CN101087801A

CN101087801A - Branched polymeric sugars and nucleotides thereof

Info

Publication number: CN101087801A
Application number: CNA2005800032641A
Authority: CN
Inventors: S·德弗雷斯; C·鲍
Original assignee: Neose Technologies Inc
Current assignee: Weisuofan Co Ltd
Priority date: 2004-01-26
Filing date: 2005-01-26
Publication date: 2007-12-12
Anticipated expiration: 2025-01-26
Also published as: CN101087801B; BRPI0507133A

Abstract

The present invention provides sugars, nucleotide sugars, activated sugars that include one or more polymeric modifying moiety within their structure. The invention is exemplified by reference to linear and branched polymers, such as the water-soluble polymer poly(ethylene glycol).

Description

Branched polymeric sugars and Nucleotide thereof

The cross reference of related application

The application requires the right of priority of following document: U.S. temporary patent application No.60/539, and 387, submit day January 26,2004 to; U.S. temporary patent application No.60/555,504, submit day March 22,2004 to; U.S. temporary patent application No.60/590,573, submit day July 23,2004 to; U.S. temporary patent application No.60/555,504, submit day March 22,2004 to; U.S. patent application No.10/997,405, submit day November 24,2004 to; U.S. temporary patent application 60/544,411, submits day February 12,2004 to; U.S. temporary patent application 60/546,631, submits day February 20,2004 to; U.S. temporary patent application, May 12,2004; U.S. day January 10,2005 is submitted in patent application ((unauthorized), attorney No.40853-01-5138US) to; PCT applies for No. ((unauthorized), attorney No.40853-01-5146WO), submits day December 3,2004 to; U.S. temporary patent application No.60/590,649, submit day July 23,2004 to; U.S. temporary patent application No.60/611,790, submit day September 20,2004 to; U.S. temporary patent application No.60/592,744, submit day July 29,2004 to; U.S. temporary patent application No.60/614,518, submit day September 29,2004 to; U.S. temporary patent application No.60/623,387, submit a day October 29,2004 to; U.S. temporary patent application No.60/626,678, submit day November 9,2004 to; With U.S. temporary patent application No. ((unauthorized), attorney No.040853-01-5150), submit day January 6,2005 to, the disclosure of every piece of document is incorporated herein by reference for all purposes in full at this.

Technical field

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

Background technology

External modification after the expression of peptide is to remedy those to depend on the absorptive strategy of being controlled the defective of glycosylated method by the through engineering approaches expression system; Comprise the modification of glycan structures or glycan the introducing at new position both.Can utilize the synthesis tool case of recombinant chou eucaryon glycosyltransferase, make the vitro enzyme of Mammals compounding sugar of glycosylation pattern with common design and glycosyl structure become possibility.Referring to, for example U.S. Patent number 5,876, and 980; 6,030,815; 5,728,554; 5,922,577; And WO/9831826; US2003180835; With WO 03/031464.

Synthetic advantage based on enzyme is regioselectivity and stereoselectivity.In addition, enzymic synthesis uses not protected substrate to carry out.The enzyme of three kinds of main types is used for synthetic carbohydrate, glycosyltransferase (as, sialytransferase, oligosaccharyl transferase, N-acetylglucosaminyl transferase), and Glycosylase.Glycosylase further be categorized as exoglycosidase (as, beta-Mannosidase, beta-glucosidase enzyme), and endoglycosidase is (as, Endo-A, Endo-M).Each of the enzyme of these classifications successfully is used to prepare carbohydrate on synthetic.As the generality summary, referring to people such as Crout, Curr.Opin.Chem.Biol.2:98-111 (1998).

Oligose structure on the glycosyltransferase modification glycopeptide.Glycosyltransferase is effective for the specific product that generation has good stereochemistry and regional chemistry control.Glycosyltransferase has been used to prepare the carbohydrate structure that oligose and the terminal N-of modification are connected with O-, on the glycopeptide that particularly produces in mammalian cell.For example, sialylated fully and/or fucosylation is to provide more consistent sugared structure with the terminal oligose of glycopeptide, and its improves glycopeptide pharmacokinetics and various other biological property.For example, β-1,4-galactotransferase are used for synthetic lactose amine, the explanation of the function of glycosyltransferase in synthetic carbohydrate (referring to, as people such as Wong, J; Org Chem.47:5416-5418 (1982)).In addition, many building-up processes use sialytransferases with from Cytidine-5 '-single phosphoric acid-N-n acetylneuraminic acid n shifts sialic acid to the 3-OH of semi-lactosi or 6-OH (referring to, as people such as Kevin, Chem.Eur.J.2:1359-1362 (1996)).Fucosyltransferase be used for from guanosine-5 '-the bisphosphate Fucose shifts the route of synthesis of Fucose unit to the specific hydroxyl of saccharide acceptor.For example, Ichikawa prepares saliva acidic group Lewis-X people such as (, J.Am.chem.Soc.114:9283-9298 (1992)) Ichikawa by relating to the method for fucosylation with the sialylated lactose amine of clone's fucosyltransferase.The discussion of the latest developments for the compounding sugar of therepic use in synthetic, referring to people such as Koeller, Nature Biotechnology 18:835-841 (2000).Also referring to U.S. Patent number 5,876,980; 6,030,815; 5,728,554; 5,922,577; With WO/98 31826.

The structure of glycosyl group, interest has been conceived to prepare glycopeptide on regulating polypeptide, and this glycopeptide is by one or more non-sugared modification groups (as water-soluble polymers) modification.Poly-(ethylene glycol) (" PEG ") is the illustrative polymkeric substance that is attached to polypeptide.The use that is used for the PEG of derived peptide therapeutical agent has shown the immunogenicity that reduces peptide.For example, U.S. Patent number 4,179,337 (people such as Davis) disclose the non-immune peptide that causes, as are attached to the enzyme and the peptide hormone of polyoxyethylene glycol (PEG) or polypropylene glycol.Every mole of polypeptide uses the 10-100 moles of polymer.Although clean-up time is extended with respect to the clean-up time of polypeptide in the body of binding substances, only keep about 15% physiologically active.Therefore, the rapid reduction payment renderd a service by peptide of the circulating half-life of prolongation.

The active loss of peptide can be directly owing to the non-selective essence of the chemistry that is used for the combination water soluble polymer.Connecting PEG (with its derivative) is non-special bonding by the peptide ammino acid residue to the main pattern of peptide.For example, U.S. Patent number 4,088,538 disclose the enzymic activity polymkeric substance-enzyme conjugates of the enzyme that is covalently bound to PEG.Similarly, U.S. Patent number 4,496,689 disclose the covalently bound mixture of α-1 protease inhibitor and polymkeric substance (as PEG).(J.Biol.Chem.252:3578 (1977) discloses MPEG covalently bound to the amine groups of bovine serum albumin to people such as Abuchowski.U.S. Patent number 4,414,147 acid anhydrides (as poly-(ethene succinyl oxide)) that disclose by Interferon, rabbit being connected to dicarboxylic acid make Interferon, rabbit reduce hydrophobic method.PCT WO87/00056 discloses PEG and has gathered the combination of (oxygen ethylization) polyvalent alcohol to for example protein (as interferon-beta, interleukin II and immunotoxin).EP 154,316 discloses the lymphokine with claimed chemical modification, as comprises the IL-2 of the PEG that is bonded directly to plain at least one primary amino of lymph.U.S. Patent number 4,055,635 disclose the pharmaceutical composition of the water-soluble compound of covalently bound proteolytic ferment to polymer material (as polysaccharide).

Connecting PEG is non-specific oxidation by glycosyl residue on the glycopeptide to the another kind of pattern of peptide.Oxosugar is used as and connects the position of peg moiety to peptide.For example M ' Timku1u (WO94/05332) discloses the purposes that amino-PEG is used for PEG is added to glycoprotein.Glycosyl part is oxidized to corresponding aldehyde at random, and it is coupled to amino-PEG subsequently.

In every kind of aforesaid method, will gather (ethylene glycol) with at random, non-special mode joins on the reactive residue of peptide main chain.For the production of therapeutic peptide, clearly, need to adopt cause specific mark, can characterize easily, the basic strategy of deriving of the formation of product uniformly.The promising approach of preparation specific marker peptide is by using enzyme (as glycosyltransferase) that modified sugar moieties is appended on the peptide.Modified sugar moieties must play the function of glycosyltransferase substrate and suitably be activated.Therefore, the route of synthesis in the easy path that obtains the activatory modified sugars need be provided.The invention provides such approach.

Summary of the invention

The invention provides polymer material, be attached to the sugar of these polymer materials and the nucleotide sugar that activates sugar and comprise these polymkeric substance.Polymer material comprises water-soluble and water-insoluble substance.In addition, polymkeric substance is branching or straight-chain polymer.Illustrative sugar moieties comprises straight chain and ring texture and aldose and ketose.

The polymer modification group can connect in any position of sugar moieties.In the following discussion, the present invention illustrates by the reference implementation scheme, and wherein the polymer modification group is connected to the C-5 of furanose or the C-6 of pyranose.The focus that those skilled in the art will recognize that discussion is for clearer explanation, uses in the method for this explanation and art-recognized method, and polymer moieties can be connected to other position of pyranose and furanose.

In illustrative embodiment, the invention provides the sugar or the sugar nucleotide that are attached to polymkeric substance:

In general formula I and I I, R ¹Be H, CH ₂OR ⁷, COOR ⁷Or OR ⁷, R wherein ⁷Expression H, replacement or unsubstituted alkyl or replacement or unsubstituted assorted alkyl.R ²Be H, OH or the group that comprises Nucleotide.Illustrative R according to this embodiment ²Have following general formula:

R wherein ⁸It is nucleosides.

Symbol R ³, R ⁴, R ⁵, R ⁶And R ⁶' represent H, replacement or not substituted alkyl, OR independently ⁹, NHC (O) R ¹⁰Mark d is 0 or 1.R ⁹And R ¹⁰Be independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl or sialic acid.R ³, R ⁴, R ⁵, R ⁶And R ⁶' at least one comprise polymer modification part (as PEG).In illustrative embodiment, R ⁶And R ⁶' the carbon that connects with their is the component of sialic acid side chain.In further illustrative embodiment, it is functionalized that this side chain is aggregated thing modification part.

In illustrative embodiment, polymer moieties is attached to sugar nuclear usually by the heteroatoms on the nuclear by connecting basic L, and is as follows:

R ¹¹It is polymer moieties and L is selected from key and linking group.Mark w represents to be selected from 1-6, preferably 1-3 and the more preferably integer of 1-2.Illustrative linking group comprises replacement or unsubstituted alkyl, replacement or unsubstituted assorted moieties and sialic acid.The illustrative component that connects base is a carboxyl groups.

When L was a key, it was at R ¹¹Precursor on reactive functional groups and the reactive functional groups of the postreaction on the L precursor between form.With R ¹¹Reaction before, L can be in place on sugar nuclear.Perhaps, R ¹¹Can be introduced into preformed molectron (cassette) with L, this preformed molectron is connected to sugar nuclear subsequently.Such as described in this, have appropriate reaction functional group precursor selection and the preparation in those skilled in the art's limit of power.In addition, the combination of precursor is carried out with chemistry well known in the art.

In illustrative embodiment, L is the linking group that forms from amino acid or little peptide (as, 1-4 amino-acid residue), and modified sugars is provided, and wherein polymer modification partly connects base by the alkyl that replaces and connects.Illustrative connection base is a glycine.

In illustrative embodiment, R ⁶Comprise the polymer modification part.In another illustrative embodiment, R ⁶Comprise the polymer modification part and be connected basic L, connect basic L partly arrives molecule in conjunction with modification remainder.

In illustrative embodiment, polymer modification partly is to comprise two or more branched structures that are connected to the polymer chain of centre portions.The illustrative structure of the useful polymer modification part precursor of this embodiment has following general formula according to the present invention:

Sugar and nucleotide sugar according to this general formula are the pure water soluble polymer substantially.X ³' be the group that comprises ionizable (as, COOH etc.) or other reactive functional groups (referring to, for example hereinafter).C is a carbon.X ⁵Preferably non-reacted group (as H, substituted alkyl does not replace assorted alkyl).R ¹²And R ¹³Be the polymeric arms of selecting independently, as non-peptide, the non-reactive polymer arm.X ²And X ⁴Be preferred junction fragment non-reacted substantially under physiological condition, they can be identical or different.Perhaps, these connections can comprise be designed to one or more groups of degrading under the physiology correlated condition, as ester, disulphide etc.X ²And X ⁴Conjugated polymer arm R ¹²And R ¹³To C.Work as X ³' when being connected base, sugar or connecting the reactive functional groups reaction of postreaction on base-sugar combination, X ³' change into junction fragment X ³Component.

By precursor and suitable sugar or the sugared reaction that is connected base, the invention provides sugar and nucleotide sugar with following general formula:

With

Wherein by the discriminating of the group of various symbolic representations with discussed above identical.L ^aBe to replace or not substituted alkyl or replacement or replace assorted alkyl group.In illustrative embodiment, L ^aBe sialic side-chain radical, the polymer modification part shown in its quilt is functionalized.

Polymer modification comprises that partly two or more can be water-soluble or water-fast substantially repeating unit.The illustrative water-soluble polymers that is used for compound of the present invention comprises PEG (as m-PEG), PPG (as m-PPG), Polysialic acid, polyglutamic acid esters, polyaspartate, polylysine, poly-inferior alkene imines, biodegradable polymer (as, polylactide, polyglycerol ester) and functionalized PEG, as the PEG of end-functionalization.

The sugar moieties of polymer conjugates of the present invention is selected from natural and non-natural furanose and hexose.Non-natural sugar randomly comprises alkylation or acylations hydroxyl and/or amine groups, as ether, ester and the amide substituents on the ring.Other non-natural sugar is included in H, hydroxyl, ether, ester or the amide substituents that ring is gone up the position, does not have such substituting group in this position in natural sugar.Perhaps, carbohydrate lacks the substituting group that should find in the carbohydrate of its title of deriving, as desoxy sugar.Still further illustrative non-natural steamed bun stuffed with sugar draw together oxidation (as ,-ketone acid and-uronic acid) and the reduction (sugar alcohol) carbohydrate.Sugar moieties can monose, oligose and polysaccharide.

Be used for illustrative natural sugar of the present invention and comprise glucose, glycosamine, semi-lactosi, GalN, Fucose, seminose, mannosamine, wood sugar, ribose, N-acetyl glucosamine, N-acetyl-glucosamine, N-acetyl semi-lactosi, N-acetylgalactosamine and sialic acid.

Illustrative have following general formula based on sialic binding substances:

Wherein AA be do not comprise carboxylic group amino-acid residue that part and NP is a nucleotide phosphodiesterase.ONP also can be substituted to form activation sugar by activated partial.Such as persons skilled in the art will recognize, polymer modification part-connection base also can be at C-6, and C-7 and/or C-9 are connected to the sialic acid side chain.

The synthetic method of producing activation sialic acid-PEG binding substances also is provided, and this binding substances is the suitable substrate of enzyme (as glycosyltransferase).This method comprises the steps: that (a) is in the amino acid that is suitable for contact mannosamine and activatory N-protected under the condition that forms the acid amides binding substances between the amino acid of mannosamine and N-protected (or be aggregated thing modification part, be connected based precursor or connection base-functionalized amino acid of polymer modification part molectron); (b) be suitable for the acid amides binding substances is changed into contact acid amides binding substances and pyruvate salt and acetylneuraminate aldolase under the condition of sialic acid acid amides binding substances; (c) be suitable for forming contact sialic acid acid amides binding substances and cytidine triphosphate(CTP) and synthetic enzyme under the condition of cytidine monophosphate sialic acid acid amides binding substances; (d) remove the N-protected group from cytidine monophosphate sialic acid acid amides binding substances, produce unhindered amina thus; (e) contact ionization amine and activatory PEG (straight chain or branching) form cytidine monophosphate sialic acid-poly-(ethylene glycol) thus.

