide sugars according to this general formula are essentially pure water-soluble polymers. ^X3 ' is a group that includes ionizable (eg, COOH, etc.) or other reactive functional groups (see, eg, below). C is carbon. X ⁵ is preferably a non-reactive group (eg H, unsubstituted alkyl, unsubstituted heteroalkyl). R ¹² and R ¹³ are independently selected polymer arms, such as non-peptide, non-reactive polymer arms. ^X2 and ^X4 are preferably substantially non-reactive linking fragments under physiological conditions, which may be the same or different. Alternatively, these linkages may include one or more groups, such as esters, disulfides, etc., designed to degrade under physiologically relevant conditions. ^X2 and ^X4 bind polymer arms ^R12 and ^R13 to C. When ^X3 ' is reacted with a complementary reactive functional group on the linker, sugar or linker-sugar combination, ^X3 ' is converted to a component of the linker fragment ^X3 .

The invention provides sugars and nucleotide sugars of the general formula by reaction of precursors with a suitable sugar or sugar linker:

Where the identification of the groups represented by the various symbols is the same as discussed above. L ^a is a substituted or unsubstituted alkyl or a substituted or unsubstituted heteroalkyl group. In the illustrated embodiment, ^La is a pendant group of sialic acid which is functionalized with the polymer modifying moiety as shown.

The polymer modifying moiety comprises two or more repeating units which may be water soluble or substantially water insoluble. Exemplary water-soluble polymers useful in the compounds of the invention include PEG (such as m-PEG), PPG (such as m-PPG), polysialic acid, polyglutamate, polyaspartate, polylysine Acids, polyalkyleneimines, biodegradable polymers (eg, polylactides, polyglycerides), and functionalized PEGs, such as end-functionalized PEGs.

The sugar moieties of the polymer conjugates of the invention are selected from both natural and unnatural furanoses and hexoses. Non-natural sugars optionally include alkylated or acylated hydroxyl and/or amine groups, such as ring ether, ester and amide substituents. Other unnatural sugars include H, hydroxyl, ether, ester or amide substituents at ring positions that do not exist in natural sugars. Alternatively, a carbohydrate lacks a substituent that should be found in the carbohydrate from which its name is derived, such as a deoxysugar. Still further exemplary unnatural sugars include both oxidized (eg, -keto acids and -uronic acids) and reduced (sugar alcohols) carbohydrates. Sugar moieties can be monosaccharides, oligosaccharides and polysaccharides.

Exemplary natural sugars useful in the present invention include glucose, glucosamine, galactose, galactosamine, fucose, mannose, mannosamine, xylose, ribose, N-acetylglucose, N-acetylglucosamine , N-acetylgalactose, N-acetylgalactosamine, and sialic acid.

Exemplary sialic acid-based conjugates have the general formula:

where AA is that portion of the amino acid residue that does not include the carboxyl group and NP is the nucleotide phosphate. ONPs can also be replaced by activating moieties to form activated sugars. As will be appreciated by those skilled in the art, the polymer modifying moiety-linker may also be attached to the sialic acid side chain at C-6, C-7 and/or C-9.

Also provided are synthetic methods for producing activated sialic acid-PEG conjugates which are suitable substrates for enzymes such as glycosyltransferases. The method comprises the steps of: (a) contacting mannosamine with an activated N-protected amino acid (or modified by a polymer) under conditions suitable to form an amide conjugate between the mannosamine and the N-protected amino acid moiety, linker precursor, or linker-polymer-modified moiety combination functionalized amino acid); (b) contacting the amide conjugate with pyruvate under conditions suitable for converting the amide conjugate into a sialic acid amide conjugate salt and sialic acid aldolase; (c) contacting the sialic acid amide conjugate with cytidine triphosphate and synthetase under conditions suitable for the formation of cytidine monophosphate sialic acid amide conjugate; (d) from cytidine monophosphate The sialic acid amide conjugate removes the N-protecting group, thereby generating a free amine; and (e) contacting the free amine with activated PEG (linear or branched), thereby forming cytidine monophosphate sialic acid-poly (ethylene glycol).

Nucleosides can be selected from both natural and unnatural nucleosides. Exemplary natural nucleosides for use in the invention include cytosine, thymine, guanine, adenine, and uracil. The structure of unnatural nucleosides and methods for their preparation are well documented in the art.

Exemplary modified sugar nucleotides of the present invention include polymer-modified GDP-Man, GDP-Fuc, UDP-Gal, UDP-GalNAc, UDP-Glc, UDP-GlcNAc, UDP-Glc, UDP-GlcUA and CMP -SA etc. Examples include UDP-Gal-2'-NH-PEG, UDP-Glc-2'NH-PEG, CMP-5'-PEG-SA and the like. Compounds covered by the present invention include those wherein the ^LR11 moiety is bound to the furanose or pyranose, such as at C-5 of the furanose or at C-6 of the pyranose, usually through a heteroatom attached to this carbon atom.

When the compound of the invention is a nucleotide sugar or an activated sugar, the polymer conjugate of the nucleotide sugar is usually a substrate for an enzyme which transfers the sugar moiety and its polymer substituent to an appropriate acceptor moiety of the substrate superior. Accordingly, the present invention also provides substrates modified by sugar conjugation using polymer conjugates of nucleotide sugars or activated sugars and appropriate enzymes. Substrates that can be used for carbohydrate conjugation using the compounds of the present invention include peptides (eg, glycopeptides, peptides), lipids (eg, glycolipids), and ligands (eg, laminol, ceramides).

Description of drawings

Figure 1 is a table of sialyltransferases for which selected modified sialic acid nucleotides and activated sugars are substrates.

Figure 2 is a general synthetic scheme of the invention for the preparation of sialic acid-poly(ethylene glycol) conjugates.

Figure 3 is a synthetic scheme of the invention for the preparation of sialic acid-glycyl-poly(ethylene glycol) conjugates.

Detailed Description and Embodiments of the Invention

abbreviation

Branched and unbranched PEG, poly(ethylene glycol), such as m-PEG, methoxy-poly(ethylene glycol); branched and unbranched PPG, poly(propylene glycol), such as m-PPG, formazan Oxy-poly(propylene glycol); Fuc, fucosyl; Gal, galactosyl; GalNAc, N-acetylgalactosamine; Glc, glucosyl; GlcNAc, N-acetylglucosamine; Man, mannosyl ; ManAc, mannosaminoacetate; Sia, sialic acid; and NeuAc, N-acetylceramino.

definition

The term "sialic acid" refers to any member of the family of nine carbon carboxylated sugars. The most common member of the sialic acid family is N-acetyl-neuraminic acid (2-keto-5-acetylamino-3,5-dideoxy-D-glycerol-D-galactonononepyranose-1- Ketoacids (often abbreviated as Neu5Ac, NeuAc, or NANA). The second member of the family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in which the N-acetyl group of NeuAc is hydroxylated. The third A member of the sialic acid family is 2-keto-3-deoxy-nonulonic acid (KDN) (Nadano et al. (1986) J. Biol. Chem. 261: 11550-11557; Kanamori et al., J. Biol. Chem. 265:21811-21819 (1990)). Also includes 9-substituted sialic acid such as 9-OC ₁ -C ₆ acyl-Neu5Ac such as 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac, 9-deoxy- 9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. For a review of the sialic acid family see, e.g., Varki, Glycobiology 2:25-40 (1992); Sialic acid: chemistry, metabolism and function, R. Schauer, Ed. (Springer-Verlag, New York (1992)). The synthesis of sialic acid compounds and their use in sialylation is disclosed in International Application WO 92/16640 published October 1,1992.

The term "modified sugar" as used herein means a natural or unnatural carbohydrate which is enzymatically added to an amino acid or glycosyl residue of a peptide in the methods of the invention. Modified sugars are selected from many enzyme substrates, including but not limited to sugar nucleotides (mono-, di-, and tri-phosphates), activated sugars (e.g., glycosyl halides, glycosyl methanesulfonates) and both A sugar that is not activated and is not a nucleotide. "Modified sugars" are covalently functionalized by "modifying groups" (modifying groups). Useful modifying groups include, but are not limited to, water soluble polymers, therapeutic moieties (or moieties), diagnostic moieties, biomolecules, and the like. The modifying group is preferably not a natural or unmodified carbohydrate. The site of functionalization by the modifying group is chosen such that it does not prevent enzymatic addition of the "modified sugar" to the peptide.

The term "water soluble" means a moiety which has some detectable degree of solubility in water. Methods of detecting and/or quantifying water solubility are well known in the art. Exemplary water soluble polymers include peptides, sugars, poly(ethers), poly(amines), poly(carboxylic acids), and the like. Peptides can have mixed sequences or consist of a single amino acid, such as poly(lysine). An exemplary polysaccharide is poly(sialic acid). An exemplary poly(ether) is poly(ethylene glycol), such as m-PEG. Poly(ethyleneimine) is an exemplary polyamine, and poly(acrylic acid) is a representative poly(carboxylic acid). Exemplary polymers typically consist of 2-8 polymer units.

The polymer backbone of the water soluble polymer can be poly(ethylene glycol) (ie PEG). It should be understood, however, that other related polymers are also suitable for use in the practice of this invention, and use of the term PEG or poly(ethylene glycol) is intended to be inclusive and not exclusive in this respect. The term PEG includes any form of poly(ethylene glycol), including alkoxy PEG, difunctional PEG, multiarm PEG, forked PEG, branched PEG, pendent PEG (i.e., containing one or more PEG or related polymers with functional groups in the main chain), or PEG with degradable linkages in it.

The polymer backbone can be linear or branched. Branched polymer backbones are generally known in the art. Typically, branched polymers contain a central branching core moiety and a plurality of linear polymer chains attached to the central branching core. PEG is typically used in a branched form, which can be prepared by the addition of ethylene oxide to various polyols such as glycerol, pentaerythritol, and sorbitol. Central branching moieties can also be derived from several amino acids, such as lysine. Branched poly(ethylene glycol) can be represented in a general form as R(-PEG-OH) _m , where R represents a core moiety, such as glycerol or pentaerythritol, and m represents the number of arms. Multi-armed PEG molecules, such as those described in US Pat. No. 5,932,462, which is hereby incorporated by reference in its entirety, can also be used as the polymer backbone.

Many other polymers are also suitable for the invention. Non-peptidic and water-soluble polymer backbones having 2 to about 300 termini are particularly suitable for use in the present 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, etc., poly(oxyethylated polyols), Poly(vinyl alcohol), poly(vinylpyrrolidone), poly(hydroxypropylmethacrylamide), poly(alpha-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazole morpholine, poly(N-acryloylmorpholine), as described in US Patent No. 5,629,384, which is hereby incorporated by reference 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 from about 100 Da to about 100,000 Da, usually from about 6,000 Da to about 80,000 Da.

The term "sugar conjugation" as used herein refers to the enzyme-mediated conjugation of a modified carbohydrate substance to an amino acid or glycosyl residue of a polypeptide such as the mutant human growth hormone of the present invention. A subclass of "sugar conjugation" is "diol-PEGylation", in which the modifying group of the modified sugar is poly(ethylene glycol), and its alkyl derivatives (e.g., m-PEG) or reactive derivatized substances (eg, H ₂ N-PEG, HOOC-PEG).

The term "glycosyl linking group" as used herein means a glycosyl residue to which a modifying group (e.g., PEG moiety, therapeutic moiety, biomolecule) is covalently attached; the glycosyl linking group binds the modifying group to the rest of the mixture. In the methods of the invention, a "glycosyl linking group" is covalently attached to a glycosylated or non-glycosylated peptide, thereby linking an agent to an amino acid and/or glycosyl residue of the peptide. A "glycosyl linking group" is typically derived from a "modified sugar" by enzymatic attachment of the "modified sugar" to an amino acid and/or glycosyl residue of a peptide. The glycosyl linking group can be a sugar-derived structure that degrades during formation of the modifying group-modified sugar combination (eg, oxidation→Schiff base formation→reduction), or the glycosyl linking group can be intact. "Intact glycosyl linking group" means a linking group derived from a glycosyl moiety wherein the glycomonomer linking the modifying group and to the remainder of the conjugate is not degraded, such as oxidized (such as by sodium metaperiodate oxidation). An "intact glycosyl linking group" of the invention may be derived from a natural oligosaccharide by the addition of a glycosyl unit or the removal of one or more glycosyl units from the parent sugar structure.

Where substituents are designated by their conventional chemical formulas written from left to right, they equally include chemically identical substituents derived from structures written from right to left, such as _-CH2O - is also written -OCH ₂ -.

