JP4871739B2 - Branched polymeric sugars and their nucleotides - Google Patents

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is related to US Provisional Patent Application No. 60 / 539,387 filed Jan. 26, 2004; US Provisional Patent Application No. 60 / 555,504 filed Mar. 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 Mar. 22, 2004; November 24, 2004; US Provisional Patent Application No. 10 / 997,405 filed; US Provisional Patent Application No. 60 / 544,411 filed on February 12, 2004; US Provisional Patent Application No. 60/604 filed on February 20, 2004; No. 546,631; U.S. provisional patent application filed on May 12, 2004; U.S. patent application filed on Jan. 10, 2005 ((unassigned), Attorney Docket No. 40853-1-01-5138 US); December 2004 3 days PCT application number of application ((unassigned), Attorney Docket No. 40853-1-01-5146WO); US Provisional Patent Application No. 60 / 590,649 filed July 23, 2004; September 20, 2004 U.S. Provisional Patent Application No. 60 / 611,790; U.S. Provisional Patent Application No. 60 / 592,744, filed July 29, 2004; U.S. Provisional Patent Application No. 60, filed September 29, 2004. US Provisional Patent Application No. 60 / 623,387, filed October 29, 2004; US Provisional Patent Application No. 60 / 626,678, filed November 9, 2004; and 2005 United States provisional patent application number ((unassigned), attorney docket number 040853-01-5150) filed January 6, 1980 (see the entire contents of each of these disclosures for all purposes) Claims priority based on more incorporated herein by reference).

The present invention relates to the field of modified sugars and their nucleotides.

Background Post-expression in vitro modification of peptides is a deficiency in methods that depend on controlling glycosylation by manipulating the expression system, including both alteration of glycan structure and introduction of glycans at new sites Is an attractive strategy to improve. A broad toolbox of recombinant eukaryotic glycosyltransferases is available to perform in vitro enzymatic synthesis of mammalian glycoconjugates with custom-designed glycosylation patterns and possible glycosyl structures It has become. For example, US Pat. No. 5,876,980; US Pat. No. 6,030,815; US Pat. No. 5,728,554; US Pat. No. 5,922,577; No./9831826; U.S. Patent Publication No. 2003180835; and WO 03/031464.

  Enzyme-based synthesis has the advantage of regioselectivity and stereoselectivity. Furthermore, enzymatic synthesis is performed using an unprotected substrate. Three major classes of enzymes are used for the synthesis of carbohydrates, glycosyltransferases (eg, sialyltransferases, oligosaccharyltransferases, N-acetylglucosaminyltransferases), and glycosidases. Glycosidases are further classified as exoglycosidases (eg, β-mannosidase, β-glucosidase) and endoglycosidases (eg, Endo-A, Endo-M). Each of these classes of enzymes has been successfully used to synthetically prepare carbohydrates. For a general review, see Crout et al., “Curr. Opin. Chem. Biol.” 2: 98-111 (1998).

  Glycosyltransferases modify the oligosaccharide structure on glycopeptides. Glycosyltransferases are effective for producing specific products with excellent stereochemical and regiochemical control. Glycosyltransferases have been used to prepare oligosaccharides and to modify N- and O-linked carbohydrate structures, particularly on glycopeptides produced in mammalian cells. For example, the terminal oligosaccharide of a glycopeptide is fully sialicated and / or fucosylated to provide a more consistent sugar structure, thereby pharmacokinetics of the glycopeptide and various other biological properties. To improve. For example, β-1,4-galactosyltransferase was used to synthesize lactosamine (a specific example of the utility of glycosyltransferases in the synthesis of carbohydrates) (eg, Wong et al., “J. Org. Chem. 47: 5416-5418 "(1982))). In addition, many synthetic procedures use α-sialyltransferase to transfer sialic acid from cytidine-5′-monophospho-N-acetylneuraminic acid to the 3-OH or 6-OH of galactose ( See, for example, Kevin et al., “Chem. Eur. J.” 2: 1359-1362 (1996)). Fucosyltransferases are used in synthetic routes to transfer fucose units from guanosine-5'-diphosphofucose to a specific hydroxyl of a saccharide acceptor. For example, Ichikawa prepared Sialyl Lewis-X (Lewis-X) by a method involving fucosylation of sialic acid-added lactosamine using a cloned fucosyltransferase (Ichikawa et al. "J. Am. Chem. Soc." 114: 9283-9298 (1992)). For a discussion of recent advances in the synthesis of glycoconjugates for therapeutic use, see Koeller et al., “Nature Biotechnology” 18: 835-841 (2000). US Pat. No. 5,876,980; US Pat. No. 6,030,815; US Pat. No. 5,728,554; US Pat. No. 5,922,577; See also / 9831826 pamphlet.

  In addition to manipulating the structure of glycosyl groups on polypeptides, there has been interest in preparing modified glycopeptides with one or more non-saccharide modifying groups (such as water-soluble polymers). Poly (ethylene glycol) (“PEG”) is a typical polymer that has been conjugated to polypeptides. The use of PEG to derivatize peptide therapeutics has been demonstrated to reduce the peptide's immunogenicity. For example, US Pat. No. 4,179,337 (Davis et al.) Discloses non-immunogenic polypeptides, such as enzymes and peptide hormones linked to polyethylene glycol (PEG) or polypropylene glycol. Yes. 10 to 100 moles of polymer are used per mole of polypeptide. Although the in vivo clearance time of the conjugate is long compared to that of the polypeptide, only about 15% of the physiological activity is maintained. Thus, the long circulating half-life is offset by a dramatic decrease in peptide efficacy.

  The loss of peptide activity is directly attributed to the non-selective nature of the chemistry utilized to bind the water soluble polymer. The main mode of attachment of PEG and its derivatives to peptides is non-specific binding by peptide amino acid residues. For example, US Pat. No. 4,088,538 discloses an enzymatically active polymer-enzyme conjugate of an enzyme covalently linked to PEG. Similarly, US Pat. No. 4,496,689 discloses a covalently attached complex of an alpha-1 proteinase inhibitor with a polymer such as PEG. Abuchowski et al. ("J. Biol. Chem." 252: 3578 (1977) discloses the covalent coupling of MPEG to the amine group of bovine serum albumin. US Patent No. 4,414,147. The specification discloses a method for making the hydrophobicity of interferon smaller by attaching it to an anhydride of a dicarboxylic acid such as poly (ethylene succinic anhydride). [0005] Pamphlet 00005 discloses the conjugation of PEG and poly (oxyethylated) polyols to proteins such as interferon-beta, interleukin 2, and immunotoxin. Contains PEG directly attached to one primary amino group US Pat. No. 4,055,635 discloses proteolysis that is covalently linked to macromolecular substances such as polysaccharides, such as IL-2, which is chemically modified, such as IL-2. Disclosed are pharmaceutical compositions of water-soluble complexes of enzymes.

  Another mode for attaching PEG to peptides is through non-specific oxidation of glycosyl residues on glycopeptides. Oxidized sugar is used as a place to attach the PEG moiety to the peptide. For example, M'Timkulu (WO 94/05332) discloses the use of amino PEG to add PEG to glycoproteins. The glycosyl moiety is randomly oxidized to the corresponding aldehyde, which is then coupled to amino-PEG.

  In each of the above methods, poly (ethylene glycol) is added in a random, non-specific manner to reactive residues on the peptide backbone. For the production of therapeutic peptides, it is clearly desirable to utilize a derivatization strategy that forms a specifically labeled, easily characterized, essentially homogeneous product. A promising route for preparing specifically labeled peptides is through the use of enzymes such as glycosyltransferases to add modified sugar moieties on the peptides. This modified sugar moiety must function as a substrate for the glycosyltransferase and must be activated appropriately. Therefore, a synthetic route that allows easy access to activated modified sugars is desirable. The present invention provides such a route.

Summary of the Invention The present invention provides polymeric species, and sugars and activated sugars attached to these polymeric species, and nucleotide sugars comprising these polymers. Polymeric species include water-soluble and water-insoluble species. Furthermore, these polymers are branched or linear polymers. Typical sugar moieties include linear and cyclic structures and aldoses and ketoses.

  The polymeric modifying group can be attached to any position of the sugar moiety. In the discussion that follows, the invention is illustrated by reference to embodiments in which the polymeric modifying group is attached to furanose C-5 or pyranose C-6. For those skilled in the art, the focus of the discussion is to clarify specific examples, and the polymer moiety can be obtained using the methods described herein and methods recognized in the art, It will be appreciated that other positions of pyranose and furanose can be bound.

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

In Formulas I and II, R ¹ is H, CH ₂ OR ⁷ , COOR ⁷ or OR ⁷ where R ⁷ represents H, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl. R ² is a moiety that includes H, OH, or nucleotides. An exemplary R ² species according to this embodiment has the formula:

In the formula, R ⁸ 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 . The index d is 0 or 1. R ⁹ and R ¹⁰ are each independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, or sialic acid. At least one of R ³ , R ⁴ , R ⁵ , R ⁶ or R ^{6 ′} includes a polymeric modifying moiety (eg, PEG). In an exemplary embodiment, R ⁶ and R ^{6 ′} are components of the sialic acid side chain, along with the carbon to which they are attached. In a further exemplary embodiment, this side chain is functionalized with a polymeric modifying moiety.

In an exemplary embodiment, the polymeric moiety is attached to the sugar center via a linker (L), usually through a central heteroatom, as shown below:

R ¹¹ is a polymer moiety and L is selected from a bond and a linking group. The index w represents an integer selected from 1 to 6, preferably 1 to 3, more preferably 1 to 2. Typical linking groups include substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl moieties, and sialic acid. A typical component of a linker is an acyl moiety.

When L is a bond, it is formed between a functional group reactive to the precursor of R ¹¹ and a reactive functional group having complementary reactivity to the precursor of L. L can be present at an appropriate location on the saccharide center prior to reaction with R ¹¹ . Alternatively, R ¹¹ and L can be incorporated into a preformed cassette that is subsequently attached to the saccharide center. As described herein, the selection and preparation of precursors with appropriate reactive functional groups is within the ability of those skilled in the art. Furthermore, linking the precursors proceeds by chemistry that is well understood in the art.

  In an exemplary embodiment, L is a linking group formed from an amino acid or small molecule peptide (eg, 1-4 amino acid residues) that provides a modified sugar. However, the polymeric modifying moiety is attached via a substituted alkyl linker. A typical linker is glycine.

In an exemplary embodiment, R ⁶ includes a polymeric modifying moiety. In another exemplary embodiment, R ⁶ includes a polymeric modifying moiety and a linker (L) that connects the modifying moiety to the remainder of the molecule.

In an exemplary embodiment, the polymeric modifying moiety is a branched structure comprising two or more polymeric chains attached to the central portion. An exemplary structure of a useful polymer-modified moiety precursor according to this embodiment of the invention has the following formula:

Sugars and nucleotide sugars according to this formula are essentially pure water-soluble polymers. X ^{3 ′} is a moiety that is ionizable (eg, COOH, etc.) or contains 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 (eg, non-peptidic, non-reactive polymer arms). X ² and X ⁴ are ligation fragments that are preferably essentially non-reactive under physiological conditions, which may be the same or different. Alternatively, these linkages can include one or more moieties that are designed to degrade under physiologically relevant conditions, such as esters, disulfides, and the like. X ² and ^{X 4,} connects the polymeric arms ^{R l2} and ^{R 13} to C. X ^{3 ′} is converted to a component of ligation fragment X ³ upon reaction with a reactive functional group having complementary reactivity to the linker, sugar, or linker-sugar cassette.

By reaction of the precursor with the appropriate sugar or sugar linker species, the present invention provides sugars and nucleotide sugars having the formula:

In the formula, the identity of radicals represented by various symbols is the same as described above. L ^a is a substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl moieties. In an exemplary embodiment, L ^a is a part of the side chain of sialic acid to be functionalized with the polymeric modifying moiety as shown.

  The polymeric modifying moiety comprises two or more repeating units (which may be water soluble or essentially insoluble in water). Typical water-soluble polymers used in the compounds of the present invention include PEG (eg m-PEG), PPG (eg m-PPG), polysialic acid, polyglutamic acid, polyaspartic acid, polylysine, polyethyleneimine, biodegradation Functional polymers (eg, polylactic acid, polyglycerides), and functionalized PEGs (eg, terminally functionalized PEG).

  The sugar moiety of the polymer conjugate of the present invention is selected from natural and non-natural furanose and hexanose. Non-natural saccharides optionally include alkylated or acylated hydroxyl and / or amine moieties (eg, ethers, esters, and amide substituents on the ring). Other non-natural saccharides include H, hydroxyl, ether, ester, or amide substituents on the ring such that such substituents are not present in natural saccharides. Alternatively, the carbohydrate has lost a substituent, such as a deoxy sugar, that would be found in the carbohydrate from which the name is derived. Additional exemplary non-natural sugars include oxidized (eg, -onic and -uronic acids) and reduced (sugar alcohol) carbohydrates. The sugar moiety can be a mono (monosaccharide), oligo (oligosaccharide), or polysaccharide.

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

A typical sialic acid-based conjugate has the following formula:

In the formula, AA is a portion of an amino acid residue that does not contain a carboxyl moiety, and NP is a nucleotide phosphate. ONP can also be replaced by an activating moiety to form an activated sugar. As will be appreciated by those skilled in the art, the polymeric modifying moiety-linker can also be attached to the sialic acid side chain at C-6, C-7 and / or C-9.

  Also provided are synthetic methods for producing activated sialic acid-PEG conjugates that are suitable substrates for enzymes (eg, glycosyltransferases). This method includes the following steps: (a) Functionalization using an activated, N-protected amino acid (or a polymeric modifying moiety, linker precursor, or linker-polymer modifying moiety cassette). Contacting the exposed amino acid) with mannosamine under conditions suitable to form an amide conjugate between the mannosamine and the N-protected amino acid; (b) the amide conjugate is converted to a sialic acid amide. Contacting with pyruvate and sialic acid aldolase under conditions suitable for conversion to the conjugate; (c) the sialic acid conjugate is suitable for forming a cytidine monophosphate sialic acid conjugate Contacting with cytidine triphosphate and synthetase under conditions; (d) cytidine Removing the N-protecting group from the phosphate sialic acid amide conjugate, thereby producing a free amine; and (e) contacting the free amine with an activated PEG (which may be linear or branched). Thereby forming cytidine monophosphate sialic acid-poly (ethylene glycol).