Nucleosides can be selected from natural and non-natural nucleoside.Be used for illustrative natural nucleus glycoside of the present invention and comprise cytosine(Cyt), thymus pyrimidine, guanine, VITAMIN B4 and uridylic.The structure of non-natural nucleoside and their preparation method have fully been showed in this area.

Illustrative modified sugars Nucleotide of the present invention comprises the GDP-Man of polymer modification, GDP-Fuc, UDP-Gal, UDP-GalNAc, UDP-Glc, UDP-GlcNAc, UDP-Glc, UDP-GlcUA and CMP-SA etc.Example comprises UDP-Gal-2 '-NH-PEG, UDP-Glc-2 '-NH-PEG, CMP-5 '-PEG-SA etc.The compound that is covered by the present invention comprises wherein L-R ¹¹Part is attached to those of furanose or pyranose, as at the C-5 of furanose or at the C-6 of pyranose, usually by being connected to the heteroatoms of this carbon atom.

When compound of the present invention was nucleotide sugar or activation sugar, the polymer conjugates of nucleotide sugar is the substrate of enzyme normally, and this enzyme transfer sugar moieties and its polymeric substituents are to the suitable acceptor portion of substrate.Therefore, the present invention also provides the polymer conjugates that uses nucleotide sugar or activation sugar and suitable enzyme by the substrate of sugar in conjunction with modification.Can use compound of the present invention to carry out sugared bonded substrate and comprise peptide (as glycopeptide, peptide), lipid (as glycolipid) and aglucon (saddle ammonia alcohol, ceramide).

Description of drawings

Fig. 1 is the table of sialytransferase, and for this sialytransferase, the modification sialic acid Nucleotide of selection and activation sugar are substrates.

Fig. 2 is the of the present invention general synthetic schemes of preparation sialic acid-poly-(ethylene glycol) binding substances.

Fig. 3 is the synthetic schemes of the present invention of preparation sialic acid-glycyl-poly-(ethylene glycol) binding substances.

Detailed Description Of The Invention and embodiment

Abbreviation

Branching and branching PEG not, PEG, such as m-PEG, methoxyl group-PEG; Branching and branching PPG not, poly-(propane diols), such as m-PPG, methoxyl group-poly-(propane diols); Fuc, rock The algae glycosyl; Gal, galactosyl; Gal NAc, N-acetylgalactosamine base; Glc, glucosyl group; GlcNAc, the N-Acetyl-D-glucosamine base; Man, mannose group; ManAc, the mannosamine guanidine-acetic acid Ester; Sia, sialic acid; And NeuAc, N-acetyl is neural amino.

Definition

Any member of nine carbon carboxylated sugar of term " sialic acid " expression family. Sialic acid family The most common member is NeuAc (2-ketone group-5-acetylaminohydroxyphenylarsonic acid 3,5-dideoxy-D-Glycerine-D-gala nonanone pyranose-1-ketone acid (usually being abbreviated as Neu5Ac, NeuAc, or NANA). Second member of family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), wherein NeuAc The N-acetyl group be hydroxylated. The 3rd sialic acid family member is 2-ketone group-3-deoxidation-ninth of the ten Heavenly Stems Onosic acid (KDN) (people (1986) J.Biol.Chem.261:11550-11557 such as Nadano; The people such as Kanamor i, J.Biol.Chem.265:21811-21819 (1990)). Also comprise 9-Replace sialic acid such as 9-O-C₁-C ₆Acyl group-Neu5Ac such as 9-O-lactyl-Neu5Ac or 9-O-second Acyl group-Neu5Ac, 9-deoxidation-9-fluoro-Neu5Ac and 9-azido-9-'-deoxy-n eu5Ac. For The summary of sialic acid family referring to, such as Varki, Glycobiology 2:25-40 (1992); Saliva Liquid acid: chemistry, metabolism and function, R.Schauer, Ed. (Springer-Yerlag, New York (1992)). Synthetic and the purposes in sialylated of sialylated compound is disclosed in 1992 10 Month disclosed International Application No. WO 92/16640 on the 1st.

Term " modified sugars " (modified sugar) is illustrated in method of the present invention as used herein Middle enzyme process adds to the amino acid of peptide or the natural or non-natural carbohydrate on the glycosyl residue. Change The property sugar be selected from many zymolytes, include but not limited to ribotide (single-, two-and three-phosphoric acid), Activation sugar (as, glycosyl halide, glycosyl methanesulfonates) and both activated neither nucleotides Sugar. " modified sugars " is by " modified group " (modifying group, or title modification group) covalency official Energyization. Useful modified group includes but not limited to water-soluble polymer, treatment part (moieties, or title group), diagnosis of partial, biomolecule etc. Modified group preferably is not Natural or unmodified carbohydrate. Selection by the functionalized position of modified group so that it is anti-End " modified sugars " and added to peptide by enzyme process.

Term " water-soluble " but be illustrated in the part that has the solubility of certain detection level in the water. Inspection The method of survey and/or quantification water solubility is well known in the art. Illustrative water-soluble polymer Comprise peptide, sugar, poly-(ether), poly-(amine), poly-(carboxylic acid) etc. Peptide can have mixed sequence or by list Monoamino-acid forms, such as poly-(lysine). Illustrative polysaccharide is poly-(sialic acid). Illustrative poly-(ether) PEG, such as m-PEG. Poly-(aziridine) is illustrative polyamine and poly-(acrylic acid) Representational poly-(carboxylic acid). Illustrative polymer typically is made up of 2-8 polymer unit.

The main polymer chain of water-soluble polymer can be PEG (being PEG). Yet, should Also be applicable to enforcement of the present invention when understanding other related polymer, and term PEG or poly-(second Glycol) use wish be in this regard comprising property and be not exclusive. Term PEG comprises Any type of PEG comprises alkoxyl PEG, two sense PEG, and multi-arm PEG, forked PEG, branching PEG, (pendent) PEG that dangles (namely contains one or more polymer that overhang The PEG of the functional group of main chain or related polymer), or wherein have the PEG of degradable linkage.

Main polymer chain can be linearity or branching. Normally this area has been for the branched polymer main chain Know. Typically, branched polymer contains center branching nuclear part and a plurality of center branching that is connected to The linear polymer chain of nuclear. PEG uses with the branching form usually, and it can pass through oxirane Add to various polyalcohols (such as glycerine, pentaerythrite and sorbierite) and prepare. Center branching section Dividing also can be derived from several seed amino acid, such as lysine. The branching PEG can be used general shape Formula is expressed as R (PEG-OH)_m, wherein R represents to examine part, such as glycerine or pentaerythrite, and m The number of expression arm. Multi-arm PEG molecule, as at U.S. Patent number 5,932, that describe in 462 Also can be used as main polymer chain a bit, which is hereby incorporated by reference for the document.

Many other polymer also are suitable for the present invention. Non-peptide and water-soluble has about 300 ends of 2-The main polymer chain of end is specially adapted to the present invention. The example of suitable polymer includes but not limited to Other poly-(aklylene glycol) is such as the copolymerization of poly-(propane diols) (" PPG "), ethylene glycol and propane diols Thing etc., poly-(oxygen ethylization polyalcohol), poly-(olefinic alcohol), PVP, poly-(hydroxyl The propyl methyl acid amides), poly-('alpha '-hydroxy acids), poly-(vinyl alcohol), polyphosphazene (polyphosphazene), poly-_ the azoles quinoline, poly-(N-acryloyl morpholine) is such as United States Patent (USP) Numbers 5,629,384 described, and which is hereby incorporated by reference for the document, and copolymer, three Membered copolymer and mixture. Although the molecular weight of each chain of main polymer chain can change, its allusion quotation Type ground is about 100 for about 100Da-, and 000Da is common about 6, and 000Da-is about 80,000Da.

As used herein term " sugar in conjunction with " expression modified sugars material to polypeptide (such as sudden change of the present invention Human growth hormone (HGH)) amino acid or the combination of the enzyme of glycosyl residue mediation. The subclass of " sugar combination " Be " glycol-PEGization ", wherein the modified group of modified sugars is PEG, and alkyl is derived Thing (as, m-PEG) or reactive derivatives (such as, H₂N-PEG，HOOC-PEG)。

As used herein term " glycosyl linking group " expression modified group (as, peg moiety is controlled Treat part, biomolecule) covalently bound glycosyl residue on it; The glycosyl linking group is in conjunction with changing The property group is to the remainder of bond. In the method for the invention, " glycosyl linking group " covalency Be connected to glycosylation or non-glycosylated peptide, thus reagent be connected to amino acid and/or the glycosyl of peptide Residue. " glycosyl linking group " usually by " modified sugars " to the amino acid of peptide and/or glycosyl residue Enzyme connects and derived from " modified sugars ". The glycosyl linking group can be in modified group-modified sugars group The sugared derived structure of degraded during fit the formation (as, oxidation → schiff bases forms → reduction), or sugar The base linking group can be complete. " complete glycosyl linking group " expression is derived from glycosyl part Linking group, wherein connect modified group and do not fall to the sugar monomer of the remainder of bond Separate, such as oxidation (as by the sodium metaperiodate oxidation). " complete glycosyl linking group " of the present invention Can spread out from removing of the sugared structure of mother by addition or one or more glycosyl units of glycosyl units Be conigenous natural compound sugar.

Under the chemical formula particular cases of their routines that substituting group is from left to right write, they comprise chemically consistent substituting group comparably, and this chemically consistent substituting group is from the structure of writing from right to left, as-CH₂O-is also by writing-OCH₂-。

Term " alkyl " self or as another substituent part, unless otherwise indicated, expression straight or branched or cyclic hydrocarbon group or its combination, they can be fully saturated, single or polyunsaturated and can comprise divalence and multivalence order group that the carbon atom number with appointment (is C₁-C ₁₀Represent 1-10 carbon). The example of saturated hydrocarbyl includes but not limited to group such as methyl, second Base, n-pro-pyl, isopropyl, normal-butyl, the tert-butyl group, isobutyl group, sec-butyl, cyclohexyl, (ring Hexyl) methyl, cyclopropyl methyl, (for example n-pentyl, n-hexyl, n-heptyl, n-octyl) Homologue and isomers etc. Unsaturated alkyl is the alkyl with one or more pairs of keys or triple bond. The example of unsaturated alkyl includes, but are not limited to vinyl, 2-acrylic, crotyl, 2-isoamyl Thiazolinyl, 2-(butadienyl), 2,4-pentadienyl, 3-(Isosorbide-5-Nitrae-pentadienyl), acetenyl, 1-With 3-propinyl, 3-butynyl and higher homologue and isomers. Unless otherwise indicated, term " alkyl " also is used for comprising the alkyl derivative of following more specific definition, such as " assorted alkyl ". Be limited to hydrocarbon The alkyl of base is called " all alkyl ".

Term " alkylidene " self or as the divalent group of another substituent a part of expression derived from alkane as illustrative is, but is not limited to-CH₂CH ₂CH ₂CH ₂-, and further comprise following Be described as those groups of " assorted alkylidene ". Typically, alkyl (or alkylidene) contains 1-24 Carbon atom, and contain 10 or still less those groups of carbon atom be preferred in the present invention. " low alkyl group " or " low-grade alkylidene " is more short-chain alkyl or alkylidene, usually contains 8 or more Few carbon atom.

Term " alkoxyl ", " alkyl amino " and " alkylthio " (or thio alkoxy) are normal with them The meaning of rule is used, and represents to pass through respectively oxygen atom, amino, or sulphur atom is connected to molecule Those alkyl of remainder.

Term " assorted alkyl " self or be combined with another term, unless otherwise indicated, the expression straight chain Or side chain or cyclic hydrocarbon group or its combination, assorted former by carbon atom and at least one of described number Subgroup becomes, and this hetero atom is selected from O, N, Si and S, and wherein nitrogen and sulphur atom optionally by Oxidation and nitrogen heteroatom are randomly by season. One or more hetero atom O, N and S and Si can Be positioned at any interior location of assorted alkyl or be positioned at the position that alkyl is connected to the molecule remainder. Example includes, but are not limited to-CH₂-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 (CH₃)-CH ₃ Two hetero atoms can be continuous at the most, for example-and CH₂-NH-OCH ₃With-CH₂-O-Si(CH ₃) ₃ Similarly, term " assorted alkylidene " is by self or as another replacement The part of base represents as illustrative to be, but to be not limited to derived from the divalent group of assorted alkyl-CH₂-CH ₂-S-CH ₂-CH ₂-and-CH₂-S-CH ₂-CH ₂-NH-CH ₂-. For assorted alkylidene, hetero atom Also can occupy one or two chain end (as, alkylene oxide group, alkylenedioxy group, alkylidene ammonia Base, alkylidene diaminourea etc.). Still further, for alkylidene and assorted alkylidene linking group, The orientation of linking group is not write the direction hint of the general formula of linking group. For example, general formula-C (O)₂R '-expression-C (o)₂R '-and-R ' C (O)₂-both.

Term " cycloalkyl " and " Heterocyclylalkyl " are by they self or be combined with other term, unless in addition External declaration, the respectively annular form of expression " alkyl " and " assorted alkyl ". In addition, for Heterocyclylalkyl, Hetero atom can occupy the position that heterocycle is connected to the molecule remainder. The example of cycloalkyl comprises, but Be not limited to cyclopenta, cyclohexyl, 1-cyclohexenyl group, 3-cyclohexenyl group, suberyl etc. Heterocycle alkane The example of base includes, but are not limited to 1-(1,2,5,6-tetrahydro pyridyl), 1-piperidyl, 2-piperidines Base, 3-piperidyl, 4-morpholinyl, morpholinyl, oxolane-2-base, oxolane-3-base, Thiophane-2-base, thiophane-3-base, 1-piperazinyl, 2-piperazinyl etc.

Term " halo " or " halogen " remove by they self or as another substituent part Non-other explanation, expression fluorine, chlorine, bromine or iodine atom. In addition, term is intended to such as " haloalkyl " Comprise single haloalkyl and multi-haloalkyl. For example, term " halo (C₁-C ₄) alkyl " be intended to comprise, But be not limited to trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl etc.

Term " aryl " unless otherwise indicated, expression can be single ring or a plurality of ring (preferred 1-3 Individual ring) how unsaturated, fragrant substituting group, they condense together or are covalently bound. Term " Heteroaryl " expression comprises 1-4 heteroatomic aryl (or ring), and this hetero atom is selected from N, O and S, wherein nitrogen and sulphur atom are randomly oxidized, and nitrogen-atoms is randomly by season. Heteroaryl can To be connected to the remainder of molecule by hetero atom.

The non-limitative example of aryl and heteroaryl comprises phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl Base, 1-pyrrole radicals, 2-pyrrole radicals, 3-pyrrole radicals, 3-pyrazolyl, 2-imidazole radicals, 4-imidazole radicals, Pyrazinyl, 2-_ azoles base, 4-_ azoles base, 2-phenyl-4-_ azoles base, 5-_ azoles base, 3-be different _ Azoles base, 4-be different _ and azoles base, 5-be different _ azoles base, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-Furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridine radicals, 3-pyridine radicals, 4-pyrrole The pyridine base, 2-pyrimidine radicals, 4-pyrimidine radicals, 5-benzothiazolyl, purine radicals, 2-benzimidazolyl, 5-indyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinoline Quinoline base, tetrazole radical, benzo [b] furyl, benzo [b] thienyl, 2,3-dihydrobenzo [Isosorbide-5-Nitrae] Two _ English-6-base, benzo [1,3] dioxole-5-base and 6-quinolyl. More than each The substituting group of the aryl of mentioning and heteroaryl ring system is selected from the above-mentioned substituting group accepted.

For simplicity, term " aryl " when with other term (such as, aryloxy group, aryl sulphur oxygen base, The aryl and the heteroaryl ring that comprise above definition when aryl alkyl) being used in combination. Therefore, art Language " aryl alkyl " is used for comprising those groups, wherein aryl be connected to alkyl (as, benzyl, benzene Ethyl, pyridylmethyls etc.), described alkyl comprises those alkyl, and wherein carbon atom is (such as, methylene Base) substituted by for example oxygen atom (as, phenoxymethyl, 2-pyridine oxygen ylmethyl, 3-(1-naphthalene oxygen Basic) propyl group etc.).