The term "alkyl" by itself or as part of another substituent, unless otherwise stated, denotes straight or branched chain, or cyclic, hydrocarbon groups, or combinations thereof, which may be fully saturated, mono- or polyunsaturated and may be Included are divalent and polyvalent groups, having the indicated number of carbon atoms (ie, C ₁ -C ₁₀ means 1-10 carbons). Examples of saturated hydrocarbon groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl , cyclopropylmethyl, (eg n-pentyl, n-hexyl, n-heptyl, n-octyl) homologues and isomers, etc. An unsaturated alkyl group is an alkyl group having one or more double 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 higher homologues and isomers. Unless otherwise stated, the term "alkyl" is also intended to include derivatives of alkyl as defined in more detail below, such as "heteroalkyl". Alkyl groups limited to hydrocarbyl groups are referred to as "homo-alkyl groups".

The term "alkylene" by itself or as part of _another substituent _denotes a divalent group derived from an alkane, as exemplified by, but not limited to, _{-CH2CH2CH2CH2-} , and further _includes the following descriptions as Those groups of "heteroalkylene". Typically, alkyl (or alkylene) groups contain 1-24 carbon atoms, and those groups containing 10 or fewer carbon atoms are preferred in the present invention. "Lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, usually containing 8 or fewer carbon atoms.

The terms "alkoxy", "alkylamino" and "alkylthio" (or thioalkoxy) are used in their conventional sense and denote attachment to the remainder of a molecule through an oxygen, amino, or sulfur atom, respectively. Part of those alkyl groups.

The term "heteroalkyl", by itself or in combination with another term, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon group, or a combination thereof, consisting of the stated number of carbon atoms and at least one heteroatom, the heteroatom The atoms are selected from O, N, Si and S, and wherein the nitrogen and sulfur atoms are optionally oxidized and nitrogen heteroatoms are optionally quaternized. One or more heteroatoms O, N and S and Si can be located at any internal position of the heteroalkyl or at the position where the alkyl is attached to the remainder of the molecule. Examples include, but are not limited to -CH ₂ -CH ₂ -O-CH ₃ , -CH ₂ -CH ₂ -NH-CH ₃ , -CH ₂ -CH ₂ -N(CH ₃ )-CH ₃ , -CH ₂ - S-CH ₂ -CH ₃ , -CH ₂ -CH ₂ , -S(O)-CH ₃ , -CH ₂ -CH ₂ -S(O) ₂ -CH ₃ , -CH-CH-O-CH ₃ , -Si(CH ₃ ) ₃ , -CH ₂ -CH=N-OCH ₃ , and -CH=CH-N(CH ₃ )-CH ₃ . Up to two heteroatoms can be consecutive, eg -CH ₂ -NH-OCH ₃ and -CH ₂ -O-Si(CH ₃ ) ₃ . Similarly, the term "heteroalkylene" denotes a divalent radical derived from a heteroalkyl by itself or as part of another substituent, as exemplified by, but not limited to, _-CH2 - _CH2 -S-CH ₂ -CH ₂ - and -CH ₂ -S-CH ₂ -CH ₂ -NH-CH ₂ -. For heteroalkylene groups, heteroatoms may also occupy one or both chain termini (eg, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Still further, for alkylene and heteroalkylene linking groups, the orientation of the linking group is not implied by the direction in which the general formula of the linking group is written. For example, the general formula -C(O) _2R'- represents both -C(O) _2R'- and -R'C(O) _2- .

The terms "cycloalkyl" and "heterocycloalkyl" by themselves or in combination with other terms, unless otherwise indicated, denote 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-morpholine base, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-piperazinyl, 2-piperazinyl, etc.

The terms "halo" or "halogen" by themselves or as part of another substituent, unless otherwise stated, denote a fluorine, chlorine, bromine or iodine atom. Additionally, terms such as "haloalkyl" are intended to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(C ₁ -C ₄ )alkyl" is intended to include, but is not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl wait.

The term "aryl" unless otherwise stated, denotes a polyunsaturated, aromatic substituent which may be a single ring or multiple rings (preferably 1-3 rings), which are fused together or linked covalently. The term "heteroaryl" means an aryl group (or ring) comprising 1-4 heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally The land is seasonalized. 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-pyrazole Base, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazole Base, 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-pyrimidinyl, 4-pyrimidinyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1 -isoquinolinyl, 5-isoquinolinyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolinyl, tetrazolyl, benzo[b]furanyl, benzo[b] Thienyl, 2,3-dihydrobenzo[1,4]dioxin-6-yl, benzo[1,3]dioxol-5-yl and 6-quinolinyl. Substituents for each of the above-mentioned aryl and heteroaryl ring systems are selected from the acceptable substituents described above.

For convenience, the term "aryl" when used in conjunction with other terms (eg, aryloxy, arylsulfoxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term "arylalkyl" is intended to include those groups in which the aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, etc.), including those in which A carbon atom (eg, methylene) is replaced by, for example, an oxygen atom (eg, phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, etc.).

Each of the above terms (eg, "alkyl," "heteroalkyl," "aryl," and "heteroaryl") is intended to include both substituted and unsubstituted forms of the indicated group. Preferred substituents for each type of radical are provided below.

Alkyl and heteroalkyl (including those commonly referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl ) substituents are generally referred to as "alkyl substituents", and they may be one or more selected from, but not limited to, the following various groups: - OR', =O, =NR', =N-OR', -NR'R", -SR', -halogen, -SiR'R"R'", -OC(O)R', -C(O )R', -CO ₂ R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R'", -NR"C(O) ₂ R', -NR-C(NR'R"R'")=NR"", -NR-C(NR'R")=NR'", -S(O)R ', -S(O) _2R ', -S(O) _2NR'R ", _-NRSO2R ', -CN, and _-NO2 , where m' is the total number of carbon atoms in such a group. R', R", R'" and R"" each preferably independently represent hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, such as aryl substituted by 1-3 halogen, substituted or Unsubstituted alkyl, alkoxy or thioalkoxy, or arylalkyl. When a compound of the invention includes more than one R group, for example, each R group is independently selected, as are each R', R", R'" and R"" groups (when more than one of these group exists). When R' and R" are attached to the same nitrogen atom, they can combine with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR'R" is intended to include, but is not limited to, 1- Pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, those skilled in the art will understand that the term "alkyl" is intended to include groups comprising carbon atoms bonded to groups other than hydrogen atoms, such as haloalkyl (e.g., -CF ₃ and -CH ₂ CF ₃ ) and acyl groups (eg, -C(O)CH ₃ , -C(O)CF ₃ , -C(O)CH ₂ OCH ₃ , etc.).

Similar to the substituents described for alkyl groups, substituents for aryl and heteroaryl groups are generally referred to as "aryl substituents". Substituents are selected from, for example: halogen, -OR', =O, =NR', =N-OR', -NR'R", -SR', -halogen, -SiR'R"R'", -OC (O)R', -C(O)R', -CO ₂ R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'- C(O)NR"R'", -NR"C(O) ₂ R', -NR-C(NR'R"R'")=NR"", -NR-C(NR'R")= NR'", -S(O)R', -S(O) ₂ R', -S(O) ₂ NR'R", -NRSO ₂ R', -CN and -NO ₂ , -R', - N ₃ , -CH(Ph) ₂ , fluoro(C ₁ -C ₄ )alkoxy, and fluoro(C ₁ -C ₄ )alkyl, in numbers ranging from zero to the total number of open valencies on the aromatic ring system; And wherein 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 base. When a compound of the invention includes more than one R group, for example, each R group is independently selected, as are each R', R", R'" and R"" groups (when more than one of these group exists). In the schemes below, the symbol X represents the above-mentioned "R".

Two substituents on adjacent atoms of an aryl or heteroaryl ring may optionally be replaced by substituents of the general formula -TC(O)-(CRR') _q -U-, where T and U are independently is -NR-, -O-, -CRR'- or a single bond, and q is an integer of 0-3. Alternatively, two substituents on adjacent atoms of an aryl or heteroaryl ring may optionally be replaced by substituents of the general formula -A-(CH ₂ ) _r -B-, where A and B are independently is -CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O) _2- , -S(O) _2NR'- or a single bond, and r is 1 An integer of -4. One of the single bonds of the new ring thus formed may optionally be replaced by a double bond. Alternatively, two substituents on adjacent atoms of an aryl or heteroaryl ring may optionally be replaced by substituents of the general formula -(CRR') _s -X-(CR"R'") _d- , wherein s and d are independently integers from 0 to 3, and X is -O-, -NR'-, -S-, -S(O)-, -S(O) ₂ -, or -S(O ) ₂ NR′-. The substituents R, R', R" and R'" are preferably independently selected from hydrogen or substituted or unsubstituted (C ₁ -C ₆ )alkyl groups.

The term "heteroatom" as used herein is intended to include oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).

The use of reactive derivatives of PEG (or other linkers) to link one or more peptide moieties to the linker is within the scope of the present invention. The present invention is not limited by the properties of the reactive PEG analogs. Many activated derivatives of poly(ethylene glycol) are commercially available and available in the literature. It is within the ability of a person skilled in the art to select and (if necessary) synthesize an appropriate activated PEG derivative (which is employed to prepare a substrate for use in the present invention). See Abuchowski et al. Cancer Biochem. Bophys., 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 derivative active ester (Buckmann et al., Makromol.Chem., 182:1379-1384 (1981); Joppich et al. People, 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), carbonate (Zalipsky et al., poly (Ethylene Glycol) Chemistry: Biotechnology 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 carboxylate (Beauchamp et al., Anal.Biochem., 131:25-33 (1983); Berger et al. , Blood, 71:1641-1647 (1988)), 4-dithiopyridine (Woghiren et al., Bioconjugate Chem., 4:314-318 (1993)), isocyanate (Byun et al., ASAIOJournal, M649-M- 653 (1992)) and epoxides (US Patent No. 4,806,595 to Noishiki et al., (1989). Their Its linking group consists of a urethane bond between the amino group and activated PEG. See Veronese et al., Appl. Biochem. Biotechnol., 11:141-152 (1985).

The term "amino acid" denotes natural and synthetic amino acids, as well as amino acid analogs and amino acid mimetics. Natural amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as natural amino acids, ie, the alpha carbon bonded to hydrogen, carboxyl, amino, and R groups, such as homoserine, proleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs contain modified R groups (eg, proleucine) or modified peptide backbones, but retain the same basic chemical structure as a natural amino acid. "Amino acid mimetic"means a compound that differs in structure from the general chemical structure of an amino acid, but functions in a manner similar to natural amino acids.

"Peptide"means a polymer in which the monomers are amino acids, amino acid analogs and/or amino acid mimetics and are held together by amide bonds, or referred to as a polypeptide. Additionally, unnatural amino acids such as beta-alanine, phenylglycine, and homoarginine are also included. Amino acids that are not genetically encoded are also useful in the present invention. In addition, amino acids modified to include reactive groups, glycosylation sites, polymers, therapeutic moieties, biomolecules, etc. are also useful in the present invention. All amino acids used in the present invention may be D- or L-isomers. Usually the L-isomer is preferred. In addition, other peptidomimetics can also be used in the present invention. "Peptide" as used herein refers to both glycosylated and non-glycosylated peptides. Also included are peptides that are incompletely glycosylated by the system expressing the peptide. For a general review see Spatola, A.F., Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983).

The term "nucleoside" refers to a glycosylamine which is a component of nucleic acids and includes links to β-D-ribofuranose to form ribonucleosides, or to 2-deoxy-β-D-ribofuranose to form The nitrogenous base of a deoxyribonucleoside. The base may be a purine such as adenine or guanine, or a pyrimidine such as thymidine, cytidine, uridine or pseudouridine. Nucleosides also include unusual nucleosides used by microorganisms.

The term "targeting moiety" as used herein means a species that is selectively localized in a particular tissue or region of the body. Localization is mediated by specific recognition of molecular determinants, molecular size of targeting agents or conjugates, ionic interactions, hydrophobic interactions, etc. Other mechanisms for targeting agents to specific tissues or regions are known to those skilled in the art. Exemplary targeting moieties include antibodies, antibody fragments, transferrin, HS-glycoproteins, coagulation factors, serum proteins, beta-glycoproteins, G-CSF, GM-CSF, M-CSF, EPO, and the like.

As used herein, "therapeutic moiety" means any agent useful in therapy, including, but not limited to, antibiotics, anti-inflammatory agents, antineoplastic agents, cytotoxins, and radioactive agents. "Therapeutic moieties" include prodrugs of biologically active agents, constructs in which more than one therapeutic moiety is bound to a carrier, such as a multivalent agent. Therapeutic moieties also include proteins and constructs comprising 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-α, -β, - gamma), interleukins (eg, interleukin II), serum proteins (eg, factors VII, VIIa, VIII, IX, and X), human chorionic gonadotropin (HCG), follicle-stimulating hormone (FSH), and Luteinizing hormone (LH) and antibody fusion proteins such as tumor necrosis factor receptor ((TNFR)/Fc domain fusion protein).