  The nucleoside can be selected from natural and non-natural nucleosides. Typical natural nucleosides used in the present invention include cytosine, thymine, guanine, adenine, and uracil. This technology is full of unnatural nucleoside structures and methods for making them.

Exemplary modified sugar nucleotides of the present invention include polymerizable modified GDP-Man, GDP-Fuc, UDP-Gal, UDP-GalNAc, UDP-Glc, UDP-GlcNAc, UDP-Glc, UDP- GlcUA, CMP-SA and the like are included. Examples include UDP-Gal-2′-NH-PEG, UDP-Glc-2′-NH-PEG, CMP-5′-PEG-SA, and the like. In the compounds encompassed by the present invention, the LR ¹¹ moiety is attached to a furanose or pyranose (eg, to furanose C-5 or pyranose C-6), usually a heteroatom bonded to this carbon atom. Which are connected via

  When the compound of the invention is a nucleotide sugar or an activated sugar, a polymeric conjugate of the nucleotide sugar is usually a substrate for an enzyme that transfers the sugar moiety and its polymeric substituents onto an appropriate acceptor moiety of the substrate. It is. Thus, the present invention also provides a substrate that is modified by a sugar conjugate using a polymeric conjugate of a nucleotide sugar, or an activated sugar, and a suitable enzyme. Substrates that can be glycoconjugated using the compounds of the present invention include peptides (eg, glycopeptides, peptides), lipids (eg, glycolipids, aglycones (sphingosine, ceramide), and the like.

Detailed Description of the Invention and Embodiment Abbreviations Branched and unbranched PEG, poly (ethylene glycol), eg, m-PEG, methoxy-poly (ethylene glycol); Branched and unbranched PPG, poly (propylene glycol) ), For example, m-PPG, methoxy-poly (propylene glycol); Fuc, fucosyl; Gal, galactosyl; GalNAc, N-acetylgalactosaminyl; Glc, glucosyl; GlcNAc, N-acetylglucosaminyl; Man, mannosyl; Mannosaminyl acetate; Sia, sialic acid; and NeuAc, N-acetylneuraminyl.

Definitions The term “sialic acid” refers to any member of the family of 9-carbon carboxylated sugars. The most common member of the sialic acid family is N-acetyl-neuraminic acid (2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-one -Onic) acid (often abbreviated as Neu5Ac, NeuAc or NANA) The second member of this family is N-glycolyl-neuraminic acid (N-acetyl group of NeuAc hydroxylated) A third sialic acid family member is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al. (1986) "J. Biol. Chem. "261: 11550-1155. 7; Kanamori et al., “J. Biol. Chem.” 265: 21811-2121819 (1990)). 9-substituted sialic acids such as 9-O-lactyl-Neu5Ac or 9-O-acetyl- Neu5Ac _{like, 9-O-C 1 ~C} 6 acyl-Neu5Ac, 9-deoxy-9-fluoro-Neu5Ac, and 9 such azido-9-deoxy-Neu5Ac is also included. for the sialic acid family of review For example, Varki, “Glycobiology” 2: 25-40 (1992); “Sialic Acids: Chemistry, Metabolism and Function”, edited by R. Schauer (Springer Publishing (Springer) ), New York (1992)) The synthesis and use of sialic acid compounds in sialylation procedures is described in WO 92/16640 published Oct. 1, 1992. Is disclosed.

  As used herein, the term “modified sugar” refers to a naturally occurring or non-naturally occurring carbohydrate that is enzymatically added to an amino acid or glycosyl residue of a peptide within the process of the invention. Modified sugars include, but are not limited to, sugar nucleotides (mono, di, and triphosphates), activated sugars (eg, glycosyl halides, glycosyl mesylate), and unactivated , Selected from several enzyme substrates, including sugars that are not nucleotides. A “modified sugar” is covalently functionalized using a “modifying group”. Useful modifying groups include, but are not limited to, water soluble polymers, therapeutic moieties, diagnostic moieties, biomolecules, and the like. The modifying group is preferably a non-naturally occurring or modified carbohydrate. The position of functionalization with the modifying group is selected such that the “modified sugar” is not prevented from being enzymatically added to the peptide.

  The term “water-soluble” refers to a moiety that has some detectable degree of solubility in water. Methods for detecting and / or quantifying water solubility are well known in the art. Typical water soluble polymers include peptides, saccharides, poly (ethers), poly (amines), poly (carboxylic acids) and the like. Peptides may have mixed sequences or may consist of a single amino acid, such as poly (lysine). A typical polysaccharide is poly (sialic acid). A typical poly (ether) is poly (ethylene glycol), such as m-PEG. Poly (ethyleneimine) is a typical polyamine and poly (acrylic) acid is a typical poly (carboxylic acid). Typical polymers generally consist of 2-8 polymeric units.

  The polymer backbone of the water soluble polymer can be poly (ethylene glycol) (ie PEG). However, other related polymers are also suitable for use in the practice of the present invention, and the use of the term PEG or poly (ethylene glycol) is inclusive and not exclusive in this regard It should be understood that is intended. The term PEG includes alkoxy PEG, bifunctional PEG, PEG with multiple arms, bifurcated PEG, branched PEG, pendant PEG (ie, PEG having one or more functional groups hanging from the polymer backbone) Or related polymers), or any form of poly (ethylene glycol), including PEG with degradable linkages therein.

The polymer main chain may be linear or branched. Branched polymer backbones are generally known in the art. Generally, a branched polymer has a central portion of a central branch and a plurality of linear polymer chains linked to the center of the central branch. PEG is commonly used in branched forms that can be prepared by addition of ethylene oxide to various polyols such as glycerol, pentaerythritol, and sorbitol. The central branch portion can also be obtained from several amino acids such as lysine. Branched poly (ethylene glycol) can be represented in general form as R (-PEG-OH) _m . Here, R represents a central portion such as glycerol or pentaerythritol, and m represents the number of arms. PEG molecules with multiple arms, such as those described in US Pat. No. 5,932,462, the entire contents of which are incorporated herein by reference, may also be used as the polymer backbone. it can.

  Many other polymers are also suitable for the present invention. Polymer backbones that are non-peptidic and water-soluble and have 2 to about 300 termini are particularly useful 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 such as ethylene glycol and propylene glycol, poly (oxyethylated polyols), Poly (olefin alcohol), poly (vinyl pyrrolidone), poly (hydroxypropyl methacrylamide), poly (α-hydroxy acid), poly (vinyl alcohol), polyphosphazene, polyoxazoline, poly (N-acryloylmorpholine) (contents) And those copolymers, terpolymers, and mixtures thereof), and the like, as described in US Pat. No. 5,629,384, which is incorporated herein by reference in its entirety. The molecular weight of each chain of the polymer backbone can vary but is generally in the range of about 100 Da to about 100,000 Da, often about 6,000 Da to about 80,000 Da.

The term “sugar conjugate” as used herein refers to the enzymatically mediated complexation of a modified sugar species to an amino acid or glycosyl residue of a polypeptide (eg, a mutant human growth hormone of the invention). Point to. A subgenus of “sugar conjugates” is “glycol-PEGylation”. However, modified sugar modifying groups are poly (ethylene glycol) and alkyl derivatives (eg, m-PEG) or reactive derivatives thereof (eg, H ₂ N-PEG, HOOC-PEG).

  The term “glycosyl linking group” as used herein refers to a glycosyl residue to which a modifying group (eg, a PEG moiety, a therapeutic moiety, a biomolecule) is covalently attached, A modifying group is linked to the remainder. In the methods of the invention, a “glycosyl linking group” becomes covalently attached to a peptide, whether or not glycosylated, thereby attaching an agent to an amino acid and / or glycosyl residue on the peptide. Are concatenated. 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 the peptide. The glycosyl linking group may be a structure derived from a saccharide that is cleaved during the formation of a sugar cassette that is modified with a modifying group (eg, oxidation → Schiff base formation → reduction) or may remain intact. Good. “Complete glycosyl linking group” refers to a linking group derived from a glycosyl moiety in which the saccharide monomer linking the modifying group to the remainder of the conjugate is not degraded (eg, oxidized by, for example, sodium metaperiodate). “Complete glycosyl linking groups” of the present invention can be obtained from naturally occurring oligosaccharides by the addition of glycosyl units or the removal of one or more glycosyl units from the parent saccharide structure.

When a substituent is specified by its normal chemical formula (written from left to right), this is equivalent to a chemically identical substituent that would result from writing a right-to-left structure. include (e.g., _-CH 2 O-is -OCH ₂ - also intended to enumerate).

The term “alkyl” means a straight or branched chain or cyclic hydrocarbon radical, or a combination thereof, as such or as part of another substituent, unless otherwise stated. It can be fully saturated or monovalent or polyunsaturated, divalent and polyvalent with the specified number of carbon atoms (ie C ₁ -C ₁₀ means 1 to 10 carbons). It can contain radicals. Examples of saturated hydrocarbon radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropylmethyl, Groups such as homologues and isomers (eg, n-pentyl, n-hexyl, n-heptyl, n-octyl, etc.) are included. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2,4-pentadienyl, 3- (1,4-pentadienyl), Ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers are included. The term “alkyl” will also include derivatives of alkyl, as defined in more detail below, eg, “heteroalkyl”, unless otherwise stated. Alkyl groups that are limited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent is divalent derived from an alkane, as exemplified by, but not limited to, —CH ₂ CH ₂ CH ₂ CH ₂ —. Means a radical and further includes those described below as "heteroalkylene". Generally, an alkyl (or alkylene) group will have 1 to 24 carbon atoms, with groups having 10 or fewer carbon atoms being preferred in the present invention. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, usually having eight or fewer carbon atoms.

  The terms “alkoxy”, “alkylamino”, and “alkylthio” (or thioalkoxy) are used in their conventional sense and are attached to the remainder of the molecule through an oxygen, amino, or sulfur atom, respectively. Refers to an alkyl group.

The term “heteroalkyl”, unless stated otherwise, by itself or in combination with another term, at least one selected from the group consisting of a predetermined number of carbon atoms and O, N, Si, and S It means a stable straight chain or branched chain or cyclic hydrocarbon radical composed of heteroatoms, or a combination thereof. However, the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatoms may optionally be quaternized. The heteroatoms O, N, and S and Si can be placed at any internal position of the heteroalkyl group or at the position where the alkyl group 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 are included. Up to two heteroatoms may be consecutive, such as, for example, —CH ₂ —NH—OCH ₃ and —CH ₂ —O—Si (CH ₃ ) ₃ . Similarly, the term “heteroalkylene” means a divalent radical derived from a heteroalkyl, by itself or as part of another substituent, including, but not limited to, —CH ₂ —CH ₂ — Illustrated by S—CH ₂ —CH ₂ — and —CH ₂ —S—CH ₂ —CH ₂ —NH—CH ₂ —. For heteroalkylene groups, the heteroatom can also occupy one or both of the chain ends (eg, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Further, for alkylene and heteroalkylene linking groups, the direction of the linking group is not implied by the direction in which the formula of the linking group is written. For example, the formula —C (O) ₂ R′— corresponds to both —C (O) ₂ R′— and —R′C (O) ₂ —.

  The terms “cycloalkyl” and “heterocycloalkyl” by themselves or in combination with other terms represent cyclic forms of “alkyl” and “heteroalkyl”, respectively, unless otherwise specified. Further, for heterocycloalkyl, the heteroatom can occupy the position where the heterocycle is attached to the rest of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1- (1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran 2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl and the like are included.

The term “halo” or “halogen”, unless otherwise stated, means a fluorine, chlorine, bromine, or iodine atom by itself or as part of another substituent. Furthermore, terms such as “haloalkyl” will include monohaloalkyl and polyhaloalkyl. For example, the term “halo (C ₁ -C ₄ ) alkyl” includes, but is not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like. It becomes.

  The term “aryl”, unless stated otherwise, can be a single ring or multiple rings (preferably 1 to 3 rings) fused together or covalently linked together, Means a polyunsaturated aromatic substituent. The term “heteroaryl” refers to 1 to 4 selected from N, O, and S, in which the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. An aryl group (or ring) containing a heteroatom. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4- Imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2- Furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1 -Isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5 Quinoxalinyl, 3-quinolyl, tetrazolyl, benzo [b] furanyl, benzo [b] thienyl, 2,3-dihydrobenzo [1,4] dioxin-6-yl, benzo [1,3] dioxol-5-yl, and 6-quinolyl is included. The substituents for each of the above aryl and heteroaryl ring systems are selected from the group of acceptable substituents set forth below.

  For brevity, the term “aryl” when used in combination with other terms (eg, aryloxy, arylthioxy, arylalkyl) is defined as aryl and heteroaryl rings as defined above. Including both. Thus, the term “arylalkyl” means, for example, an alkyl group in which a carbon atom (eg, methylene group) is replaced by an oxygen atom (eg, phenoxymethyl, 2-pyridyloxymethyl, 3- (1-naphthyloxy) propyl, etc.) It includes a radical in which an aryl group is attached to an alkyl group including, for example, benzyl, phenethyl, pyridylmethyl, and the like.

  Each of the above terms (eg, “alkyl”, “heteroalkyl”, “aryl”, and “heteroaryl”) will include both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.