Each above-mentioned term (as, " alkyl, the " " alkyl of mixing, " " aryl " and " heteroaryl ") is intended to comprise The replacement of the group of appointment and do not replace form. The preferred of every type of group below is provided Substituting group.

Alkyl and assorted alkyl (comprise and are commonly referred to alkylidene, thiazolinyl, assorted alkylidene, assorted thiazolinyl, alkynyl, cycloalkyl, Heterocyclylalkyl, cycloalkenyl group, with those of heterocycloalkenyl) substituting group be commonly referred to " alkyl substituent ", and they can be to be selected from, but are not limited in the following various groups one or more: number is zero (2m '+the 1)-OR ' that arrives,=O,=NR ',=N-OR ',-NR ' R " ;-SR ' ;-halogen ;-SiR ' R " R_,-OC (O) R ',-C (O) R ',-CO₂R′、-CONR′R″、 -OC(O)NR′R″、-NR″C(O)R′、-NR′-C(O)NR″R_、-NR″C(O) ₂R′、 -NR-C(NR′R″R_)＝NR″″、-NR-C(NR′R″)＝NR_、-S(O)R′、-S(O) ₂R′、 -S(O) ₂NR′R″、-NRSO ₂R ' ,-CN and-NO₂, wherein m ' is the total of carbon atom in such group Number. R ', R ", R_ and R " " each preferably represent independently hydrogen, replacement or replace assorted alkyl, Replace or unsubstituting aromatic yl, as by the aryl of 1-3 halogen replacement, replacement or not substituted alkyl, Alkoxyl or thio alkoxy or aryl alkyl. When compound of the present invention comprises more than a R During group, for example each R group is selected independently, resembles each R ', R ", R_ and R " " group that Sample (when existing more than these groups of one). As R ' and R " when being connected to identical nitrogen-atoms, They can be combined with nitrogen-atoms to form 5-, 6-, or 7-unit ring. For example ,-NR ' R " be intended to the bag Draw together, but be not limited to 1-pyrrolidinyl and 4-morpholinyl. More than substituent, this area is discussed The technical staff is to be understood that term " alkyl " is intended to comprise following groups, and described group comprises and being attached to The carbon atom of the group beyond the hydrogen atom, as haloalkyl (as ,-CF₃With-CH₂CF ₃) and acyl group (as ,-C (O) CH₃、-C(O)CF ₃、-C(O)CH ₂OCH ₃Deng).

Similar in appearance to for the described substituting group of alkyl, usually claim for the substituting group of aryl and heteroaryl Be " aryl substituent ". Substituting group is selected from, for example: halogen ,-OR ' ,=O ,=NR ' ,=N-OR ',-NR ' R " ,-SR ' ,-halogen ,-SiR ' R " R_ ,-OC (O) R ' ,-C (O) R ' ,-CO₂R′、-CONR′R″、 -OC(O)NR′R″、-NR″C(O)R′、-NR′-C(O)NR″R_、-NR″C(O) ₂R′、 -NR-C(NR′R″R_)＝NR″″、-NR-C(NR′R″)＝NR_、-S(O)R′、-S(O) ₂R′、 -S(o) ₂NR′R″、-NRSO ₂R ' ,-CN and-NO₂、-R′、-N ₃、-CH(Ph) ₂, fluorine (C₁-C ₄) alkoxyl and fluorine (C₁-C ₄) alkyl, number is to the open valent sum of aromatic ring system from zero Order; And R ' wherein, R ", R_ and R " " preferably be independently selected from hydrogen, replacement or not substituted alkyl, Replace or replace assorted alkyl, replacement or unsubstituting aromatic yl and replacement or substituted heteroaryl not. When this When the compound of invention comprised more than a R group, for example each R group was selected independently, resembles Each R ', R ", R ' " and R " " group is (when existing more than these groups of one) like that. In the following scheme, " R " that sign X is above-mentioned.

Two substituting groups on the adjacent atom of aryl or heteroaryl ring optionally by general formula are-T-C (O)-(CRR ')_qThe substituting group of-U-substitutes, wherein T and U be independently-NR-,-O-,-CRR '-Or singly-bound, and q is the integer of 0-3. Perhaps, on the adjacent atom of aryl or heteroaryl ring Two substituting groups optionally by general formula are-A-(CH₂) _rThe substituting group of-B-substitutes, wherein A and B be independently-CRR '-,-O-,-NR-,-S-,-S (O)-,-S (O)₂-、-S(O) ₂NR '-or single Key, and r is the integer of 1-4. One in the singly-bound of the new ring that forms so optionally by two Key substitutes. Perhaps, two substituting groups on the adjacent atom of aryl or heteroaryl ring optionally By general formula be-(CRR ')_s-X-(CR″R′″) _d-substituting group substitute, wherein s and d are the integer of 0-3 independently, and X is-O-,-NR '-,-S-,-S (O)-,-S (O)₂-or-S (O)₂NR '-. Get Basic R of generation, R ', R " and R ' " preferably are independently selected from hydrogen or replacement or unsubstituted (C₁-C ₆) alkyl.

Term " hetero atom " is intended to comprise oxygen (O), nitrogen (N), sulphur (S) and silicon (Si) as used herein.

The reactive derivatives of PEG (or other connects base) connects one or more peptide moieties to connecting The purposes of base within the scope of the invention. The present invention is not by the characteristic limitations of reactive PEG analog. Many activated derivatives of PEG are can obtain in commercially available and the document. Select and (if Needs) synthetic suitable activated PEG derivative is in those skilled in the art's limit of power (adopt this derivative with for the preparation of substrate of the present invention). Referring to people such as Abuchowski Cancer Biochem.Bophys., 7:175-186 (1984); The people such as Abuchowski, J. Biol.Chem., 252:3582-3586 (1977); The people Anal.Biochem. such as Jackson, 165:114-127 (1987); The people such as Koide, Biochem Biophys.Res.Commun., 111: 659-667 (1983)), tresylate (people such as Nilsson, Methods Enzymol., 104: 56-69 (1984); The people such as Delgado, Biotechnol.Appl.Biochem., 12: 119-128 (1990)); N-hydroxy-succinamide derivatives active ester (Buckmann etc. The people, Makromol.Chem., 182:1379-1384 (1981); The people such as Joppich, Makromol. Chem., 180:1381-1384 (1979); The people such as Abuchowski, Cancer Biochem. Biophys., 7:175-186 (1984); The people Proc.Natl.Acad.Sci.U.S. such as Katre A., 84:1487-1491 (1987); The people such as Kitamura, Cancer Res., 51: 4310-4315 (1991); The people such as Boccu, Z.Naturforsch., 38C:94-99 (1983), Carbonic ester (people such as Zalipsky, the PEG chemistry: biotechnology and biologic medical are used, Harris, Ed., P1enum Press, New York, 1992, pp.347-370; Zalipsky Deng the people, Biotechnol.Appl.Biochem., 15:100-114 (1992); Veronese Deng the people, Appl.Biochem.Biotech., 11:141-152 (1985)), the imidazole radicals formic acid esters (people such as Beauchamp, Anal.Biochem., 131:25-33 (1983); The people such as Berger, Blood, 71:1641-1647 (1988)), 4-two sulfo-pyridines (people such as Woghiren, Biocon jugate Chem., 4:314-318 (1993)), isocyanates (people such as Byun, ASAIO Journal, M649-M-653 (1992)) and epoxides (U.S. Patent number 4,806,595 is awarded Give the people such as Noishiki, (1989). Other linking group is included between amino and the activated PEG Urethane bond. Referring to people such as Veronese, Appl.Biochem.Biotechnol., 11: 141-152 (1985).

Natural and the synthesizing amino acid of term " amino acid " expression, and amino acid analogue and amino acid Analogies. Natural amino acid is by those of genetic code coding, and with those ammonia of post-modification Base acid is such as hydroxy-proline, Gla ester and O-phosphoserine. Amino acids seemingly Thing represents to have with natural amino acid the compound of identical basic chemical structure, namely α carbon be attached to hydrogen, Carboxyl, amino and R group are such as homoserine, norleucine, methionine sulfoxide, methionine The methyl sulfonium. Such analog contains modification R group (such as, norleucine) or modification peptide main chain, But keep the basic chemical structure identical with natural amino acid. " amino acid analog thing " expression structure is not Be same as amino acid whose general chemical constitution, but adopt the mode similar to natural amino acid to work Compound.

" peptide " expression polymer, wherein monomer is amino acid, amino acid analogue and/or amino acid Analogies and combine by amido link perhaps are called polypeptide. In addition, also comprise non-day Right amino acid, for example Beta-alanine, phenylglycine and homoarginine. Not the ammonia of gene code Base acid also can be used for the present invention. In addition, modification is to comprise reactive group, glycosylation position, poly-The amino acid of compound, treatment part, biomolecule etc. also can be used for the present invention. Be used for of the present invention All amino acid can be D-or L-isomers. Usually L-isomers preferably. In addition, other Peptide mimics also can be used for the present invention. " peptide " represents glycosylation and non-glycosylated peptide two as used herein The person. Also comprise the incomplete glycosylated peptide of system by expression of peptides. For general summary, referring to Spatola, A.F., amino acid, the chemistry of peptides and proteins and biochemistry, B. Weinstein, eds., Marcel Dekker, New York, p.267 (1983).

Term " nucleosides " expression glycosyl amine, this glycosyl amine are that the component of nucleic acid and comprising is connected to β-D-RIBOSE to be forming ribonucleotide, or is connected to 2-deoxidation-β-D-RIBOSE with shape Become the nitrogenous base of dezyribonucleoside. Base can be purine as, adenine or guanine, or Pyrimidine as, thymidine, cytidine, uridine or pseudouridine. Nucleosides also comprises by microorganism and using seldom See nucleosides.

Term " targeting moiety " is illustrated in the kind of selectivity location in the particular organization of health or the zone as used herein.The location is by mediations such as the molecular size of specific identification, target agent or the binding substances of molecule determiner, ionic interaction, hydrophobic interactions.Targeting agent is well known by persons skilled in the art to other mechanism in particular organization or zone.Illustrative targeting moiety comprises antibody, antibody fragment, siderophilin, HS-glycoprotein, thrombin, serum protein, β-glycoprotein, G-CSF, GM-CSF, M-CSF, EPO etc.

Any reagent of being used for the treatment of of " treatment part " expression as used herein includes, but are not limited to, microbiotic, anti-inflammatory agent, antitumour drug, cytotoxin and radioreagent." treatment part " comprises the prodrug of biologically active agent, construction (one of them above treatment partly is attached to carrier, as multivalence reagent).The treatment part also comprises protein and comprises proteinic construction.Illustrative protein comprises, but be not limited to erythropoietin (EPO), granulocyte colony-stimulating factor (GCSF), rHuGM-CSF (GMCSF), Interferon, rabbit (as, interferon-' alpha ' ,-β ,-γ), interleukin-(as, interleukin-II), serum protein (as, factor VII, VIIa, VIII, IX, and X), human chorionic gonadotropin (HCG), follicle stimulating hormone (FSH) and lutropin (LH) and antibody fusion protein (as Tumor Necrosis Factor Receptors ((TNFR)/Fc domain fusion protein)).

" antitumour drug " expression is used to resist any reagent of cancer as used herein, include, but are not limited to cytotoxin and reagent (as metabolic antagonist, alkylating reagent, anthracycline compound, microbiotic, antimitotic agent, Procarbazine, hydroxyurea, Asparaginase, cortin, Interferon, rabbit and radioreagent).Being also included within the scope of term " antitumour drug " is the peptide with anti-tumor activity, as the binding substances of TNF-α.Those that binding substances includes, but are not limited to form between therapeutic protein and glycoprotein of the present invention.Representational binding substances forms between PSGL-1 and TNF-α.

" cytotoxin or cell toxicant reagent " represents the deleterious any reagent of pair cell as used herein.Example comprises taxol, cytochalasin B, Gramicidin D, ethidium bromide, Hemometine, mitomycin, Etoposide, teniposide, vincristine(VCR), vinealeucoblastine(VLB), colchicine, Dx, daunorubicin, dihydroxyl anthracin diketone (authracinedione), mitoxantrone, duomycin, Actinomycin D, 1-dehydrotestosterone, glucocorticosteroid, PROCAINE HCL, PHARMA GRADE, tetracaine, lignocaine, Proprasylyte and tetracycline and analogue or homologue.Other toxin comprises, for example Ricin, CC-1065 and analogue, times ganmycin (duocarmycin).Further other toxin comprises diptheria toxin and snake venom (as, cobra-venom).

" radioreagent " comprises any radio isotope of efficient diagnosis or destruction tumour as used herein.Example includes, but are not limited to indium-111, cobalt-60.In addition, natural radioactive element such as uranium, radium and thorium (the radioisotopic mixture of their ordinary representations) are the suitable example of radioreagent.Metal ion typically with organic chelated part chelating.

Many useful chelation groups, crown ether, cryptand etc. be known in the art and can introduce compound of the present invention (as, EDTA, DTPA, DOTA, NTA, HDTA etc. and their phosphonate analogs such as DTPP, EDTP, HDTP, NTP etc.).Referring to, people such as Pitt for example, " be used for the design of sequestrant of the treatment of iron overload, " in INORGANIC CHEMISTRY INBIOLOGY AND MEDICINE; Martell, Ed.; American Chemical Society, Washington, D.C., 1980, pp.279-312; Lindoy, the chemistry of macrocyclic ligand mixture; CambridgeUniversity Press, Cambridge, 1989; Dugas, BIOORGANIC CHEMISTRY; Springer-Verlag, New York, 1989, and the reference that wherein comprises.

In addition, allowing sequestrant, crown ether and cyclodextrin is that those skilled in the art are obtainable to the various approach that is connected of other molecule.Referring to, people such as the Meares for example, " performance of inner complex-labelled protein and polypeptide in the body." in MODIFICATION OF PROTEINS:FOOD, NUTRITIONAL, AND PHARMACOLOGICAL ASPECTS; " Feeney waits the people, Eds., American Chemical Society, Washington, D.C., 1982, pp.370-387; People such as Kasina, Biocon jugate Chem., 9:108-117 (1998); People such as Song, Biocon jugateChem., 8:249-255 (1997).

Introduce

The invention provides polymer material, and the sugar, activation sugar and the nucleotide sugar that are attached to these polymkeric substance.The polymer conjugates of nucleotide sugar is the substrate of enzyme normally, and described enzyme transfer sugar moieties and its polymeric substituents are to the suitable acceptor portion of substrate.Therefore, the present invention also provides the polymer conjugates that uses nucleotide sugar and suitable enzyme by the substrate of sugar in conjunction with modification.Can use compound of the present invention to carry out sugared bonded substrate and comprise peptide, as glycopeptide, lipid, as join sugar ester and aglucon (saddle ammonia alcohol, ceramide).

As discussing in the part formerly, the chemical process of art-recognized covalency PEGization depends on by the Chemical bond of reactive group on amino acid or carbohydrate.By careful design, use the useful binding substances of combination strategy preparation of chemistry mediation to binding substances and reaction conditions.Polymkeric substance is the selectivity that lacks activated polymer to the chemically combined main drawback of protein or glycoprotein, and it causes the connection at the polymkeric substance that relates to protein or the bioactive position of glycoprotein usually.Developed several schemes with solution position selective binding chemistry, yet, a kind of universal method that is suitable for various recombinant proteins only developed.

Form contrast with art-recognized method, the invention provides compound, this compound is used for the high selectivity of branched water-soluble polymers, the sugar combination of position orientation with the strategy of novelty, as sugar-PEGization.In illustrative embodiment of the present invention, directed vitro enzyme glycosylation, use nucleotide sugar of the present invention or the activation sugar that connects by special peptide sequence in the position of branched water-soluble polymers is finished.Sugar-combination can adopt glycosyltransferase (as sialytransferase) to carry out with enzyme process, and this transferring enzyme can arrive glycosylation position (" sugar-PEGization ") for transfer of material branched water-soluble polymers-sugar (as the PEG-sialic acid).