As used herein, "antineoplastic agent" means any agent useful in the fight against cancer, including, but not limited to, cytotoxins and agents (e.g., antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotics, propanes, carbazide, hydroxyurea, asparaginase, corticosteroids, interferons, and radioactive agents). Also included within the scope of the term "antineoplastic agent" are peptides having antineoplastic activity, such as conjugates of 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 formed between PSGL-1 and TNF-α.

"Cytotoxin or cytotoxic agent" as used herein means any agent that is harmful to cells. Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin Bixin, daunorubicin, authracinedione, mitoxantrone, aureomycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine , tetracaine, lidocaine, propranolol, and puromycin and its analogs or homologues. Other toxins include, for example, ricin, CC-1065 and analogs, duocarmycin. Still other toxins include diptheria toxins, and snake venoms (eg, cobra vipers).

As used herein, "radioactive agent" includes any radioactive isotope effective in diagnosing or destroying tumors. Examples include, but are not limited to Indium-111, Cobalt-60. Additionally, naturally occurring radioactive elements such as uranium, radium, and thorium, which generally represent mixtures of radioactive isotopes, are suitable examples of radioactive agents. Metal ions are typically chelated with organic chelating moieties.

Many useful chelating groups, crown ethers, cryptands, etc. 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, e.g., Pitt et al., "Design of chelating agents for the treatment of iron overload," in INORGANIC CHEMISTRY INBIOLOGY AND MEDICINE; Martell, Ed.; American Chemical Society, Washington, D.C., 1980, pp.279-312; Lindoy, Chemistry of Macrocyclic Ligand Complexes; CambridgeUniversity Press, Cambridge, 1989; Dugas, BIOORGANIC CHEMISTRY; Springer-Verlag, New York, 1989, and references contained therein.

In addition, various routes allowing attachment of chelators, crown ethers and cyclodextrins to other molecules are available to those skilled in the art. See, for example, Meares et al., "Properties of Chelate-Labeled Proteins and Peptides in Vivo." 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).

introduce

The present invention provides polymeric substances, as well as sugars, activated sugars, and nucleotide sugars bound to these polymers. Polymeric conjugates of nucleotide sugars are often substrates for enzymes that transfer the sugar moiety and its polymeric substituent to an appropriate acceptor moiety of the substrate. Accordingly, the present invention also provides substrates modified by sugar conjugation using polymer conjugates of nucleotide sugars and appropriate enzymes. Substrates that can be used for carbohydrate conjugation using the compounds of the present invention include peptides, such as glycopeptides, lipids, such as glycosides and ligands (andol, ceramides).

As discussed in the previous section, the art-recognized chemistry of covalent PEGylation relies on chemical attachment via reactive groups on amino acids or carbohydrates. Chemically mediated conjugation strategies are used to prepare useful conjugates through careful design of conjugates and reaction conditions. A major disadvantage of chemical conjugation of polymers to proteins or glycoproteins is the lack of selectivity for activating the polymer, which often results in attachment of the polymer at sites involved in the biological activity of the protein or glycoprotein. Several approaches have been developed to address site-selective conjugation chemistry, however, only one general approach has been developed for a variety of recombinant proteins.

In contrast to art-recognized approaches, the present invention provides compounds that employ novel strategies for highly selective, site-directed carbohydrate conjugation, such as carbohydrate-PEGylation, of branched water-soluble polymers. 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 nucleotide sugars or activated sugars of the invention. Sugar-conjugation can be performed enzymatically using glycosyltransferases (such as sialyltransferases) capable of transferring substances branched water-soluble polymer-sugars (such as PEG-sialic acid) to glycosylation sites ("sugar -PEGylated").

As discussed above, the present invention provides glycoconjugates having any desired carbohydrate structure partially modified by polymers. Sugar nucleotides and activated sugars based on these sugar structures are also part of the invention. The polymer modification moiety is attached to the sugar moiety enzymatically, chemically, or a combination thereof, thereby producing a modified nucleotide sugar. Sugars are substituted by polymer modifying moieties at any desired position. In an exemplary embodiment, the sugar is a furanose substituted at one or more of C-1, C-2, C-3, C-4 or C-5. In another embodiment, the present invention provides a pyranose substituted at one or more of C-1, C-2, C-3, C-4, C-5 or C-6 by a polymer modifying moiety . Preferably, the polymer modifying moiety is directly attached to the oxygen, nitrogen or sulfur pendant from the carbon. Alternatively, the polymer modifying moiety is attached to a linker located between the sugar and the modifying moiety. The linker is attached to oxygen, nitrogen or sulfur pendant from the selected carbon.

In a presently preferred embodiment, the polymer modifying moiety is appended to a position selected such that the resulting conjugate functions as a substrate for the enzyme used to bind the modified sugar moiety to another substance, such as a peptide , glycopeptides, lipids, glycolipids, etc. Exemplary enzymes are discussed in more detail herein and include glycosyltransferases (sialyltransferase, glucosyltransferase, galactosyltransferase, N-acetylglucosyltransferase, N-acetylgalactosyltransferase, mannosyltransferase, transferase, fucosyltransferase, etc.). Exemplary sugar nucleotides 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.

In exemplary embodiments, the conjugates of the invention comprise a sugar, an activated sugar, or a nucleotide sugar bound to one or more polymers, such as a branched polymer. Exemplary polymers include both water-soluble and water-insoluble classes.

In an exemplary embodiment, the polymer modifying group is directly or indirectly attached to a pyranose or furanose. For example:

In general formulas I and II, R ¹ is H, CH ₂ OR ⁷ , COOR ⁷ or OR ⁷ , wherein R ⁷ represents H, substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl. ^R2 is H, OH, NH or a moiety comprising nucleotides. Exemplary ^R according to this embodiment has the general formula:

where ^X1 represents O or NH and ^R8 is a nucleoside.

The symbols R ³ , R ⁴ , R ⁵ , R ⁶ and R ^6′ independently represent H, substituted or unsubstituted alkyl, OR ⁹ , NHC(O)R ¹⁰ . Flag d is 0 or 1. ^R9 and ^R10 are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, or sialic acid. At least one of ^R3 , ^R4 , ^R5 , ^R6 , and R6 ^' includes a polymer modifying moiety (eg, PEG). In an exemplary embodiment, R ⁶ and R ^6' together with the carbon to which they are attached are components of the sialic acid side chain. In still further exemplary embodiments, this side chain is modified at one or more of C-6, C-7 or C-9 by a polymer modifying moiety (or linker-polymer modifying moiety).

The symbols R ³ , R ⁴ , R ⁵ and R ⁶ independently represent H, OR ⁹ , NHC(O)R ¹⁰ . R ⁹ and R ¹⁰ are independently selected from H, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl. At least one of R ³ , R ⁴ , R ⁵ , R ⁶ , and R ^6' includes a polymer modifying moiety.

In another exemplary embodiment, the sugar moiety is a sialic acid moiety oxidized and bound to a polymer modifying moiety, as described in commonly assigned U.S. Provisional Patent Application No. ___ (Attorney Docket No. 040853-01- 5150), filed January 6, 2005.

In an exemplary embodiment, the polymer modifying moiety is bound to the sugar core via a linker:

wherein R ¹¹ is a polymer moiety and L is selected from a bond and a linking group, and w is an integer of 1-6, preferably 1-3 and more preferably 1-2.

When L is a bond, it is formed between a reactive functional group on the precursor of R ¹¹ and a complementary reactive reactive functional group on the L precursor. As illustrated herein, the selection and preparation of precursors with appropriate reactive functional groups is within the purview of those skilled in the art. In addition, precursor conjugation is performed according to chemistry well known in the art.

In an exemplary embodiment, L is a linker formed from an amino acid, an amino acid mimetic, or a small peptide (e.g., 1-4 amino acid residues), providing a modified sugar wherein the polymer is modified by a substituted Alkyl linker connection. The linker is formed by the reaction of the amine moiety of the amino acid having complementary reactive groups on the L and ^R11 precursors with a carboxylic acid (or reactive derivative such as active ester, acid halide, etc.). The elements of the combination may be combined in essentially any convenient order. For example, the precursor of L may be in place on the sugar core prior to binding ^R11 and the precursor of L. Alternatively, a ^R11 -L cassette with a reactive functionality on L and subsequent attachment to the sugar via a complementary reactive functional group on this material.

In an illustrated embodiment, the polymer modifying moiety is ^R3 and/or ^R6 . In another exemplary embodiment, ^R3 and/or ^R6 include both a polymer modifying moiety and a linker L that binds the polymer moiety to the remainder of the molecule. In another exemplary embodiment, the polymer modifying moiety is ^R3 . In yet another exemplary embodiment, ^R3 includes both a polymer modifying moiety and a linker L that binds the polymer moiety to the remainder of the molecule. In yet another exemplary embodiment, wherein the sugar is sialic acid, the polymer modifying moiety is attached at ^R5 or at the position of the sialic acid side chain, such as C-9.

linear polymer conjugate

In exemplary embodiments, the invention provides sugar or activated sugar conjugates or nucleotide sugar conjugates formed with linear polymers such as water soluble or water insoluble polymers. In the conjugates of the invention, the polymer is linked to a sugar, activated sugar or sugar nucleotide. As discussed herein, the polymer is attached to the sugar moiety either directly or through a linker.

Exemplary compounds according to this embodiment have a structure according to general formula I or II, wherein at least one R ¹ , R ³ , R ⁴ , R ⁵ or R ⁶ has the following general formula:

Another example according to this embodiment has the general formula:

wherein s is an integer from 0-20 and R ¹¹ is a linear polymer modification moiety.

PEG moieties of any molecular weight (eg, 2Kda, 5Kda, 10Kda, 20Kda, 30Kda and 40Kda) can be used in the present invention.

branched polymer conjugates

In an exemplary embodiment, the polymer modifying moiety is a branched structure comprising two or more polymer chains attached to a central moiety having the general formula:

wherein R ¹¹ and L are as discussed above and w' is an integer of 2-6, preferably 2-4 and more preferably 2-3.

Exemplary precursors for forming conjugates according to this embodiment of the invention have the general formula:

Branched polymers according to this general formula are substantially pure water-soluble polymers. ^X3' is _a moiety that includes ionizable (eg, COOH, _H2PO4 , _HSO3 , _HPO3, etc.) or other reactive functional groups, as described below. C is carbon. ^X5 is preferably a non-reactive group (eg, H, unsubstituted alkyl, unsubstituted heteroalkyl), and can be a polymer arm. R ¹² and R ¹³ are independently selected polymer arms, such as non-peptidic, non-reactive polymer arms. ^X2 and ^X4 are preferably substantially non-reactive linking fragments under physiological conditions, which may be the same or different. Alternatively, these linkages may include one or more moieties, such as esters, disulfides, etc., designed to degrade under physiologically relevant conditions. ^X2 and ^X4 bind polymer arms ^R12 and ^R13 to C. When X ^3' is reacted with a complementary reactive functional group on the linker, sugar or linker-sugar combination, X ^3' is converted to a component of the linker fragment ^X3 .

Exemplary linker fragments for ^X2 and ^X4 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 ₂ S, CH ₂ O, CH ₂ CH ₂ O, CH ₂ CH ₂ S, (CH ₂ ) _a O, (CH ₂ ) _a S or (CH ₂ ) _a Y′- PEG or (CH ₂ ) _a Y′-PEG, wherein Y′ is S or O and a is an integer of 1-50.

In an exemplary embodiment, precursor (III), or an activated derivative thereof, is bound to the sugar, activated sugar or sugar nucleotide via a reaction between X ^3' and a complementary reactive group on the sugar moiety. Alternatively, X ^3' reacts with a reactive functional group on the precursor to form a linker L. One or more of R ¹ , R ³ , R ⁴ , R ⁵ or R ⁶ of formulas I and II may include branched polymer modifying moieties.

In the illustrated embodiment, the following sections:

is the connecting base arm L. In this embodiment, exemplary linkers are derived from natural or unnatural amino acids, amino acid analogs or mimetics, or small peptides formed from one or more of such substances. For example, certain branched polymers found in the compounds of the present invention have the general formula:

^Xa is the linking moiety formed by the reaction of the branched polymer modifying part and the reactive functional group on the precursor of the sugar moiety, or the reaction of the precursor to the linking group. For example, when ^X3 ' is a carboxylic acid, it can be activated and bond directly to an amine group pendant from an amino-sugar (eg, _GalNH2 , _GlcNH2 , _ManNH2, etc.) to form ^Xa which is an amide. Additional exemplary reactive functional groups and activated precursors are described below. Mark c represents an integer of 1-10. Other symbols have the same identity as those discussed above.