Substituents for alkyl and heteroalkyl radicals (often including groups called alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl and heterocycloalkenyl) are generally “alkyl Referred to as “group substituents”, which include, but are not limited to, a number ranging from zero to (2m ′ + 1), where m ′ is the sum of carbon atoms in such radicals, It can be one or more of various groups selected from: -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′— (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 ′, R ″, R ′ ″ and R ″ ″ are preferably each independently hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl (eg 1 to 3 Halogen, substituted or unsubstituted alkyl, alkoxy, or thioalkoxy group, or aryl substituted with an arylalkyl group). Where a compound of the present invention contains more than one R group, for example, as with each R ′, R ″, R ″ ′, and R ″ ″ group when more than one such group is present. , Each R group is independently selected. When R ′ and R ″ are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5, 6 or 7 membered ring. For example, —NR′R ″ will include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above description of substituents, one of skill in the art will recognize that the term “alkyl” includes haloalkyl (eg, —CF ₃ and —CH ₂ CF ₃ ) and acyl (eg, —C (O) CH ₃ , It will be understood that groups containing carbon atoms bonded to groups other than hydrogen groups, such as —C (O) CF ₃ , —C (O) CH ₂ OCH ₃ will be included.

Similar to the described substituents for alkyl radicals, substituents for aryl and heteroaryl groups are commonly referred to as “aryl group substituents”. For example, these substituents are selected from: a number ranging from zero to the sum of open valences on the aromatic ring system, 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 —NOR ₂ , —R ′, —N ₃ , — CH _{(Ph) 2,} fluoro _(C 1 _{~C 4)} alkoxy, and fluoro _(C 1 _{~C 4)} alkyl. Where R ′, R ″, R ″ ′, and R ″ ″ are hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted Each is preferably independently selected from heteroaryl. For example, if a compound of the present invention contains two or more R groups, each R ′, R ″, R ″ ′, and R ″ ″ group when two or more such groups are present, and Similarly, each R group is independently selected. In the scheme below, the symbol X corresponds to “R” as described above.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ring can be optionally replaced with a substituent of the formula -TC (O)-(CRR ') _q- U-. In the formula, T and U are independently —NR—, —O—, —CRR′—, or a single bond, and q is an integer of 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring, with the formula -A- (CH ₂₎ r _-B- substituents can be replaced optionally. In the formula, A and B are independently —CRR′—, —O—, —NR—, —S—, —S (O) —, —S (O) ₂ —, —S (O) _2. NR′— or a single bond, and r is an integer of 1 to 4. One of the new ring single bonds thus formed can optionally be replaced by a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring are optional substituents of the formula — (CRR ′) _s —X— (CR ″ R ′ ″) _d — Can be replaced. Here, s and d are each independently an integer of 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.

  As used herein, the term “heteroatom” is meant to include oxygen (O), nitrogen (N), sulfur (S), and silicon (Si).

  The use of a reactive derivative of PEG (or other linker) that attaches one or more peptide moieties to the linker is within the scope of the invention. The present invention is not limited by the identity of the reactive PEG analog. Many activated derivatives of poly (ethylene glycol) are commercially available and also exist in the literature. It is well within the ability of those skilled in the art to select and optionally synthesize appropriate activated PEG derivatives that are used to prepare substrates useful in the present invention. 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)); active esters derived from N-hydroxysuccinimide (Buckmann et al.," Makromol. Chem. ", 182: 1379-1384 (1981); Joppic et al., “Makromol. Chem.”, 180: 1381-1384 (1979); Abuchowski et al., “Cancer Biochem. Biophys.”, 7: 175-186 (1984); Katre) et al., "Proc. Natl. Acad. Sci. USA", 84: 1487-1491 (1987); Kitamura et al., "Cancer Res.", 51: 4310-431. (1991); Boccu et al., "Z.Naturforsch.", 38C: 94-99 (1983), carbonate (Zalippsky et al., POLY (ETHYLENE GLYCOL) CHEMISTRY: BIOTECHNICAL AND BIOMETICALSPLIS). 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 formate (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., “ASAIO Journal”). , M649-M-653 (1992)), and epoxides (US Pat. No. 4,806,595 issued to Noishiki et al. (1989)). Other linking groups include urethane linkages between amino groups and activated PEG. See Veronese et al., “Appl. Biochem. Biotechnol.” 11: 141-152 (1985).

  The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified (eg, hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine). Amino acid analogs are compounds having the same basic chemical structure as a naturally occurring amino acid (ie, hydrogen, carboxyl group, amino group, and α carbon bonded to R group), such as homoserine, norleucine, methionine sulfoxide, methionine Refers to methylsulfonium. Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. “Amino acid mimetics” refers to compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

  “Peptide” refers to a polymer in which the monomers are amino acids, amino acid analogs, and / or amino acid mimetics and are linked together through amide bonds, also referred to as a polypeptide. In addition, unnatural amino acids such as β-alanine, phenylglycine, and homoarginine are also included. Amino acids that are not gene-encoded can also be used in the present invention. In addition, amino acids modified to include reactive groups, glycosylation sites, polymers, therapeutic moieties, biomolecules, and the like can also be used in the present invention. All amino acids used in the present invention can be either the D- or L-isomer. The L-isomer is usually preferred. In addition, other peptidomimetics are also useful in the present invention. As used herein, “peptide” refers to both glycosylated and non-glycosylated peptides. Also included are peptides that are incompletely glycosylated by a system that expresses the peptide. For a general review, see Spatra, A. F. (Spatola, A.F.), “CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS”, “PEPTIDES AND PROTEINS”, B.C. See B. Weinstein, Marcel Dekker, New York, page 267 (1983).

  The term “nucleoside” is a component of a nucleic acid and linked to β-D-ribofuranose (which forms a ribonucleoside) or 2-deoxy-β-D-ribofuranose (which forms a deoxyribonucleoside). Refers to a glycosylamine containing a base containing nitrogen. The base can be a purine (eg, adenine or guanosine) or a pyrimidine (eg, thymidine, cytidine, uridine, or pseudouridine). Nucleosides also include unusual nucleosides used by microorganisms.

  The term “targeting moiety” as used herein refers to a species that is selectively localized to 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, and the like. Other mechanisms for directing drugs to specific tissues or areas are known to those skilled in the art. Typical targeting moieties include antibodies, antibody fragments, transferrin, HS-glycoprotein, clotting factors, serum proteins, β-glycoprotein, G-CSF, GM-CSF, M-CSF, EPO, and the like.

  As used herein, “therapeutic moiety” means any agent useful in a therapeutic method, including but not limited to antibiotics, anti-inflammatory agents, anti-tumor agents, cytotoxins and radioactive agents. . “Therapeutic moiety” includes a prodrug of a bioactive agent, a construct in which two or more therapeutic moieties are attached to a carrier (eg, a multivalent agent). The therapeutic portion also includes 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), interferons (eg, interferon-α, -β, -Γ), interleukins (eg interleukin II), serum proteins (eg VII, VIIa, VIII, IX and factor X), human chorionic gonadotropin (HCG), follicle stimulating hormone (FSH) and Luteinizing hormone (LH), as well as antibody fusion proteins (eg, tumor necrosis factor receptor (TNFR) / Fc domain fusion proteins) are included.

  As used herein, “antitumor agent” includes, but is not limited to, antimetabolite, alkylating agent, anthracycline, antibiotic, antimitotic agent, procarbazine, hydroxyurea, asparaginase, corticosteroid Any drug useful to fight cancer, including cytotoxins and drugs such as, interferon, and radioactive drugs. Also included within the scope of the term “anti-tumor agent” are conjugates of peptides having anti-tumor activity (eg, TNF-α). Conjugates include, but are not limited to, those formed between therapeutic proteins and glycoproteins of the invention. A typical conjugate is that formed between PSGL-1 and TNF-α.

  As used herein, “cytotoxin or cytotoxic agent” means any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione (anthracindiedion) Santron, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologues thereof are included. Other toxins include, for example, ricin, CC-1065 and analogs, duocarmycin. Still other toxins include diphtheria toxin, and snake venom (eg, cobra venom).

  As used herein, “radioactive agent” includes any radioisotope that is effective in diagnosing or destroying a tumor. Examples include, but are not limited to, indium-111 and cobalt-60. In addition, naturally occurring radioactive elements such as uranium, radium, and thorium (generally corresponding to a mixture of radioisotopes) are also suitable examples of radioactive agents. Metal ions are generally chelated using an organic chelating moiety.

  Many useful chelating groups, crown ethers, cryptands, etc. are known in the art, and compounds of the present invention (such as EDTA, DTPA, DOTA, NTA, HDTA, and DTPP, EDTP, HDTP, NTP, etc.) Its phosphonic acid analog). For example, in Pitt et al., “The Design of Cheating Agents for the Treatment of Iron Over”, “INORGANIC CHEMISTRY IN BIOLOGY AND MEDICINE”, edited by Martell (Mart. Washington DC), 1980, pp. 279-312; Lindoy, “THE CHEMISTRY OF MACROCYCLIC LIGAND COMPLEXES”, Cambridge University Press, Cambridge, 198, Cambridge Year; Deyugasu (Dugas), "BIOORGANIC CHEMISTRY", Springer Publishing (Springer-Verlag), New York (New York), 1989 years, and a bibliography of reference be included in that.

  In addition, numerous routes are available to those skilled in the art that allow attachment of chelating agents, crown ethers, and cyclodextrin to other molecules. For example, Meares et al., "Properties of In Vivo Chelate-Tagged Proteins and Polypeptides." Washington, DC, 1982, pages 370-387; Kasina et al., “Bioconjugate Chem.” 9: 108-117 (1998); Song et al., “Bioconjugate”. Chem. ", 8: 249-255. 1997) a reference to that.

Introduction The present invention provides macromolecular species and sugars, activated sugars, and nucleotide sugars that are attached to these polymers. A polymeric conjugate of a nucleotide sugar is usually a substrate for an enzyme that transfers the sugar moiety and its polymeric substituents onto an appropriate acceptor moiety of the substrate. Thus, the present invention also provides a substrate that is modified by a sugar conjugate using a polymer conjugate of a nucleotide sugar and a suitable enzyme. Substrates that can be glycoconjugated using the compounds of the present invention include peptides (eg, glycopeptides), lipids (eg, glycolipids), and aglycones (sphingosine, ceramide).

  As stated in the previous section, the art recognized chemical method of covalent PEGylation relies on chemical conjugation via reactive groups on amino acids or carbohydrates. Useful conjugates were prepared using a chemically mediated binding strategy through careful design of the conjugates and reaction conditions. The main drawback of chemical coupling of polymers to proteins or glycoproteins is the lack of selectivity of the activated polymers, often at sites related to the biological activity of the protein or glycoprotein. Results in adhesion. Several strategies have been developed to address site-selective conjugation chemistry, but only one universal method suitable for various recombinant proteins has evolved.

  In contrast to methods recognized in the art, the present invention is used in a novel strategy for highly selective, site-specific sugar conjugates (eg, sugar PEGylation) of branched water-soluble polymers. A compound is provided. In an exemplary embodiment of the invention, site-specific attachment of a branched water-soluble polymer is performed by in vitro enzymatic glycosylation of specific peptide sequences using the nucleotide sugars or activated sugars of the invention. Achieved by: Sugar conjugates can be performed enzymatically utilizing a glycosyltransferase (eg, sialyltransferase) capable of transferring a branched water-soluble polymer-sugar species (eg, PEG-sialic acid) to a glycosylation site (eg, sialyltransferase). “Sugar PEGylation”).

  As described above, the present invention provides conjugates between sugars having any desired carbohydrate structure that have been modified with a polymeric moiety. Sugar nucleotides and activated sugars based on these sugar structures are also components of the present invention. The polymeric modifying moiety is attached to the sugar moiety by enzymatic means, chemical means or combinations thereof, thereby producing a modified nucleotide sugar. The sugar is replaced with a polymeric modifying moiety at any desired position. In an exemplary embodiment, the sugar is a furanose substituted with one or more of C-1, C-2, C-3, C-4, or C-5. In another embodiment, the invention replaces one or more of C-1, C-2, C-3, C-4, C-5, or C-6 with a polymeric modifying moiety. Serving pyranose. Preferably, the polymeric modifying moiety is attached directly to oxygen, nitrogen or sulfur hanging from the carbon. Alternatively, the polymeric modifying moiety is attached to a linker placed between the sugar and the modifying moiety. This linker is attached to oxygen, nitrogen, or sulfur hanging from the selected carbon.

  In presently preferred embodiments, the resulting conjugate is a substrate for an enzyme used to link the modified sugar moiety to another species (eg, peptide, glycopeptide, lipid, glycolipid, etc.). A polymer-modifying moiety is added at a position selected to function as Exemplary enzymes are described in more detail herein, including glycosyltransferases (sialyltransferases, glucosyltransferases, galactosyltransferases, N-acetylglucosyltransferases, N-acetylgalactosyltransferases, mannosyltransferases, fucosyltransferases, etc. ) Is included. Exemplary sugar nucleotides and activated sugar conjugates of the present invention also include substrates for mutant glycosidases and mutant glycoceramidases that have been modified to have synthetic activity rather than hydrolytic activity.

  In an exemplary embodiment, the conjugates of the invention comprise a sugar, activated sugar, or nucleotide sugar attached to one or more polymers (eg, branched polymers). Typical polymers include both water-soluble and water-insoluble species.

In typical embodiments, the polymeric modifying group is attached directly or indirectly to the pyranose or furanose.
For example:

And in Formulas I and II, R ¹ is H, CH ₂ OR ⁷ , COOR ⁷ or OR ⁷ . Here, R ⁷ represents H, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl. R ² is a moiety containing H, OH, NH, or a nucleotide. An exemplary R ² species according to this embodiment has the formula:

In the formula, X ¹ represents O or NH, and R ⁸ 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 . The index d is 0 or 1. R ⁹ and R ¹⁰ are each independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, or sialic acid. At least one of R ³ , R ⁴ , R ⁵ , R ⁶ , and R ^{6 ′} includes a polymeric modifying moiety (eg, PEG). In an exemplary embodiment, R ⁶ and R ^{6 ′} are components of the side chain of sialic acid, along with the carbon to which they are attached. In further exemplary embodiments, the side chain is modified with one or more of C-6, C-7, or C-9 using a polymeric modifying moiety (or linker polymeric modifying moiety). Is done.

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 each 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 , or R ^{6 ′} includes a polymeric modifying moiety.

  In another exemplary embodiment, the sugar moiety is described in US Provisional Patent Application Number (Attorney Docket No. 040853-1-01-5150) filed Jan. 6, 2005 and assigned to the assignee of the present invention. A sialic acid moiety that has been oxidized and combined with a polymeric modifying moiety, such as

In an exemplary embodiment, the polymeric modifying moiety is linked to the sugar center via a linker:

In the formula, R ¹¹ is a polymer moiety, L is selected from a bond and a linking group, and w is an integer of 1 to 6, preferably 1 to 3, preferably 1 to 2.

When L is a bond, it is formed between a reactive functional group with respect to the precursor of R ¹¹ and a reactive functional group with complementary reactivity with respect to the precursor of L. As described herein, the selection and preparation of precursors using appropriate reactive functional groups is within the ability of those skilled in the art. Furthermore, combining the precursors proceeds by chemistry well understood in the art.

In an exemplary embodiment, L is an amino acid, amino acid mimetic or small molecule peptide (eg 1 to 1) that provides a modified sugar such that the polymeric modifying moiety is attached via a substituted alkyl linker. A linking group formed from 4 amino acid residues). The linker is through the reaction of an amine moiety of an amino acid and a carboxylic acid (or a reactive derivative such as an active ester, acid halide, etc.) with a group that is complementary to the precursor to L and R ¹¹ . It is formed. The elements of the conjugate can be combined in essentially any convenient order. For example, a precursor to L can be present at a suitable location on the saccharide center prior to conjugation with the R ¹¹ and L precursors. Alternatively, an R ¹¹ -L cassette with a functional group reactive to L can be prepared and then linked to a saccharide via a reactive functional group with complementary reactivity to this species.

In an exemplary embodiment, the polymeric modifying moiety is R ³ and / or R ⁶ . In another exemplary embodiment, R ³ and / or R ⁶ include both a polymeric modifying moiety and a linker (L) that connects the polymeric moiety to the remainder of the molecule. In another exemplary embodiment, the polymeric modifying moiety is R ³ . In a further exemplary embodiment, R ^{3 also} includes both a polymeric modifying moiety and a linker (L) that connects the polymeric moiety to the remainder of the molecule. In yet another exemplary embodiment where the sugar is sialic acid, the polymeric modifying moiety is R ⁵ or is attached to a sialic acid side chain position (eg, C-9).

Linear Polymer Conjugates In an exemplary embodiment, the invention provides a nucleotide sugar conjugate formed between a sugar or an activated sugar conjugate, or a linear polymer (such as a water soluble or water insoluble polymer). Provide a gate. In the conjugates of the invention, the polymer is attached to a sugar, activated sugar, or sugar nucleotide. As described herein, the polymer is linked to the sugar moiety either directly or through a linker.

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

Another example according to this embodiment has the following formula:

In the formula, s is an integer of 0 to 20, and R ¹¹ is a linear polymer modifying moiety.

  PEG moieties of any molecular weight, such as 2Kda, 5Kda, 10Kda, 20Kda, 30Kda, and 40Kda, are used in the present invention.

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

In the formula, R ¹¹ and L are as described above, and w ′ is an integer of 2 to 6, preferably 2 to 4, preferably 2 to 3.

A typical precursor used to form a conjugate according to this embodiment of the invention has the following formula:

Branched polymer species according to this formula are essentially pure water-soluble polymers. X ^{3 ′} is a moiety that includes ionizable (eg, COOH, H ₂ PO ₄ , HSO ₃ , HPO ₃ , etc.) or other reactive functional groups (eg, below). C is carbon. X ⁵ is preferably a non-reactive group (eg, H, unsubstituted alkyl, unsubstituted heteroalkyl), and may be a polymer arm. R ¹² and R ¹³ are independently selected polymer arms, for example, non-peptidic and non-reactive polymer arms. X ² and X ⁴ are ligation fragments that are preferably essentially non-reactive under physiological conditions, which may be the same or different. Alternatively, these linkages can include one or more moieties that are designed to degrade under physiologically relevant conditions, such as esters, disulfides, etc. X ² and X ⁴ are high The molecular arms R ¹² and R ¹³ are linked to C. X ^{3 ′} is converted to a component of ligation fragment X ³ upon reaction with a reactive functional group with complementary reactivity to the linker, sugar, or linker-sugar cassette.

Exemplary ligation fragments for X ² and X ⁴ include S, SC (O) NH, HNC (O) S, SC (O) O, O, NH, NHC (O), (O) CNH and NHC (O) O and OC (O) NH, CH 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, a is an integer from 1 to 50) are included.

In an exemplary embodiment, precursor (III), or an activated derivative thereof, is a saccharide, activated, via a reaction between X ^{3 ′} and a group that has complementary reactivity to the sugar moiety. Bound to a sugar or sugar nucleotide. Alternatively, X ^{3 ′} reacts with a functional group that is reactive towards a precursor to the linker (L). One or more of R ¹ , R ³ , R ⁴ , R ⁵ , or R ⁶ of Formulas I and II can include a branched polymeric modifying moiety.

In an exemplary embodiment, the following parts:

Is an arm (L). In this embodiment, typical linkers are derived from natural or non-natural amino acids, amino acid analogs, or amino acid mimetics, or small peptides derived from one or more such species. For example, certain branched polymers found in the compounds of the present invention have the formula:

^Xa is a linking moiety formed by reaction of a reactive functional group with a precursor of a branched polymer modifying moiety and a sugar moiety, or a precursor to a linker. For example, if X ^{3 ′} is a carboxylic acid, it can be activated and an amine group hanging from an amino-saccharide (eg, GalNH ₂ , GlcNH ₂ , ManNH _2, etc.) that forms the amide X ^a Can be directly bound to Further exemplary reactive functional groups and activated precursors are described below. The index c represents an integer from 1 to 10. Other symbols have the same identity as described above.

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

Wherein X ^b is a linking moiety and each independently selected from the groups described for X ^a , and L ¹ is a bond, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl.

Typical species 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 are included.

For example,

In the formula, s is an integer from 0 to 20, and R ¹¹ is a linear polymer modifying moiety.

In another exemplary embodiment, X ⁴ is a peptide bond to R ¹³ (the alpha-amine moiety and / or the side chain heteroatom is an amino acid, dipeptide, or tripeptide modified with a polymer). It is.

In a further exemplary embodiment, R ⁶ comprises a branched polymeric modifying group, and the modified sugar or nucleotide sugar has a formula selected from:

In the formula, the identity of radicals represented by various symbols is the same as described above. L ^a is a substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl moieties. In an exemplary embodiment, L ^a is a part of the side chain of sialic acid that is functionalized with the polymeric modifying moiety as shown.

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

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

  Embodiments of the invention described above refer to species where the polymer is a water soluble polymer (especially polyethylene glycol) (“PEG”), eg, methoxy-poly (ethylene glycol) (“m-PEG”). This is further exemplified. For those skilled in the art, the focus in the following sections is for clarity of illustration, and the various themes described using PEG as a typical polymer are other than PEG. It will be appreciated that the polymer is equally applicable to the species in which it is utilized.

Water-Soluble Polymers Many water-soluble polymers are known to those skilled in the art and are useful for practicing the present invention. The term “water-soluble polymer” refers to sugars (eg, dextran, amylose, hyaluronic acid, poly (sialic acid), heparan, heparin, etc.); poly (amino acids) such as poly (aspartic acid) and poly (glutamic acid); nucleic acids; synthesis Polymers (eg, poly (acrylic acid), poly (ether), eg, poly (ethylene glycol)); species such as peptides, proteins, and the like. The polymer generally comprises at least two polymeric units. In typical embodiments, the polymer is from 2 to 25 units. In another exemplary embodiment, the polymer comprises 2-8 polymeric units. The present invention can be practiced with any water soluble polymer, with the only limitation that the polymer must include a position where the remainder of the conjugate can be attached.

  Methods for polymer activation are also described in WO 94/17039, US Pat. No. 5,324,844, WO 94/18247, WO 94/04193, US Pat. No. 5,219,564, US Pat. No. 5,122,614, WO 90/13540, US Pat. No. 5,281,698, and more No. 93/15189, and methods for conjugation between activated polymers and peptides include, for example, coagulation factor VIII (WO 94/15625), hemoglobin (WO 94/09027). Pamphlet), oxygen-carrying molecules (US Pat. No. 4,412,989), ribonuclease Ze and superoxide dismutase: are on the (Veronese (Veronese), et al., 11 "App.Biochem.Biotech." 141-45 (1985)).

  Preferred water-soluble polymers are such that a significant percentage of the polymer molecules in the polymer sample are of approximately the same molecular weight, such polymers are “homodiperse”.

  The invention is further illustrated by reference to poly (ethylene glycol) conjugates. Several reviews and research papers on PEG functionalization and conjugation are available. For example, Harris, “Macronol. Chem. Phys.” C25: 325-373 (1985); Scouten, “Methods in Enzymology” 135: 30-65 (1987); Wong et al. "Enzyme Microb. Technol." 14: 866-874 (1992); Delgado et al., "Critical Reviews in Therapeutic Drug Carrier Systems" 9: 249-304 (p. 1992); Chem. "6: 150-165 (1995); and Bhadra et al.," Pharmacizie ", 57: To 29 of the reference that (2002). Routes for preparing reactive PEG molecules, and using reactive molecules to form conjugates are known in the art. For example, US Pat. No. 5,672,662 is from linear or branched poly (alkylene oxide), poly (oxyethylated polyol), poly (olefin alcohol), and poly (acrylomorpholine). Disclosed are water-soluble and isolatable conjugates of active esters of selected polymeric acids.

  U.S. Pat. No. 6,376,604 describes the reaction of water-soluble and non-peptidic polymer water-soluble 1-by reacting di (1-benzotriazolyl) carbonate with the terminal hydroxyl of the polymer in an organic solvent. Describes a process for preparing benzotriazolyl carbonate. The active ester is used to form a conjugate with a biologically active agent (such as a protein or peptide).

  WO 99/45964 is a conjugate comprising a biologically active agent comprising a polymer backbone having at least one terminus linked to the polymer backbone via a stable bond and an activated water-soluble polymer. Describe the gate. However, at least one end includes a branching portion having a proximal reactive group linked to the branching portion, and the biologically active agent is at least one of the proximal reactive groups. Connected to one. Other branched poly (ethylene glycols) are described in WO 96/21469, US Pat. No. 5,932,462 includes branched ends containing reactive functional groups. Describes conjugates formed using branched PEG molecules. Free reactive groups can be used to react with biologically active species such as proteins or peptides to form conjugates of poly (ethylene glycol) with biologically active species . US Pat. No. 5,446,090 describes a bifunctional PEG linker and its use in forming conjugates having peptides at each PEG linker terminus.

  Conjugates containing degradable PEG linkages are described in WO 99/34833, and WO 99/14259, and US Pat. No. 6,348,558. Such a releasable connection is applicable to the present invention.

  The art-recognized methods of activating the polymers described above are those in the formation of the branched polymers described herein, and their separation into other species such as sugars, sugar nucleotides, and the like. Useful in the present invention for the attachment of branch polymers.

  Typical modifying groups are described below. The modifying group can be selected for its ability to confer one or more desirable properties to the peptide. Typical properties include, but are not limited to, enhanced pharmacokinetics, enhanced pharmacodynamic action, improved biodistribution, provision of multivalent species, improved water solubility, enhanced or attenuated. Includes lipophilicity, and tissue targeting.

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

Wherein A ² is H, OH, NH ₂ , substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroalkyl , for example, _{acetal, OHC-, H 2 N- (CH} 2) q -, HS- (CH 2) q, or _{- (CH} 2) a ^{q C (Y b) Z b} . The index “e” represents an integer from 1 to 2500. The indices b, d, and q independently represent an integer from 0 to 20. The symbols Z ^a and Z ^b are independently OH, NH ₂ , a leaving group such as imidazole, p-nitrophenyl, HOBT, tetrazole, halide, S—R ^a , the alcohol moiety of an activated ester; ^{_{CH 2) p C (Y b}} ) V _{or, - (CH 2) p U} (CH 2) represents an S ^{C (Y} _{b) V.} Symbol ^{Y a} is, H (2), = O , represents a ^{= S, = N-R b} . The symbols X ^a , Y ^a , Y ^b , A ¹ , and U independently represent the moieties O, S, N—R ^C. The symbol V represents OH, NH ₂ , halogen, S—R ^a , the alcohol component of the activated ester, the amine component of the activated amide, the sugar nucleotide, and the protein. The indices p, q, s, and v are members independently selected from integers from 0 to 20. The symbols R ^a , R ^b , and R ^c are independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl, and substituted or Represents unsubstituted heteroaryl.

Particular embodiments of linear and branched polymers (eg, PEG) used in the present invention include:

Carbonate esters and active esters of these species such as:

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

Other exemplary activating groups or leaving groups suitable for activating the linear PEG used to prepare the compounds described herein include, but are not limited to, The following species are included:

It is well within the ability of those skilled in the art to select an appropriate activating group for a selected moiety on a precursor to a polymeric modifying moiety.

  PEG molecules that are activated using these and other species, and methods of making activated PEGs are described in WO 04/083259.

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

The indices e and f are each independently selected from 1 to 2500.

In yet another embodiment, the branched PEG moiety is based on a tri-lysine peptide. Tri-lysine can be mono-, di-, tri- or tetra-PEGylated. A typical species according to this embodiment has the formula:

Where e, f and f ′ are integers independently selected from 1 to 2500, and q, q ′ and q ″ are integers independently selected from 0 to 20.

In an exemplary embodiment of the invention, the PEG is m-PEG (5 kd, 10 kd, 20 kd, 30 kd, or 40 kd). A typical branched PEG species is lysine, serine- or cysteine- (m-PEG) ₂ where m-PEG is 20 kd m-PEG.

  As will be appreciated by those skilled in the art, the branched polymers used in the present invention include variations on the above-mentioned issues. For example, the di-lysine-PEG conjugate described above may contain three macromolecular subunits, the third coupled with an α-amine that has been shown to be unmodified in the structure described above. The Similarly, the use of tri-lysine functionalized with three or four polymeric subunits is also within the scope of the present invention.

Those of skill in the art, one or more of m-PEG arms of the branched polymer, different terminal, for _{example, OH, COOH, NH 2,} C 2 ~C 10 - replaced by a PEG moiety with alkyl and You will understand that you can. Furthermore, the above structure is easily modified by inserting an alkyl linker (or removing a carbon atom) between the side chain α-carbon atom and the functional group. Thus, “homo” derivatives and higher homologues, as well as lower homologues, are within the core for the branched PEG used in the present invention.

The branched PEG species described herein are readily prepared by methods such as those described in the scheme below:

In the formula, ^Xb is O, NH, or S, and r is an integer of 1 to 10. The indices e and f are integers independently selected from 1 to 2500. Typical 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 1 by alkylating the side chain heteroatom ^Xb . A monofunctionalized m-PEG amino acid is subjected to N-acylation conditions with a reactive m-PEG derivative, thereby assembling a branched m-PEG2. One skilled in the art will appreciate that the tosylate leaving group can be replaced with any suitable leaving group, such as halogen, mesylate, triflate, and the like. Similarly, the reactive carbonate utilized to acylate the amine can be replaced with an active ester such as N-hydroxysuccinimide. Alternatively, the acid can be activated in situ using a dehydrating agent (dicyclohexylcarbodiimide, carbonyldiimidazole, etc.).

  In the exemplary scheme described above, the modifying group is a linear PEG moiety, but any modifying group (eg, a water soluble polymer, a water insoluble polymer, a branched polymer, a therapeutic moiety, etc.) Can be incorporated into the glycosyl moiety.

Further branched macromolecular species used in the compounds of the present invention are exemplified by branch centers that are functionalized with PEG, such as the examples described below:

Where R ¹⁴ is OH or another reactive functional group. A typical reactive functional group is C (O) Q ′. Here, Q ′ is selected such that C (O) Q ′ is a reactive functional group. Typical species for Q ′ include halogen, NHS, pentafluorophenyl, HOBT, HOAt, and p-nitrophenyl. The index “e” and the index “f” are integers independently selected from 1 to 2500.

The branched compounds mentioned above, and further branched compounds used in the compounds of the present invention, are readily prepared from starting materials such as:

Polymer Modified Sugar Species The sugar moiety of the nucleotide sugars of the present invention can be selected from both natural and non-natural furanose and hexanose. Non-natural saccharides optionally include alkylated or acylated hydroxyl and / or amine moieties (eg, ethers, esters, and amide substituents on the ring). Other non-natural saccharides include H, hydroxyl, ether, ester, or amide substituents at ring positions such that such substituents are not present in natural saccharides. The sugar moiety can be a monosaccharide, oligosaccharide, or polysaccharide.

  Typical natural sugars used in the present invention include glucose, galactose, fucose, mannose, xylanose, ribose, N-acetylglucose, sialic acid, and N-acetylgalactose.

  Similarly, nucleosides can be selected from both natural and non-natural or unusual nucleosides. Typical natural nucleosides used in the present invention include cytosine, thymine, guanine, adenine, and uracil. Unusual nucleosides can include, but are not limited to, molecules such as spongouridine and spongothymidine. This technology is full of unnatural and unusual nucleoside structures, as well as methods of making them.

Exemplary modified sugar nucleotide of the present _{invention, 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-sialic acid are included. Similar to the sugars of the invention described above, the sugar nucleotides of the invention can be substituted with a polymeric modifying moiety (or linker modifying moiety) at any position of the saccharide. For example, compounds encompassed by the present invention include those in which the LR ¹¹ moiety is linked to C-5 of a furanose-based nucleotide sugar or C-6 of a pyranose-based nucleotide sugar.

Exemplary moieties attached to the conjugates disclosed herein include, but are not limited to, PEG derivatives (eg, alkyl-PEG, acyl-PEG, acyl-alkyl-PEG, alkyl-acyl-PEG carbamoyl). -PEG, aryl PEG), PPG derivatives (eg, alkyl-PPG, acyl-PPG, acyl-alkyl-PPG, alkyl-acyl-PPG carbamoyl-PPG, aryl PPG), therapeutic moieties, diagnostic moieties, mannose-6 - phosphoric acid, heparin, heparan, SLe _x, mannose, mannose 6-phosphate, sialyl Lewis (sialyl Lewis) X, FGF, VFGF, protein, chondroitin, keratan, dermatan, albumin, integrins, branched (antennary) oligosaccharide, Peptide, and the like. Methods for attaching various modifying groups to the saccharide moiety are readily available to those skilled in the art ("POLY (ETHYLENE GLYCOL CHEMISTRY: BIOTECHNICAL AND BIOMODICAL APPLICATIONS", edited by J. Milton Harris). Plenum Pub. Corp., 1992; “POLY (ETHYLENE GLYCOL) CHEMICAL AND BIOLOGICAL APPLICATIONS”, edited by J. Milton Harris (ACS No. 6 Symposium Series (ACS Symos 80). , American Chemical Society, 1997, -Hermanson, “BIOCONJUGATE TECHNIQUES”, Academic Press, San Diego, 1996; and Dunn et al., “POLYMERIC DRUGS AND DRUGS EL ST Series 469, American Chemical Society, Washington, DC (Washington, DC), 1991).

  In their modified form, typical sugar nucleotides of the invention include nucleotide mono-, di-, or triphosphates, or UDP-glycosides, CMP-glycosides, or analogs of GDP-glycosides. 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 used in the methods of the invention.

  In other embodiments, the modified sugar is an activated sugar. Activated modified sugars that are useful in the present invention are generally glycosides that have been synthetically altered to include an activated leaving group. As used herein, the term “activated leaving group” refers to a moiety that is readily displaced by an enzymatically controlled nucleophilic substitution reaction. Many activated sugars are known in the art. For example, in Vocadlo et al., “Carbohydrate Chemistry and Biology”, Volume 2, edited by Ernst et al., Wiley-VCH Verlag: Weinheim, Germany, 2000 (Korea; Kodama et al., “Tetrahedron Lett.” 34: 6419 (1993); Lockheed et al., “J. Biol. Chem.” 274: 37717 (1999)).

  Examples of the activating group (leaving group) include fluoro, chloro, bromo, tosylate ester, mesylate ester, triflate ester and the like. Preferred activated leaving groups for use in the present invention are those which do not significantly interfere with the enzymatic transfer of the glycoside to the acceptor. Accordingly, preferred embodiments of activated glycoside derivatives include glycosyl fluoride and glycosyl mesylate, with glycosyl fluoride being particularly preferred. Among the glycosyl fluorides, α-galactosyl fluoride, α-mannosyl fluoride, α-glucosyl fluoride, α-fucosyl fluoride, α-xylosil fluoride, α-sialyl fluoride, α-N-acetyl fluoride Glucosaminyl, α-N-fluorinated acetylgalactosaminyl, β-fluorinated galactosyl, β-fluorinated mannosyl, β-fluorinated glucosyl, β-fucosyl fluoride, β-fluorinated xylosyl, β-sialyl fluoride , Β-N-fluorinated acetylglucosaminyl, and β-N-fluorinated acetylgalactosaminyl are most preferred.

  As a specific example, glycosyl fluoride can be prepared from the free sugar by first acetylating the sugar and then treating it with HF / pyridine. This produces the thermodynamically most stable anomer of protected (acetylated) glycosyl fluoride (ie α-glycosyl fluoride). If a less stable anomer (i.e., β-glycosyl fluoride) is desired, this can be accomplished using HBr / HOAc or using HCI to convert the peracetylated sugar to produce an anomeric bromide or It can be prepared by producing chloride. This intermediate is reacted with a fluoride salt such as silver fluoride to produce glycosyl fluoride. Acetylated glycosyl fluoride can be deprotected by reaction with a weak (catalytic) base (eg, NaOMe / MeOH) in methanol. 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 a fully benzylated hemiacetal form of sugar with mesyl chloride followed by catalytic hydrogenation to remove the benzyl group.

  In a further exemplary embodiment, the modified sugar is an oligosaccharide having a branched structure. In another embodiment, one or more of the ends of the branches have a modifying moiety. When more than one modifying moiety is attached to an oligosaccharide having a branched structure, the oligosaccharide is useful for “amplifying” the modifying moiety, and each oligosaccharide unit attached to the peptide is attached to the peptide. Multiple copies of the modifying group are attached. The general structure of a typical conjugate of the present invention as shown in the above drawings includes multivalent species that will prepare the conjugate of the present invention utilizing a branched structure. Many branched saccharide structures are known in the art and the methods of the invention can be practiced with them without limitation.

  In an exemplary embodiment, the activated modified sugar is a substrate for a mutant enzyme that transfers the sugar to an appropriate acceptor moiety of the substrate. Exemplary mutant enzymes include, for example, those described in PCT Publication WO 03/046150 and WO 03/045980, assigned to the assignee of the present invention.

  Sugars modified with water-soluble polymers, activated sugars, and nucleotide sugar species whose sugar moieties are modified with water-soluble polymers (eg, water-soluble polymers) are used in the present invention. A typical modified sugar nucleotide has a modified sugar group through an amine moiety on the sugar. Modified sugar nucleotides, such as saccharyl-amine derivatives of sugar nucleotides, are also used in the methods of the invention. For example, a saccharylamine (without a modifying group) can be enzymatically coupled to a peptide (or other species), and then the free saccharylamine moiety can be coupled to the desired modifying group. Alternatively, the modified sugar nucleotide can function as a substrate for an enzyme that transfers the modified sugar to a saccharyl acceptor on a substrate (eg, a peptide, glycopeptide, lipid, aglycone, glycolipid, etc.).

  In one embodiment, the sugar is attached to a branched macromolecular species such as those described below.

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

In another embodiment where the sugar center 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. As shown below for N-acetylgalactosamine, the 6-amino-sugar moiety is readily prepared by standard methods.

  In the above scheme, the index n represents an integer of 1 to 2500, preferably 10 to 1500, preferably 10 to 1200. The symbol “A” represents an activating group such as halo, an activated ester component (eg, N-hydroxysuccinimide ester), a carbonate ester component (eg, p-nitrophenyl carbonate), and the like. One skilled in the art will appreciate that other PEG-amide nucleotide sugars are readily prepared by this and similar methods. Further, as described herein, the branched polymer can be replaced with linear PEG.

Another exemplary polymericly modified nucleotide sugar of the present invention in which the C-6 position is modified has the following formula:

In the formula, X ⁶ is a bond or O, J is S or O, and y is 0 or 1. The indices e and f are each independently selected from 1 to 2500.

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

  In the discussion that follows, some specific examples of modified sugars that are useful in practicing the present invention are described. In an exemplary embodiment, the sialic acid derivative is utilized as the sugar center to which the modifying group is attached. The focus of the discussion on sialic acid derivatives is for clarity of illustration only and should not be construed to limit the scope of the invention. Those skilled in the art will appreciate that various other sugar moieties can be activated and derivatized in a manner similar to that described using sialic acid as an example. For example, numerous methods are available to modify galactose, glucose, N-acetylgalactosamine, and fucose (to name a few sugar substrates) that are easily modified by methods recognized in the art. is there. See, for example, Elhalabi et al., “Curr. Med. Chem.” 6:93 (1999) and Schaffer et al., “J. Org. Chem.” 65:24 (2000). .

  In FIG. 2, a general scheme according to the invention is described. Thus, according to FIG. 2, the protection wherein an amide conjugate between mannosamine and a protected amino acid is formed by contacting an N-protected amino acid with mannosamine under conditions suitable to form the conjugate. The carboxyl terminus of the rendered amino acid is activated in situ, or it is optionally converted to a reactive group that is stable to storage (eg, N-hydroxy-succinimide). The amino acid can be selected from any natural or non-natural amino acid. Those skilled in the art will understand how to protect side chain amino acids from undesired reactions in the methods of the present invention. The amide conjugate is reacted with pyruvate and sialic acid aldolase under conditions suitable to convert the amide conjugate to a sialic acid amide conjugate, which is then reacted with a nucleotide phosphate precursor and a sialic acid amide. Conversion to a nucleotide phosphate-sialic acid conjugate by reaction of the conjugate and the appropriate enzyme. In an exemplary embodiment, the precursor is cytidine triphosphate and the enzyme is a synthetase. After formation of the nucleotide sugar, the amino acid amine is deprotected to provide the free reactive amine amine. The amine serves as a site for attaching the modifying moiety to the nucleotide sugar. In FIG. 2, the modifying moiety is exemplified by, for example, a water-soluble polymer, ie poly (ethylene glycol), such as PEG, m-PEG, and the like.

  The invention is further illustrated in FIG. 3, which describes a scheme for preparing sialic acid-glycyl-PEG-cytidine monophosphate. Similar to the scheme described in FIG. 2, the scheme of FIG. 3 also starts with mannosamine. The sugar is coupled to FMOC-glycine using an N-hydroxysuccinimide activated derivative of the protected amino acid. The resulting amide conjugate is converted to the corresponding sialic acid by the action of sialic acid aldolase on the conjugate and pyruvate. The resulting sialic acid conjugate is converted to a cytidine monophosphate analog using cytidine triphosphate and synthetase. The CMP-analog is deprotected by removing the protecting group from the amino acid amine moiety, which is converted to a reactive site for conjugation. The amine moiety is reacted with an activated PEG species, m-PEG-O-nitrophenyl carbonate, thereby forming sialic acid-glycyl-PEG-cytidine monophosphate.

A typical sugar center based on sialic acid has the following formula:

In the formula, D is —OH or (R ¹¹ ) _{W ′} -L—. Symbol G represents H, a ^{(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 present invention provides a sugar, activated sugar, or sugar nucleotide comprising the following structure:

Wherein L ² is, for example, a bond, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl group as described above for L. The index e represents an integer from 1 to about 2500.

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

Where s is selected from an integer from 0 to 20 and e is from 1 to 2500.

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

Where AA is an amino acid residue, PEG is poly (ethylene glycol) or methoxy-poly (ethylene glycol), and NP is a glycosyl via a phosphodiester bond (“nucleotide phosphate”). A nucleotide linked to a moiety. One skilled in the art will appreciate that ONP can be replaced by an activating moiety as described herein.

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

In the formula, X ⁶ is a bond or O, and J is S or O. The indices a, b and c are each independently selected from 0 to 20, and e and f are each independently selected from 1 to 2500.

Furthermore, as described above, the present invention provides nucleotide sugars that are modified with water-soluble polymers that are linear or branched. For example, compounds having the formula shown below are within the scope of the invention:

In the formula, X ⁶ is O or a bond, and J is S or O. The indices e and f are each independently selected from 1 to 2500.

Also provided are conjugates of peptides and glycopeptides, lipids and glycolipids, including the compositions of the invention. The conjugate includes a nucleotide sugar of the invention or an activated sugar, and a substrate having an appropriate acceptor moiety for the sugar moiety, and (the modified nucleotide sugar has been modified from the nucleotide sugar onto the acceptor moiety. Formed by combining the enzyme (which is a substrate for it) under conditions suitable to transfer sugars. For example, the present invention provides a conjugate having the following formula:

In the formula, J and X ⁶ are as described above. The indices a, b, c, e, and f are as described above.

Selected compounds of the present invention are based on species having the stereochemistry of mannose, galactose, and glucose. The general formula of these compounds is as follows:

Wherein one of R ^{3 to} R ⁶ is a modifying moiety, such as a polymeric modifying moiety or a polymeric modifying moiety-linker construct.

  As mentioned above, certain compounds of the present invention are sugar nucleotides modified with macromolecules. In their modified form, typical sugar nucleotides used in the present invention include nucleotide mono-, di-, or triphosphates or analogs thereof. In preferred embodiments, the modified sugar nucleotide is selected from UDP-glycosides, CMP-glycosides, or GDP-glycosides. 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, the nucleotide mono, di, or triphosphate is attached to C-1.

  Saccharyl-amine derivatives of sugar nucleotides are also used in the methods of the invention. For example, a saccharylamine (without a modifying group) can be enzymatically coupled to a peptide (or other species) and the free saccharylamine moiety can then be coupled to the desired modifying group.

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

In the formula, the symbols represent groups as described above. When the sugar center is mannose, the polymeric modifying moiety is preferably R ³ , R ⁴ , or R ⁶ . For glucose, the polymeric modifying moiety is optionally R ⁵ or R ⁶ . The index “u” is 0, 1, or 2.

A more typical nucleotide sugar of the present invention based on GDP mannose has the following structure:

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

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

In each of the three aforementioned formulas, the identity of the radical and index is as described above.

  As will be apparent to those skilled in the art, the linear PEG moiety can be replaced by a branched polymer or other linear polymer species as described herein.

In one embodiment where the saccharide center is galactose or glucose, R ⁵ is NHC (O) Y.

Water Insoluble Polymer In another embodiment similar to that described above, the modified sugar comprises a water insoluble polymer rather than a water soluble polymer. A water-insoluble polymer, like a water-soluble polymer, generally comprises at least two polymer units. In one exemplary embodiment, the polymer consists of 2 to 25 polymeric units. In another exemplary embodiment, the polymer consists of 2 to 8 polymeric units. The conjugates of the invention can also include one or more water-insoluble polymers. This embodiment of the invention is illustrated by using the conjugate as a vehicle used to deliver therapeutic peptides in a controlled manner. Polymeric drug delivery systems are known in the art. For example, edited by Dunn et al., "POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS", ACS Symposium Series Vol. 469, American Chemical Society, Washington City, 91. . Those skilled in the art will appreciate that virtually any known drug delivery system is applicable to the conjugates of the present invention.

  Representative water-insoluble polymers include, but are not limited to, polyphosphazine, poly (vinyl alcohol), polyamide, polycarbonate, polyalkylene, polyacrylamide, polyalkylene glycol, polyalkylene oxide, polyalkylene terephthalate, polyvinyl Ether, polyvinyl ester, polyvinyl halide, polyvinylpyrrolidone, polyglycolide, polysiloxane, polyurethane, poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (methacrylic) Acid hexyl), poly (isodecyl methacrylate), poly (lauryl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate) Poly (isopropyl acrylate), poly (isobutyl acrylate), poly (octadecyl acrylate) polyethylene, polypropylene, poly (ethylene glycol), poly (ethylene oxide), poly (ethylene terephthalate), poly (vinyl acetate), polyvinyl chloride , Polystyrene, polyvinylpyrrolidone, pluronic, and polyvinylphenol, and copolymers thereof.

  Synthetically modified natural polymers used in the conjugates of the present invention include, but are not limited to, alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, and nitrocellulose. A particularly preferred member of the broad class of synthetically modified natural polymers includes, but is not limited to, methylcellulose, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxybutylmethylcellulose, cellulose acetate, cellulose propionate, butyric acetate Cellulose, cellulose acetate phthalate, carboxymethyl cellulose, cellulose triacetate, cellulose sulfate sodium salt, and polymers of acrylic and methacrylic acid esters and alginic acid are included.

  These and other polymers described herein are available from Sigma Chemical Co. (St. Louis, Missouri), Polysciences (Warrenton, Pa.), Aldrich (Aldrich) Can be readily obtained from commercial sources such as Milwaukee, Wisconsin, Fluka (Ronkonkoma, New York), and BioRad (Richmond, Calif.), Or from these sources From the resulting monomer, it is synthesized using standard techniques.

  Representative biodegradable polymers used in the conjugates of the invention include, but are not limited to, polylactic acid, 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 compositions that form gels, such as those including collagen, pluronics, and the like.

  The polymers used in the present invention include “hybrid” polymers that include water-insoluble materials that have bioresorbable molecules in at least a portion of their structure. Examples of such polymers are those that comprise a water-insoluble copolymer with a bioresorbable region, a hydrophilic region, and a plurality of crosslinkable functional groups for each polymer chain.

  For the purposes of the present invention, a “water-insoluble material” includes a material that is substantially insoluble in water or a hydrous environment. Thus, even if certain regions or segments of the copolymer are hydrophilic or water soluble, the polymer molecule as a whole is “not soluble in water” for any substantial measurement.

  For the purposes of the present invention, the term “bioresorbable molecule” includes a region that can be metabolized or degraded and eliminated and / or reabsorbed by the body via normal excretion pathways. Such metabolites or degraded products are preferably substantially non-toxic to the body.

  The bioresorbable region may be hydrophobic or hydrophilic so long as the copolymer composition as a whole is not water soluble. Thus, the bioresorbable region is selected based on the priority that the polymer as a whole remains water insoluble. Thus, the relative properties, i.e. the type of functional groups contained, and the relative proportion of the bioresorbable region, and the hydrophilic region, ensure that the useful bioresorbable composition is water insoluble. Selected to remain.

  Typical resorbable polymers include, for example, synthetically produced resorbable block copolymers of poly (α-hydroxy-carboxylic acid) / poly (oxyalkylene) (Cohn). Et al., U.S. Pat. No. 4,826,945). These copolymers are not crosslinked and are water soluble so that the body can expel the degraded block copolymer composition. Younes et al., “J Biomed. Mater. Res.” 21: 1301-1316 (1987); and Cohn et al., “J Biomed. Mater. Res.” 22: 993-1009 (1988). checking ...

  Presently preferred bioresorbable polymers include poly (ester), poly (hydroxy acid), poly (lactone), poly (amide), poly (ester-amide), poly (amino acid), poly (anhydride). , Poly (orthoester), poly (carbonate), poly (phosphadine), poly (phosphate ester), poly (thioester), polysaccharide, and mixtures thereof. Preferably, the bioresorbable polymer includes a poly (hydroxy) acid component. Of the poly (hydroxy) acids, polylactic acid, polyglycolic acid, polycaproic acid, polybutyric acid, polyvaleric acid, and copolymers and mixtures thereof are preferred.

  In addition to forming fragments that are absorbed in vivo (“bioresorbed”), preferred polymeric coatings for use in the methods of the present invention are also excretable and / or metabolic. Can be formed.

  Higher degrees of copolymer can also be used in the present invention. For example, Casey et al., US Pat. No. 4,438,253, issued March 20, 1984, is a transesterification reaction of poly (glycolic acid) and hydroxyl-terminated poly (alkylene glycol). Discloses tri-block copolymers. Such compositions are disclosed for use as resorbable monofilament sutures. The flexibility of such compositions is controlled 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 can also be used. For example, Spinu, US Pat. No. 5,202,413, issued April 13, 1993, describes the opening of lactide and / or glycolide to either oligomeric diol or diamine residues. Having sequentially ordered blocks of polylactic acid and / or polyglycolide produced by ring polymerization followed by chain extension using a difunctional compound (e.g. diisocyanate, diacyl chloride, or dichlorosilane), Biodegradable multi-block copolymers are disclosed.

  The bioresorbable region of the coating useful in the present invention can be designed to be hydrolytically and / or enzymatically cleavable. For the purposes of the present invention, “hydrolytically cleavable” refers to the copolymer, especially the bioresorbable region, being sensitive to hydrolysis in water or a hydrous environment. Similarly, as used herein, “enzymatically cleavable” refers to a copolymer, particularly a bioresorbable region, that is sensitive to cleavage by endogenous or exogenous enzymes.

  When placed in the body, the hydrophilic region may be processed into excretable and / or metabolic fragments. Thus, hydrophilic regions include, for example, polyethers, polyalkylene oxides, polyols, poly (vinyl pyrrolidines), poly (vinyl alcohols), poly (alkyloxazolines), polysaccharides, carbohydrates, peptides, proteins, and copolymers thereof. And mixtures may be included. Furthermore, the hydrophilic region can also be, for example, a poly (alkylene) oxide. Such poly (alkylene) oxides can include, for example, poly (ethylene) oxide, poly (propylene) oxide, and mixtures and copolymers thereof.

  Polymers that are components of hydrogels are also useful in the present invention. Hydrogels are polymeric materials that can absorb relatively large amounts of water. Examples of compounds that form hydrogels include, but are not limited to, polyacrylic acid, sodium carboxymethyl cellulose, polyvinyl alcohol, polyvinyl pyrrolidine, gelatin, carrageenan and other polysaccharides, hydroxyethylene methacrylic acid (HEMA), and their Derivatives and the like are included. A stable, biodegradable, bioresorbable hydrogel can be produced. In addition, the hydrogel composition can include one or more subunits that exhibit such properties.

  Biocompatible hydrogel compositions are known whose integrity can be controlled via cross-linking, which is presently preferred for use in the methods of the present invention. For example, Hubbell et al., US Pat. No. 5,410,016 issued April 25, 1995, US Pat. No. 5,529,914 issued June 25, 1996. Discloses a water soluble system which is a cross-linked block copolymer having a water soluble central block segment sandwiched between two hydrolytically variable extensions. Such copolymers are further end-capped with photopolymerizable acrylic acid functional groups. When crosslinked, these systems become hydrogels. The water-soluble central block of such copolymers can include poly (ethylene glycol), but the hydrolytically variable extension is poly (α-hydroxy acid) (such as polyglycolic acid or polylactic acid). possible. See Sawney et al., “Macromolecules” 26: 581-587 (1993).

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

  In yet another exemplary embodiment, the conjugate of the invention comprises a component of a liposome. Liposomes can be prepared according to well-known methods, for example, as described in Epstein et al., US Pat. No. 4,522,811, issued Jun. 11, 1985. For example, liposomal formulations can dissolve one or more suitable lipids (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent, which is then evaporated, It can be prepared by leaving a dry lipid film on the surface. An aqueous solution of the active compound or pharmaceutically acceptable salt thereof is then introduced into the container. The container is then turned by hand to peel the lipid material from the side of the container and disperse the lipid aggregates, thereby forming a liposome suspension.

  The foregoing microparticles and methods for preparing such microparticles are provided as examples, but this is not intended to define the range of microparticles used in the present invention. It will be apparent to those skilled in the art that a series of microparticles made by different methods are useful in the present invention.

  In water-soluble polymers (both linear and branched), the structural forms described above are usually applicable to water-insoluble polymers as well. Thus, for example, cysteine, serine, dilysine, and trilysine branch centers can be functionalized using two water-insoluble polymer moieties. The methods used to generate such species are usually closely similar to the methods used to generate water-soluble polymers.

  The in vivo half-life of therapeutic glycopeptides can also be enhanced with PEG moieties such as polyethylene glycol (PEG). For example, chemical modification (PEGylation) of a protein with PEG increases its molecular size and its surface- and functional group-contactability (which depend on the size of the PEG attached to the protein, respectively). Decrease. This results in improved plasma half-life and proteolytic-stability, and reduced immunogenicity and liver uptake (Chaffee et al. “J. Clin. Invest.” 89: 1643-1651 (1992). ); Pyatak et al., “Res. Commun. Chem. Pathol Pharmacol.” 29: 113-127 (1980)). PEGylation of interleukin 2 has been reported to increase its anti-tumor efficacy in vivo (Kater et al., Proc. Natl. Acad. Sci. USA. 84: 1487-1491 (1987). )), PEGylation of F (ab ′) 2 obtained from monoclonal antibody A7 has improved its tumor localization (Kitamura et al. “Biochem. Biophys. Res. Commun.” 28: 1387. ~ 1394 (1990)). Thus, in another embodiment, the in vivo half-life of a peptide derivatized with a PEG moiety by the method of the invention is increased relative to the in vivo half-life of a non-derivatized peptide. The

  The increase in peptide half-life in vivo is best expressed as a percentage of this amount increase. The lower end of the increase rate range is about 40%, about 60%, about 80%, about 100%, about 150%, or about 200%. The upper end of this range is about 60%, about 80%, about 100%, about 150%, or more than about 250%.

Preparation of modified sugars In general, the sugar moiety and the modifying group are linked together by using a reactive group, which is typically linked to a new organic functional group or non-reactive species by a linking process. Converted. The sugar-reactive functional group is located at any position on the sugar moiety. The reactive groups and classes of reactions useful in practicing the present invention are generally those well known in the field of bioconjugate chemistry. The currently convenient class of reactions available with reactive sugar moieties are those that proceed under relatively mild conditions. This includes, but is not limited to, nucleophilic substitution (eg, acyl halides, amine and alcohol reactions using active esters), electrophilic substitution (eg, enamine reaction), and carbon-carbon and Additions to carbon-heteroatom multiple bonds (eg, Michael reaction, Diels-Alder addition) These reactions and other useful reactions include, for example, March, “ADVANCED ORGANIC CHEMISTRY”, 3rd edition, John Wiley & Sons, New York, New York, 1985; Hermanson, “BIOCONJU GATE TECHNIQUES ", Academic Press, San Diego, 1996; and Feney et al.," MODIFICATION OF PROTEINICS ", Advanced in Chemistry, USA, 198 Washington, DC, 1982.

Useful reactive functional groups dangling from a sugar center, linker precursor, or polymer-modified moiety precursor include, but are not limited to:
(A) including but not limited to N-hydroxysuccinimide ester, N-hydroxybenztriazole ester, acid halide, acylimidazole, thioester, p-nitrophenyl ester, alkyl, alkenyl, alkynyl, and aromatic ester, A carboxyl group and various derivatives thereof;
(B) a hydroxyl group that can be converted into, for example, an ester, ether, aldehyde, etc .;
(C) A haloalkyl group. However, halides can be replaced later with nucleophilic groups such as amines, carboxylate anions, thiol anions, carbanions, or alkoxide ions, thereby sharing new groups with functional groups of halogen atoms. Results in a bond;
(D) a dienophile group (eg, a maleimide group) that may participate in the Diels-Alder reaction;
(E) Aldehyde or ketone group. However, subsequent derivatization can be through, for example, the formation of carbonyl derivatives such as imines, hydrazones, semicarbazones, or oximes, or through such mechanisms as Grignard addition or alkyllithium addition. thing;
(F) a sulfonyl halide group for subsequent reactions with amines, for example to form sulfonamides;
(G) a thiol group that can be converted, for example, to a disulfide or reacted with an acyl halide;
(H) amine or sulfhydryl groups that can be acylated, alkylated, or oxidized;
(I) alkenes capable of undergoing, for example, cycloaddition, acylation, Michael addition; and (j) epoxides capable of reacting with, for example, amines and hydroxyl compounds.

  The reactive functional groups can be selected such that they do not participate in or interfere with the reactions necessary to assemble the reactive sugar center or modifying group. Alternatively, reactive functional groups can be protected from participating in the reaction by the presence of protecting groups. Those skilled in the art will understand how to protect a particular functional group so as not to interfere with the set of reaction conditions selected. For examples of useful protecting groups, see, for example, Greene et al., “PROTECTIVE GROUPS IN ORGANIC SYNTHESIS”, John Wiley & Sons, New York, 1991.

  In the discussion that follows, some specific examples of modified sugars that are useful in practicing the present invention are described. In an exemplary embodiment, the sialic acid derivative is utilized as the sugar center to which the modifying group is attached. The focus of the discussion on sialic acid derivatives is for clarity of illustration only and should not be construed to limit the scope of the invention. Those skilled in the art will appreciate that various other sugar moieties can be activated and derivatized in a manner similar to that described using sialic acid as an example. For example, numerous methods are available to modify galactose, glucose, N-acetylgalactosamine, and fucose (to name a few sugar substrates) that are easily modified by methods recognized in the art. is there. See, for example, Elhalabi et al., “Curr. Med. Chem.” 6:93 (1999); and Schaffer 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. This adduct is treated with aldolase to form α-hydroxycarboxylic acid ester 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 through the formation of glycine adducts are utilized as positions for attachment of PEG or PPG and activated (m-) PEG or (m-) PPG derivatives (eg PEG-C (O) NHS, Reaction of PPG-C (O) NHS) with compound 3 yields 4 or 5, respectively.
As those skilled in the art will appreciate, the polymeric modifying moiety may also be a branched moiety such as those described herein.

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

Another exemplary scheme for preparing the polymerizable modified sugars of the present invention is described below:

  Table 1 describes representative examples of sugar monophosphates derivatized with polymeric modifying moieties (eg, branched or linear PEG or PPG moieties). Certain compounds in Table 1 are prepared by the method of Scheme 1. Other derivatives are prepared by methods recognized in the art. See, for example, Kepler et al., “Glycobiology” 11: 11R (2001); and Charter et al., “Glycobiology” 10: 1049 (2000). Other amine-reactive polymeric modifying moiety precursors and components, such as PEG and PPG analogs, are commercially available, or they can be produced by methods readily available to those skilled in the art. Can be prepared.

Where R is a polymeric (branched or straight chain) modifying moiety.

The modified sugar phosphate used to practice the invention can be substituted not only at the positions mentioned above, but also at other positions. The presently preferred substitution of sialic acid is described in the following formula:

Wherein one or more of X ^c , Y ^a , Y ^b , Y ^c , and Z are preferably —O—, —N (H) —, —S, CH ₂ —, and N (R ) A linking group selected from ₂ . When X ^c , Y ^a , Y ^b , Y ^c , and Z are linking groups, this is a polymeric modifying moiety as represented by R ^c , R ^d , R ^e , R ^f , and R ^g . To be attached to. Alternatively, these symbols represent a linker that is attached to a branched- or linear-water-soluble or water-insoluble polymer, therapeutic moiety, biomolecule, or other moiety. When R ^c , R ^d , R ^e , R ^f , or R ^g is not a polymer modifying moiety, the combination of X ^c R ^c , Y ^a R ^d , Y ^b R ^e , Y ^c R ^f , or ZR ^g is , H, OH, or NC (O) CH ₃ .

  Synthetic methods for producing activated sialic acid-polymer modifying group conjugates that are suitable substrates for enzymes that transfer modified sugar moieties onto acceptors (eg, glycosyltransferases) Is also provided. This method includes the following steps: (a) Functionalization with an activated N-protected amino acid (alternatively with a polymeric modifying moiety, linker precursor, or linker-polymer modifying moiety cassette). The amino acid) and mannosamine under conditions suitable to form an amide conjugate between mannosamine and the N-protected amino acid; (b) the amide conjugate and the amide conjugate are sialic acid amide conjugates; Contacting pyruvic acid and sialic acid aldolase under conditions suitable for conversion to a gate; (c) conditions suitable for forming a sialic acid conjugate to form a cytidine monophosphate sialic acid conjugate. Under contacting with cytidine triphosphate and synthetase; (d) cytidine mono Removing the N-protecting group from the acid sialic acid amide conjugate, thereby producing a free amine; and (e) contacting the free amine with an activated PEG (either linear or branched). , Thereby forming cytidine monophosphate sialic acid-poly (ethylene glycol).

Bridging groups Preparation of modified sugars for use in the methods of the present invention involves the attachment of modifying groups to sugar residues and the formation of stable adducts that are substrates for glycosyltransferases. Sugars and modifying groups can be linked by zero- or higher-order crosslinkers. Exemplary bifunctional compounds that can be used to attach the modifying group to the carbohydrate moiety include, but are not limited to, bifunctional poly (ethylene glycol), polyamide, polyether, polyester, and the like. General techniques for linking carbohydrates to other molecules are known in the literature. For example, Lee et al., “Biochemistry” 28: 1856 (1989); Bhatia et al., “Anal. Biochem.” 178: 408 (1989); Janda et al., “J. Am. Chem. Soc. "112: 8886 (1990); and Bednarski et al., WO 92/18135. In the discussion that follows, reactive groups are treated as benign on the sugar moiety of the nascent modified sugar. The focus of the discussion is to clarify specific examples. One skilled in the art will appreciate that this discussion is equally relevant for reactive groups on the modifying group.

  A typical strategy is to use the heterobifunctional crosslinker SPDP (n-succinimidyl-3- (2-pyridyldithio) propionic acid to incorporate a protected sulfhydryl into the sugar, followed by another sulfhydryl on the modifying group. Used to deprotect sulfhydryls for the formation of disulfide bonds.

  Various reagents are used to modify the modified sugar components using intramolecular chemical cross-linking (for review of cross-linking reagents and cross-linking procedures, see: Waldo, F. (Wold, F.), “Meth. Enzymol.” 25: 623-651, 1972; Weetall, H. H. (Weetall, H. H.) and Cooney, D. A. (Cooney, D.). A.) In “ENZYMES AS DRUGS.”: (Edited by Holsenberg and Roberts) pages 395-442, Wiley, New York, 1981; , TH), “Meth. Enzymol.” 91: 580-609, 1983; n) et al., 17 "Mol.Biol.Rep.": 167-183, which is incorporated in 1993 (these all, herein by reference)). Preferred cross-linking reagents are derived from a variety of zero-length, homobifunctional, and heterobifunctional cross-linking reagents. Zero-length cross-linking reagents involve the direct binding of two endogenous chemical groups without the introduction of external materials.

Conjugation of modified sugars to peptides Modified sugars are attached to glycosylated or non-glycosylated peptides using an appropriate enzyme to mediate the attachment. Accordingly, the compounds of the invention, particularly nucleotide sugars, are preferably substrates for enzymes that transfer sugar moieties from nucleotide sugars to amino acid, glycosyl, or aglycone acceptor moieties. Acceptors, such as galactosyl acceptors (eg, GalNAc, Galβ1,4GlcNAc, Galβ1,4GalNAc, Galβ1,3GalNAc, lacto-N-tetraose, Galβ1,3GlcNAc, Galβ1,3Ara, Galβl, 6GlcNAc, Galβ1,4Gc, etc.) And nucleotide sugars that serve as sugar donors for other acceptors known to those skilled in the art (see Paulson et al., “J. Biol. Chem.” 253: 5617-5624 (1978). )

  Exemplary enzymes for which the modified nucleotide sugars of the present invention are substrates include glycosyltransferases. The glycosyltransferase can be cloned or isolated from any source. Many cloned glycosyltransferases are known for their polynucleotide sequences. For example, see “The WWW Guide To Clone Glycosyltransferases” (http://www.vei.co.uk/TGN/gt_guide.htm). Glycosyltransferase amino acid sequences and nucleotide sequences encoding glycosyltransferases from which amino acid sequences can be deduced are also found in a variety of publicly available databases, including GenBank, Swiss-Prot, EMBL, and others.

  The glycosyltransferase for which the compound of the present invention is a substrate includes, but is not limited to, galactosyltransferase, fucosyltransferase, glucosyltransferase, N-acetylgalactosaminyltransferase, N-acetylglucosaminyltransferase, glucuronyltransferase, sialyl. Included are transferases, mannosyl transferases, glucuronic acid transferases, galacturonic acid transferases, and oligosaccharyl transferases. Suitable glycosyltransferases include those obtained from eukaryotes as well as prokaryotes.

  In certain embodiments, the compounds of the invention are substrates for fucosyl tranferases. Fucosyltransferases are generally known to those skilled in the art and are exemplified by enzymes that transfer L-fucose from GDP-fucose to the hydroxy position of the acceptor sugar.

  In another group of embodiments, the compound is a substrate for a galactosyltransferase. Exemplary galactosyltransferases include α (1,3) galactosyltransferase (EC No. 2.4.1.11, for example, Dabkowski et al., “Transplant Proc.” 25: 2921 (1993). ) And Yamamoto et al. “Nature” 345: 229-233 (1990)), GenBank j04989, Joziasse et al., “J. Biol. Chem.” 264: 14290-14297 (see FIG. 1989), rat (GenBank m26925; Larsen et al., "Proc. Nat'l. Acad. Sci. USA" 86: 8227-8231 (1989), pig (GenBank L3615). 2, Strahan et al., “Immunogenetics” 41: 101-105 (1995) Another suitable α1,3 galactosyltransferase is involved in the synthesis of blood group B antigens (EC 2 4.1.37, Yamamoto et al., “J. Biol. Chem.” 265: 1146-1151 (1990) (human)) A more typical galactosyltransferase is the core Gal-T1. Further examples include β (1,4) galactosyltransferase, which includes, for example, EC 2.4.4.1.90 (LacNAc synthetase) and EC 2.4.1.22 (lactose synthetase). ) (D'Agostaro et al., “ Eur. J. Biochem. "183: 211-217 (1989), human (Masri et al., Biochem. Biophys. Res. Commun. 157: 657-663, 1988, mouse (Nakazawa et al., “J. Biochem.” 104: 165-168 (1988), and EC 2.4.4.18, and ceramide galactosyltransferase (EC 2.4.4.15, Stahl et al., “ J. Neurosci.Res. ”38: 234-242 (1994) Other suitable galactosyltransferases include, for example, α1,2 galactosyltransferases (eg, Schizosaccharomyces pombe). pombe), Chapel et al., “Mol. Biol. Cell "5: 519-528 (1994). Soluble forms of α1,3-galactosyltransferases, such as those reported by Cho et al., “J. Biol. Chem.” 272: 13622-13628 (1997), are also practiced according to the present invention. Suitable for

a) Sialyltransferases Sialyltransferases are another type of glycosyltransferase in which the compounds of the present invention are substrates. Examples include ST3Gal III (eg, rat or human ST3Gal III), ST3Gal IV, ST3Gal I, ST6Gal I, ST3Gal V, ST6Gal II, ST6GalNAc I, ST6GalNAc II, and ST6GalNAc III (as used herein). The sialyltransferase nomenclature is as described in Tsuji et al., “Glycobiology” 6: v-xiv (1996)). A typical α (2,3) sialyltransferase called α (2,3) sialyltransferase (EC 2.4.99.6) transfers sialic acid to the Galβ1 → 3Glc disaccharide or the non-reducing terminal Gal of the glycoside. To do. Van den Eijdenden 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 Realick et al., “J. Biol. Chem.” 254: 4444 (1979), and Gillespie et al., “J. Biol. Chem.” 267: 21004 (1992). Further typical enzymes include Gal-β-1,4-GlcNAc α-2,6 sialyltransferase (Kurosawa et al., “Eur. J. Biochem.” 219: 375-381 (1994). )checking). Other sialyltransferases on which the compounds of the present invention are substrates include those that form polysialic acid. Examples include α-2,8-polysialyltransferases such as ST8SiaI, ST8SiaII, ST8SiaIII, ST8SiaIV, and ST8SiaV. For example, Angata et al. “J. Biol. Chem.” 275: 18594-18601 (2000); Kono et al., “J. Biol. Chem.” 271: 29366-29371 (1996); (Greiner et al., “Infect. Immun.” 72: 4249-4260 (2004); and Jones et al., “J. Biol. Chem.” 277: 14598-14611 (2002).

  An example of a sialyltransferase that is useful for the claimed method is ST3Gal III, also referred to as α (2,3) sialyltransferase (EC 2.4.99.6). This enzyme catalyzes the transfer of sialic acid to Galβ1,3GlcNAc or Galβ1,4GlcNAc glycoside to Gal (eg, Wen et al., “J. Biol. Chem.” 267: 21011 (1992); (See Van den Eijnden et al., “J. Biol. Chem.” 256: 3159 (1991)). Additional sialyltransferases include those isolated from Campylobacter jejuni, including α (2,3). For example, see WO99 / 49051 pamphlet.

  Preferably, the compounds of the invention are modified to the sequence Galβ1,4GlcNAc—the most common penultimate sequence underlying terminal sialic acid on a fully sialylated carbohydrate structure. It is a substrate for enzymes that transfer sialic acid.

b) GalNAc transferase Selected compounds of the present invention are substrates for N-acetylgalactosaminyltransferase. Exemplary N-acetylgalactosaminyltransferases include, but are not limited to, α (1,3) N-acetylgalactosaminyltransferase, β (1,4) N-acetylgalactosaminyltransferase (Nagata ) Et al., “J. Biol. Chem” 267: 12082-12089 (1992), and Smith et al., “J. Biol Chem.” 269: 15162 (1994), and the polypeptide N-acetylgalactosa. Minyltransferase (Homa et al., “J. Biol. Chem.” 268: 12609 (1993) is included.

c) Glycosidases The present invention also encompasses the use of substrates for wild type and mutant glycosidases. Mutant β-galactosidase enzymes have been demonstrated to catalyze the formation of disaccharides through the binding of α-fluorinated sugars to galactosyl acceptor molecules (Withers, US Pat. No. 6,284). No. 494, issued on September 4, 2001). Other glycosidases used in the present invention include, for example, β-glucosidase, β-galactosidase, β-mannosidase, β-acetylglucosaminidase, β-N-acetylgalactosaminidase, β-xylosidase, β-fucosidase, cellulase, Xylanase, galactanase, mannanase, hemicellulase, amylase, glucoamylase, α-glucosidase, α-galactosidase, α-mannosidase, α-N-acetylglucosaminidase, α-N-acetylgalactose-aminidase, α-xylosidase, α-fucosidase And neuraminidase / sialidase, endoglycoceramidase.

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

Example 1
Preparation of UDP-GalNAc-6′-CHO UDP-GalNAc (200 mg, 0.30 mmole) 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 with balloons was charged with oxygen and stirred at room temperature for 7 days. The reaction mixture was then filtered (spin cartridge, MWCO 5K) and the filtrate (about 40 mL) was stored at 4 ° C. until needed. TLC (silica, EtOH / water (7/2), R _f = 0.77, visualized with anisaldehyde dye).

Example 2
Preparation of UDP-GalNAc-6′-NH ₂ :
A solution of ammonium acetate (15 mg, 0.194 mmole) and NaBH ₃ CN (1M in THF, 0.17 mL, 0.17 mmole) was added to UDP-GalNAc-6′-CHO (2 mL or about 20 mg) from above at 0 ° C. In addition, it was allowed to warm to room temperature overnight. The reaction was filtered through a G-10 column with water and the collected product. Appropriate fractions were lyophilized and stored frozen. TLC (silica, ethanol / water (7/2), R _f = 0.72, visualized with ninhydrin reagent).

Example 3
Preparation of _{UDP-GalNAc-6-NHCO (} CH 2) 2 -O-PEG-OMe (1KDa).
Galactosaminyl-1-phosphate _{-2-NHCO (CH 2) 2} -O-PEG-OMe (1KDa) (58mg, 0.045mmole) was dissolved in DMF (6 mL) and pyridine (1.2 mL). UMP-morpholide (60 mg, 0.15 mmole) 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 between 10 to 80%, methanol / water). The desired fractions were collected and dried under reduced pressure to yield a white solid. TLC (silica, propanol / H ₂ O / NH ₄ OH, (30/20/2), R _f = 0.54). MS (MALDI): observed, 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 powder) was added to a solution of L-cysteine (93.7 mg, 0.75 mmol) in anhydrous methanol (20 L) under argon. . The mixture was stirred for 30 minutes at room temperature, after which mPEG-O-tosylate (Ts, 1.0 g, 0.05 mmol) with a molecular weight of 20 kilodalton was added in several portions over 2 hours. The mixture was stirred for 5 days at room temperature and concentrated by rotary evaporation. The residue was diluted with water (30 mL) and stirred for 2 hours at room temperature to destroy some excess 20 kilodalton mPEG-O-tosylate. The solution was then neutralized with acetic acid and the pH was adjusted to pH 5.0 and loaded onto a reverse phase chromatography (C-18 silica) column. The column is eluted using a methanol / water gradient (the product elutes at approximately 70% methanol), product elution is monitored by evaporative light scattering, the appropriate fractions are collected, and water (500 mL) is collected. Diluted. This solution was color layer analyzed (ion exchange, XK 50 Q, BIG Beads, 300 ml, hydroxide form, water to water / acetic acid-0.75N gradient) and the pH of the appropriate fractions adjusted. To 6.0 using acetic acid. This solution was then captured on a reverse phase column (C-18 silica) and eluted with a methanol / water gradient as described above. The product fractions were pooled, concentrated, redissolved in water and lyophilized to give a white solid (1). The structural data for the compound were as follows: ¹ H-NMR (500 MHz, D ₂ O) δ 2.83 (t, 2H, O—C—CH ₂ —S), 3.05 ( q, 1H, S-CHH- CHN), 3.18 (q, 1H, (q, 1H, S-CHH-CHN), 3.38 (s, 3H, CH 3 O), 3.7 (t, OCH ₂ CH ₂ O), 3.95 (q, 1H, CHN) The purity of the product was confirmed by SDS PAGE.

4.2 Synthesis of Compound 2 (Cysteine-PEG ₂ ) Triethylamine (about 0.5 mL) is a solution of Compound 1 (440 mg, 22 μmol) in anhydrous CH ₂ Cl ₂ (30 mL) until the solution is basic. It was dripped in. A solution of 20 kilodalton mPEG-Op-nitrophenyl carbonate (660 mg, 33 μmol) and N-hydroxysuccinimide (3.6 mg, 30.8 μmol) in CH ₂ Cl ₂ (20 ml) was added over a period of several hours. Add in portions at room temperature. The reaction mixture was stirred for 24 hours at room temperature. The solvent was then removed by rotary evaporation, the residue was dissolved in water (100 mL) and the pH was adjusted to 9.5 using 1.0 N NaOH. The basic solution was stirred for 2 hours at room temperature and then neutralized to pH 7.0 with acetic acid. This solution was then loaded onto a reverse phase chromatography (C-18 silica) column. The column is eluted using a methanol / water gradient (the product elutes at about 70% methanol), product elution is monitored by evaporative light scattering, the appropriate fractions collected and water (500 mL) Diluted with This solution was color layer analyzed (ion exchange, XK 50 Q, BIG Beads, 300 mL, hydroxide form, water to water / acetic acid-0.75N gradient) and the pH of the appropriate fraction was Reduced to 6.0 with acetic acid. This solution was then captured on a reverse phase column (C-18 silica) and eluted using a methanol / water gradient as described above. Product fractions were pooled, concentrated, redissolved in water and lyophilized to give a white solid (2). The structural data for this compound were as follows: ¹ H-NMR (500 MHz, D ₂ O) δ 2.83 (t, 2H, O—C—CH ₂ —S), 2.95 ( _{t, 2H, O-C-} CH 2 -S), 3.12 (q, 1H, S-CHH-CHN), 3.39 (s, 3HCH 3 O), 3.71 (t, OCH 2 CH 2 O). The purity of the product was confirmed by SDS PAGE.

Example 5
_{UDP-GalNAc-6-NHCO (} CH 2) 2 -O-PEG-OMe Preparation of (1 KDa) galactosaminyl-1-phosphate _{-2-NHCO (CH 2) 2} -O-PEG-OMe (1 kilodalton) ( 58 mg, 0.045 mmole) was dissolved in DMF (6 mL) and pyridine (1.2 mL). UMP-morpholidate (60 mg, 0.15 mmole) 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 between 10 to 80%, methanol / water). The desired fractions were collected and dried under reduced pressure to give a white solid. TLC (silica, propanol / H ₂ O / NH ₄ OH (30/20/2), R _f = 0.54). MS (MALDI): observed, 1485, 1529, 1618, 1706.

SDS PAGE Procedure The purity of the products (1 and 2) was confirmed by SDS PAGE. 4-20% Tris Glycine SDS PAGE gel (Invitrogen) was used. Samples were mixed 1: 1 with SDS sample buffer (Sample Buffer), 1 hour 50 minutes at constant voltage (125V) in Tris-Glycine Running Buffer (LC2675-5). I ran it. After electrophoresis, the gel was washed with water (100 mL) for 10 minutes, followed by 10 minutes with 5% aqueous barium chloride (100 mL). Product 1 or 2 was visualized by staining the gel with 0.1 N iodine solution (4.0 mL) at room temperature and the staining process was stopped by washing the gel with water. Visualized product bands were scanned using HP Scanjet 7400C and gel images were optimized using HP Precision Scan Program.

  Although the present invention has been disclosed in terms of particular embodiments, other embodiments and variations of the invention can be devised by other skilled artisans without departing from the true spirit and scope of the invention. It is clear that there is a possibility.

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

10 is a table of sialyltransferases on which selected modified sialic acid nucleotides and activated sugars are substrates. 1 is a general synthetic scheme of the present invention for preparing sialic acid-poly (ethylene glycol) conjugates. Is a synthetic scheme of the present invention for preparing sialic acid-glycyl-poly (ethylene glycol) conjugates.

Claims (10)

A compound represented by the formula selected from:
(Where
R ¹ is H, CH ₂ OR ⁷ , COOR ⁷ or OR ⁷ ;
here,
d is 0 or 1;
R ⁷ represents H, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl,
R ² is a member selected from a moiety comprising H, OH, an activating group, and a nucleotide, wherein the activating group is from fluoro, chloro, bromo, tosylate ester, mesylate ester, and triflate ester A group selected from the group consisting of
R ³ , R ⁴ , R ⁵ , R ⁶ , and R ^{6 ′} are each independently selected from H, substituted or unsubstituted alkyl, OR ⁹ , and NHC (O) R ¹⁰ ;
Wherein R ⁹ and R ¹⁰ are each 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 ′} has the formula:
(Where
s is an integer from 0 to 20,
R ¹¹ is a polymer modifying moiety represented by the following formula:
here,
X ² and X ⁴ are 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 ) _g Y ″ each independently selected;
g is an integer from 1 to 50;
Y ″ is a member selected from O, S, and NH;
X ⁵ is a non-reactive group, and the non-reactive group is a group selected from the group consisting of H, unsubstituted alkyl, and unsubstituted heteroalkyl;
R ¹² and R ¹³ are each independently selected from the group consisting of PEG, methoxy PEG, PPG, methoxy PPG, polysialic acid, polyglutamic acid, polyaspartic acid, polylysine, polyethyleneimine, polylactic acid, and polyglyceride). The compound according to claim 1, wherein R ² is represented by the following formula :
(Wherein R ⁸ is a nucleoside) . The compound of claim 2, wherein R ⁸ is a member selected from cytidine , uridine, guanosine, adenosine, and thymidine. X ⁴ is a peptide bond,
The compound according to any one of claims 1 to 3, wherein R ¹³ is an amino acid residue. The compound of any one of Claim 1 to 4 represented by following Formula.
(Where
D is a member selected from —OH and (R ¹¹ ) _{w ′} -L—;
G is a member selected from ( R ¹¹ ) _{w ′} -L—, and —C (O (C ₁ -C ₆ ) alkyl;
w ′ is an integer from 2 to 6;
At least one of D and G is (R ¹¹ ) _{w ′} -L-;
L is selected from a single bond and a linking group selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl ).   6. The substrate of any one of claims 1 to 5, wherein the compound is a substrate for an enzyme that transfers a sugar moiety from a member selected from activated sugars, nucleotide sugars, and combinations thereof to an acceptor moiety of the substrate. The compound according to item.   7. A compound according to claim 6, wherein the acceptor moiety is a member selected from a glycosyl residue, an amino acid residue, and an aglycon.   The compound according to any one of claims 1 to 7, represented by the following formula:
(Wherein AA is —CH ₂ And
  NP is a nucleotide phosphate).   9. The compound according to any one of claims 1 to 8, wherein the polymer modifying moiety is selected from the following formula:
(Wherein f is independently selected from an integer of 1 to 2500). A method for preparing cytidine monophosphate sialic acid-poly (ethylene glycol) comprising:
(A) contacting the activated N-protected amino acid and mannosamine under conditions suitable to form an amide conjugate between the mannosamine and the N-protected amino acid;
(B) contacting the amide conjugate with pyruvate and sialic acid aldolase under conditions suitable to convert the amide conjugate to a sialic acid amide conjugate;
(C) contacting the sialic acid conjugate with cytidine triphosphate and synthetase under conditions suitable to form a cytidine monophosphate sialic acid conjugate;
(D) removing the N-protecting group from the cytidine monophosphate sialic acid amide conjugate, thereby producing a free amine;
(E) the free amine is contacted with the activated PEG and thereby, the cytidine monophosphate sialic acid - see including a step of forming a poly (ethylene glycol),
The activated N-protected amino acid has the formula:
Having a method.