As discussed above, the invention provides and be aggregated the partially modified glycoconjugate of thing with any required carbohydrate structure.Sugar nucleotide and also be integral part of the present invention based on the activation sugar of these sugared structures.Polymer modification part is by enzyme process, and chemical method or its sugar moieties that is connected produces modified ribonucleotide sugar thus.Sugar is aggregated thing modification part and replaces in any desired location.In illustrative embodiment, sugar is at one or more C-1, C-2, C-3, the furanose that C-4 or C-5 place replace.In another embodiment, the invention provides and be aggregated thing modification part, C-2, C-3, C-4, the pyranose of C-5 or C-6 replacement at one or more C-1.Preferably, polymer modification partly is directly connected to oxygen, nitrogen or the sulphur that dangles from carbon.Perhaps, polymer modification partly is connected to the connection base between sugar and modification part.Connect base and be connected to oxygen, nitrogen or the sulphur that dangles from the carbon of selecting.

In present embodiment preferred, polymer modification partly appends to certain position, selects this position to make the binding substances that obtains play enzyme substrates, and this enzyme is used in conjunction with modified sugar moieties to another kind of material, as peptide, glycopeptide, lipid, glycolipid etc.Illustrative enzyme discusses in more detail and comprises glycosyltransferase (sialytransferase, glucanotransferase, galactotransferase, N-acetyl glucosamine based transferase, N-acetyl galactotransferase, mannose transferase, fucosyltransferase etc.) at this.Illustrative sugar nucleotide of the present invention and activation glycoconjugate also comprise the substrate of mutant Glycosylase and mutant glycosyl ceramide enzyme, and this substrate of modification is to have synthetic rather than hydrolytic activity.

In illustrative embodiment, binding substances of the present invention comprises sugar, activation sugar or the nucleotide sugar that is attached to one or more polymkeric substance (as branched polymer).Illustrative polymkeric substance comprises water-soluble and the water-insoluble classification.

In illustrative embodiment, the polymer modification group directly or indirectly is connected to pyranose or furanose.For example:

With

In general formula I and II, R ¹Be H, CH ₂OR ⁷, COOR ⁷Or OR ⁷, R wherein ⁷Expression H, replacement or unsubstituted alkyl or replacement or unsubstituted assorted alkyl.R ²Be H, OH, NH or the part that comprises Nucleotide.Illustrative R according to this embodiment ²Have following general formula:

X wherein ¹Expression O or NH and R ⁸It is nucleosides.

Symbol R ³, R ⁴, R ⁵, R ⁶And R ⁶' represent H, replacement or not substituted alkyl, OR independently ⁹, NHC (O) R ¹⁰Mark d is 0 or 1.R ⁹And R ¹⁰Be independently selected from H, replacement or not substituted alkyl, replacement or replace assorted alkyl or sialic acid.R ³, R ⁴, R ⁵, R ⁶, and R ⁶' at least one comprise polymer modification part (as PEG).In illustrative embodiment, R ⁶And R ⁶' in the carbon that connects with their be the component of sialic acid side chain.In further illustrative embodiment still, this side chain is aggregated thing modification part (or connecting base-polymer modification part) at one or more C-6, C-7 or C-9 modification.

Symbol R ³, R ⁴, R ⁵And R ⁶Represent H, OR independently ⁹, NHC (O) R ¹⁰R ⁹And R ¹⁰Be independently selected from H, replacement or unsubstituted alkyl or replacement or unsubstituted assorted alkyl.R ³, R ⁴, R ⁵, R ⁶And ⁶' at least one comprise the polymer modification part.

In another illustrative embodiment, sugar moieties is oxidation and the sialic acid part that is attached to the polymer modification part, as be described in the U.S. temporary patent application No._____ (attorney No.040853-01-5150) of common transfer, submit day on January 6th, 2005 to.

In illustrative embodiment, the polymer modification part is attached to sugar nuclear by connecting base:

R wherein ¹¹Be polymer moieties and L is selected from key and linking group, and w is 1-6, preferred 1-3 and the more preferably integer of 1-2.

When L was key, it was at R ¹¹Precursor on reactive functional groups and the reactive functional groups of the postreaction on the L precursor between form.Such as described in this, have appropriate reaction functional group precursor selection and the preparation in those skilled in the art's limit of power.In addition, carry out the precursor combination according to chemistry well known in the art.

In illustrative embodiment, L is the linking group that forms from amino acid, amino acid analog thing or little peptide (as, 1-4 amino-acid residue), and modified sugars is provided, and wherein polymer modification partly connects base by the alkyl that replaces and connects.Connect the reaction of base by amino acid whose amine moiety and carboxylic acid (or reactive derivatives, as active ester, carboxylic acid halides etc.) and form, this amino acid has L and R ¹¹The group of the postreaction on the precursor.The element of binding substances can adopt the combination of any basically order easily.For example in conjunction with R ¹¹Before the precursor of L, the precursor of L can be in place on sugar nuclear.Perhaps, the R that has reactive functionalities on the L ¹¹-L molectron (cassette) also is connected to sugar by the reactive functional groups of postreaction on this material subsequently.

In illustrative embodiment, polymer modification partly is R ³And/or R ⁶In another illustrative embodiment, R ³And/or R ⁶Comprise the polymer modification part and be connected basic L, connect the remainder that basic L conjugated polymer partly arrives molecule.In another illustrative embodiment, polymer modification partly is R ³In another illustrative embodiment, R ³Comprise polymer modification part and connector L, connector L conjugated polymer partly arrives the remainder of molecule.In another illustrative embodiment, wherein sugar is sialic acid, and the polymer modification part is at R ⁵The place or in the position of sialic acid side chain, connect as the C-9 place.

The linear polymerization combination

In illustrative embodiment, the invention provides and linear polymer (as water-soluble or insoluble polymer) between the sugar or activation glycoconjugate or the nucleotide sugar binding substances that form.In binding substances of the present invention, polymkeric substance is connected to sugar, activation sugar or sugar nucleotide.Such as in this discussion, polymkeric substance directly or by connecting base is connected to sugar moieties.

Have structure according to general formula I or II, wherein at least one R according to the illustrative compound of this embodiment ¹, R ³, R ⁴, R ⁵Or R ⁶Have following general formula:

Another example according to this embodiment has following general formula:

Wherein s is the integer of 0-20 and R ¹¹It is linear polymer modification part.

The peg moiety of any molecular weight (as 2Kda, 5Kda, 10Kda, 20Kda, 30Kda and 40Kda) all can be used for the present invention.

The branched polymer binding substances

In illustrative embodiment, polymer modification partly is to comprise two or more branched structures with following general formula that are connected to the polymer chain of centre portions:

R wherein ¹¹With L as discussed above and w ' be 2-6, preferred 2-4 and the more preferably integer of 2-3.

The illustrative precursor that is used to form the binding substances of this embodiment according to the present invention has following general formula:

Branched polymer according to this general formula is pure substantially water-soluble polymers.X ³' be comprise ionizable (as COOH, H ₂PO ₄, HSO ₃, HPO ₃Deng) or the part of other reactive functional groups, as mentioned below.C is a carbon.X ⁵Preferably non-reacted group (as, H, substituted alkyl does not replace assorted alkyl), and can be polymeric arms.R ¹²And R ¹³Be the polymeric arms of selecting independently, as non-peptide, non-reactive polymer arm.X ²And X ⁴Non-reacted substantially junction fragment under physiological condition preferably, they can be identical or different.Perhaps, these keys can comprise one or more parts of degrading of being designed under the physiology correlated condition, as ester, disulphide etc.X ²And X ⁴Conjugated polymer arm R ¹²And R ¹³To C.Work as X ³' when being connected base, sugar or connecting the reactive functional groups reaction of postreaction on base-sugar combination, X ³' change into junction fragment X ³Component.

X ²And X ⁴Illustrative junction fragment comprise 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 ₂S, CH ₂O, CH ₂CH ₂O, CH ₂CH ₂S, (CH ₂) _aO, (CH ₂) _aS or (CH ₂) _aY '-PEG or (CH ₂) _aY '-PEG, wherein Y ' is S or O and a is the integer of 1-50.

In illustrative embodiment, precursor (III), or its activated derivatives passes through at X ³' and sugar moieties on reaction bonded between the group of postreaction to sugar, activation sugar or sugar nucleotide.Perhaps, X ³' be reacted into and be connected basic L with reactive functional groups on the precursor.One or more R of general formula I and II ¹, R ³, R ⁴, R ⁵Or R ⁶Can comprise branched polymer modification part.

In illustrative embodiment, as the lower section:

Be to connect basic arm L.In this embodiment, the illustrative base that connects is derived from natural or alpha-non-natural amino acid, amino acid analogue or amino acid analog thing or the little peptide that forms from one or more such materials.For example, some branched polymer of finding in compound of the present invention has following general formula:

X ^aBe that reaction by the reactive functional groups on the precursor of branched polymer modification part and sugar moieties or precursor are to the connection portion that is connected basic reaction formation.For example, work as X ³' when being carboxylic acid, it can activate and directly be attached to from amino-sugar (as, GalNH ₂, G1cNH ₂, ManNH ₂Deng) amine groups of dangling, formation is the X of acid amides ^aOther illustrative reactive functional groups and activation precursor are below described.Mark c represents the integer of 1-10.Other symbol has and those identical identity discussed above.

In another illustrative embodiment, X ^aBe to adopt another to connect the connection portion that base forms:

X wherein ^bBe the connection portion and be independently selected from for X ^aThose groups that illustrate, and L ¹Be key, replacement or unsubstituted alkyl or replacement or unsubstituted assorted alkyl.

X ^aAnd X ^bIllustrative material comprise S, SC (O) NH, HNC (O) S, SC (O) O, O, NH, NHC (O), (O) CNH and NHC (O) O and OC (O) NH.

For example,

In another illustrative embodiment, X ⁴Be to be attached to R ¹³Peptide bond, it is an amino acid, two-peptide or three-peptide, wherein α-amine moiety and/or side chain heteroatoms are aggregated the thing modification.

In further illustrative embodiment, R ⁶Comprising that branched polymer modification group and modified sugars or nucleotide sugar have is selected from following general formula:

With

Wherein by the characteristic of the group of various symbolic representations with discussed above identical.L ^aBe replacement or unsubstituted alkyl or replacement or unsubstituted assorted moieties.In illustrative embodiment, L ^aBe the part of sialic acid side chain, the polymer modification shown in the sialic acid quilt is partially modified.

In another illustrative embodiment still, the invention provides sugar and nucleotide sugar with following general formula:

By the characteristic of the group of various symbolic representations with discussed above identical.As the skilled person will recognize, the base arm that is connected among general formula VI and the VIII is equally applicable to other modified sugars in this explanation.

More than Shuo Ming embodiment of the present invention further illustrate with reference to following material, and polymkeric substance is a water-soluble polymers in the described material, and particularly poly-(ethylene glycol) (" PEG ") is as methoxyl group-poly-(ethylene glycol) (" m-PEG ").Those skilled in the art will recognize that the emphasis in the following content is for clearly illustrating and using PEG to be equally applicable to wherein adopt the material of PEG polymkeric substance in addition as the various motifs of illustrative polymkeric substance.

Water-soluble polymers

Many water-soluble polymerss are well known by persons skilled in the art and can be used for implementing the present invention.The term water-soluble polymers comprises that material is as sugar (as dextran, the straight chain powder forms sediment, hyaluronic acid, poly-(sialic acid), heparitin, heparin etc.); Poly-(amino acid) is as poly-(Aspartic Acid) and poly-(L-glutamic acid); Nucleic acid; Synthetic polymer is (as poly-(vinylformic acid), poly-(ether), as poly-(ethylene glycol); Peptide, protein etc.Polymkeric substance typically comprises at least two polymer units.In illustrative embodiment, polymkeric substance is 2-25 unit.In another illustrative embodiment, polymkeric substance comprises 2-8 polymer unit.The present invention can adopt any water-soluble polymers to implement, and only polymkeric substance that is limited in must comprise that the binding substances remainder can connect point thereon.

The activation method of polymkeric substance also can see WO 94/17039, U.S. Patent number 5,324,844, WO 94/18247, and WO 94/04193, U.S. Patent number 5,219,564, U.S. Patent number 5,122,614, WO 90/13540, U.S. Patent number 5,281,698, with other WO93/15189, and be used at activated polymer and peptide, as blood coagulation factor VIII (WO94/15625), oxyphorase (WO 94/09027), fortune oxygen molecule (U.S. Patent number 4,412,989), the combination between rnase and the superoxide dismutase (people such as Veronese, App.Biochem.Biotecb.11:141-45 (1985)).

Preferred water-soluble polymers be those wherein in the polymer samples essential part of polymer molecule have the polymkeric substance of approximately identical molecular weight; Such polymkeric substance is " homodisperse ".

The present invention is further by poly-(ethylene glycol) binding substances explanation of reference.Can obtain PEG functionalized and several pieces of summaries of bonded and feature article.Referring to, Harris for example, Macronol.Chem.Phys.C25:325-373 (1985); Scouten, Enzymology method 135:30-65 (1987); People such as Wong, Ehzyme Microb.Technol.14:866-874 (1992); People such as Delgado, the key summary 9:249-304 (1992) in the curative drug carrier system; Zalipsky, Biocon jugate Chem.6:150-165 (1995); And Bhadra, wait the people, Pharmazie, 57:5-29 (2002).It is known in the art using the approach of reactive molecule preparation feedback PEG molecule and formation binding substances.For example; U.S. Patent number 5; 672,662 disclose the water-soluble and separable binding substances of the active ester that is selected from following polymeric acid: linearity or branching are gathered (epoxy alkane), poly-(oxygen ethylization polyvalent alcohol), poly-(olefinic alcohol) and poly-(acryloyl morpholine).

U.S. Patent number 6,376,604 have illustrated the method for preparing the water-soluble 1-benzotriazole base carbonic ether of water-soluble and nonpeptidic polymer in organic solvent by terminal hydroxyl and two (the 1-benzotriazole base) carbonate reaction that makes polymkeric substance.Active ester is used to form the binding substances with biologically active agent (as protein or peptide).

WO 99/45964 has described the binding substances that comprises biologically active agent and activatory water-soluble polymers, this water-soluble polymers comprises main polymer chain, this main chain contains at least one end that is connected to main polymer chain by appropriate key, wherein at least one end comprises the branching part that contains the proximal response group that is connected to the branching part, and wherein biologically active agent is connected at least one proximal response group.Other branching poly-(ethylene glycol) is described in WO 96/21469, U.S. Patent number 5,932, and 462 have described the binding substances that adopts branching PEG molecule to form, and this PEG molecule comprises the branching end, and this end comprises reactive functional groups.Free reactive group can be used for and biologically active substance (as protein or peptide) reaction, forms binding substances between poly-(ethylene glycol) and biologically active substance.U.S. Patent number 5,446,090 has described difunctionality PEG connects base and its purposes in forming binding substances, and this binding substances connects basic end at each PEG and has peptide.

Comprise that the binding substances that degradable PEG connects is described in WO 99/34833; And WO99/14259, and be described in U.S. Patent number 6,348,558.Such degradable connection is applicable to the present invention.

More than Shuo Ming art-recognized polymer activation method is used in the formation of the branched polymer of introducing of the present invention and the combination that these branched polymers arrive other material (as sugar, sugar nucleotide etc.) here.

Illustrative modification group below is discussed.Can select the modification group to the ability that they give one or more desired properties to peptide.The pharmacokinetics that illustrative performance includes but not limited to improve, pharmacodynamics, the improved bio distribution of raising, provide multivalence material, improved water-soluble, the lipophilicity that improves or reduce and tissue target to.