In another exemplary embodiment, ^X is a linking moiety formed using another linker:

wherein ^Xb is a linking moiety and is independently selected from those groups specified for ^Xa , and ^L1 is a bond, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl.

Exemplary substances for Xa and ^Xb 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.

For example,

wherein s is an integer from 0-20 and R ¹¹ is a linear polymer modification moiety.

In another exemplary embodiment, ^X4 is a peptide bond bound to ^R13 , which is an amino acid, di-peptide or tri-peptide, wherein the α-amine moiety and/or the side chain heteroatom is modified by the polymer.

In further exemplary embodiments, ^R comprises a branched polymer modifying group and the modified sugar or nucleotide sugar has a general formula selected from:

The identity of the groups represented by the various symbols therein is the same as discussed above. L ^a is a substituted or unsubstituted alkyl or a substituted or unsubstituted heteroalkyl moiety. In an exemplary embodiment, ^La is a moiety of the side chain of sialic acid, the sialic acid being modified by the polymer modifying moiety as shown.

In yet another exemplary embodiment, the invention provides sugars and nucleotide sugars having the general formula:

The identity of the groups represented by the various symbols is the same as discussed above. As will be recognized by those skilled in the art, the linker arms in Formulas VI and VIII are equally applicable to the other modified sugars described herein.

The embodiments of the invention described above are further exemplified with reference to materials in which the polymer is a water-soluble polymer, particularly poly(ethylene glycol) ("PEG"), such as methoxy-poly(ethylene glycol) alcohol) ("m-PEG"). Those skilled in the art will recognize that the emphasis in the following is for clarity and that the various motifs using PEG as an exemplified polymer are equally applicable to species in which polymers other than PEG are employed.

water soluble polymer

Many water soluble polymers are known to those skilled in the art and can be used in the practice of this invention. The term water-soluble polymer includes substances such as sugars (eg, dextran, amylose, hyaluronic acid, poly(sialic acid), heparinoids, heparin, etc.); poly(amino acids), such as poly(aspartic acid) and poly(glutamic acid); nucleic acids; synthetic polymers such as poly(acrylic acid), poly(ethers), such as poly(ethylene glycol); peptides, proteins, etc. Polymers typically comprise at least two polymer units. In an illustrated embodiment, the polymer is 2-25 units. In another illustrated embodiment, the polymer includes 2-8 polymer units. The present invention can be implemented using any water-soluble polymer, only The limitation is that the polymer must include points to which the remainder of the conjugate can be attached.

Methods of activation of polymers can also be found in WO 94/17039, U.S. Patent No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Patent No. 5,219,564, U.S. Patent No. 5,122,614, WO 90/13540, U.S. Patent No. 5,281,698, and others WO93/15189, and for the activation of polymers and peptides, such as coagulation factor VIII (WO94/15625), hemoglobin (WO 94/09027), oxygen transport molecules (US Patent No. 4,412,989), ribonuclease and superoxide Binding between enzymes (Veronese et al., App. Biochem. Biotech. 11:141-45 (1985)).

Preferred water-soluble polymers are those in which a substantial portion of the polymer molecules in a sample of polymer have about the same molecular weight; such polymers are "uniformly dispersed".

The invention is further illustrated by reference to poly(ethylene glycol) conjugates. Several reviews and monographs on PEG functionalization and conjugation are available. See, e.g., Harris, Macronol.Chem.Phys.C25:325-373 (1985); Scouten, Methods in Enzymology 135:30-65 (1987); Wong et al., Ehzyme Microb.Technol.14:866-874 (1992 ); Delgado et al., Critical Review 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 reactive molecules are known in the art. For example, U.S. Patent No. 5,672,662 discloses water-soluble and isolatable combinations of active esters of polymeric acids selected from the group consisting of linear or branched poly(alkylene oxides), poly(oxyethylated polyols), poly(oxyethylated polyols), poly(oxyethylated polyols), (alkenyl alcohol), and poly(acryloylmorpholine).

U.S. Patent No. 6,376,604 describes the preparation of water-soluble and non-peptidic polymers of water-soluble 1-benzotriazolylcarbonate by reacting the terminal hydroxyl groups of the polymer with bis(1-benzotriazolyl)carbonate in an organic solvent. ester method. Active esters are used to form conjugates with biologically active agents such as proteins or peptides.

WO 99/45964 describes a combination comprising a bioactive agent and an activated water-soluble polymer comprising a polymer backbone comprising at least one terminus attached to the polymer backbone by a suitable bond, Wherein at least one end includes a branched portion comprising a proximal reactive group attached to the branched portion, wherein the bioactive agent is attached to the at least one proximal reactive group. Other branched poly(ethylene glycol)s are described in WO 96/21469, and U.S. Pat. No. 5,932,462 describes conjugates formed using branched PEG molecules that include branched ends that include reactive functional groups. Free reactive groups can be used to react with biologically active substances, such as proteins or peptides, to form conjugates between poly(ethylene glycol) and biologically active substances. US Patent No. 5,446,090 describes a bifunctional PEG linker and its use in forming conjugates having a peptide at the end of each PEG linker.

Conjugates comprising degradable PEG linkages are described in WO 99/34833; and WO 99/14259, and in U.S. Patent No. 6,348,558. Such degradable linkages are suitable for use in the present invention.

The art-recognized polymer activation methods described above can be used for the formation of branched polymers and the incorporation of these branched polymers into other species (eg, sugars, sugar nucleotides, etc.) according to the invention described herein.

Exemplary modifying groups are discussed below. Modifying groups can be selected for their ability to impart one or more desired properties to the peptide. Exemplary properties include, but are not limited to, improved pharmacokinetics, enhanced pharmacodynamics, improved biodistribution, provision of multivalent substances, improved water solubility, increased or decreased lipophilicity, and tissue targeting.

Exemplary poly(ethylene glycol) molecules useful in the present invention include, but are not limited to, those having the general formula:

wherein ^A2 is H, OH, _NH2 , substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroalkyl, Such as acetal, OHC-, H ₂ N-(CH ₂ ) _q -, HS-(CH ₂ ) _q , or -(CH ₂ ) _q C(Y ^b )Z ^b . The notation "e" represents an integer of 1-2500. The notations b, d, and q independently represent integers from 0-20. The symbols Z ^a and Z ^b independently represent OH, NH ² , leaving groups such as imidazole, p-nitrophenyl, HOBT, tetrazole, halides, SR ^a , alcohol moieties of activated esters; -(CH ₂ ) _p C(Y ^b )V, or -(CH ₂ ) _p U(CH ₂ )SC(Y ^b ) _v . The symbol Y ^a represents H(2), =O, =S, =NR ^b . The symbols X ^a , Y ^a , Y ^b , A ¹ , and U independently represent the groups O, S, NR ^c . The symbol V represents OH, _NH2 , halogen, ^SRa , the alcohol component of activated esters, the amine component of activated amides, sugar-nucleotides, and proteins. The labels p, q, s and v are integers independently selected from 0-20. The symbols R ^a , R ^b , and R ^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.

Specific embodiments of linear and branched polymers (such as PEG) useful in the present invention include:

and carbonates and active esters of these substances, such as:

Can be used to form linear and branched polymeric species, linker arm conjugates of these species and conjugates between these compounds and sugars and nucleotide sugars. Indices e and f are independently selected from 1-2500.

Other exemplary activating, or leaving groups suitable for activating linear PEG for the preparation of compounds described herein include, but are not limited to, the following:

It is within the ability of those skilled in the art to choose the appropriate activating group for the selected moiety on the precursor to the polymer modifying moiety.

PEG molecules activated with these and other substances and methods of making activated PEGs are described in WO 04/083259.

In an exemplary embodiment, the branched polymer is a cysteine, serine, lysine, di- or tri-lysine core based PEG. Thus, further exemplary branched PEGs include:

Indices e and f are independently selected from 1-2500.

In yet another embodiment, the branched PEG moieties are based on tris-lysine peptides. Tris-lysine can be mono-, di-, tri-, or tetra-PEG-ylated. Exemplary substances according to this embodiment have the general formula:

wherein e, f and f' are independently selected from integers of 1-2500; and q, q' and q" are independently selected from integers of 0-20.

In an exemplary embodiment of the invention, the PEG is m-PEG (5kD, 1OkD, 2OkD, 3OkD or 4OkD). Exemplary branched PEG species are lysine-, serine- or cysteine-(m-PEG) ₂ , where m-PEG is 20kD m-PEG.

It will be apparent to those skilled in the art that the branched polymers useful in the present invention include variations of what has been described above. For example the di-lysine-PEG conjugate shown above may comprise three polymer subunits, the third bonded to the unmodified α-amine shown in the structure above. Similarly, tris-lysine functionalized with three or four polymer subunits are within the scope of the invention.

Those skilled in the art will recognize that one or more m-PEG arms of a branched polymer may be replaced by a PEG moiety with a different end (eg OH, COOH, NH ₂ , C ₂ -C ₁₀ -alkyl, etc.). In addition, it is easy to modify the above structure by inserting an alkyl linker (or removing a carbon atom) between the α-carbon atom and the functional group of the side chain. Thus, "homo" derivatives and higher homologues, as well as lower homologues, are within the core range of branched PEGs useful in the present invention.

The branched PEG species described here are readily prepared by the methods illustrated in the following schemes:

wherein X ^b is O, NH or S and r is an integer of 1-10. Indices e and f are independently selected from integers from 1-2500. Exemplary branched PEG species are 10,000, 15,000, 20,000, 30,000 and 40,000 Daltons.

Thus, according to this scheme, a natural or unnatural amino acid is contacted with an activated m-PEG derivative (in this case tosylate) to form compound 1 by alkylating the side chain heteroatom ^Xb . Branched m-PEG 2 is assembled by subjecting mono-functionalized m-PEG amino acids and reactive m-PEG derivatives to N-acylation conditions. As will be recognized by those skilled in the art, the tosylate leaving group can be changed to any suitable leaving group, such as halo, mesylate, triflate, and the like. Similarly, the reactive carbonates used to acylate amines can be changed to active esters, such as N-hydroxysuccinimide, etc., or the acids can use dehydrating agents (such as dicyclohexylcarbodiimide, carbonyldiimidazole, etc.) In situ activation.

In the exemplified scheme described above, the modifying group is a linear PEG moiety, however, any modifying group such as water soluble polymers, water insoluble polymers, branched polymers, therapeutic moieties, etc. part.

Further branched polymer species for the compounds of the invention are exemplified by PEG functionalized branched cores, as illustrated in the following examples:

where ^R14 is OH or another reactive functional group. An exemplary reactive functional group is C(O)Q', where 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 label "e" and the label "f" are integers independently selected from 1-2500.

The branched compounds described above, and additional branched compounds useful in the compounds of the present invention, are readily prepared from:

Polymer Modified Sugar Substances

The sugar moiety of the nucleotide sugars of the invention may be selected from both natural and unnatural furanose and hexose sugars. Non-natural sugars optionally include alkylated or acylated hydroxyl and/or amine moieties such as ring ether, ester and amide substituents. Other non-natural sugars include H, hydroxyl, ether, ester, or amide substituents at ring positions that do not exist in natural sugars at that position. The sugar moieties can be monosaccharides, oligosaccharides or polysaccharides.

Exemplary natural sugars for use in the present invention include glucose, galactose, fucose, mannose, xylose, ribose, N-acetylglucose, sialic acid, and N-acetylgalactose.

Similarly, nucleosides can be selected from both natural and unnatural (or rare) nucleosides. Exemplary natural nucleosides for use in the invention include cytosine, thymine, guanine, adenine, and uracil. Rare nucleosides may include, but are not limited to, such molecules as arabouridine and arabyothymidine. Unnatural and unusual nucleosides and methods for their preparation are well-documented in the art.

Exemplary modified sugar nucleotides of the present invention include GDP-Man, GDP-Fuc, UDP-Gal, UDP-Gal-NH ₂ , UDP-GalNAc, UDP-Glc, UDP-Glc-NH 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 polymer modifying moieties (or linker-modifying moieties) at any position on the sugar. For example, compounds covered by the present invention include those wherein the LR ¹¹ moiety is bound to C-5 of the furanose nucleotide sugar or C-6 of the pyranose nucleotide sugar.