Be used for illustrative poly-(ethylene glycol) molecule of the present invention and include, but are not limited to have those of following general formula:

A wherein ²Be H, OH, NH ₂, replace or not substituted alkyl, replacement or unsubstituting aromatic yl, replacement or not substituted heteroaryl, replacement or unsubstituting heterocycle alkyl, replacement or replace assorted alkyl, as acetal, OHC-, H ₂N-(CH ₂) _q-, HS-(CH ₂) _q, or-(CH ₂) _qC (Y ^b) Z ^bThe integer of mark " e " expression 1-2500.Mark b, d and q represent the integer of 0-20 independently.Symbols Z ^aAnd Z ^bRepresent OH, NH independently ₂, leavings group, as imidazoles, p-nitrophenyl, HOBT, tetrazolium, halogenide, S-R ^a, Acibenzolar alcohol moiety;-(CH ₂) _pC (Y ^b) V or-(CH ₂) _pU (CH ₂) _sC (Y ^b) _vSymbol Y ^aExpression H (2) ,=O ,=S ,=N-R ^bSymbol X ^a, Y ^a, Y ^b, A ¹And U represents group O, S, N-R independently ^cSymbol V represents OH, NH ₂, halogen, S-R ^a, the alkoxide component of Acibenzolar, amine component, sugar-Nucleotide and the protein of activating terephthalamide amine.Mark p, q, s and v are the integers that is independently selected from 0-20.Symbol R ^a, R ^b, and R ^cRepresent H independently, replace or substituted alkyl not, replace or replace assorted alkyl, replace or unsubstituting aromatic yl, replace or unsubstituting heterocycle alkyl and replacement or substituted heteroaryl not.

The specific embodiments that is used for linearity of the present invention and branched polymer (as PEG) comprises:

With the carbonic ether and the active ester of these materials, as:

Can be used for forming linear and branched polymer material, the connection base arm binding substances of these materials and the binding substances between these compounds and sugar and nucleotide sugar.Mark e and f are independently selected from 1-2500.

Be suitable for activating other illustrative activation or the leavings group that straight chain PEG is used to prepare at the compound of this explanation and include but not limited to following material:

In those skilled in the art's limit of power is to assign to polymer modification for the selection portion on the precursor partly to select suitable activating group.

Adopt these and other material activatory PEG molecule and the method for preparing activated PEG to be illustrated in WO 04/083259.

In illustrative embodiment, branched polymer is based on halfcystine, Serine, Methionin, two-or the PEG of three-Methionin nuclear.Therefore, further illustrative branching PEG comprises:

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

In another embodiment, the branching peg moiety is based on three-lysine peptide.Three-Methionin can be singly-, two-, three-, or four-PEG-baseization.Illustrative material according to this embodiment has following general formula:

E wherein, f and f ' are independently selected from the integer of 1-2500; And q, q ' and q " are independently selected from the integer of 0-20.

In illustrative embodiment of the present invention, PEG is m-PEG (5kD, 10kD, 20kD, 30kD or 40kD).Illustrative branching PEG material be Methionin-, Serine-or halfcystine-(m-PEG) ₂, wherein m-PEG is 20kD m-PEG.

It is apparent that for those skilled in the art, be used for the version that branched polymer of the present invention comprises the situation of above explanation.The for example above two-Methionin-PEG binding substances that shows can comprise three polymkeric substance subelements, and the 3rd is bonded to the α-amine that is shown as non-modification in above structure.Similarly, within the scope of the invention by the functionalized three-Methionin of three or four polymkeric substance subelements.

The one or more m-PEG arms that those skilled in the art will recognize that branched polymer can be different terminal (as OH, COOH, NH by having ₂, C ₂-C ₁₀-alkyl etc.) peg moiety substitutes.In addition, be easy to connect base (or removing carbon atom) structure more than the modification by between the functional group of alpha-carbon atom and side chain, inserting alkyl.Therefore, " high (homo) " derivative and higher homologue, and lower homologue is being used in the nuclear scope of branching PEG of the present invention.

Branching PEG material described here is the method preparation by illustrating in the following route easily:

X wherein ^bBe O, NH or S and r is the integer of 1-10.Mark e and f are independently selected from the integer of 1-2500.Illustrative branching PEG material is 10,000,15,000,20,000,30,000 and 40,000 dalton.

Therefore, according to this scheme, natural or alpha-non-natural amino acid are contacted with activatory m-PEG derivative (tosylate in the case), by alkylation side chain heteroatoms X ^bForm compound 1.List-functionalized m-PEG amino acid and reactive m-PEG derivative are stood N-acylations condition, assemble branching m-PEG 2 thus.As the skilled person will recognize, the tosylate leavings group can change any suitable leavings group into, as halogen, methanesulfonates, triflate etc.Similarly, the reactive carbonic ether that is used for acylations amine can change active ester into, and as N-hydroxy-succinamide etc., perhaps acid can be used dewatering agent (as dicyclohexyl carbodiimide, N,N'-carbonyldiimidazole etc.) in-situ activation.

In the illustrative scheme of above explanation, the modification group is linear peg moiety, yet any modification group can be by introducing glycosyl part as water-soluble polymers, insoluble polymer, branched polymer, treatment part etc.

The further branched polymer material that is used for compound of the present invention illustrates example as described below by the functionalized branching nuclear of PBG:

R wherein ¹⁴Be OH or another reactive functional groups.Illustrative reactive functional groups is C (O) Q ', wherein selects Q ' to make that C (O) Q ' is a reactive functional groups.The illustrative material that is used for Q ' comprises halogen, NHS, pentafluorophenyl group, HOBT, HOAt and p-nitrophenyl.Mark " e " and mark " f " are the integers that is independently selected from 1-2500.

More than Shuo Ming hyperbranched compounds and the other hyperbranched compounds that is used for The compounds of this invention are easily from following material preparation:

The polymer modification sugar substance

The sugar moieties of nucleotide sugar of the present invention can be selected from natural and non-natural furanose and hexose.Non-natural sugar randomly comprises alkylation or acylations hydroxyl and/or amine moiety, as ether, ester and the amide substituents on the ring.The certain position of other non-natural sugar on ring comprises H, hydroxyl, ether, ester or amide substituents, do not have such substituting group in this position in natural sugar.Sugar moieties can be monose, oligose or saccharan.

Be used for illustrative natural sugar of the present invention and comprise glucose, semi-lactosi, Fucose, seminose, wood sugar, ribose, N-acetyl glucosamine, sialic acid and N-acetyl semi-lactosi.

Similarly, nucleosides can be selected from natural and non-natural (or rare) nucleosides.Be used for illustrative natural nucleus glycoside of the present invention and comprise cytosine(Cyt), thymus pyrimidine, guanine, VITAMIN B4 and uridylic.Rare nucleosides can include but not limited to such molecule such as ara U and thymine arabinoside.Non-natural and rare nucleosides and their preparation method have fully been showed in this area.

Illustrative modified sugars Nucleotide of the present invention comprises GDP-Man, GDP-Fuc, UDP-Gal, UDP-Gal-NH ₂, UDP-GalNAc, UDP-Glc, UDP-Glc-NH ₂, UDP-GlcNAc, UDP-Glc, UDP-GlcUA and CMP-Sia.Sugar of the present invention as discussed above, sugar nucleotide of the present invention can be aggregated thing modification part (or connecting base-modification part) and replace in any position of sugar.For example, the compound that is covered by the present invention comprises those wherein L-R ¹¹Part is attached to the compound of the C-6 of the C-5 of furanose class nucleotide sugar or pyranose class nucleotide sugar.

The illustrative part that is connected to binding substances disclosed herein include, but are not limited to the PEG derivative (as, alkyl-PEG, acyl group-PEG; acyl group-alkyl-PEG, alkyl-acyl group-PEG formamyl-PEG, aryl-PEG); the PPG derivative (as, alkyl-PPG, acyl group-PPG; acyl group-alkyl-PPG, alkyl-acyl group-PPG formamyl-PPG, aryl-PPG); the treatment part, diagnosis part, Man-6-P ester; heparin, heparitin, SLe _x, seminose, Man-6-P ester, saliva acidic group Lewis X, FGF, VFGF, protein, chrondroitin, keratin, dermatan, albumin, integrin (integrins), feeler oligose, peptide etc.Is that those skilled in the art obtain easily that (poly-(ethylene glycol) chemistry: biotechnology and biologic medical are used, J.Milton Harris, Ed., Plenum Pub.Corp., 1992 in conjunction with various modification groups to the method for sugar moieties; Poly-(ethylene glycol) chemistry and medical applications, J.Milton Harris, Ed., ACS symposium series No.680, American Chemical Society, 1997; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, SanDiego, 1996; With people such as Dunn, Bds. polymeric medicine delivery system, ACS symposium series Vol.469, American Chemical Society, Washington, D.C.1991).

With their the of the present invention illustrative sugar nucleotide of modified form comprise the Nucleotide list-, two-or triguaiacyl phosphate or its UDP-glucosides, CMP-glucosides, or the analogue of GDP-glucosides.Even more preferably, modified sugars Nucleotide is selected from the UDP-semi-lactosi, UDP-GalN, UDP-glucose, UDP-glycosamine, GDP-seminose, GDP-Fucose, cmp sialic acid, or CMP-NeuAc.The N-acetyl amine derivative of sugar nucleotide also is used for method of the present invention.

In other embodiments, modified sugars is an activation sugar.Being used for activation modification sugar of the present invention is changed to comprise the glucosides that activates leavings group by synthetic typically.Those parts represented in term " activation leavings group " as used herein, and they shift in the nucleophilic substitution reaction that enzyme is regulated easily.Many activation sugar are known in the art.Referring to, people such as Vocadlo for example, carbohydrate chemistry and biology, Vol.2, people Ed. such as Erns t, Wiley-VCH Verlag:Weinheim, Germany, 2000; People such as Kodama, Tetrahedron Lett.34:6419 (1993); Lougheed waits the people, J.Biol.Chem.274:37717 (1999)).

The example of activating group (leavings group) comprises fluorine, chlorine, bromine, tosylate, methanesulfonates, triflate etc.Be used for preferred activation leavings group of the present invention and be steric restriction glucosides indistinctively shifts to the enzyme of acceptor those.Therefore, the preferred embodiment of activation glycosides derivatives comprises glycosyl fluorochemical and glycosyl methanesulfonates, preferred especially glycosyl fluorochemical.In the glycosyl fluorochemical, α-galactosyl fluorochemical most preferably, α-mannose group fluorochemical, alpha-glucose-based fluorochemical, α-fucosido fluorochemical, α-xylosyl fluorochemical, α-saliva acidic group fluorochemical, α-N-acetyl glucosamine amido fluorochemical, α-N-acetylgalactosamine base fluorochemical, beta galactose base fluorochemical, β-mannose group fluorochemical, β-glucosyl group fluorochemical, β-fucosido fluorochemical, β-xylosyl fluorochemical, β-saliva acidic group fluorochemical, β-N-acetyl glucosamine amido fluorochemical, β-N-acetylgalactosamine base fluorochemical.

According to explanation, the glycosyl fluorochemical can be from free sugar by the sugar of ethanoylization at first and adopt the HF/ pyridine to handle it then to prepare.The stable end groups isomer of the thermodynamics of this generation protected (ethanoylization) glycosyl fluorochemical (that is salmefamol fluorochemical).Stablize less anomer (that is, β-glycosyl fluorochemical) if desired, it can be prepared by following mode: adopt HBr/HOAc or adopt HCl to transform the sugar of ethanoylization to produce anomer bromide or muriate.This intermediate and fluoride salt (as silver fluoride) reaction is to produce the glycosyl fluorochemical.Ethanoyl glycosyl fluorochemical can be by separating protection with the reaction of gentle (catalysis) alkali in methyl alcohol (as NaOMe/MeOH).In addition, many glycosyl fluorochemicals are commercially available.

Other activatory glycosyl derivatives can use ordinary method preparation well known by persons skilled in the art.For example, the glycosyl methanesulfonates can be prepared by following mode: the complete benzyl hemiacetal form of sugar is handled with methylsulfonyl chloride, and subsequent catalytic hydrogenation is to remove benzyl.

In further illustrative embodiment, modified sugars is the oligose with feeler structure.In another embodiment, one or more ends of feeler have the modification part.When partly being connected to oligose with feeler structure more than one modification, " amplification " modification part that oligose can be used for; Each the oligose unit that is attached to peptide is connected to the modification group of multiple copied on the peptide.The universal architecture of the typical binding substances of the present invention that illustrates among the above figure comprises by adopting the feeler structure to prepare the multivalence material that binding substances of the present invention produces.Many feeler sugar structures are known in the art, and present method can adopt them to implement ad lib.

In illustrative embodiment, the activatory modified sugars is the substrate of many variant enzymes, and this enzyme shifts sugar to the suitable receptor site of substrate.Illustrative mutant enzyme comprises, those as describing among open WO03/046150 of the PCT of common transfer and the W003/045980.

Wherein (water-soluble polymers modified sugars, activation sugar and nucleotide sugar material as the water-soluble polymers modification are used for the present invention to sugar moieties by water-soluble polymers.Illustrative modified sugars Nucleotide has the glycosyl by amine moiety modification on the sugar.Modified sugars Nucleotide (as the glycosyl-sulfonamide derivatives of sugar nucleotide) also is applicable to method of the present invention.For example, glycosyl amine (not having the modification group) can enzyme process be attached to peptide (or other material) and free glycosyl amine moiety is attached to required modification group subsequently.Perhaps, modified sugars Nucleotide can play enzyme substrates, and this enzyme shifts the glycosyl acceptor of modified sugars to the substrate, as peptide, glycopeptide, lipid, aglycon (aglycone), glycolipid etc.

In one embodiment, sugar is attached to the branched polymer material, as described in this those.

In another embodiment, sugar moieties is the modification sialic acid.When sialic acid is sugar, sialic acid be modified group on acetonyl (pyruvyl) side chain the 9-position or the 5-position modification on amine moiety, the acetylize in sialic acid normally of this amine moiety.

In another embodiment, wherein sugar nuclear is semi-lactosi or glucose, R ⁵Be NHC (O) Y.

In illustrative embodiment, modified sugars is based on 6-amino-N-ethanoyl-glycosyl part.For shown in the N-acetylgalactosamine, 6-amino-sugar moieties is prepared by standard method easily as following:

(a. galactose oxidase; NH ₄OAc, NaBH ₃CN; B.

In above scheme, mark n represents 1-2500, preferably 10-1500 and the more preferably integer of 10-1200.Symbol " A " expression activating group is as the component of halogen, the Acibenzolar component of (as, N-hydroxy-succinamide ester), carbonic ether (as, p-nitrophenyl carbonic ether) etc.Those skilled in the art will recognize that other PEG-acid amides nucleotide sugar prepares with similar approach easily thus.In addition, as described herein, branched polymer can replace linear PEG.

The illustrative polymer modification nucleotide sugar of another kind of the present invention has following general formula, and wherein the C-6 position is modification:

X wherein ⁶Be key or O, J is S or O, and y is 0 or 1.Mark e and f are independently selected from 1-2500.

In other illustrative embodiment, amide moieties is replaced by group (as urethane or urea).

In the following discussion, to being applicable to that many object lessons of implementing modified sugars of the present invention are described.In illustrative embodiment, the sugar nuclear that sialic acid derivative is connected thereto as the modification group.Discussion emphasis to sialic acid derivative is the scope that only should not be construed as limiting the invention in order clearly to illustrate.Those skilled in the art will recognize that various other sugar moieties can adopt is similar to the mode of using sialic acid to illustrate as an example and activates and derive.For example, many methods can be used for the modification semi-lactosi, glucose, and N-acetylgalactosamine and Fucose are to name some sugared substrates, and they are easy to by art-recognized method modification.Referring to, people such as Elhalabi for example, Curr.Med.Chem.6:93 (1999); With people such as Schafer, J.Org.Chem.65:24 (2000)).

In Fig. 2, described according to general scheme of the present invention.Therefore, according to Fig. 2, the acid amides binding substances between mannosamine and shielded amino acid is formed by following mode: the amino acid that contacts mannosamine and N-protected under suitable condition is to form binding substances.Shielded amino acid whose C-terminal in-situ activation or it randomly change into the reactive group of shelf-stable, as N-hydroxyl-succinimide.Amino acid can be selected from any natural or alpha-non-natural amino acid.How those skilled in the art's understanding protects side chain amino acid to avoid undesirable reaction in the method for the invention.The acid amides binding substances becomes sialic acid acid amides binding substances with the acetylneuraminate aldolase reaction to transform the acid amides binding substances with pyruvate salt under suitable condition, it by sialic acid acid amides binding substances and nucleotide phosphodiesterase precursor and the suitably reaction of enzyme, changes into nucleotide phosphodiesterase sialic acid acid amides binding substances subsequently.In illustrative embodiment, precursor is a cytidine triphosphate(CTP) and enzyme is a synthetic enzyme.After the formation of nucleotide sugar, amino acid amide is separated protection, and free, reactive amine are provided.Amine is used as the position of partly arriving nucleotide sugar in conjunction with modification.In Fig. 2, illustrative modification partly is a water-soluble polymers, i.e. poly-(ethylene glycol), as PEG, m-PEG etc.

The present invention is further in Fig. 3 illustrated, and it has described the scheme of preparation sialic acid-glycyl-PEG-cytidine monophosphate.Similar in appearance to scheme illustrated in fig. 2, Fig. 3 is derived from mannosamine.Sugar adopts the FMOC-glycine, uses the combination of shielded amino acid whose N-hydroxy-succinamide activated derivatives.The effect of binding substances and pyruvate salt is changed into the acid amides binding substances that obtains the sialic acid of correspondence by acetylneuraminate aldolase.Use cytidine triphosphate(CTP) and synthetic enzyme that the sialic acid conjugate that obtains is changed into the cytidine monophosphate analogue.Separate protection CMP-analogue by remove blocking group from the amino acid amine moiety, this part is changed into the reactive moieties that is used for association reaction (conjugation).Amine moiety and activated PEG material (m-PEG-O-nitrophenyl carbonate) reaction forms sialic acid-glycyl-PEG-cytidine monophosphate thus.

Have following general formula based on sialic illustrative sugar nuclear:

Wherein D is-OH or (R ¹¹) _w'-L-.Symbol G represents H, (R ¹¹) _w'-L-or-C (O) (C ₁-C ₆) alkyl.R ¹¹As previously discussed.Among D and the G at least one is R ¹¹-L-.

In another embodiment, the invention provides sugar, activation sugar or the sugar nucleotide that comprises following structure:

L wherein ²As to described in the argumentation of L, as key, replacement or unsubstituted alkyl or replacement or unsubstituted assorted alkyl.Mark e represents the integer of 1-about 2500.

In another embodiment, sugar or sugar nucleotide comprise following structure:

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

Have following general formula by the functionalized selection of branched polymer based on sialic nucleotide sugar:

Wherein AA is an amino-acid residue, and PEG is poly-(ethylene glycol) or methoxyl group-poly-(ethylene glycol) and NP is a Nucleotide, and it is connected to glycosyl part by phosphodiester bond (" nucleotide phosphodiesterase ").Those skilled in the art will recognize that ONP can be substituted by the activated partial in this discussion.

In further embodiment, the structure of sialic acid derivative is to be selected from following member:

X wherein ⁶Be key or O, and J is S or O.Mark a, b and c are independently selected from 0-20, and e and f are independently selected from 1-2500.

In addition, as discussed above, the invention provides nucleotide sugar by the water-soluble polymers modification, it is straight chain or branching.For example, the compound with general formula shown below is within the scope of the invention:

X wherein ⁶Be key or O, and J is S or O.Mark e and f are independently selected from 1-2500.

The peptide that comprises the present composition and the binding substances of glycopeptide, lipid and glycolipid also are provided.Binding substances is formed by following mode; Under suitable condition in conjunction with the suitable acceptor portion and the enzyme (modified ribonucleotide sugar is the substrate of this enzyme) of nucleotide sugar of the present invention or activation sugar and substrate and sugar moieties, so that modified sugars is transferred on the acceptor portion from nucleotide sugar.For example, the invention provides binding substances with following general formula:

Wherein J and X ⁶As discussed above.Mark a, b, c, e and f are as discussed above.

The compound-base of selection of the present invention is in the stereochemical material with seminose, semi-lactosi and glucose.The general formula of these compounds is:

R wherein ³-R ⁶One of be the modification part, as the basic construction of polymer modification part or polymer modification part-connect.

As discussed above, some compound of the present invention is the sugar nucleotide of polymer modification.With their modified form be used for illustrative sugar nucleotide of the present invention comprise the Nucleotide list-, two-or triphosphoric acid or its analogue.In preferred embodiments, modified sugars Nucleotide is selected from the UDP-glucosides, CMP-glucosides, or GDP-glucosides.Even more preferably, modified sugars Nucleotide is selected from the UDP-semi-lactosi, UDP-GalN, UDP-glucose, UDP-glycosamine, GDP-seminose, GDP-Fucose, cmp sialic acid, or CMP-Sia.In illustrative embodiment, Nucleotide list-two-or three-phosphoric acid be connected to C-1.

Glycosyl-the sulfonamide derivatives of sugar nucleotide also is applicable to method of the present invention.For example, glycosyl amine (not having the modification group) can enzyme process be attached to peptide (or other material) and free glycosyl amine moiety is attached to required modification group subsequently.

Sugar nucleotide binding substances of the present invention is described by following general formula usually:

Symbol expression group as discussed above wherein.When sugar nuclear was seminose, the polymer modification part was preferably at R ³, R ⁴Or R ⁶For glucose, the polymer modification part is randomly at R ⁵Or R ⁶Mark " u " is 0,1 or 2.

Of the present invention further illustrative nucleotide sugar based on the GDP seminose has following structure:

In further illustrative embodiment, the invention provides the binding substances that has following structure based on the UDP semi-lactosi:

In another illustrative embodiment, nucleotide sugar is based on glucose and have following general formula:

In each of three aforementioned formula, the identity of group and mark as discussed above.

It will be apparent for a person skilled in the art that linear peg moiety can be substituted by branched polymer or other linear polymer material described herein.

In one embodiment, wherein sugar nuclear is semi-lactosi or glucose, R ⁵Be NHC (O) Y.

Insoluble polymer

In another embodiment, be similar to discussed above those, modified sugars comprises insoluble polymer, rather than water-soluble polymers.Insoluble polymer as water-soluble polymers, typically is made up of at least two polymer units.In an illustrative embodiment, polymkeric substance is made up of 2-25 polymer unit.In another illustrative embodiment, polymkeric substance is made up of 2-8 polymer unit.Binding substances of the present invention also can comprise one or more insoluble polymers.This embodiment of the present invention illustrates as carrier by using binding substances, adopts this carrier delivering therapeutic peptide in a controlled manner.(transmit, delivery) system is known in the art in the polymeric medicine conveying.Referring to, people such as Dunn for example, Eds. polymeric medicine and delivery system, ACS symposium series Vol.469, American Chemical Society, Washington, D.C.1991.Those skilled in the art will recognize that any basically known drug delivery system all is applicable to binding substances of the present invention.

Representative insoluble polymer includes, but are not limited to poly-phosphine piperazine, poly-(vinyl alcohol), polymeric amide, polycarbonate, polyalkylene, polyacrylamide, polyalkylene glycol, polyalkylene oxide, polyalkylene terephthalate, polyvingl ether, polyvinylesters, polyvinylhalide, polyvinylpyrrolidone, polyglycolide, polysiloxane, urethane, poly-(methyl methacrylate), poly-(Jia Jibingxisuanyizhi), poly-(butyl methacrylate), poly-(Propenoic acid, 2-methyl, isobutyl ester), poly-(N-Hexyl methacrylate), poly-(isodecyl methacrylate), poly-(lauryl methacrylate(LMA)), poly-(phenyl methacrylate), poly-(methyl acrylate), poly-(isopropyl acrylate), poly-(isobutyl acrylate), poly-(vinylformic acid stearyl), polyethylene, polypropylene, poly-(ethylene glycol), poly-(oxyethane), poly-(ethylene glycol terephthalate), poly-(vinyl acetate), polyvinyl chloride, polystyrene, Polyvinylpyrolidone (PVP), poloxamer class and polyvinylphenol and multipolymer thereof.

The synthesis modification natural polymer that is used for binding substances of the present invention includes, but are not limited to alkylcellulose, hydroxy alkyl cellulose, ether of cellulose, cellulose ester and Nitrocellulose.The preferred especially member of one big class synthesis modification natural polymer includes, but are not limited to the polymkeric substance and the Lalgine of methylcellulose gum, ethyl cellulose, hydroxypropylcellulose, Vltra tears, hydroxy butyl methyl cellulose, rhodia, cellulose propionate, cellulose acetate butyrate, Cellacefate, carboxymethyl cellulose, cellulose triacetate, cellulose sulfate sodium salt and vinylformic acid and methacrylic ester.

These and other polymkeric substance in this discussion can be easily from commercial source such as SigmaChemical Co. (St.Louis, MO.), Polysciences (Warrenton, PA.), Aldrich (Milwaukee, WI.), Fluka (Ronkonkoma, NY), and BioRad (Richmond, CA) obtain, or use standard technique synthetic from the monomer that these suppliers obtain.

The representative biodegradable polymer that is used for binding substances of the present invention comprises, but be not limited to polylactide, polyglycolide and multipolymer thereof, gather (ethylene glycol terephthalate), gather (butyric acid), gather (valeric acid), gather (lactide-be total to-caprolactone), gather (lactide-be total to-glycollide), gather acid anhydrides, poe, its blend and multipolymer.Useful especially is the composition that forms gel, as comprises collagen, those of poloxamer class etc.

Be used for polymkeric substance of the present invention and comprise " heterozygosis " polymkeric substance, this heterozygosis polymkeric substance is included in and contains the biological water-soluble material that absorbs molecule again at least a portion of their structures.The example of such polymkeric substance is the polymkeric substance that comprises water-insoluble copolymer, each polymer chain of this multipolymer contain can biology absorption region, hydrophilic region and a plurality of crosslinkable functionality again.

For purpose of the present invention, " water-insoluble material " comprises the material of water insoluble substantially or aqueous environment.Therefore, although some zone of multipolymer or segment can be wetting ability or even water miscible, polymer molecule as a whole any basic measure water insoluble.

For purpose of the present invention, term " can biology absorb molecule again " comprises the zone (part) of can metabolism or destroying and absorbing and/or eliminate by the normal excretion pathway of health.Such meta-bolites or degraded product are atoxic substantially to health preferably.

Can biology again the absorption region can be hydrophobicity or hydrophilic, as long as multipolymer does not become water miscible as a whole.Therefore, can make polymkeric substance water insoluble as a whole according to preferential the selection in the absorption region again by biology.Therefore, select relative performance, promptly by the kind of the functional group that can biological absorption region again comprises and can biological absorption region again with the relative proportion of hydrophilic region with guarantee useful can biology the absorbing composition maintenance is water-insoluble again.

Illustrative resorbable polymers comprises, the synthetic production of for example poly-(Alpha-hydroxy-carboxylic acid)/poly-(oxyalkylene) can absorb segmented copolymer (referring to people such as Cohn, U.S. Patent number 4,826,945) again.These multipolymers are not crosslinked and be water miscible, thereby make health can drain the block copolymer composition of degraded.Referring to people such as Younes, J Biomed.Mater.Res.21:1301-1316 (1987); With people such as Cohn, J Biomed.Mater.Res.22:993-1009 (1988).

At present preferred bioresorbable polymer comprises that one or more are selected from following component: poly-(ester), poly-(alcohol acid), poly-(lactone), poly-(acid amides), poly-(ester-acid amide), poly-(amino acid), poly-(acid anhydrides), poly-(ortho ester), poly-(carbonic ether), poly-(phosphine piperazine), poly-(phosphoric acid ester), poly-(monothioester), polysaccharide and composition thereof.Still more preferably, bioresorbable polymer comprises poly-(hydroxyl) acid constituents.In poly-(hydroxyl) acid, preferably poly(lactic acid), polyglycolic acid, poly-caproic acid, poly-butyric acid, poly-valeric acid and multipolymer and mixture.

Except that absorbing the fragment of (" biology absorbs again ") in the organizer,, can secrete and/or the metabolism fragment preferred polymers dressing that is used for the inventive method but also can forming.

More high-grade multipolymer also can be used for the present invention.For example, people such as Casey, U.S. Patent number 4,438,253, it is open on March 20th, 1984, discloses three-segmented copolymer of producing from the transesterify of poly-(oxyacetic acid) and hydroxy-end capped poly-(aklylene glycol).Such composition is disclosed as absorbing monofilament linea suturalis again.The handiness of composition is by the introducing control of aromatics orthocarbonic ester (as four-p-methylphenyl orthocarbonic ester) in copolymer structure like this.

Also can adopt other polymkeric substance based on lactic acid and/or oxyacetic acid.For example; Spinu; U.S. Patent number 5,202,413; it is open on April 13rd, 1993; disclose biodegradable segmented copolymer, this multipolymer contains the stereo-block in order of polylactide and/or polyglycolide, by the ring-opening polymerization on oligomer diol or the diamine of lactide and/or glycollide; adopt difunctional's compound subsequently, as the chain extension of vulcabond, diacyl chlorine or dichlorosilane and produce.

What be used for dressing of the present invention can biological absorption region again can be designed as hydrolyzable and/or enzymatic lysis.For purpose of the present invention, " hydrolyzable cracked " expression multipolymer, especially can biological absorption region again to the susceptibility of hydrolysis in water or the aqueous environment.Similarly, " but enzymatic lysis " expression multipolymer as used herein, particularly can biological absorption region again to cracked susceptibility by endogenous or exogenous enzyme.

When putting into body,, can drain and/or the metabolism fragment hydrophilic region but can being processed into.Therefore, hydrophilic region can comprise, for example polyethers, polyalkylene oxide, polyvalent alcohol, poly-(vinyl pyrrolidone), poly-(vinyl alcohol), poly-(alkyl _ azoles quinoline), polysaccharide, carbohydrate, peptide, protein and multipolymer and mixture.In addition, hydrophilic region also, polyalkylene oxide for example.Such polyalkylene oxide can comprise, for example gathers (oxyethane), poly-(propylene oxide) and composition thereof and multipolymer.

The polymkeric substance that is the hydrogel component also is used for the present invention.Hydrogel is the polymer materials that can absorb the relatively large number water gaging.The example that hydrogel forms compound includes, but are not limited to polyacrylic acid, Xylo-Mucine, polyvinyl alcohol, Polyvinylpyrolidone (PVP), gelatin, carrageenin and other polysaccharide, hydroxy ethylene methacrylic acid (HEMA), with and derivative etc.Can produce stablely, can biological reduce and can biological resorbent hydrogel.In addition, hydrogel composition can comprise the subunit that shows one or more these performances.

Its globality can be known by the bio-compatibility hydrogel composition of crosslinked control and be preferred for method of the present invention at present.For example, people such as Hubbell, U.S. patent Nos.5,410,016, it is in April 25,1995 and 5,529,914, it is open on June 25th, 1996, discloses water-soluble system, and this system is to contain the water-soluble central block segmental cross-linked block copolymer that sandwiches between two hydrolytically unstable extensions.Such multipolymer is further by the acrylate-functional groups end-blocking of photopolymerization.When crosslinked, these systems become hydrogel.The water-soluble central block of multipolymer can comprise poly-(ethylene glycol) like this; Yet the hydrolytically unstable continuation can be poly-(alpha hydroxy acid), as polyglycolic acid or poly(lactic acid).Referring to people such as Sawhney, Macromolecules 26:581-587 (1993).

In another embodiment, gel is a heat reversible gel.Preferably comprise component at present, as the heat reversible gel of poloxamer class, collagen, gelatin, hyaluronic acid, polysaccharide, polyurethane hydrogel, polyurethane-urea hydrogel and combination thereof.

In another illustrative embodiment still, binding substances of the present invention comprises the component of liposome.Liposome can prepare according to method known to those skilled in the art, for example people such as Eppstein, and U.S. Patent number 4,522, described in 811, the document is open on June 1st, 1985.For example; Liposomal formulation can be prepared by following mode: dissolve suitable lipid (as stearyl-phosphatidylethanolamine, stearyl-phosphatidylcholine in inorganic solvent; peanut acyl phospholipids phatidylcholine; and cholesterol); evaporate this solvent then, on vessel surface, stay the film of dried lipid.Then the aqueous solution of active compound or its pharmaceutical salts is introduced container.By hand container is eddied with from container side release matrix material and dispersion lipid aggregate then, therefore form liposome suspension.