Exemplary moieties attached to 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 Moiety, Mannose-6-Phosphate, Heparin, Heparanoid, SLe _x , Mannose, Mannose-6-Phosphate, Sialyl Lewis X, FGF, VFGF, Protein, Chondroitin, Keratin, Dermatin , albumin, integrins (integrins), antennal oligosaccharides, peptides, etc. Methods for incorporating various modifying groups to sugar moieties are readily available to those skilled in the art (Poly(ethylene glycol) Chemistry: Biotechnology and Biomedical Applications, J. Milton Harris, Ed., Plenum Pub. Corp., 1992; Poly(ethylene glycol) Chemistry and Medical 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 Drug Delivery Systems, ACS Symposium Series Vol. 469, American Chemical Society, Washington, DC 1991).

Exemplary sugar nucleotides of the invention in their modified forms include nucleotide mono-, di- or triphosphates or UDP-glycoside, CMP-glycoside, or GDP-glycoside analogs thereof. Even more preferably, the modified sugar nucleotide is selected from UDP-galactose, UDP-galactosamine, UDP-glucose, UDP-glucosamine, GDP-mannose, GDP-fucose, CMP-sialic acid, or CMP-NeuAc. N-acetylamine derivatives of sugar nucleotides are also useful in the methods of the invention.

In other embodiments, the modified sugar is an activated sugar. Activated modified sugars for use in the present invention are typically glycosides that are synthetically converted to include an activated leaving group. The term "activating leaving group" as used herein refers to those moieties which are readily transferred in an enzyme-mediated nucleophilic substitution reaction. Many activated sugars are known in the art. See, for example, Vocadlo et al., 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)).

Examples of activating groups (leaving groups) include fluorine, chlorine, bromine, tosylate, mesylate, triflate, and the like. Preferred activating leaving groups for use in the present invention are those that do not significantly sterically hinder 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, α-galactosyl fluoride, α-mannosyl fluoride, α-glucosyl fluoride, α-fucosyl fluoride, α-xylosyl fluoride, α - Sialyl fluoride, α-N-acetylglucosamine fluoride, α-N-acetylgalactosamine fluoride, β-galactosyl fluoride, β-mannosyl fluoride, β-glucosyl fluoride Fluoride, β-fucosyl fluoride, β-xylosyl fluoride, β-sialyl fluoride, β-N-acetylglucosamine fluoride, β-N-acetylgalactosamine fluoride .

As indicated, glycosyl fluorides can be prepared from free sugars by first acetylating the sugar and then treating it with HF/pyridine. This produces the most thermodynamically stable anomer of the protected (acetylated) glycosyl fluoride (ie, the alpha-diol fluoride). If stabilization of the smaller anomer (i.e., β-glycosyl fluoride) is desired, it can be prepared by converting a peracetylated sugar with HBr/HOAc or with HCl to produce the anomer bromide or chloride. This intermediate is reacted with a fluoride salt such as silver fluoride to produce glycosyl fluoride. Acetylated glycosyl fluorides can be deprotected by reaction with a mild (catalyzed) base in methanol such as NaOMe/MeOH. In addition, many glycosyl fluorides are commercially available.

Other activated glycosyl derivatives can be prepared using conventional methods known to those skilled in the art. For example, glycosyl mesylate can be prepared by treating the fully benzylated hemiacetal form of the sugar with methanesulfonyl chloride, followed by catalytic hydrogenation to debenzylate.

In a further exemplary embodiment, the modified sugar is an oligosaccharide having an antenna structure. In another embodiment, one or more ends of the antennae carry a modified moiety. Oligosaccharides can be used to "amplify" the modifying moiety when more than one modifying moiety is attached to the oligosaccharide with the antenna structure; each oligosaccharide unit bound to the peptide attaches multiple copies of the modifying group to the peptide. The general structures of typical conjugates of the invention illustrated in the above figures include polyvalent species produced by employing the antennae structure to prepare conjugates of the invention. Many antennal saccharide structures are known in the art, and the method can be practiced using them without limitation.

In an exemplary embodiment, the activated modified sugar is a substrate for a polymorphic enzyme which transfers the sugar to an appropriate acceptor site on the substrate. Exemplary mutant enzymes include, for example, those described in commonly assigned PCT Publications WO03/046150 and WO03/045980.

Water-soluble polymer-modified sugars, activated sugars, and nucleotide sugar substances in which the sugar moiety is modified by a water-soluble polymer (such as a water-soluble polymer) are used in the present invention. Exemplary modified sugar nucleotides have Amine moiety-modified sugar groups. Modified sugar nucleotides (such as sugar-amine derivatives of sugar nucleotides) are also suitable for use in the method of the invention. For example, sugar amines (without modifying groups) can be enzymatically method to bind to a peptide (or other substance) and the free glycosylamine moiety is subsequently bound to the desired modifying group. Alternatively, the modified sugar nucleotide can act as a substrate for an enzyme that transfers the modified sugar to the end Glycosyl acceptors on substances, such as peptides, glycopeptides, lipids, aglycone (aglycone), glycolipids, etc.

In one embodiment, the sugar is bound to a branched polymeric material, such as those described herein.

In another embodiment, the sugar moiety is a modified sialic acid. When sialic acid is a sugar, the sialic acid is modified by a modifying group at the 9-position on the pyruvyl side chain or at the 5-position on the amine moiety that is normally acetylated in sialic acid .

In another embodiment, wherein the sugar nucleus is galactose or glucose, R ⁵ is NHC(O)Y.

In an exemplary embodiment, the modified sugar is based on a 6-amino-N-acetyl-glycosyl moiety. The 6-amino-sugar moiety is readily prepared by standard methods as shown below for N-acetylgalactosamine:

(a. Galactose oxidase; NH ₄ OAc, NaBH ₃ CN; b.

In the above schemes, the notation n represents an integer of 1-2500, preferably 10-1500, and more preferably 10-1200. The symbol "A" represents an activating group, such as a halogen, a component of an activated ester (eg, N-hydroxysuccinimide ester), a component of a carbonate (eg, p-nitrophenyl carbonate), and the like. Those skilled in the art will recognize that other PEG-amide nucleotide sugars are readily prepared by this and similar methods. Additionally, branched polymers can be substituted for linear PEG, as described herein.

Another exemplary polymer-modified nucleotide sugar of the present invention has the following general formula, wherein the C-6 position is modified:

where ^X6 is a bond or O, J is S or O, and y is 0 or 1. Indices e and f are independently selected from 1-2500.

In other exemplary embodiments, the amide moiety is replaced by a group such as urethane or urea.

In the following discussion, a number of specific examples of modified sugars suitable for use in the practice of the present invention are described. In an exemplary embodiment, a sialic acid derivative is used as the sugar core to which the modifying group is attached. The focus of the discussion of sialic acid derivatives is for clarity of illustration only and should not be construed as limiting the scope of the invention. Those skilled in the art will recognize that various other sugar moieties can be activated and derivatized in a manner similar to that illustrated using sialic acid as an example. For example, many methods can be used to modify galactose, glucose, N-acetylgalactosamine and fucose to name some sugar substrates, which are easily modified by methods recognized in the art. See, eg, Elhalabi et al., Curr. Med. Chem. 6:93 (1999); and Schafer et al., J. Org. Chem. 65:24 (2000)).

In Fig. 2, a general scheme according to the invention is depicted. Thus, according to Figure 2, an amide conjugate between mannosamine and a protected amino acid is formed by contacting mannosamine with an N-protected amino acid under appropriate conditions to form the conjugate. The carboxyl terminus of the protected amino acid is activated in situ or it is optionally converted into a storage stable reactive group such as N-hydroxy-succinimide. Amino acids may be selected from any natural or unnatural amino acid. Those skilled in the art understand how to protect side chain amino acids from undesired reactions in the methods of the invention. The amide conjugate is reacted with pyruvate and sialic acid aldolase under appropriate conditions to convert the amide conjugate to a sialic acid amide conjugate, which is subsequently synthesized from the sialic acid amide conjugate with a nucleotide phosphate precursor and an appropriate enzyme. Reaction, conversion into nucleotide phospho-sialic acid amide conjugates. In an exemplary embodiment, the precursor is cytidine triphosphate and the enzyme is a synthetase. Following formation of the nucleotide sugar, the amino acid amine is deprotected to provide a free, reactive amine. Amines are used as sites for attachment of modifying moieties to nucleotide sugars. In FIG. 2, the exemplified modification moieties are water-soluble polymers, namely poly(ethylene glycol), such as PEG, m-PEG, and the like.

The invention is further illustrated in Figure 3, which depicts a scheme for the preparation of sialic acid-glycyl-PEG-cytidine monophosphate. Similar to the scheme illustrated in Figure 2, Figure 3 was derived from mannosamine. The sugar is conjugated using FMOC-glycine, using an N-hydroxysuccinimide activated derivative of a protected amino acid. The action of sialic acid aldolase on the conjugate and pyruvate converts the obtained amide conjugate into the corresponding sialic acid. The obtained sialic acid conjugates are converted to cytidine monophosphate analogs using cytidine triphosphate and synthetase. Deprotection of the CMP-analogue by removal of the protecting group from the amino acid amine moiety converts this moiety into a reactive site for conjugation. The amine moiety reacts with an activated PEG species (m-PEG-O-nitrophenyl carbonate), thereby forming sialic acid-glycyl-PEG-cytidine monophosphate.

Exemplary sugar cores based on sialic acid have the general formula:

wherein D is -OH or (R ¹¹ ) _w '-L-. The symbol G represents H, (R ¹¹ ) _w '-L- or -C(O)(C ₁ -C ₆ )alkyl. R ¹¹ is as described above. At least one of D and G is R ¹¹ -L-.

In another embodiment, the invention provides sugars, activated sugars or sugar nucleotides comprising the structure:

wherein ^L2 is as described in the discussion for L, such as a bond, a substituted or unsubstituted alkyl, or a substituted or unsubstituted heteroalkyl. The symbol e represents an integer from 1 to about 2500.

In another embodiment, the sugar or sugar nucleotide comprises the structure:

Wherein s is selected from the integer of 0-20, and e is 1-2500.

Selected sialic acid-based nucleotide sugars functionalized with branched polymers have the general formula:

where AA is an amino acid residue, PEG is poly(ethylene glycol) or methoxy-poly(ethylene glycol) and NP is a nucleotide, which is attached to the sugar by a phosphodiester bond ("nucleotide phosphate") base part. Those skilled in the art will recognize that ONP can be replaced by the activating moieties discussed herein.

In a further embodiment, the structure of the sialic acid derivative is a member selected from:

where ^X6 is a bond or O, and J is S or O. The labels a, b and c are independently selected from 0-20, while e and f are independently selected from 1-2500.

In addition, as discussed above, the present invention provides nucleotide sugars modified by water-soluble polymers, which are linear or branched. For example, compounds having the general formula shown below are within the scope of the invention:

where ^X6 is a bond or O, and J is S or O. Indices e and f are independently selected from 1-2500.

Conjugates of peptides and glycopeptides, lipids and glycolipids comprising compositions of the invention are also provided. The conjugate is formed by a suitable acceptor moiety and an enzyme (the modified nucleotide sugar is the substrate of the enzyme) that binds a nucleotide sugar or activated sugar of the invention and a substrate and sugar moiety under appropriate conditions , to transfer the modified sugar from the nucleotide sugar to the acceptor moiety. For example, the invention provides conjugates having the general formula:

where J and X ⁶ are as discussed above. Labels a, b, c, e and f are as discussed above.

Selected compounds of the present invention are based on substances with the stereochemistry of mannose, galactose and glucose. The general formula of these compounds is:

wherein one of R ³ -R ⁶ is a modifying moiety, such as a polymer modifying moiety or a polymer modifying moiety-linker construct.

As discussed above, certain compounds of the invention are polymer-modified sugar nucleotides. Exemplary sugar nucleotides for use in the present invention in their modified form include nucleotide mono-, di- or triphosphates or analogs thereof. In preferred embodiments, the modified sugar nucleotides are selected from UDP-glycosides, CMP-glycosides, or GDP-glycosides. Even more preferably, the modified sugar nucleotide is selected from UDP-galactose, UDP-galactosamine, UDP-glucose, UDP-glucosamine, GDP-mannose, GDP-fucose, CMP-sialic acid, or CMP-Sia. In an exemplary embodiment, a nucleotide mono-di- or tri-phosphate is attached to C-1.

Glyco-amine derivatives of sugar nucleotides are also suitable for use in the methods of the invention. For example, a glycosylamine (without a modifying group) can be enzymatically conjugated to a peptide (or other substance) with the free glycosylamine moiety subsequently conjugated to the desired modifying group.

The sugar nucleotide conjugates of the present invention are generally described by the following general formula:

where the symbols represent groups as discussed above. When the sugar core is mannose, the polymer modification part is preferably at R ³ , R ⁴ or R ⁶ . For glucose, the polymer modification moiety is optionally at ^R5 or ^R6 . The token "u" is 0, 1 or 2.

A further exemplary nucleotide sugar of the present invention based on GDP mannose has the following structure:

In a further exemplary embodiment, the invention provides a conjugate based on UDP galactose having the following structure:

In another exemplary embodiment, the nucleotide sugar is glucose-based and has the general formula:

In each of the three preceding formulas, the identity of the groups and labels is as discussed above.

It will be apparent to those skilled in the art that the linear PEG moieties may be replaced by branched polymers or other linear polymeric species as described herein.

In one embodiment, wherein the sugar core is galactose or glucose, ^R is NHC(O)Y.

water insoluble polymer

In another embodiment, similar to those discussed above, the modified sugar comprises a water insoluble polymer instead of a water soluble polymer. Water insoluble polymers, like water soluble polymers, typically consist of at least two polymer units. In an exemplary embodiment, the polymer consists of 2-25 polymer units. In another exemplary embodiment, the polymer consists of 2-8 polymer 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 conjugates as vehicles with which to deliver therapeutic peptides in a controlled manner. Polymeric drug delivery systems are known in the art. See, eg, Dunn et al., Eds. Polymeric Drugs and Drug Delivery Systems, ACS Symposium Series Vol. 469, American Chemical Society, Washington, D.C. 1991. Those skilled in the art will recognize that essentially any known drug delivery system is suitable for use in the conjugates of the invention.

Representative water-insoluble polymers include, but are not limited to, polyphosphazines, poly(vinyl alcohol), polyamides, polycarbonates, polyalkylenes, polyacrylamides, polyalkylene glycols, polyalkylene oxides, poly Alkylene terephthalate, polyvinyl ether, polyvinyl ester, polyvinyl halide, polyvinyl pyrrolidone, polyglycolide, polysiloxane, polyurethane, poly(methyl methacrylate), Poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(methacrylate lauryl acrylate), 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, polyvinylpyrrolidone , poloxamers and polyvinylphenol and its copolymers.

Synthetically modified natural polymers useful in the conjugates of the present invention include, but are not limited to, alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, and nitrocelluloses. Particularly preferred members of the large class of synthetically modified natural polymers include, but are not limited to, methylcellulose, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxybutylmethylcellulose, cellulose acetate cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxymethyl cellulose, cellulose triacetate, sodium cellulose sulfate, and polymers of acrylic and methacrylates and seaweed acid.

These and other polymers discussed herein are readily available from commercial sources such as Sigma Chemical Co. (St. Louis, MO.), Polysciences (Warrenton, PA.), Aldrich (Milwaukee, WI.), Fluka (Ronkonkoma, NY) , and BioRad (Richmond, CA), or monomers obtained from these suppliers were synthesized using standard techniques.

Representative biodegradable polymers for use in the conjugates of the invention include, but are not limited to, polylactide, polyglycolide 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. Particularly useful are gel-forming compositions, such as those comprising collagen, poloxamers, and the like.

Polymers useful in the present invention include "hybrid"polymers that include water-soluble materials that contain bioresorbable molecules in at least a portion of their structure. Examples of such polymers are polymers comprising water insoluble copolymers containing bioresorbable regions, hydrophilic regions and multiple crosslinkable functional groups per polymer chain.

For purposes of the present invention, "water-insoluble materials" include materials that are substantially insoluble in water or aqueous environments. Thus, while certain regions or segments of the copolymer may be hydrophilic or even water soluble, the polymer molecule as a whole is insoluble in water by any substantial measure.

For the purposes of the present invention, the term "bioresorbable molecule" includes regions (moieties) capable of being metabolized or destroyed and reabsorbed and/or eliminated by the body's normal excretory pathways. Such metabolites or degradation products are preferably substantially non-toxic to the body.

The bioresorbable regions can be hydrophobic or hydrophilic as long as the copolymer as a whole does not become water soluble. Thus, the bioresorbable regions are preferentially selected to render the polymer as a whole insoluble in water. Accordingly, the relative properties, ie, the type of functional groups comprised by the bioresorbable regions, and the relative ratio of bioresorbable regions to hydrophilic regions are selected to ensure that useful bioresorbable compositions remain water-insoluble.

Exemplary resorbable polymers include, for example, synthetically produced resorbable block copolymers of poly(alpha-hydroxy-carboxylic acid)/poly(oxyalkylene) (see Cohn et al., US Patent No. 4,826,945). These copolymers are not crosslinked and are water soluble, allowing the body to excrete the degraded block copolymer composition. See Younes et al., J Biomed. Mater. Res. 21:1301-1316 (1987); and Cohn et al., J Biomed. Mater. Res. 22:993-1009 (1988).

Presently preferred bioresorbable polymers comprise one or more components selected from the group consisting of poly(esters), poly(hydroxy acids), poly(lactones), poly(amides), poly(ester-amides) , poly(amino acids), poly(anhydrides), poly(orthoesters), poly(carbonates), poly(phosphazines), poly(phosphates), poly(thioesters), polysaccharides, and mixtures thereof. Still more preferably, the bioresorbable polymer includes a poly(hydroxy)acid component. Among the poly(hydroxy) acids, polylactic acid, polyglycolic acid, polycaproic acid, polybutyric acid, polyvaleric acid, and copolymers and mixtures thereof are preferred.

In addition to forming fragments that are absorbed in vivo ("bioresorbable"), preferred polymer coatings for use in the methods of the invention may also form secretable and/or metabolizable fragments.

Higher grade copolymers are also useful in this invention. For example, Casey et al., U.S. Patent No. 4,438,253, which issued March 20, 1984, discloses tri-blocks produced from the transesterification of poly(glycolic acid) and hydroxyl-terminated poly(alkylene glycols). copolymer. Such compositions are disclosed for use as resorbable monofilament sutures. The flexibility of such compositions is governed by the incorporation of aromatic orthocarbonates such as tetra-p-tolyl orthocarbonate into the copolymer structure.

Other polymers based on lactic acid and/or glycolic acid may also be used. For example, Spinu, U.S. Patent No. 5,202,413, which issued April 13, 1993, discloses biodegradable multi-block copolymers containing sequentially oriented blocks of polylactide and/or polyglycolide. segment by ring-opening polymerization of lactide and/or glycolide to oligomeric diols or diamines, followed by chain extension with difunctional compounds such as diisocyanates, diacid chlorides, or dichlorosilanes Production.

The bioresorbable regions of the coatings used in the present invention can be designed to be hydrolyzable and/or enzymatically cleavable. For the purposes of the present invention, "hydrolytically cleavable" means the susceptibility of the copolymer, particularly the bioresorbable region, to hydrolysis in water or an aqueous environment. Similarly, "enzymatically cleavable" as used herein refers to the susceptibility of the copolymer, and in particular the bioresorbable region, to cleavage by endogenous or exogenous enzymes.

When placed in the body, the hydrophilic regions can be processed into excretable and/or metabolizable fragments. Thus, hydrophilic regions may include, for example, polyethers, polyalkylene oxides, polyols, poly(vinylpyrrolidones), poly(vinyl alcohols), poly(alkyloxazolines), polysaccharides, carbohydrates, peptides, Proteins and their copolymers and mixtures. In addition, hydrophilic regions may also be, for example, polyalkylene oxides. Such polyalkylene oxides may include, for example, poly(ethylene oxide), poly(propylene oxide), and mixtures and copolymers thereof.

Polymers that are components of hydrogels are also useful in the present invention. Hydrogels are polymeric materials capable of absorbing relatively large quantities of water. Examples of hydrogel-forming compounds include, but are not limited to, polyacrylic acid, sodium carboxymethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, gelatin, carrageenan and other polysaccharides, hydroxyethylene methacrylic acid (HEMA ), and its derivatives, etc. Stable, biodegradable and bioresorbable hydrogels can be produced. Additionally, hydrogel compositions may include subunits that exhibit one or more of these properties.

Biocompatible hydrogel compositions whose integrity can be controlled by crosslinking are known and presently preferred for use in the methods of the invention. For example, Hubbell et al., U.S. Patent Nos. 5,410,016, which was published on April 25, 1995, and 5,529,914, which was published on June 25, 1996, disclose water-soluble systems containing A cross-linked block copolymer of water-soluble central block segments sandwiched between them. Such copolymers are further terminated with photopolymerizable acrylate functional groups. When crosslinked, these systems become hydrogels. The water-soluble central block of such copolymers may comprise poly(ethylene glycol); however the hydrolytically labile extender may be a poly(alpha-hydroxy acid), such as polyglycolic acid or polylactic acid. See Sawhney et al., Macromolecules 26:581-587 (1993).

In another embodiment, the gel is a thermoreversible gel. Presently preferred are thermoreversible gels comprising components such as poloxamers, collagen, gelatin, hyaluronic acid, polysaccharides, polyurethane hydrogels, polyurethane-urea hydrogels, and combinations thereof.

In yet another exemplary embodiment, the conjugates of the invention comprise components of liposomes. Liposomes can be prepared according to methods known to those skilled in the art, such as described in Eppstein et al., US Patent No. 4,522,811, published June 1, 1985. For example, liposomal formulations can be prepared by dissolving an appropriate lipid (such as stearoylphosphatidylethanolamine, stearoylphosphatidylcholine, arachidylphosphatidylcholine, and cholesterol) in an inorganic solvent, and then Evaporation of the solvent leaves a thin film of dry lipids on the vessel surface. An aqueous solution of the active compound, or a pharmaceutically acceptable salt thereof, is then introduced into the container. The container is then swirled by hand to release lipid material from the sides of the container and to disperse lipid aggregates, thus forming a liposomal suspension.

The foregoing microparticles and methods of making microparticles are provided by way of example and they are not intended to limit the scope of microparticles useful in the present invention. It will be apparent to those skilled in the art that arrays of microparticles made by different methods are useful in the present invention.

The structural formats discussed above in the discussion of both linear and branched water-soluble polymers are also generally applicable to water-insoluble polymers. Thus, eg cysteine, serine, dilysine and trilysine branched cores can be functionalized with two water insoluble polymer moieties. The methods used to produce these materials are generally very similar to those used to produce water soluble polymers.

The in vivo half-life of therapeutic glycopeptides can also be increased by PEG moieties such as polyethylene glycol (PEG). For example, chemical modification of proteins with PEG (PEGylation) increases their molecular size and reduces their surface and functional group-accessibility, each of which depends on the size of the PEG attached to the protein. This leads to an improvement in plasma half-life and to proteolytic stability, as well as a reduction in immunogenicity and hepatic absorption (Chaffee et al. J. Clin. Invest. 89:1643-1651 (1992); Pyatak et al. Res. Commun. Chem. Pathol Pharmacol. 29:113-127 (1980)). It has been reported that PEGylation of interleukin-2 increases its antitumor efficacy in vivo (Katre et al. Proc.Natl.Acad.Sci.USA.84:1487-1491 (1987)) PEGylation of (ab')2 improves 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 by the method of the invention using a PEG moiety is increased relative to the in vivo half-life of a non-derivatized peptide.

An increase in the in vivo half-life of a peptide is best expressed as a percentage increase of this amount. 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 greater than about 250%.

Preparation of modified sugars

Typically, the sugar moiety and modifying group are linked together through the use of a reactive group that is typically converted by the linking process into a new organic functional group or non-reactive species. The sugar-reactive functional group is located anywhere on the sugar moiety. Reactive groups and reactive classes useful in the practice of the invention are generally those well known in the art of bioconjugate chemistry. Presently advantageous types of reactions available for reactive sugar moieties are those that proceed under relatively mild conditions. These include, but are not limited to, nucleophilic substitution (eg, reaction of amines and alcohols with acid halides, active esters), electrophilic substitution (eg, enamine reaction) and addition to carbon-carbon and carbon-heteroatom multiple bonds (eg, Michael reaction, Diels-Alder reaction). These and other useful reactions are discussed, for example, in March, Advanced Organic Chemistry, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson, Bioconjugate Technology, Academic Press, San Diego, 1996; and Feeney et al., Protein Modification; Chemistry Advances Series, Vol.198, American Chemical Society, Washington, D.C., 1982.

Useful reactive functional groups pendant from the sugar core, linker precursor, or polymer modifying moiety precursor include, but are not limited to:

(a) Carboxyl and its various derivatives, including, but not limited to, N-hydroxysuccinimide esters, N-hydroxybenzotriazole esters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters , alkyl, alkenyl, alkynyl and aromatic esters;

(b) Carboxyl, which can be converted into, for example, esters, ethers, aldehydes and the like.

(c) Haloalkyl groups, where the halide can subsequently be displaced by a nucleophilic group, such as an amine, carboxylate anion, thiolate anion, carbanion or alkoxide ion, thus resulting in the covalent attachment of a new group at the halogen atom functional group ;

(d) dienophile (dienophile) group, it can participate in Diels-Alder reaction, for example maleimido (maleimido);

(e) aldehyde or ketone groups, so that subsequent derivatization is possible by the formation of carbonyl derivatives such as, for example, imines, hydrazones, semicarbazones or oximes, or by mechanisms such as Grignard addition or alkyllithium addition;

(f) is a sulfonyl halide for subsequent reaction with an amine (for example to form a sulfonamide);

(g) thiol groups, which can, for example, be converted into disulfides or reacted with acid halides;

(h) amines or mercapto groups, which may, for example, be acylated, alkylated or oxidized;

(i) alkenes, which can undergo, for example, cycloaddition, addition, Michael addition, etc.; and

(j) Epoxides, which can react with, for example, amines and hydroxyl compounds.

Reactive functional groups can be chosen such that they do not participate in, or interfere with, reactions necessary to assemble a reactive sugar core or modifying group. Alternatively, a reactive functional group can be protected from participation in the reaction by the presence of a protecting group. Those skilled in the art understand how to protect a particular functional group so that it does not interfere with the chosen set of reaction conditions. For examples of useful protecting groups see, eg, Greene et al., Protecting Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.

In the following discussion, specific examples of modified sugars useful in the practice of the present invention are illustrated. In an exemplary embodiment, a sialic acid derivative is used as the sugar core to which the modifying group is attached. The focus of the discussion of sialic acid derivatives is for clarity of illustration only and should not be construed as limiting the scope of the invention. Those skilled in the art recognize that various other sugar moieties can be activated and derivatized in a manner similar to that illustrated using sialic acid as an example. For example, many 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, eg, Elhalabi et al., Curr. Med. Chem. 6:93 (1999); and Schafer et al., J. Org. Chem. 65:24 (2000)).

In Scheme 1 below, aminoglycoside 1 is treated with an active ester of a protected amino acid (eg, glycine) derivative to convert the sugar amine residue to the corresponding protected amino acid amide adduct. The adduct is treated with aldolase to form the α-hydroxycarboxylate 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 . Amines introduced via the formation of glycine adducts by reaction of compound 3 with activated (m-)PEG or (m-)PPG derivatives (e.g., PEG-C(O)NHS, PPG-C(O)NHS) Used as a site for PEG or PPG attachment, yielding 4 or 5 , respectively.

plan 1

As will be appreciated by those skilled in the art, the polymer modifying moieties may also be branched moieties, such as those described herein.

An exemplary scheme for preparing the branched polymer-modified sugars of the present invention is provided below:

Another exemplary scheme for preparing the polymer-modified sugars of the present invention is illustrated below:

Table 1 illustrates representative examples of sugar monophosphates derived from polymer modifying moieties such as branched- or linear PEG or PPG moieties. Certain compounds of Table 1 were prepared by the method of Scheme 1 . Other derivatives are prepared by art recognized methods. See, eg, Keppler et al., Glycobiology 11:11R (2001); and Charter et al., Glycobiology 10:1049 (2000)). Other amine-reactive polymer-modifying moiety precursors and components, such as PEG and PPG analogs, are commercially available, or they can be prepared by methods readily available to those skilled in the art.

Table 1

where R is a polymer (branched or linear) modifying moiety.

The modified sugar phosphates used in the practice of this invention may be substituted at other positions as well as those described above. Presently preferred substitutions for sialic acid are illustrated in the following general formula:

wherein one or more of X ^c , Y ^a , Y ^b , Y ^c and Z are linking groups, which are preferably selected from the group consisting of -O-, -N(H)-, -S, CH ₂ -, and N(R) ₂ . When ^Xc , ^Ya , ^Yb , ^Yc and Z are linking groups, it is linked to the polymer modifying moiety as indicated by ^Rc , ^Rd , ^Re , ^Rf and ^Rg . Alternatively, these symbols represent linkers to branched- or linear water-soluble or water-insoluble polymers, therapeutic moieties, biomolecules or other moieties. When R ^c , R ^d , ^Re , R ^f or R ^g are not polymer modifying moieties, the combination of X ^c R ^c , Y ^a R ^d , Y ^{b Re} , Y ^c R ^f or ZR ^g is H, OH or NC(O) _CH3 .

Also provided are methods of producing activated sialic acid-polymer modifying group conjugates that are suitable substrates for enzymes that transfer modified sugar moieties to acceptors, such as glycosyltransferases. The method comprises the steps of: (a) subjecting mannosamine to an activated N-protected amino acid (or a polymer-modifying moiety, a linker precursor or a linker-polymer-modifying moiety combination) under suitable conditions functionalized amino acid) to form an amide conjugate between the mannosamine and the N-protected amino acid; (b) contact the amide conjugate with pyruvate and sialic acid aldolase under appropriate conditions to convert the amide The conjugate becomes a sialic acid amide conjugate; (c) contacting the sialic acid amide conjugate with cytidine triphosphate and synthetase under appropriate conditions to form a cytidine monophosphate sialic acid amide conjugate; (d) from cytidine The monophosphosialic acid amide conjugate removes the N-protecting group, thereby generating a free amine; and (e) contacting the free amine with activated PEG (linear or branched), thereby forming cytidine monophosphosialic acid- Poly(ethylene glycol).

Crosslinking group

The preparation of modified sugars for use in the methods of the invention involves attachment of modifying groups to sugar residues and formation of stable adducts, which are substrates for glycosyltransferases. Sugars and modifying groups can be coupled via zero- or higher-order crosslinkers. Exemplary difunctional compounds that can be used to attach the modifying group to the carbohydrate moiety include, but are not limited to, difunctional poly(ethylene glycol), polyamides, polyethers, polyesters, and the like. General procedures for attaching 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 following discussion, reactive groups are treated as benign on the sugar moiety of the nascent modified sugar. Discussion is focused for clarity of illustration. Those skilled in the art will recognize that the discussion is also relevant to reactive groups on modifying groups.

An exemplary strategy involves the introduction of protected thiols onto sugars using the heterobifunctional crosslinker SPDP (n-succinimidyl-3-(2-pyridyldithio)propionate, followed by deprotection of the thiols, thereby Forms a disulfide bond with another sulfhydryl group on the modifying group.

A number of reagents are available for modifying components of modified sugars using intramolecular chemical crosslinkers (for a review of crosslinking reagents and crosslinking processes see: Wold, F., Meth. Enzymol. 25:623-651, 1972; Weetall , H.H., and Cooney, D.A., In: ENZYMES ASDRUGS. (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 hereby incorporated by reference). Preferred crosslinking reagents are derived from various zero-length, homo-bifunctional, and hetero-bifunctional crosslinking reagents. Zero-length crosslinking reagents involve the direct incorporation of two intrinsic chemical groups (without the introduction of external materials).

Modified sugar to peptide conjugation

The modified sugar is conjugated to the glycosylated or non-glycosylated peptide using the appropriate enzyme that mediates the conjugation. Accordingly, compounds of the invention, particularly nucleotide sugars, are preferably substrates for enzymes that transfer a sugar moiety from a nucleotide sugar to an amino acid, glycosyl, or aglycone acceptor moiety. Nucleotide sugars useful as sugar donors for acceptors such as Galactosyl acceptors such as GalNAc, Galβ1, 4GlcNAc, Galβ1, 4GalNAc, Galβ1, 3GalNAc, lactose-N-tetraose , Galβ1, 3GlcNAc, Galβ1, 3Ara, Galβ1, 6GlcNAc, Galβ1, 4Glc (lactose), and other receptors known to those skilled in the art (see, e.g., Paulson et al., J. Biol. Chem. 253:5617-5624( 1978)).

Exemplary enzymes for which the modified nucleotide sugar of the present invention is a substrate include glycosyltransferases. Glycosyltransferases can be cloned, or isolated from any source. Many cloned glycosyltransferases are known, as are their polynucleotide sequences. See, eg, "The WWW Guide to Cloning Glycosyltransferases," ( http://www.vei.co.uk/TGN/gtguide.htm ). Glycosyltransferase amino acid sequences and nucleotide sequences encoding glycosyltransferases from which amino acid sequences can be deduced are also found in various publicly available databases, including GenBank, Swiss-Prot, EMBL, and others.

Glycosyltransferases for which the compounds of the present invention are substrates include, but are not limited to, galactosyltransferase, fucosyltransferase, glucosyltransferase, N-acetylgalactosylaminotransferase, N-acetylglucosaminylase Transferase, glucuronyltransferase, sialyltransferase, mannosyltransferase, glucuronyltransferase, galacturonyltransferase, and oligosaccharyltransferase. Suitable glycosyltransferases include those obtained from eukaryotes, as well as prokaryotes.

In some embodiments, compounds of the invention are substrates of fucosyltransferases. Fucosyltransferases are generally known to those skilled in the art, exemplified by enzymes that transfer L-fucose from GDP-fucose to the hydroxyl position of an acceptor sugar.

In another set of embodiments, the compound is a substrate for galactosyltransferase. Exemplary galactosyltransferases include α(1,3) galactosyltransferase (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 α1,3 galactosyltransferase is involved in the detection of B blood group antigens Synthetic (EC 2.4.1.37, Yamamoto et al., J.Biol.Chem.265:1146-1151 (1990) (human)). Still a further exemplified galactosyltransferase is nuclear Gal-T1. Still further examples include β(1,4) galactosyltransferases, which include, for example, EC 2.4.1.90 (LacNAc synthetase) and EC 2.4.1.22 (lactose synthase) (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)), and E.C.2.4.1.38 and ceramide galactosyltransferase (EC 2.4.1.45, Stahl et al., J.Neurosci.Res.38:234-242 (1994)). Other suitable galactosyltransferases Including, for example, α1,2-galactosyltransferase (as from, Schizosaccharomyces pombe, Chapell et al., Mol.Biol.Cell 5:519-528 (1994)). Also suitable in the practice of the present invention is α1,3-semi A soluble form of lactosyltransferase as reported by Cho et al., J. Biol. Chem., 272:13622-13628 (1997).

a) Sialyltransferase

Sialyltransferases are another type of glycosyltransferase for which the compounds of the invention are substrates. Examples include ST3Gal III (e.g., murine or human ST3Gal III), ST3Gal IV, ST3Gal I, ST6Gal I, ST3Gal V, ST6Gal II, ST6GalNAc I, ST6GalNAc II, and ST6GalNAc III (sialyltransferase nomenclature used herein is as described In Tsuji et al., Glycobiology 6:v-xiv (1996)). An exemplary α(2,3) sialyltransferase known as α(2,3) sialyltransferase (EC 2.4.99.6) transfers sialic acid to the non-reducing terminal Gal of the Galβ1→3Glc disaccharide or glycoside. See Vanden 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 α2,3-sialyltransferase (EC 2.4.99.4) transfers sialic acid to the non-reducing terminal Gal of a 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-GlcNAca-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 acid. Examples include alpha-2,8-polysialyltransferases such as ST8SiaI, ST8SiaII, ST8SiaIII, ST8SiaIV and ST8SiaV. See, eg, 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).

An example of a sialyltransferase for use in the claimed method is ST3Gal III, which is also known as α(2,3) sialyltransferase (EC 2.4.99.6). This enzyme catalyzes the transfer of sialic acid to Gal of the class Galβ1, 3GlcNAc or class Galβ1, 4GlcNAc glycosides (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 alpha(2,3). See eg WO99/49051.

Preferably, the compound of the invention is a substrate for an enzyme that transfers a modified sialic acid to the sequence Galβ1,4GlcNAc-, the next most common sequence below the terminal sialic acid on fully sialylated carbohydrate structures.

b) GalNAc transferase

Selected compounds of the present invention are substrates of N-acetylgalactose aminotransferase. Exemplary N-acetylgalactose aminotransferases include, but are not limited to α(1,3) N-acetylgalactose aminotransferase, β(1,4) N-acetylgalactose aminotransferase (Nagata et al., J .Biol.Chem.267:12082-12089 (1992) and Smith et al., J.Biol.Chem.269:15162 (1994) and polypeptide N-acetylgalactosaminotransferase (Homa et al., J.Biol. Chem. 268:12609 (1993)).

c) Glycosidase

The invention also includes substrates for wild-type and mutant glycosidases. Mutant β-galactosidases have been shown to catalyze the formation of disaccharides through the coupling of α-glycosyl fluorides to galactosyl acceptor molecules. (Withers, U.S. Pat. No. 6284494; September 4, 2001). Other glycosidases suitable for use in the present invention include, for example, β-glucosidase, β-galactosidase, β-mannosidase, β-acetylglucosaminidase, β-N-acetylgalactosaminidase, β-xylinase Glycosidase, β-fucosidase, cellulase, xylanase, galactanase, mannanase, hemicellulase, amylase, glucosaminidase, α-glucosidase, α- Galactosidase, α-mannosidase, α-N-acetylglucosaminidase, α-N-acetylgalactosamidase, α-xylosidase, α-fucosidase, and neuraminidase/saliva acidase, endoceramidase.

Detailed ways

The following examples are provided to illustrate selected embodiments of the invention and should not be construed as limiting its scope.

Example

Example 1

Preparation of UDP-GalNAc-6′-CHO

UDP-GalNAc (200 mg, 0.30 mmol) was dissolved in 1 mM CuSO ₄ solution (20 mL) and 25 mM NaH ₂ PO ₄ solution (pH 6.0; 20 mL). Galactose oxidase (240 U; 240 μL) and catalase (13000 U; 130 μL) were then added, and the reaction system equipped with an air chamber was filled with oxygen and stirred at room temperature for several days. The reaction mixture was then filtered (spin box; MWCO 5K) and the filtrate (-40 mL) was stored at 4°C until needed. TLC (silica; EtOH/water (7/2); _Rf = 0.77; visualized by anisaldehyde staining).

Example 2

Preparation of UDP-GalNAc-6'-NH ₂ ):

Ammonium acetate (15 mg, 0.194 mmol) and NaBH ₃ CN (1M in THF; 0.17 mL, 0.17 mmol) were added to the above obtained UDP-GalNAc-6'-CHO solution (2 mL or ~20 mg) at 0 °C and allowed to warm to room temperature overnight. The reaction was filtered through a G-10 cartridge and the water and product collected. Appropriate fractions were lyophilized and stored frozen. TLC (silica; ethanol/water (7/2); _Rf = 0.72; visualized with Ninhidelin's reagent).

Example 3

Preparation of UDP-GalNAc-6-NHCO(CH ₂ ) ₂ -O-PEG-OMe (1 KDa).

Galactosamido-1-phosphate-2-NHCO( _CH2 ) ₂ -O-PEG-OMe (1 KDa) (58 mg, 0.045 mmol) was dissolved in DMF (6 mL) and pyridine (1.2 mL). Then UMP-morpholinate (60 mg, 0.15 mmol) was added and the resulting mixture was stirred at 70° C. for 48 h. The solvent was removed by bubbling nitrogen through the reaction mixture and the residue was purified by reverse phase chromatography (C-18 silica, step gradient 10-80%, methanol/water). The desired fractions were collected and dried under reduced pressure to give a white solid. TLC (silica, propanol/ _H2O / _NH4OH , (30/20/2), _Rf = 0.54). MS (MALDI): Observations, 1485, 1529, 1618, 1706.

Example 4

Preparation of Cysteine-PEG ₂ (2)

4.1 Synthesis of compound 1

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 dry methanol (20 L) under argon. The mixture was stirred at room temperature for 30 min, and then mPEG-O-tosylate (Ts; 1.0 g, 0.05 mmol) with a molecular weight of 20 kDa 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 kDa mPEG-O-tosylate. The solution was then neutralized with acetic acid, the pH adjusted to pH 5.0, and loaded onto a reverse phase chromatography (C-18 silica) column. The column was eluted with a methanol/water gradient (product eluted at about 70% methanol), product elution was monitored by evaporative light scattering, and appropriate fractions were collected and diluted with water (500 mL). This solution was chromatographed (ion exchange, XK 50Q, BIG Beads, 300ml, hydroxide form; water to water/acetic acid gradient - 0.75N) and the pH of the appropriate fraction was lowered to 6.0 with acetic acid. This solution was then trapped on a reverse phase column (C-18 silica) and eluted with the methanol/water gradient described above. The product fractions were pooled, concentrated, redissolved in water and lyophilized to afford a white solid (1). The structural data of the compound are as follows: ¹ H-NMR (500MHz; D ₂ O) δ 2.83(t, 2H, OCC H ₂ -S), 3.05(q, 1H, SC H H-CHN), 3.18(q, 1H , ( _q , 1H, SCHH - _CHN ), 3.38 ₍ s , 3H, CH3O), 3.7 ( t , OCH2CH2O), 3.95(q, 1H, CHN ). Product The purity of was confirmed by SDS PAGE.

4.2 Synthesis of compound 2 (cysteine-PEG ₂ )

Triethylamine (-0.5 mL) was added dropwise to a solution of compound 1 (440 mg, 22 μmol) dissolved in _{anhydrous CH2Cl2} (30 mL) until the solution was basic. A solution of 20 kilodaltons of mPEG-O-p-nitrophenyl carbonate (660 mg, 33 μmol) and N-hydroxysuccinimide (3.6 mg, 30.8 μmol) in _CH2Cl2 ( ₂₀ mL) was divided in several portions Each portion was added 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 was adjusted to 9.5 with 1.0 N NaOH. The basic solution was stirred at room temperature for 2 hours, then neutralized to pH 7.0 with acetic acid. The solution was then loaded onto a reverse phase chromatography (C-18 silica) column. The column was eluted with a methanol/water gradient (product eluted at about 70% methanol), product elution was monitored by evaporative light scattering, and appropriate fractions were collected and diluted with water (500 mL). This solution was chromatographed (ion exchange, XK 50Q, BIG Beads, 300ml, hydroxide form; water to water/acetic acid gradient - 0.75N) and the pH of the appropriate fraction was lowered to 6.0 with acetic acid. This solution was then trapped on a reverse phase column (C-18 silica) and eluted using the above methanol/water gradient. The product fractions were pooled, concentrated, redissolved in water and lyophilized to afford a white solid (2). The structural data of the compound are as follows: ¹ H-NMR (500MHz; D ₂ O) δ 2.83(t, 2H, OC-CH ₂ -S), 2.95(t, 2H, OCC H ₂ -S), 3.12(q, 1H, SCHH - _CHN ), 3.39 ( s _, 3HCH3O ), 3.71 (t, _OCH2CH2O ) . The purity of the product was confirmed by SDS PAGE.

Example 5

Preparation of UDP-GalNAc-6-NHCO(CH ₂ ) ₂ -O-PEG-OMe (1 KDa).

Galactosamido-1-phosphate-2-NHCO(CH ₂ ) ₂ -O-PEG-OMe (1 kilodalton) (58 mg, 0.045 mmol) was dissolved in DMF (6 mL) and pyridine (1.2 mL) . UMP-morpholinate (60 mg, 0.15 mmol) was then added and the resulting mixture was stirred at 70°C for 48 hours. The solvent was removed by bubbling nitrogen through the reaction mixture and the residue was purified by reverse phase chromatography (C-18 silica, step gradient 10-80%, methanol/water). The desired fractions were collected and dried under reduced pressure to give a white solid. TLC (silica, propanol/ _H2O / _NH4OH , (30/20/2), _Rf = 0.54). MS (MALDI): Observations, 1485, 1529, 1618, 1706.

SDS PAGE process

The purity of products 1 and 2 was confirmed by SDS PAGE. 4-20% Tris-glycine SDS PAGE gels (Invitrogen) were used. The samples were mixed 1:1 with SDS sample buffer and run in Tris-glycine running buffer (LC2675-5) at constant voltage (125V) for 1 hour and 50 min. After electrophoresis, the gel was washed with water (100 mL) for 10 min, followed by 5% aqueous barium chloride (100 mL) for 10 min. Products 1 or 2 were visualized by staining the gel with 0.1 N iodine solution (4.0 mL) at room temperature, the staining process was stopped by washing the gel with water. The displayed product spectrum was scanned with HP Scanjet 7400C, and the image of the gel was optimized with HP Precision ScanProgram.

Although the invention has been disclosed with reference to specific embodiments, it is evident that other embodiments and variations of the invention can be devised by those skilled in the art without departing from the true spirit and scope of the invention.

All patents, patent applications, and other publications cited in this application are hereby incorporated by reference in their entirety for all purposes.

Claims (11)

1. general formula is the compound that is selected from following member:

Wherein

R ¹Be H, CH ₂OR ⁷, COOR ⁷Or OR ⁷

D is 0 or 1;

R ⁷Expression H, replacement or unsubstituted alkyl or replacement or unsubstituted assorted alkyl;

R ²It is the part that is selected from following member: H, OH, activating group and comprises Nucleotide;

R ³, R ⁴, R ⁵, R ⁶And R ^{6 '}Be independently selected from H, replacement or unsubstituted alkyl, OR ⁹, and NHC (O) R ¹⁰

R wherein ⁹And R ¹⁰Be independently selected from H, replacement or unsubstituted alkyl or replacement or unsubstituted assorted alkyl;

And R ³, R ⁴, R ⁵, R ⁶And R ^{6 '}At least one comprise part with following formula:

Wherein s is the integer of 0-20; And

R ¹¹Be the polymer modification part, it has following general formula:

Wherein

X ²And X ⁴Be 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 ₂) _gY "; Wherein g is the integer of 1-50, and Y " is the member who is selected from O, S and NH;

X ⁵It is non-reactive group; And

R ¹²And R ¹³Be the polymeric arms of selecting independently;

Wherein " assorted alkyl " expression is selected from down the group of group: acetal, OHC-, H ₂N-(CH ₂) _q-, HS-(CH ₂) _q, or-(CH ₂) _qC (Y ^b) Z ^bWherein each q that occurs represents the integer of 0-20 independently; Y ^bExpression is selected from O, S and N-R ^cGroup; Each R that occurs ^cRepresent H, replacement or unsubstituted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted Heterocyclylalkyl and replacement or unsubstituted heteroaryl independently; Z ^bExpression is selected from OH, NH ₂Group with leavings group.

2. according to the compound of claim 1, Z wherein ^bExpression leavings group imidazoles or halogenide.

3. according to the compound of claim 1, R wherein ²Have following general formula:

R wherein ⁸It is nucleosides.

4. according to the compound of claim 3, R wherein ⁸Be the member who is selected from cytidine, uridine, guanosine, adenosine and thymidine.

5. according to each compound of claim 1-4, wherein

X ⁴It is peptide bond; With

R ¹³It is amino-acid residue.

6. according to each compound of claim 1-4, have general formula:

Wherein

D is selected from-OH and (R ¹¹) _wThe member of '-L-;

G represents to be selected from H, (R ¹¹) _w'-L-and-C (O) (C ₁-C ₆) member of alkyl;

W ' is the integer of 2-6, and

At least one of D and G is (R ¹¹) _w'-L-,

Wherein L is the member who is selected from key and linking group, and described linking group is selected from the part of replacement or unsubstituted alkyl and replacement or unsubstituted assorted alkyl.

7. according to each compound of claim 1-4, wherein said compound is an enzyme substrates, and described enzyme shifts sugar moieties to the acceptor portion of substrate from the member who is selected from activation sugar, nucleotide sugar and combination thereof.

8. according to the compound of claim 6, wherein said acceptor portion is to be selected from glycosyl residue, the member of amino-acid residue and aglycon.

9. according to the compound of claim 1, it has following formula:

Wherein AA is-CH ₂-; With

NP is a nucleotide phosphodiesterase.

10. according to the compound of claim 1, wherein said polymer modification partly has and is selected from following formula:

Wherein e and f are independently selected from 1 to 2500.

11. a method for preparing cytidine monophosphate sialic acid-poly-(ethylene glycol), this method comprises:

(a) mannosamine is contacted to form the acid amides binding substances between the amino acid of this mannosamine and N-protected with the amino acid of activatory N-protected;

(b) this acid amides binding substances and pyruvate salt are contacted to transform this acid amides binding substances with acetylneuraminate aldolase and become sialic acid acid amides binding substances;

(c) this sialic acid acid amides binding substances and cytidine triphosphate(CTP) are contacted to form cytidine monophosphate sialic acid acid amides binding substances with synthetic enzyme;

(d) remove the N-protected group from this cytidine monophosphate sialic acid acid amides binding substances, produce unhindered amina thus; With

(e) this unhindered amina is contacted with activatory PEG, form described cytidine monophosphate sialic acid-poly-(ethylene glycol) thus; Wherein the amino acid of this activatory N-protected has following structural formula:

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