Above-mentioned micropartical and the atomic method of preparation provide with them by example does not wish to be defined for atomic scope of the present invention.To it will be apparent to one skilled in the art that the atomic array by the different methods manufacturing is used for the present invention.

Architecture discussed above also is suitable for usually for insoluble polymer in the argumentation of the water-soluble polymers of straight chain and branching.Therefore, for example halfcystine, Serine, two Methionins and three Methionin branching are endorsed with functionalized by two insoluble polymer parts.The method that is used to produce these materials is similar to those that are used to produce water-soluble polymers usually very much.

The transformation period also can be improved by peg moiety such as polyoxyethylene glycol (PEG) in the body of therapeutic glycopeptide.For example, protein has been increased their molecular size by the chemical modification (PEGization) of PEG and has reduced their surface and functional group-accessibility, they each depend on the size that is connected to proteinic PEG.This causes the improvement of plasma half-life and causes proteolysis stability, and the reduction that absorbs of immunogenicity and liver (people J.Clin.Invest.89:1643-1651 (1992) such as Chaffee; People Res.Commun.Chem.Pathol Pharmacol.29:113-127 (1980) such as Pyatak).There is the PEGization of report interleukin II to increase anti-knurl effectiveness in its body people Proc.Natl.Acad.Sci.USA.84:1487-1491 (1987) such as () Katre and derived from its tumor-localizing of PEGization improvement of the F (ab ') 2 of monoclonal antibody A7 people Biochem.Biophys.Res.Commun.28:1387-1394 (1990) such as () Kitamura.Therefore, in another embodiment, adopt peg moiety to increase to some extent with respect to the transformation period in the body of non-derived peptide with the transformation period in the body of method deutero-peptide of the present invention.

The per-cent increase of quantity is for this reason preferably expressed in the increase of transformation period in the peptide body.The lower limit that per-cent increases scope is about 40%, about 60%, about 80%, about 100%, about 150% or about 200%.The upper limit of scope is about 60%, and is about 80%, about 100%, about 150%, or greater than about 250%.

The preparation of modified sugars

Usually, sugar moieties and modification group link together by using reactive group, and this reactive group typically is transformed into new organo-functional group or non-reactive material by connection procedure.The sugar reactive functional groups is positioned at any position on the sugar moieties.Be used to implement reactive group of the present invention and the reaction classification normally the bioconjugation chemical field known those.The present favourable reaction type that can be used for the reactive sugars part is those that carry out under relative mild conditions.These include, but are not limited to nucleophilic substitution (as, amine and alcohol and carboxylic acid halides, the reaction of active ester), electrophilic substitution (as, enamine reaction) and to the addition of carbon-to-carbon and carbon-heteroatoms multikey (as, Michael reaction, Diels-Alder reaction).These and other useful reaction is discussed at, March for example, senior organic chemistry, 3rd Ed., John Wiley ﹠amp; Sons, New York, 1985; Hermanson, bioconjugation technology, Academic Press, San Diego, 1996; With people such as Feeney, protein-modified; Chemical progress series, Vol.198, American Chemical Society, Washington, D.C., 1982.

The useful reactive functional groups that dangles from sugar nuclear, connection based precursor or polymer modification part precursor includes, but are not limited to:

(a) carboxyl and various derivative thereof include, but are not limited to the N-hydroxy-succinamide ester, N-hydroxybenzotriazole ester, carboxylic acid halides, acylimidazole, monothioester, p-nitrophenyl ester, alkyl, thiazolinyl, alkynyl and aromatic ester;

(b) carboxyl, it can change into, as ester, ether, aldehyde etc.

(c) haloalkyl, wherein halogenide can after by nucleophilic group, therefore for example amine, carboxylate anion, mercaptan negatively charged ion, carbanion or alkoxide ion displacement causes new group covalently bound in halogen atom functional group;

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

(e) aldehydes or ketones base makes deriving by carbonyl derivative as, imines for example subsequently, hydrazone, and the formation of semicarbazone or oxime, or be possible by mechanism as Ge Shi addition or lithium alkylide addition;

(f) be sulfonic acid halide with amine with afterreaction (for example to form sulphonamide);

(g) thiol group, it can, for example change into disulphide or react with carboxylic acid halides;

(h) amine or sulfydryl, it can, acylations for example, alkylation or oxidation;

(i) alkene, it can experience, for example cycloaddition, addition, Michael addition etc.; With

(j) epoxide, it can with, for example amine and oxy-compound the reaction.

Can selective reaction functional group make them not participate in, or disturb the reaction that to assemble reactive sugars nuclear or modification group.Perhaps, can by blocking group exist protective reaction functional group with do not participate in the reaction.How those skilled in the art's understanding protects specific functional group to make it not disturb the complete reaction conditions of selection.For the example of useful blocking group referring to, people such as Greene for example, the blocking group in the organic synthesis, John Wiley ﹠amp; Sons, New York, 1991.

In the following discussion, the object lesson that is used to implement modified sugars of the present invention is described.In illustrative embodiment, the sugar nuclear that sialic acid derivative is connected thereto as the modification group.Discussion emphasis to sialic acid derivative is the scope that only should not be construed as limiting the invention in order clearly to illustrate.Those skilled in the art recognize that various other sugar moieties can adopt is similar to the mode of using sialic acid to illustrate as an example and activates and derive.For example, many methods can be used for the modification semi-lactosi, glucose, and N-acetylgalactosamine and Fucose are to name several sugared substrates, and they are easily by art-recognized method modification.Referring to, people such as Elhalabi for example, Curr.Med.Chem.6:93 (1999); With people such as Schafer, J.Org.Chem.65:24 (2000)).

In following scheme 1, with aminoglycoside 1Adopt the active ester of shielded amino acid (as, glycine) derivative to handle, the osamine residue is changed into corresponding shielded amino acid amide adducts.Adopt zymohexase to handle adducts to form alpha-hydroxycarboxylic ester 2.With compound 2Effect by the CMP-SA synthetic enzyme changes into corresponding CMP derivative, and the catalytic hydrogenation of carrying out the CMP derivative subsequently is with the production compound 3Pass through compound 3With activatory (m-) PEG or (m-) the PPG derivative (as, PEG-C (O) NHS, PPG-C (O) NHS) reaction, the amine that the formation by glycine adduct is introduced produces respectively as the position that PEG or PPG connect 4Or 5

Scheme 1

As those skilled in the art recognize that, polymer modification part also can be the branching part, as described here those.

The illustrative scheme for preparing branched polymer modified sugars of the present invention below is provided:

Below explanation prepares another illustrative scheme of polymer modification sugar of the present invention:

Table 1 explanation by the polymer modification part (as branching-or straight chain PEG or PPG part) representative example of deutero-sugar phosplate.Some compound of table 1 is by the method preparation of scheme 1.Other derivative is by art-recognized method preparation.Referring to, people such as Keppler for example, Glycobiology11:11R (2001); With people such as Charter, Glycobiology 10:1049 (2000)).Other amine reactive polymer modification part precursor and component are commercially available as PEG and PPG analogue, and perhaps they can be prepared by the method that those skilled in the art obtain easily.

Table 1

Wherein R is polymkeric substance (branching or straight chain) modification part.

Being used to implement modified sugars phosphoric acid ester of the present invention can be substituted at those of other position and above explanation.Sialic preferably being substituted in the following general formula at present illustrates:

Wherein one or more X ^c, Y ^a, Y ^b, Y ^cWith Z be linking group, it is preferably selected from-O-,-N (H)-,-S, CH ₂-, and N (R) ₂Work as X ^c, Y ^a, Y ^b, Y ^cWhen being linking group with Z, it is connected to the polymer modification part, as R ^c, R ^d, R ^e, R ^fAnd R ^gShown in.Perhaps, these symbolic representations are attached to that branching-or straight chain is water-soluble or the connection base of insoluble polymer, treatment part, biomolecules or other parts.Work as R ^c, R ^d, R ^e, R ^fOr R ^gWhen not being the polymer modification part, X ^cR ^c, Y ^aR ^d, Y ^bR ^e, Y ^cR ^fOr ZR ^gCombination be H, OH or NC (O) CH ₃

The method of producing activation sialic acid-polymer modification group binding substances also is provided, and this binding substances is the suitable substrate of enzyme, and this enzyme shifts modified sugar moieties to acceptor, as glycosyltransferase.This method comprises the steps: that (a) makes amino acid (or by the polymer modification part, connect based precursor or connect base-functionalized amino acid of the polymer modification part molectron) contact of mannosamine and activatory N-protected to form the acid amides binding substances between the amino acid of mannosamine and N-protected under suitable condition; (b) acid amides binding substances and pyruvate salt are contacted to transform the acid amides binding substances with acetylneuraminate aldolase and become sialic acid acid amides binding substances; (c) sialic acid acid amides binding substances and cytidine triphosphate(CTP) are contacted to form cytidine monophosphate sialic acid acid amides binding substances with synthetic enzyme; (d) remove the N-protected group from cytidine monophosphate sialic acid acid amides binding substances, therefore produce unhindered amina; (e) unhindered amina is contacted with activatory PEG (straight chain or branching), form cytidine monophosphate sialic acid-poly-(ethylene glycol) thus.

Crosslinked group

The preparation that is used for the modified sugars of the inventive method comprises the modification group to the connection of saccharide residue with form and stablize adducts, and it is the substrate of glycosyltransferase.Sugar and modification group can be by zero-or more senior linking agent couplings.Can be used for connecting the modification group and include but not limited to that to the illustrative difunctional compound of carbohydrate part difunctionality gathers (ethylene glycol), polymeric amide, polyethers, polyester etc.The connection carbohydrate is known in the document to the universal process of other molecule.Referring to, people such as Lee for example, Biochemistry 28:1856 (1989); People such as Bhatia, Anal.Biochem.178:408 (1989); People such as Janda, people such as J.Am.Chem.Soc.112:8886 (1990) and Bednarski, WO 92/18135.In the following discussion, reactive group is treated on the sugar moieties of newborn modified sugars benign.The emphasis of discussing is for clearly explanation.Those skilled in the art will recognize that discussion is also relevant with reactive group on the modification group.

Illustrative strategy relate to use Heterobifunctional crosslinking aid S PDP (n-succinimido-3-(2-pyridyl dithio) propionic ester is incorporated into protected sulfydryl on the sugar, separates the protection sulfydryl then, thus form with the modification group on the disulfide linkage of another sulfydryl.

Many reagent are applicable to that the component that adopts intramolecularly chemically crosslinked thing modification modified sugars is (for the summary of cross-linking reagent and cross-linking process referring to Wold, F., Meth.Enzymol.25:623-651,1972; Weetall, H.H., and Cooney, D.A., In:ENZYMES ASDRUGS. (Holcenberg, and Roberts, eds.) pp.395-442, Wiley, New York, 1981; Ji, T.H., Meth.Enzymol.91:580-609,1983; People such as Mattson, Mol.Biol.Rep.17:167-183,1993, all documents are hereby incorporated by).Preferred cross-linking reagent is derived from various distance of zero mark degree, equal-difunctionality and assorted-difunctionality cross-linking reagent.Zero-length cross-linking reagent comprises the direct combination (and not introducing outer material) of two inherent chemical groups.

Modified sugars is to the combination of peptide

Use the suitable enzyme of mediation bonded, modified sugars is attached to glycosylation or non-glycosylated peptide.Therefore, compound of the present invention, particularly nucleotide sugar be the substrate of enzyme preferably, and this enzyme shifts sugar moieties on amino acid, glycosyl or aglycon acceptor portion from nucleotide sugar.The sugar that is used as acceptor (as the galactosyl acceptor) is given the nucleotide sugar of body, and described galactosyl acceptor is GalNAc for example, Gal β 1,4GlcNAc, Gal β 1,4GalNAc, Gal β 1,3GalNAc, lactose-N-tetrose, Gal β 1,3GlcNAc, Gal β 1,3Ara, Gal β 1,6GlcNAc, Gal β 1,4Glc (lactose), and well known to a person skilled in the art other acceptor (referring to,, J.Biol.Chem.253:5617-5624 (1978)) as people such as Paulson.

Modified ribonucleotide sugar of the present invention is that the illustrative enzyme of its substrate comprises glycosyltransferase.Glycosyltransferase can be cloned, or separates from any source.Many clones' glycosyltransferase is known, as their polynucleotide sequence.Referring to, as " WWW of clone's glycosyltransferase instructs, " ( Http:// www.vei.co.uk/TGN/gt guide.htm).Glycosyltransferase aminoacid sequence and also be found in various disclosed available databases from its nucleotide sequence that can reason out the encoding glycosyl transferring enzyme of aminoacid sequence comprises GenBank, Swiss-Prot, and EMBL, and other.

Compound of the present invention comprises for the glycosyltransferase of its substrate; but be not limited to galactotransferase, fucosyltransferase, glucanotransferase; the N-acetyl-galactosaminyl transferase; the N-acetylglucosaminyl transferase, glucuronyl transferase, sialytransferase; mannose transferase; glucuronyl transferase, galacturonic acid transferring enzyme, and oligosaccharyl transferase.Suitable glycosyltransferase comprises from eukaryote, and prokaryotic organism obtain those.

In some embodiments, compound of the present invention is the substrate of fucosyltransferase.Fucosyltransferase is that those skilled in the art are known usually, and illustrative is to shift the enzyme of L-Fucose to the acceptor saccharide hydroxy position from the GDP-Fucose.

In another group embodiment, compound is the substrate of galactotransferase.Illustrative galactotransferase comprises α (1,3) galactotransferase (E.C.No.2.4.1.151, referring to, as people such as Dabkowski, people Nature 345:229-233 (1990) such as Transplant Proc.25:2921 (1993) and Yamamoto, (GenBank j04989, the people such as Joziasse of ox, J.Biol.Chem.264:14290-14297 (1989)), muroid (GenBank m26925; People such as Larsen, Proc.Nat ' l.Acad.Sci.USA 86:8227-8231 (1989)), (the GenBank L36152 of pig; People such as Strahan, Immunogenetics 41:101-105 (1995)).Another kind of suitable α 1,3 galactotransferase relates to synthetic (EC2.4.1.37, people such as Yamamoto, the J.Biol.Chem.265:1146-1151 (1990) (mankind)) of Blood group antigen B.Still further illustrative galactotransferase is nuclear Ga1-T1.Still further example comprises β (1,4) galactotransferase, it comprises, EC2.4.1.90 (LacNAc synthetic enzyme) and EC 2.4.1.22 (lactose synthetase) ((people such as D ' Agostaro of ox for example, Eur.J.Biochem.183:211-217 (1989), human (people such as Masri, Biochem.Biophys.Res.Commun.157:657-663 (1988)), muroid (people such as Nakazawa, J.Biochem.104:165-168 (1988)), and E.C.2.4.1.38 and ceramide galactotransferase (EC 2.4.1.45, people such as Stahl, J.Neurosci.Res.38:234-242 (1994)).Other suitable galactotransferase comprises, for example α 1,2 galactotransferase (as from, Schizosaccharomyces pombe, people such as Chapell, Mol.Biol.Cell 5:519-528 (1994)).Also suitable in the invention process is α 1, the soluble form of 3-galactotransferase, and as by people such as Cho, J.Biol.Chem., 272:13622-13628 (1997) report.

A) sialytransferase

Sialytransferase is the another kind of type glycosyltransferase of compound of the present invention for its substrate.Example comprise ST3Gal III (as, mouse or human ST3Gal III), ST3Gal IV, ST3Gal I, ST6Gal I, ST3Gal V, ST6Gal II, ST6Gal NAc I, ST6Gal NAcII, with ST6Gal NAc III (the sialytransferase nomenclature is as being described in people such as Tsuji, Glycobiology 6:v-xiv (1996) as used herein).Illustrative α (2, the 3) sialytransferase that is called α (2,3) sialytransferase (EC2.4.99.6) is transferred to sialic acid the non-reduced terminal Gal of Gal β 1 → 3Glc disaccharides or glucosides.Referring to people such as Vanden Eijnden, J.Biol.Chem.256:3159 (1981), people such as Weinstein, people such as J.Biol.Chem.257:13845 (1982) and Wen, J.Biol.Chem.267:21011 (1992).Another kind of illustrative α 2,3-sialytransferase (EC 2.4.99.4) shift the non-reduced terminal Gal of sialic acid to disaccharides or glucosides.Referring to people such as Rearick, people such as J.Biol.Chem.254:4444 (1979) and Gillespie, J.Biol.Chem.267:21004 (1992).Further illustrative enzyme comprises Ga1-β-1,4-GlcNAc α-2,6 sialytransferase (referring to people Eur.J.Biochem.219:375-381 (1994) such as Kurosawa).Compound of the present invention comprises those that form Polysialic acid for other sialytransferase of its substrate.Example comprises α-2,8-Polysialic acid transferring enzyme, and as ST8SiaI, ST8SiaII, ST8SiaIII, ST8SiaIV and ST8SiaV.Referring to for example, people J.Biol.Chem.275:18594-18601 (2000) such as Angata; People such as Kono, J.Biol.Chem.271:29366-29371 (1996); People such as Greiner, Infect.Immun.72:4249-4260 (2004); With people such as Jones, J.Biol.Chem.277:14598-14611 (2002).

The example that is used for the sialytransferase of claimed method is ST3Gal III, and it is also referred to as α (2,3) sialytransferase (EC 2.4.99.6).This enzyme catalysis sialic acid arrives classification Gal β 1,3GlcNAc or classification Gal β 1, the transfer of the Gal of 4GlcNAc glucosides (referring to, as people such as Wen, J.Biol.Chem.267:21011 (1992); People such as Van den Eijnden, J.Biol.Chem.256:3159 (1991)).Still further sialytransferase comprise from campylobacter jejuni isolating those, comprise α (2,3).Referring to as W099/49051.

Preferably, compound of the present invention is the substrate of enzyme, and this enzyme shifts modification sialic acid to sequence Gal β 1,4GlcNAc-, the most common inferior final stage sequence below the sialic acid endways on complete sialylated carbohydrate structure.

B) GalNAc transferring enzyme

The compound of selection of the present invention is the substrate of N-acetyl-galactosaminyl transferase.Illustrative N-acetyl-galactosaminyl transferase comprises, but be not limited to α (1,3) N-acetyl-galactosaminyl transferase, β (1,4) N-acetyl-galactosaminyl transferase (people such as Nagata, people such as J.Biol.Chem.267:12082-12089 (1992) and Smith, J.Biol.Chem.269:15162 (1994) and Polypeptide N-acetylgalactosaminyltransferase (people such as Homa, J.Biol.Chem.268:12609 (1993)).

C) Glycosylase

The present invention also comprises the substrate for wild-type and mutant Glycosylase.The mutant beta-galactosidase enzymes has been shown as catalysis by the coupling formation disaccharides of alpha-glycosyl fluorochemical to the galactosyl acceptor molecule.(Withers, U.S.Pat.No.6284494; September 4 calendar year 2001).Be applicable to that other Glycosylase of the present invention comprises, for example beta-glucosidase, beta-galactosidase enzymes, beta-Mannosidase, β-acetylglucosamine enzyme, beta-N-Acetylgalactosaminidase, xylobiase, β-fucosidase, cellulase, zytase, Galactanase, mannase, hemicellulase, amylase, glucosaminidase, alpha-glucosidase, alpha-galactosidase, alpha-Mannosidase, α-N-acetylglucosamine enzyme, alpha-N-Acetylgalactosaminidase, α-xylosidase, Alpha-Fucosidase, and neuraminidase/sialidase, inscribe ceramide glucoside enzyme.

Embodiment

Provide following embodiment so that selectivity embodiment of the present invention to be described but should not be construed as the scope that limits it.

Embodiment

Embodiment 1

Preparation UDP-GalNAc-6 '-CHO

(200mg 0.30mmol) is dissolved in 1mM CuSO with UDP-GalNAc ₄Solution (20mL) and 25mM NaH ₂PO ₄(pH 6.0 for solution; 20mL).Add galactose oxidase (240U then; 240 μ L) and catalase (13000U; 130 μ L), the reactive system that will be equipped with air chamber is filled and was at room temperature stirred several days with oxygen.Filter reaction mixture (rotates box then; MWCO 5K) and with filtrate (～40mL) when storing down up to needs for 4 ℃.TLC (silicon-dioxide; EtOH/ water (7/2); R _f=0.77; Adopt aubepine dyeing to manifest).

Embodiment 2

Preparation UDP-Gal NAc-6 '-NH ₂):

With ammonium acetate (15mg, 0.194mmol) and NaBH ₃CN (1M THF solution; 0.17mL, 0.17mmol) 0 ℃ of UDP-Ga lNAc-6 '-CHO solution that obtains above adding down (2mL or～20mg) and allow to be warmed up to ambient temperature overnight.To react by the G-10 post and filter and collect water and product.With suitable fraction lyophilize and refrigerated storage.TLC (silicon-dioxide; Ethanol/water (7/2); R _f=0.72; Adopt ninhydrin reagent to manifest).

Embodiment 3

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

With semi-lactosi amido-1-phosphoric acid-2-NHCO (CH ₂) ₂-O-PEG-OMe (1KDa) (58mg, 0.045 mmole) is dissolved in DMF (6mL) and pyridine (1.2mL).Add UMP-morpholine acid esters (60mg, 0.15 mmole) then, the mixture that obtains is stirred 48h down at 70 ℃.Remove solvent by reaction mixture drum nitrogen, by the refining resistates (C-18 silicon-dioxide, step gradient 10-80%, methanol) of reverse-phase chromatography.Collect required fraction and drying under reduced pressure to obtain white solid.TLC (silicon-dioxide, propyl alcohol/H ₂O/NH ₄OH, (30/20/2), R _f=0.54).MS (MALDI): observed value, 1485,1529,1618,1706.

Embodiment 4

Preparation halfcystine-PEG ₂(2)

4.1 synthetic compound 1

Under argon gas, potassium hydroxide (84.2mg, 1.5mmol is as powder) added L-halfcystine (93.7mg, 0.75mmol) solution in anhydrous methanol (20L).Mixture is at room temperature stirred 30min and divide several sections in 2 hours, to add the mPEG-O-tosylate (Ts that molecular weight is 20 kilodaltons then; 1.0g, 0.05mmol).Mixture was at room temperature stirred 5 days and concentrate by rotary evaporation.Resistates is adopted water (30mL) dilution and at room temperature stirs 2 hours to destroy any 20 excessive kilodalton mPEG-O-tosylates.Solution is adopted the acetate neutralization then, and pH regulator is loaded on reverse-phase chromatography (C-18 silicon-dioxide) post to pH 5.0.Post is adopted the gradient elution (product is wash-out under about 70% methyl alcohol) of methanol, and the product wash-out is collected suitable fraction and is adopted water (500mL) to dilute by vapo(u)rability scattering of light monitoring.This solution is carried out stratographic analysis (ion-exchange, XK 50 Q, BIG Beads, 300ml, hydroxide form; Water is to the gradient of water/acetate-0.75N), adopts acetate to be reduced to 6.0 the pH of suitable fraction.Then this solution is gone up capture at reversed-phase column (C-18 silicon-dioxide), adopt above-mentioned methanol gradient elution.With the product fraction compile, concentrate, water-soluble and lyophilize to be to provide white solid (1) again.The structured data of compound is as follows: ¹H-NMR (500 MHz; D ₂O) δ 2.83 (t, 2H, O-C-C H ₂-S), 3.05 (q, 1H, S-C HH-CHN), 3.18 (q, 1H, (q, 1H, S-C HH-CHN), 3.38 (s, 3H, C H ₃O), 3.7 (t, OC H ₂C H ₂O), 3.95 (q, 1H, C HN).Degree of purity of production is confirmed by SDS PAGE.

4.2 synthetic compound 2 (halfcystine-PEG ₂)

With triethylamine (～0.5mL) be added drop-wise to and be dissolved in anhydrous CH ₂Cl ₂The solution of compound 1 (30mL) (440mg, 22 μ mol) is alkalescence up to solution.With 20 kilodalton mPEG-O-p-nitrophenyl carbonic ethers (660mg, 33 μ mol) and N-hydroxy-succinamide (3.6mg, 30.8 μ mol) at CH ₂Cl ₂Solution (20mL) divides several sections at room temperature to add in 1 hour.Reaction mixture was at room temperature stirred 24 hours.Remove solvent by rotary evaporation then,, adopt 1.0N NaOH to regulate pH to 9.5 resistates water-soluble (100mL).Basic solution was at room temperature stirred 2 hours, adopt the acetate pH 7.0 that neutralizes then.Then solution is loaded on reverse-phase chromatography (C-18 silicon-dioxide) post.Adopt methanol to carry out gradient elution (product is wash-out under about 70% methyl alcohol) on post,, collect suitable fraction and adopt water (500mL) dilution by vapo(u)rability scattering of light monitoring product wash-out.This solution is carried out stratographic analysis (ion-exchange, XK 50 Q, BIG Beads, 300ml, hydroxide form; Water is to the gradient of water/acetate-0.75N), adopts acetate to be reduced to 6.0 the pH of suitable fraction.Then this solution is captured and adopts above-mentioned methanol gradient elution on reversed-phase column (C-18 silicon-dioxide).With the product fraction compile, concentrate, water-soluble and lyophilize to be to provide white solid (2) again.The structured data of compound is as follows: ¹H-NMR (500MHz; D ₂O) δ 2.83 (t, 2H, O-C-C H ₂-S), 2.95 (t, 2H, O-C-C H ₂-S), 3.12 (q, 1H, S-C HH-CHN), 3.39 (s, 3H C H ₃O), 3.71 (t, OC H ₂C H ₂O).Degree of purity of production is confirmed by SDS PAGE.

Embodiment 5

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

With semi-lactosi amido-1-phosphoric acid-2-NHCO (CH ₂) ₂-O-PEG-OMe (1 kilodalton) (58mg, 0.045 mmole) is dissolved in DMF (6mL) and pyridine (1.2mL).Add UMP-morpholine acid esters (60mg, 0.15 mmole) then, the mixture that obtains was stirred 48 hours down at 70 ℃.Remove solvent by reaction mixture drum nitrogen, by the refining resistates (C-18 silicon-dioxide, step gradient 10-80%, methanol) of reverse-phase chromatography.Collect required fraction, drying under reduced pressure is to obtain white solid.TLC (silicon-dioxide, propyl alcohol/H ₂O/NH ₄OH, (30/20/2), R _f=0.54).MS (MALDI): observed value, 1485,1529,1618,1706.

SDS PAGE process

Product

1 and 2 purity are confirmed by SDS PAGE.Use 4-20%Tris-glycine SDSPAGE gel (Invitrogen).Sample is mixed with SDS sample buffer reagent at 1: 1, in Tris-glycine runtime buffer agent (LC2675-5), move 1 hour 50min down in constant voltage (125V).After electrophoresis, gel is adopted water (100mL) washing 10min, adopt 5% barium chloride solution (100mL) washing 10min subsequently.By adopting 0.1N iodine solution (4.0mL) gel that at room temperature dyes to manifest

product

1 or 2, stop dyeing course by washing gel with water.The production spectra that will manifest adopts HP Scanjet 7400C scanning, adopts HP Precision ScanProgram to optimize the image of gel.

Although disclose the present invention with reference to specific embodiment, other embodiment of the present invention clearly and version can not deviate from true spirit of the present invention and scope by those skilled in the art's design.

All patents, patent application and other publication quoted in this application are incorporated herein by reference for all purposes in full at this.

Claims

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

With

Wherein

R ¹Be H, CH ₂OR ⁷, COOR ⁷Or OR ⁷, wherein

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

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

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

R wherein ⁹And R ¹⁰Be independently selected from H, replacement or not substituted alkyl or replacement or unsubstituted assorted alkyl,

And R ³, R ⁴, R ⁵, R ⁶And R ⁶' at least one comprise the polymer modification part.

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

R wherein ⁸It is nucleosides.

3. according to the compound of claim 2, R wherein ⁸Be the member who is selected from cytosine(Cyt), uridine, guanosine, adenosine and thymidine.

4. according to the compound of claim 1, R wherein ³, R ⁴, R ⁵And R ⁶At least one comprise as the lower section:

R wherein ¹¹It is the polymer modification part;

L is the member who is selected from key and linking group; And

W is selected from the integer of 1-6.

5. according to the compound of claim 4, wherein this linking group is the member who is selected from replacement or unsubstituted alkyl and replacement or unsubstituted assorted moieties.

6. according to the compound of claim 5, wherein as the lower section:

Have general formula:

Wherein

X ²And X ⁴Be independently selected from junction fragment;

X ^aIt is junction fragment;

R ¹²And R ¹³It is the polymeric arms of selecting independently; And

C is the integer of 1-20.

7. according to the compound of claim 5, wherein this linking group has following general formula:

Wherein

X ^aAnd X ^bIt is the junction fragment of selecting independently; And

L ¹Be the member who is selected from key, replacement or unsubstituted alkyl or replacement or unsubstituted assorted alkyl.

8. according to the compound of claim 7, X wherein ^aAnd X ^bBe to be independently selected from following junction fragment: S, SC (O) NH, HNC (O) S, SC (O) O, O, NH, NHC (O), (O) CNH and NHC (O) O and OC (O) NH.

9. according to the compound of claim 5, wherein this connection base comprises acyl moiety.

10. according to the compound of claim 9, L-R wherein ¹¹Have following general formula:

Wherein s is the integer of 0-20; And

R ¹¹It is this polymer modification part.

11. according to the compound of claim 1, wherein this polymer modification partly has following general formula:

Wherein

X ²And X ⁴Be independently selected from junction fragment;

X ⁵It is non-reactive group; And

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

12. according to the compound of claim 11, wherein X ²And X ⁴Be to be independently selected from following junction fragment: S, SC (O) NH, HNC (O) S, SC (O) O, O, NH, NHC (O), (O) CNH and NHC (O) O, OC (O) NH and (CH ₂) _gY ",

Wherein

G is the integer of 1-50; With

Y " is the member who is selected from O, S and NH.

13. according to the compound of claim 11, wherein

X ⁴It is peptide bond; With

R ¹³It is amino-acid residue.

14. the compound according to claim 1 has general formula:

Wherein

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

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

W ' is the integer of 2-6, and

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

15. the compound according to claim 14 has general formula:

With

Wherein

L ^aBe the member who is selected from replacement or unsubstituted alkyl and replacement or unsubstituted assorted alkyl.

16. the compound according to claim 1 has general formula:

With

Wherein

L ^aBe the member who is selected from amino-acid residue and contains the peptidyl residue of 2-4 amino-acid residue;

X ²And X ⁴Be independently selected from junction fragment;

X ⁵It is non-reactive group; And

R ¹²And R ¹³It is the polymeric arms of selecting independently.

17., have following general formula according to the compound of claim 16:

Wherein

X ²And X ⁴Be independently selected from junction fragment;

X ^aIt is junction fragment;

R ¹²And R ¹³It is the polymeric arms of selecting independently; With

C is the integer of 1-20.

18., have following general formula according to the compound of claim 1:

Wherein

AA-NH is an amino-acid residue; With

P is the polymer modification group.

19. according to the compound of claim 18, wherein-AA-NH is-CH ₂NH.

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

21. according to the compound of claim 20, wherein said acceptor portion is to be selected from glycosyl residue, the member of amino-acid residue and aglycon.

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

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

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

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

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

(e) this unhindered amina is contacted with activatory PEG, form described cytidine monophosphate sialic acid-poly-(ethylene glycol) thus.

23. according to the method for claim 21, wherein the amino acid of this activatory N-protected has following structural formula: