CN1777364A

CN1777364A - Branched water-soluble polymers and their conjugates.

Info

Publication number: CN1777364A
Application number: CN 200480010689
Authority: CN
Inventors: S·德弗里斯
Original assignee: Neose Technologies Inc
Current assignee: Ratiopharm GmbH
Priority date: 2003-03-14
Filing date: 2004-03-15
Publication date: 2006-05-24
Also published as: ZA200506990B

Abstract

The present invention provides branched water-soluble polymers that allow two or more water-soluble polymers to be conjugated to another species. The branched polymers provide access to therapeutic agents that are conjugated at a single site to two or more water-soluble polymers. The branched polymers are based upon branch points that are simple branched alkyl structures, reactive side-chain amino acids and small peptides of reactive side-chain amino acids, and saccharides. Also provided is a method for preparing mono-disperse poly(ethylene glycol) of a well-defined and determinable molecular weight, and a method for the rational end-functionalization of poly(ethylene glycol). Conjugates of the branched water-soluble polymers with diverse species, e.g., peptides, lipids, glycolipids and small molecules are also provided.

Description

Branched water-soluble polymers and conjugate thereof

The cross reference of related application

The application is the US temporary patent application No.60/454 that proposed on March 14th, 2003,993, the US temporary patent application No.60/474 that on May 29th, 2003 proposed, 094, and the US temporary patent application No.60/509 of proposition on October 7th, 2003,752 non-interim submission, the content of each application is introduced for reference at this paper comprehensively.

Technical field

The conjugate that the present invention relates to branched water-soluble polymers and form by these branched polymers.

Background technology

Hydrophilic polymer, such as poly-(ethylene glycol) (abbreviation PEG, also be called poly-(oxirane), the abbreviation PEO) with molecule and the surface be conjugated in biotechnology and field of medicaments has considerable purposes.According to its most common form, PEG is at each terminal line polymer with hydroxy terminal:

HO-CH ₂CH ₂O-(CH ₂CH ₂O) _n-CH ₂CH ₂-OH

Wherein n normally about 3 to about 4000.Many end-functionalization derivatives are known in the literature, and can be commercial.For example, referring to Shearwater Polymers, Inc. " Polyethylene Glycol Derivatives " catalogue.

Having not isoplastic PEG material in each of two ends is special compounds effective.For example, Heterobifunctional PEG can be used as crosslinking agent.And, for example use the PEG molecule of alkyl " end-blocking " (such as methoxyl group) to make the C-terminal of molecule be converted into any one of a large amount of reactive organo-functional groups at an end.

Random or the block copolymer of oxirane shown below and expoxy propane is closely related with PEG on chemical constitution, and they can replace PEG in many application of PEG.

HO-CH ₂CHRO-(CH ₂CHRO) _n-CH ₂CR-OH

Wherein each R is H or CH independently ₃

The formation of the conjugate between therapeutic active substance and water-soluble polymer has proved the strategy that is full of effect of the pharmacokinetics and the pharmacodynamics that are used to improve therapeutic agent.For example, referring to Dunn and Ottenbrite, " Polymeric Drugs and Drug DeliverySystems ": ACS Symposium Series 469, American Chemical Society, Washington, D.C.1991.For example, PEG is used for that the application of derivatization peptide therapeutics is verified lowers the immunogenicity of peptide and prolong checkout time in circulation.For example, US patent No.4,179,337 (people such as Davis) relate to the non-immunogenic peptide that is coupled to polyethylene glycol (PEG) or polypropylene glycol, such as enzyme and peptide hormone.Every mole of peptide uses the 10-100 moles of polymer, and keeps at least 15% physiologically active.

Many other examples of PEG-peptide conjugate are well known in the art.The main mode that PEG and derivative thereof are connected in peptide is the non-specific bond by the peptide ammino acid residue.For example, US patent No.4,088,538 discloses the enzymic activity polymer-enzyme conjugate that is connected in the enzyme of PEG with covalent bond.Similarly, US patent No.4,496,689 disclose the compound that α-1 protease inhibitors and polymer are connected such as the covalent bond of PEG or poly-(ethylene glycol) (" mPEG ") of methoxyl group.(J.Biol.Chem.252:3578 (1977) has disclosed amido covalently bound of mPEG and bovine serum albumin(BSA) to people such as Abuchowski.WO93/15189 (people such as Veronese) relates to by proteolytic enzyme being connected in big molecule inhibitor and keeps the method for the activity of poly ethyldiol modified proteolytic enzyme.This conjugate purpose is used for medical application.US patent No.4,414,147 have disclosed by interferon being puted together the acid anhydrides in dicarboxylic acids, lower its hydrophobic method such as poly-(ethylene glycol succinic anhydride).PCT WO87/00056 discloses PEG and poly-(ethoxylation) polyalcohol and has puted together in such as beta-interferon the method for the albumen of proleulzin and immunotoxin and so on.EP 154,316 discloses and has required the chemical modification lymphokine, such as the IL-2 of the PEG that contains at least one primary amino radical that directly is bonded in this lymphokine.US patent No.4,055,635 discloses with covalent bond and has been connected in the pharmaceutical composition of polymeric material such as the water-soluble compound of the proteolytic enzyme of polysaccharide.

The another way that PEG is connected in peptide is the non-special oxidation by the glycosyl residue on peptide.Oxidized sugar is as the site that the PEG structure division is connected in peptide.For example, M ' Timkulu (WO 94/05332) disclose hydrazine-or amino-PEG be used for PEG is added to the purposes of glycoprotein.The glycosyl structure division randomly is oxidized to corresponding aldehyde, and they are coupled to amino-PEG subsequently.

In above-mentioned each method, poly-(ethylene glycol) adds to reactive residue on the peptide backbone in random, non-specific mode.Usually, caused the loss of peptide activity with the PEG derivatization, this is directly owing to the non-selective character of the chemical process that is used to put together this water-soluble polymer.

With form another relevant difficult problem of conjugate between water-soluble polymer and biomolecule is the ability of reaction water-soluble polymeric reagent in more than one site mark biomolecule.Comprise more than one water-soluble polymer structure division though wish each conjugate usually, the decreased extent of biomolecule activity usually is directly proportional with the number of the polymer architecture part that is bonded in biomolecule.Therefore, it is significant to obtain reactivity, branched substances that per molecule comprises two or more water-soluble polymer structure divisions.By using branching molecule, more than one water-soluble polymer can be puted together in biomolecule, and need not the more than one site on the interfere with biomolecules.

Branched polymer based on poly-(ethylene glycol) is well known in the art.For example, people such as Greenwald (WO 98/41562) disclose based on 1, the branching PEG of 3-diaminourea-2-propyl alcohol nuclear.Morpurgo and colleague thereof have discussed the purposes based on the branching PEG of lysine nuclear in Appl.Biochem.Biotechnol.56:59-72 (1996).Similarly based on the branching PEG of lysine by people such as Guiotto, Bioorg.Med.Chem.Lett.12:177-180 (2002) is prepared.People such as Harris (US patent No.5,932,462) have also prepared the branching PEG based on lysine.People such as Martinez (US patent No.5,643,575) have described many based on the branching PEG material of various nuclear structures and the conjugate of these materials and bioactive materials (US patent No.6,113,906).

Polymer is to exist as the assorted colony that disperses such as gathering (ethylene glycol) known, and it has comprised the polymer chain length and the molecular weight of certain limit.When preparation treatment preparation, obviously, wish to adopt polymer to guarantee uniformity and reappearance between the preparation with minimum heterodispersity.Be known in the art the method that does not almost prepare the single PEG of dispersion sample.People such as Loiseau disclose the synthetic of well-defined PEG molecule.This method adopts protects/goes the protection strategy, and this disperses the PEG for a large amount of basic list of preparation is not best.Therefore, except poly-(ethylene glycol) polymer of branching, prepare single dispersion PEG and the method that should the list dispersion be incorporated in the branched polymer is desirable especially.

The present invention has responded the demand for branched water-soluble polymers and the single PEG of dispersion material, and opened and obtained the novel therapeutic conjugate, the approach of peptide conjugate for example, and solved for more stable and treat the demand of effective therapeutant.For still existing demand such as water-soluble polymer modification treatment with the method for the industrial practicality of biomolecule with the modification group.Special meaningfully wherein conjugate have method than the improved performance of unmodified therapeutic agent.The present invention has satisfied these and other demand.

Summary of the present invention

The invention provides branched water-soluble polymers based on the nuclear of multiple structure.Branched polymer of the present invention provides the means that two or more water-soluble polymer structure divisions are connected in another material by single link position.The present invention illustrates by reference branching PEG molecule.It is for clarity that PEG concentrates description as representative water-soluble polymer, should not be considered to limit the present invention.The technical staff will appreciate that, branched substances as herein described can prepare with any water-soluble polymer basically.Except PEG, other exemplary water soluble polymer comprises poly-(propane diols).

Aspect first, the invention provides branched water-soluble polymers with following general formula:

WSP-Y-R ^x

Wherein WSP is a water-soluble polymer.Symbol Y represents to connect base, key for example, or contain acid amides, carboxylate, urea alkane, mercaptan, replacement or the structure division of substituted alkyl etc. not.Exemplary connection base comprises: key, (CH ₂) _n, (CH ₂) _mC (O) O (CH ₂) _n, (CH ₂) _mC (O) NH (CH ₂) _n, (CH ₂) _mOC (O) NH (CH ₂) _n, (CH ₂) _mO (CH ₂) _n, (CH ₂) _mNH (CH ₂) _nAnd (CH ₂) _mS (CH ₂) _n, wherein m and n are the integers that is independently selected from 0-6.R ^xBe water-soluble polymer, be connected in the replacement of water-soluble polymer or substituted alkyl structure division not; Be connected in the amino acid or the amino acid whose dimer of water-soluble polymer; Perhaps be connected in the sugar or the ribotide of water-soluble polymer.WSP and R ^xWater-soluble copolymer component can be identical water-soluble polymer or different water-soluble polymers.

The exemplary water soluble polymer that is used for compound of the present invention comprises m-PEG, PEG, m-PPG, PPG, poly sialic acid, polyglutamic acid, poly-aspartate, polylysine, polymine, biodegradable polymer (for example polyactide, polyglycerol ester), with functionalized PEG, end-functionalization PEG for example.

In an exemplary, Y is a substituted alkyl, and the invention provides the branched water-soluble polymers with following general formula:

Wherein X and Y are independently selected from OR ¹, NR ²R ³, SR ⁴, COOR ⁵, CONR ⁶R ⁷, OCONR ⁶R ⁷, replace and unsubstituted alkyl, and the member in replacement and the unsubstituted aryl.Z ¹Be to be selected from OR ¹', NR ²' R ³', SR ⁴', COOR ⁵', CONR ⁶' R ⁷', replace and substituted alkyl not, and the member in replacement and the unsubstituting aromatic yl.Symbol R ¹, R ⁴And R ⁵Represent water-soluble polymer.R ², R ³, R ⁶And R ⁷Be to be independently selected from H, replace and substituted alkyl not, replace and unsubstituting aromatic yl, replace and substituted heteroaryl not, replace and the unsubstituting heterocycle alkyl reactive functional groups, and the member in the water-soluble polymer, prerequisite is to select these groups, and feasible compound according to general formula I comprises at least two water-soluble polymer structure divisions.Symbol R ¹', R ²', R ³', R ⁴', R ⁵', R ⁶' and R ⁷' expression is independently selected from H, replaces and substituted alkyl not, replaces and unsubstituting aromatic yl, replaces and substituted heteroaryl not, replaces and the unsubstituting heterocycle alkyl reactive functional groups, the group in carrier molecule and the water-soluble polymer.

In another exemplary, Z ¹Comprise glycosyl (saccharyl) structure division.The glycosyl structure division can be activation glycosyl structure division, for example nucleotide sugar.In addition, Z ¹Can comprise the amino acid whose glycosyl structure division that is directly connected in peptide, perhaps be connected in the amino acid whose glycosyl structure division of peptide with being connected in puting together of amino acid whose glycosyl residue indirectly by it.

The present invention also provides the branched polymer based on amino acid or oligomerization amino acid (for example dipeptides, tripeptides, tetrapeptide).Exemplary amino acid type branched polymer has the general formula that is selected among following:

With

R wherein ¹¹, R ^{11 '}, R ¹², R ^{12 '}, R ¹³And R ^{13 '}Be independently selected from H, replace or not substituted alkyl and water-soluble polymer, prerequisite is to select these groups, makes above-claimed cpd comprise at least two water-soluble polymer structure divisions.R ¹⁴Be to be selected from OH, reactive functional groups contains the group of sugared structure division or is connected in a kind of in the group of carrier molecule.A is selected from NH, a kind of among O and the S.The integer of subscript " s " expression 1-5.

The chemical PEGization that can be used for other material (for example nucleic acid, peptide, sugar etc.) at each compound described in the above general formula.The method that forms the conjugate between the PEG (and contain PEG material) is well known in the art usually.For example, referring to Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; With people such as Feeney, MODIFICATION OF PROTEINS; Advances in ChemistrySeries, Vol.198, American Chemical Society, Washing ton, D.C., 1982.

In another exemplary, R ¹⁴Comprise the glycosyl structure division.This glycosyl structure division can be activation glycosyl structure division, for example nucleotide sugar.In addition, R ¹⁴Can comprise the amino acid whose glycosyl structure division that is directly connected in peptide, perhaps be connected in the amino acid whose glycosyl structure division of peptide with being connected in puting together of amino acid whose glycosyl residue indirectly by it.

In yet another aspect, the invention provides the branched water-soluble polymers of examining (" a type nuclear ") based on sugar.The technical staff is clear, and sugar is endorsed to have any structure.The exemplary steamed bun stuffed with sugar that is used for this respect of the present invention is drawn together GlcNAc, Gal, Sia, Fuc, Glc, GalNAc, GalNH ₂, GlcNH ₂Deng.

Exemplary compounds of the present invention has following general formula:

Sugar-O-(L-WSP) ₂

Wherein L is that connection base and WSP are water-soluble polymers.

In another exemplary, glycosyl branched water-soluble polymers of the present invention has following general formula:

Nucleotide-sugar-O-(L-WSP) ₂

Another exemplary compounds (based on sialic acid nuclear) according to this aspect of the present invention has following general formula:

R wherein ¹⁶And R ¹⁶' be to be selected from H, the member in acetyl group and the general formula (I):

And R ¹⁷, R ¹⁸, R ¹⁹And R ¹⁹' be to be independently selected from H, OH, NH ₂, NHAc and according to the member in the structure division of general formula I.In general formula I, Z ²Be to be selected from O, S, CH ₂A kind of with among the S.R ¹¹As mentioned above, and the integer of subscript " a " expression 0-20, prerequisite is R ¹⁶, R ^{16 '}, R ¹⁷, R ¹⁸, and R ¹⁹At least two structures that have according to general formula I.R ¹¹It can also be the key that is connected in the group of carrier molecule or is bonded in carrier molecule.R ¹⁵Be to be selected from H and activated group, a member in the phosphoric acid nucleoside acid for example.

In yet another aspect, this branched polymer is based on galactose or N-acetyl group galactose, and it has following general formula:

R wherein ¹⁵-R ¹⁹As mentioned above, R ¹⁵-R ¹⁹At least two be structure division according to general formula I.

Has structure that other exemplary sugar of general formula as implied above derives and is mannose and based on the branched water-soluble polymers of glucose.

In addition, R ¹⁵Can comprise the amino acid whose key that is connected in peptide, or be connected in the key of glycosyl structure division, this glycosyl structure division is bonded directly to the amino acid of peptide, or by puting together the amino acid that is bonded to peptide in connecting amino acid whose glycosyl residue indirectly.

The present invention also provides basic single method of disperseing colony of poly-(ethylene glycol) molecule of preparation.This method comprises allows the PEG molecule with clear and definite molecular weight, the difunctionality activated PEG that also has clear and definite molecular weight of PEG200 and at least 2 equivalents for example, and for example PEG200 contact, thus produce single dispersed sample of PEG, PEG600 for example:

G is a leaving group, such as sulphonic acid ester or tresylate ester.Single dispersed sample of PEG600 can contact with difunctionality activated PEG 200 then, forms single PPEG100 of dispersion.Perhaps, single PEG600 that disperses can be converted into corresponding Bifunctionalized derivative, and the single dihydroxy-PEG600 that disperses with at least 2 equivalents reacts again, produces single PEG1800 of dispersion.Repeat method of the present invention, till the single PEG of dispersion that obtains required size.Can design this and synthesize, make the molecular weight differences permission between initiation material and product separate the material of any unreacted or partial reaction by SEC.

And, as to preparation modified soluble polymer, such as the response of the demand of improving one's methods of gathering (ethylene glycol), the method that the invention provides chemical activation and prolong polymer backbone.Single activated PEG molecule can be used for PEG is puted together in various materials, and for example the target structure division is treated and used structure division, antineoplastic, cytotoxin, radiological agent, amino acids, sugar etc.

Therefore, in yet another aspect, the invention provides the activated water soluble polymer, especially the method for the substep of poly-(ethylene glycol) and analogue thereof assembling.This method provides the approach that makes things convenient for that obtains monofunctional and Bifunctionalized PEG molecule.

Therefore, in an exemplary, the invention provides the method for preparation poly-(ethylene glycol).Summarized this method below:

A.R-Y/ (acid or alkali); B. activation, for example tosylation, halogen-go hydroxylating, for example HX or SOX ₂, and and PEG _mReaction; C. chloro-carbonic acid p-nitrophenyl ester is for example used in activation (R ').

Wherein, subscript m and n represent 1-100,000 independently.

In step a, original glycol contacts with activated group (R-Y), the hydroxyl structure partial reaction of this activated group and glycol.Y is leaving group normally, and R is placed on one of the hydroxyl structure part of PEG molecule.In step b, the free hydroxyl group of gained adduct activates by the group that it is converted into sulfonic acid esters.The activation the PEG material with as initial PEG (" PEG _m") another PEG structure division contact of the identical or different degree of polymerization.In order to make it be connected in another material, RO-PEG _(n+m)Choose wantonly in free hydroxyl group structural portion office and activate.

Compound of the present invention can be used for by the direct chemical PEGization of one or more obtainable reactive residues on therapeutic agent, forms substrate such as therapeutic agent, peptide for example, lipid, the water-soluble polymer conjugate of glycolipid.Compound of the present invention also is incorporated into easily can be at substrate, for example in the glycoconjugate of the activation of adopting in the sugar-PEGization of the enzyme of therapeutic agent mediation.

The present invention also provides one or more branched water-soluble polymers of the present invention to put together the pharmaceutical preparation of therapeutic agent thereon.The method of treatment disease also is provided, and these diseases are improved or are cured by the conjugate that gives between therapeutic agent and the branched water-soluble polymers of the present invention.

Others of the present invention, advantage and purpose can be recognized from following detailed description.

The accompanying drawing summary

Detailed description of the present invention

Abbreviation

PEG, poly-(ethylene glycol); M-PEG, methoxyl group-poly-(ethylene glycol); PPG, poly-(propane diols); M-PPG, methoxyl group-poly-(propane diols); Fuc, fucosido; Gal, galactosyl; GalNAc, N-acetylamino galactosamine base; Glc, glucosyl group; GlcNAc, the N-acetylamino glucosyl; Man, the epichitosamine base; ManAc, the epichitosamine yl acetate; Sia, sialic acid; And NeuAc, the neural amido (N-acetylneuraminyl) of N-acetyl group.

Definition

Unless otherwise prescribed, otherwise the employed whole technology of this paper have the implication identical with those skilled in the art's general understanding usually with scientific terminology.Usually, the nomenclature of using in the experiment operation of this paper and cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and cross discipline is well-known and commonly used those in this area.The synthetic standard technique that adopts of nucleic acid and peptide.This technology and operation generally according to the commonsense method and the various general list of references of this area carry out (generally referring to, people such as Sambrook, MOLECULAR CLONING; A LABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., the document is incorporated herein for reference), they are provided in whole file.The nomenclature that adopts in the experiment operation of this paper and analytical chemistry and following organic synthesis is well-known and commonly used those in this area.Chemosynthesis and chemical analysis adopt standard technique or their improvement.

Term as used herein " sugar is puted together (glycoconjugation) " is meant the puting together of enzyme mediation of the amino acid of modified sugars material and peptide or saccharide residue.The subgenus of " sugar is puted together " is " sugar-PEGization ", and wherein the modification group of modified sugars is poly-(ethylene glycol), and its alkyl derivative (for example m-PEG) or reactive derivatives (H for example ₂N-PEG, HOOC-PEG).

Term " sialic acid " is meant any member of the family of the carboxylated sugar of nine carbon.The modal member of sialic acid family is N-acetyl group-neuraminic acid (2-ketone-5-acetylaminohydroxyphenylarsonic acid 3; 5-dideoxy-D-glycerine-D-galactose ketononose pyranose-1-acid (galactononulopyranos-1-onic acid); often be abbreviated as Neu5Ac, NeuAc or NANA).Second member of this family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), and wherein the N-acetyl group of NeuAc is by hydroxylating.The 3rd sialic acid family member is (KDN) (people (1986) such as Nadano, J.Biol.Chem.261:11550-11557 of 2-ketone-3-deoxidation-nonanone saccharic acid (nonulosonic acid); People such as Kanamori, J.Biol.Chem.265:21811-21819 (1990)).The sialic acid that has also comprised the 9-replacement is such as 9-O-C ₁-C ₆Acyl group-Neu5Ac such as 9-O-lactyl-Neu5Ac or 9-O-acetyl group-Neu5Ac, 9-deoxidation-9-fluoro-Neu5Ac and 9-azido-9-'-deoxy-n eu5Ac.About the commentary of sialic acid family, for example referring to Varki, Glycobiology 2:25-40 (1992); Sialic Acids:Chemistry, Metabolism and Function, R.Schauer, Ed. (Springer-Verlag, New York (1992)).Synthetic and the purposes of sialylated compound in sialylated operation is disclosed in the International Application No. WO 92/16640 of publishing on October 1st, 1992.

" peptide " is meant polymer, and wherein monomer is amino acid and links together by amido link, perhaps is referred to as polypeptide.In addition, alpha-non-natural amino acid, Beta-alanine for example, phenylglycine and homoarginine are also comprised.The non-genomic amino acids coding also can adopt in the present invention.In addition, being modified becomes to comprise reactive group, glycosylation site, and polymer, structure division is used in treatment, and the amino acid of biomolecule etc. also can adopt in the present invention.All amino acid of Shi Yonging can be D-or L-isomer in the present invention.The L-isomer generally is preferred.In addition, other plan peptide (peptidomimetics) also can be used for the present invention.This paper employed " peptide " is meant glycosylated peptide and non-glycosylated peptide.Also comprised the incomplete glycosylated peptide of the system that is expressed peptide.About general commentary, referring to Spatola, A.F., CHEMISTRY ANDBIOCHEMISTRY OF AMINO ACIDS, PEPTIDES AND PROTEINS, B.Weinstein, eds., Marcel Dekker, New York, p.267 (1983).

Term " peptide conjugate " is meant material of the present invention, and wherein peptide and modified sugars as described herein are carried out sugar and puted together.As representative example, this peptide is the mutant peptide with glycosylation site of the O-connection that is not present in the wild type peptide.

Term " amino acid " is meant natural existence and synthetic amino acid, and amino acid analogue and the plan amino acid to work with mode like the naturally occurring amino acids.Naturally occurring amino acid is by those coded amino acid of genetic code, and those amino acid of modification afterwards, hydroxy-proline for example, Gla, and O-phosphoserine.Amino acid analogue is meant the compound with basic chemical structure identical with naturally occurring amino acid,, has the hydrogen of being bonded in that is, carboxyl, the α carbon of amino and R group, homoserine for example, nor-leucine, methionine sulfoxide, methionine methyl sulfonium.These analogs have modification R group (for example nor-leucine) or modification peptide backbone, but have kept the basic chemical structure identical with naturally occurring amino acid.Intend amino acid and be meant to have the structure that is different from amino acid whose general chemical constitution, but the compound to work with mode like the naturally occurring amino acids.

As used herein term " modified sugars " is meant the carbohydrate that natural existence or non-natural exist, and with branched water-soluble polymers modification of the present invention, can add on the amino acid or glycosyl residue of peptide, lipid, glycolipid etc. by enzymatic.Modified sugars is selected from many zymolytes, including, but not limited to ribotide (Monophosphate, bisphosphate and triguaiacyl phosphate), and activation sugar (for example halogen-sugar, glycosyl methanesulfonates) and both activated also the not carbohydrate of nucleotidylation." modified sugars " is functionalized with " modification group " (it is a branched polymer of the present invention) covalency.Select position, make it not hinder " modified sugars " enzymatic and add on peptide or other substrate with the modification group functionalization.

Term " water-soluble " be meant in water, have can detected solvability structure division.Detection and/or quantitative water miscible method are well-known in the art.The exemplary water soluble polymer comprises peptide, carbohydrate, poly-(ether), poly-(amine), poly-(carboxylic acid) etc.Peptide can have the mixed sequence of being made up of single amino acid, for example poly-(lysine).Exemplary polysaccharide is poly-(sialic acid).Exemplary poly-(ether) is poly-(ethylene glycol), for example m-PEG.Poly-(aziridine) is exemplary polyamine, and poly-(acrylic acid) is representative poly-(carboxylic acid).

Term " poly-(ethylene glycol) ", " PEG ", " poly-(propane diols) " and " PPG " use with their general meaning, and they have also comprised the derivative of this parent compound, monoalkyl material for example, m-PEG for example, m-PPG, reactive materials, N-maloyl imines, p-nitrophenyl carbonic ester (p-NP), HOBT derivative, and amine.Also being included in these term scopes is the material that comprises two or more modifications, for example p-NP-PEG-OMe etc.

Term as used herein " glycosyl connects base " is meant glycosyl residue, and agent (for example water-soluble polymer is treated and used structure division, biomolecule) is connected in it with covalent bond.In the method for the invention, " glycosyl connect base " is connected in glycosylation or non-glycosylated peptide with covalent bond, thereby agent is connected on the amino acid and/or glycosyl residue on the peptide." glycosyl connect base " general amino acid and/or enzymatic of glycosyl residue by " modified sugars " and peptide is connected and produced by " modified sugars "." complete glycosyl connects base " is meant the connection base of being derived by the glycosyl structure division, and each sugar monomer that wherein connects conjugate is not degraded, and is for example not oxidized, for example not by the oxidation of sodium metaperiodate institute." intact glycosyl connect base " of the present invention can be by increasing glycosyl units or removing one or more glycosyl units and produced by naturally occurring oligosaccharides from parent sugar structure.

This paper employed " pharmaceutically acceptable carrier " comprises any material, when combining with conjugate, has kept the activity of conjugate, and does not react with patient's immune system.Example is any including, but not limited to the standard drug carrier, such as the saline solution of phosphate-buffered, and water, emulsion is such as oil/aqueous emulsion, and various types of wetting agent.Other carriers can also comprise thimerosal, and tablet comprises coated tablet and capsule.Typically, these carriers contain excipients such as starch, milk, sugar, the clay of some type, gelatin, stearic acid or its salt, dolomol or calcium stearate, talcum, plant fat or oil, natural gum, glycols, or other known excipients.Examples of such carriers can also comprise flavouring agent and color additives or other composition.The composition that comprises examples of such carriers is prepared by known commonsense method.

This paper employed " administration " is meant oral administration, as the suppository administration, and local contact, intravenous, in the peritonaeum, intramuscular, damage zone, or subcutaneous administration, inhalation, or implant the interior slowly-releasing equipment of patient's body, for example little osmotic pump.Administration can comprise non-enteron aisle and see through mucous membrane mode (for example oral, intranasal, transvaginal, per rectum or transdermal) by any approach, especially by sucking.Parenterai administration comprises for example intravenous, and intramuscular is subcutaneous in the corium in the arteriole, in the peritonaeum, and in the ventricle, and encephalic.And, if injection is used for the treatment of tumour, for example, bring out Apoptosis, directly tumour and/or tumour tissue are on every side given in administration.Other mode of movement is including, but not limited to using the liposome formulation agent, intravenous input, transdermal patch etc.

Term " separation " is meant substantially or is substantially free of the material of the component that is used to produce this material.For peptide conjugate of the present invention, term " separation " be meant substantially or be substantially free of normally this material of mixture that is used for preparing peptide conjugate with the material of component." separation " and " pure " can exchange use.Typically, the peptide conjugate of separation of the present invention has the certain purity of preferably representing by scope.The lower limit of peptide conjugate purity range is about 60%, about 70% or about 80%, and the upper limit of purity range is about 70%, about 80%, about 90% or surpasses about 90%.

When peptide conjugate surpassed about 90% purity, their purity was also preferably represented by scope.The lower limit of purity range is about 90%, and is about 92%, about 94%, about 96%, or about 98%.The upper limit of purity range is about 92%, about 94%, about 96%, about 98% or about 100% purity.

The analytical method that purity is generally acknowledged by any technology is measured (for example, the band intensity on silver-colored stained gel, polyacrylamide gel electrophoresis, HPLC or similar fashion).

This paper employed " each member basically of colony " has described the characteristic of the colony of peptide conjugate of the present invention, and the percentile modified sugars that is added on the polypeptide that wherein will select is added on a plurality of identical acceptor site on the peptide." each member basically of colony " mentioned and puted together in " uniformity " in the site of the peptide of modified sugars, be meant about at least 80%, preferred about at least 90% and more preferably about at least 95% uniform conjugate of the present invention.

" uniformity " is meant that modified sugars puts together the structural integrity of the colony of receptor structure part thereon.Therefore, each modified sugars structure division is puted together in the peptide conjugate of the present invention of the identical acceptor site of the acceptor site that other modified sugars of structure and each are puted together therein, and it is about 100% uniform that this peptide conjugate is said to be.Uniformity is represented by scope usually.The lower limit of the uniformity scope of peptide conjugate is about 60%, and is about 70%, or about 80%, and the upper limit of purity range is about 70%, about 80%, about 90%, or surpasses about 90%.

When peptide conjugate surpassed or equal about 90% uniformity, their uniformity was also preferably represented by scope.The lower limit of inhomogeneity scope is about 90%, about 92%, about 94%, about 96% or about 98%.The upper limit of purity range is about 92%, about 94%, about 96%, about 98% or about 100% uniformity.The purity of peptide conjugate is general by one or more methods well known by persons skilled in the art, liquid chromatography one mass spectrometry (LC-MS) for example, substance assistant laser desorpted time-of-flight mass spectrometry (MALDITOF), Capillary Electrophoresis etc.

When mentioning the glycopeptide material, " uniform substantially sugared shape " or " basic glycosylation form uniformly " is meant by the percentage of the glycosylated receptor structure part of glycosyl transferase that studied (for example fucosyltransferase).For example, at α 1, under the situation of 2-fucosyltransferase, if in peptide conjugate of the present invention the Ga1B1 of all (as following definition) basically, 4-G1cNAc-R and saliva acidic group analog thereof are then had basic fucosylation form uniformly by fucosylation.It will be apparent to those skilled in that initiation material can contain glycosylation receptor structure part (for example fucosylation Ga1 β 1,4-G1cNAc-R structure division).Therefore, the glycosylation percentage of calculating comprises with the glycosylated receptor structure part of method of the present invention, and glycosylated those receptor structure parts in initiation material.

Term " substantially " in the above definition of " even substantially " generally is meant about at least 40% of specific glycosyl transferase, at least about 70%, at least about 80%, or more preferably about at least 90%, also more preferably about at least 95% receptor structure part by glycosylation.

Term " on a large scale " and " commercial scale " are used interchangeably, and are meant to have produced at least approximately 250mg when finish a reaction time, preferably at least approximately 500mg and the more preferably reaction time of the glycoconjugate of about at least 1g.

No matter be vertical with key as key or demonstration, symbol Represent that shown structure division is connected in the remainder of molecule, the position of solid carrier etc.

Some compound of the present invention can exist with non-solvent form and solvation form, comprises hydrated form.Generally, the solvation form is equal to the non-solvent form, and comprises within the scope of the invention.Some compound of the present invention can exist with multiple crystallization or amorphous form.Generally, for by the contemplated application of the present invention, all physical form are equivalent, and are within the scope of the invention.

Some compound of the present invention has asymmetric c atom (optical centre) or two key; Racemic modification, diastereoisomer, geometric isomer and individual isomer comprise within the scope of the invention.

Compound of the present invention can be used as individual isomer (for example enantiomer is suitable-trans, position, diastereoisomer) or as mixture of isomers.In a preferred embodiment, compound is as individual isomer preparation basically.Preparing basically, the method for isomery pure compound is well known in the art.For example, it is constant or cause its reaction of changing fully to prepare with the spatial chemistry that keeps chiral centre to be rich in synthetic intermediate that the mixture of enantiomer and pure enantiomeric compounds can be by using pure enantiomer.Perhaps, the intermediate in end product or the route of synthesis can resolvedly be a single stereoisomers.Conversion or those technology that keep the constant technology in specific three-dimensional center and be used to resolve the mixture of stereoisomer are known in the art, and obviously are that those skilled in the art selects suitable method to be used in the limit of power of particular case.Usually, can be referring to people such as Furniss (eds.), VOGEL ' SENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY 5 ^THED., LongmanScientific and Technical Ltd., Essex, 1991, pp.809-816; And Heller, Acc.Chem.Res.23:128 (1990).

Compound of the present invention can also contain the atom isotope of non-natural proportion at the one or more atoms place that constitutes this compounds.For example, these compounds can be used radioisotope, for example tritium ( ³H), iodine-125 ( ¹²⁵I) or carbon-14 ( ¹⁴C) mark.No matter whether have radioactivity, all isotope modification intentions of compound of the present invention comprise within the scope of the invention.

If substituting group represents that with their the general chemistry formula of from left to right writing they have comprised the chemically uniform substituting group that is obtained by writing structural formula from right to left equally, for example-CH ₂O-can also be written as-OCH ₂-.

Unless otherwise prescribed, otherwise itself or be meant straight or branched as the term " alkyl " of another substituent part, or cyclic hydrocarbon group, perhaps their bond, it can be complete saturated, single or polyunsaturated, can comprise divalence (" alkylidene ") and multivalence group, the carbon number with appointment (is C ₁-C ₁₀Be meant 1-10 carbon atom).The example of saturated hydrocarbyl is including, but not limited to the group such as following: methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, the tert-butyl group, isobutyl group, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropyl methyl, analog and isomer, n-pentyl for example, n-hexyl, n-heptyl, the analog of n-octyl etc. and isomer.Unsaturated alkyl is the alkyl with one or more pairs of keys or triple bond.The example of unsaturated alkyl is including, but not limited to vinyl, 2-acrylic, crotyl, 2-isopentene group, 2-(butadienyl), 2,4-pentadienyl, 3-(1, the 4-pentadienyl), acetenyl, 1-and 3-propinyl, 3-butynyl, and senior analog and isomer.Unless otherwise prescribed, otherwise term " alkyl " also is intended to be included in those derivatives of the alkyl of following more specific definition, such as " assorted alkyl ".The alkyl that is limited to alkyl is called as " same alkyl (homoalkyl) ".

Be used for exemplary alkyl of the present invention and contain about 1 to about 25 carbon atoms (for example methyl, ethyl etc.).Have≤straight chain, branching or the cyclic hydrocarbon chain of 8 carbon atoms will be referred to herein as " low alkyl group ".In addition, term as used herein " alkyl " further is included in the one or more substituting groups on one or more carbon atoms of hydrocarbon chain fragment.

Term " alkoxyl ", " alkyl amino " or " alkylthio group " (or thio alkoxy) uses with their ordinary meaning, is meant those alkyl that are connected in the remainder of molecule respectively via oxygen atom, amino or sulphur atom.

Unless otherwise prescribed, itself or the term " assorted alkyl " that combines with another term are meant straight or branched, perhaps ring-type carbon-containing group, perhaps their bond, form by the carbon atom of described number and at least one hetero atom that is selected among O, N, Si, P and the S, wherein nitrogen, p and s atom are optional oxidized, and nitrogen heteroatom is optional by seasonization.Hetero atom O, N, P, S and Si can be positioned at any interior location of assorted alkyl or the position of the remainder that alkyl is connected in molecule.Example is including, but not limited to-CH ₂-CH ₂-O-CH ₃,-CH ₂-CH ₂-NH-CH ₃,-CH ₂-CH ₂-N (CH ₃)-CH ₃,-CH ₂-S-CH ₂-CH ₃,-CH ₂-CH ₂-S (O)-CH ₃,-CH ₂-CH ₂-S (O) ₂-CH ₃,-CH=CH-O-CH ₃,-Si (CH ₃) ₃,-CH ₂-CH=N-OCH ₃And-CH=CH-N (CH ₃)-CH ₃Two hetero atoms can be continuous at the most, for example, and-CH ₂-NH-OCH ₃With-CH ₂-O-Si (CH ₃) ₃Similarly, itself or be meant the divalent group of deriving as the term of another substituent part " inferior assorted alkyl " by assorted alkyl, for example but be not limited to-CH ₂-CH ₂-S-CH ₂-CH ₂-and-CH ₂-S-CH ₂-CH ₂-NH-CH ₂-.For the assorted alkyl in Asia, hetero atom can also occupy one or two (for example alkylene oxide group, alkylenedioxy group, alkylidene amino, alkylidene diaminourea etc.) of chain end.In addition, be connected base for the assorted alkyl in alkylidene and Asia, the nothing that connects base is orientated the direction of writing with the chemical formula that wherein connects base and represents.Chemical formula-C (O) for example ₂R '-expression-C (O) ₂R '-and-R ' C (O) ₂-.

Unless otherwise prescribed, itself or the term " cycloalkyl " that combines with other term and " Heterocyclylalkyl " are represented the ring-type modification of " alkyl " and " alkyl of mixing " respectively.In addition, for Heterocyclylalkyl, hetero atom can occupy the position that heterocycle is connected in the remainder of molecule.The example of cycloalkyl is including, but not limited to cyclopenta, cyclohexyl, 1-cyclohexenyl group, 3-cyclohexenyl group, suberyl etc.The example of Heterocyclylalkyl is including, but not limited to 1-(1,2,5, the 6-tetrahydro pyridyl), 1-piperidyl, the 2-piperidyl, 3-piperidyl, 4-morpholinyl, morpholinyl, oxolane-2-base, oxolane-3-base, thiophane-2-base, thiophane-3-base, 1-piperazinyl, 2-piperazinyl etc.

Unless otherwise prescribed, term " aryl " is meant how unsaturated aromatic structure part, and it can be monocycle or many rings (preferred 1-3 ring), condenses together or the covalent bond connection.Term " heteroaryl " is meant and contains 1-4 the heteroatomic aryl (or ring) that is selected among N, O and the S that wherein nitrogen and sulphur atom are optional oxidized, and nitrogen-atoms is optional by seasonization.Heteroaryl can be connected in the remainder of molecule by hetero atom.The limiting examples of aryl and heteroaryl comprises phenyl, 1-naphthyl, 2-naphthyl, 4-xenyl, the 1-pyrrole radicals, 2-pyrrole radicals, 3-pyrrole radicals, 3-pyrazolyl, the 2-imidazole radicals, 4-imidazole radicals, pyrazinyl, 2-oxazolyl, the 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, the 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, the 5-thiazolyl, 2-furyl, 3-furyl, the 2-thienyl, 3-thienyl, 2-pyridine radicals, the 3-pyridine radicals, 4-pyridine radicals, 2-pyrimidine radicals, the 4-pyrimidine radicals, 5-benzothiazolyl, purine radicals, the 2-benzimidazolyl, 5-indyl, 1-isoquinolyl, the 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, the 3-quinolyl, tetrazole radical, benzo [b] furyl, benzo [b] thienyl, 2,3-dihydrobenzo [1,4] dioxazine-6-base, benzo [1,3]-dioxole-5-base and 6-quinolyl.Above-mentioned aryl and heteroaryl ring system substituting group separately are selected from following acceptable substituting group.

For for simplicity, when being used in combination with other term (for example aryloxy group, fragrant sulphur oxygen base (arylthioxyl), aralkyl), term " aryl " comprises aryl and the heteroaryl as above definition.Therefore, term " aralkyl " comprises that aryl wherein is connected in those groups of alkyl (benzyl for example, phenethyl, pyridylmethyl etc.), comprise that carbon atom in the described alkyl (for example methylene) is by those alkyl of the displacement of oxygen atom for example (phenoxymethyl for example, 2-pyridine oxygen ylmethyl, 3-(1-naphthoxy) propyl group etc.).

More than each term (for example " alkyl ", " assorted alkyl ", " aryl " and " heteroaryl ") comprise the replacement of described group and do not replace form.The preferred substituted of all kinds of groups below is provided.

The substituting group of alkyl and assorted alkyl (comprise and usually be called alkylidene, alkenyl, inferior assorted alkyl, heterochain thiazolinyl, alkynyl, cycloalkyl, Heterocyclylalkyl, cycloalkenyl group and heterocycloalkenyl) is commonly referred to as " alkyl substituent ", they can be to be selected from but to be not limited to various groups one or more in the following group :-OR ' ,=O ,=NR ',=N-OR ',-NR ' R " ,-SR ' ,-halogen ;-SiR ' R " R ,-OC (O) R ' ,-C (O) R ' ,-CO ₂R ' ,-CONR ' R " ,-OC (O) NR ' R " and ,-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-NO ₂, number is 0 to (2m '+1), wherein m ' is the sum of the carbon atom in this group.R ', R ", the preferred separately independent expression hydrogen of R and R " " replaces or replaces assorted alkyl, replaces or unsubstituting aromatic yl the aryl that is replaced by 1-3 halogen for example, replacement or substituted alkyl not, alkoxyl or thio alkoxy, or aralkyl.When compound of the present invention comprised more than one R group, for example, when having more than one these groups, each R group was selected independently, each R ', R ", R and R " " group is selected equally independently." when being connected in same nitrogen-atoms, they can combine with this nitrogen-atoms, form 5,6 or 7 yuan of rings as R ' and R.For example ,-" intention is including, but not limited to 1-pyrrolidinyl and 4-morpholinyl for NR ' R.Discuss as can be seen more than substituent, it will be apparent to those skilled in that, term " alkyl " intention comprises the group that contains the group that is bonded in non-hydrogen base, such as haloalkyl (for example-CF ₃With-CH ₂CF ₃) and acyl group (for example ,-C (O) CH ₃,-C (O) CF ₃,-C (O) CH ₂OCH ₃Deng).

With similar for the described substituting group of alkyl, the substituting group of aryl and heteroaryl is commonly referred to as " aryl substituent ".These substituting groups for example are selected from: halogen ,-OR ' ,=O ,=NR ' ,=N-OR ' ,-NR ' R " ,-SR ' ,-halogen ,-SiR ' R " R ,-OC (O) R ' ,-C (O) R ' ,-CO ₂R ' ,-CONR ' R " ,-OC (O) NR ' R " and ,-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-NO ₂,-R ' ,-N ₃,-CH (Ph) ₂, fluorine (C ₁-C ₄) alkoxyl, and fluorine (C ₁-C ₄) alkyl, number is 0 to the valent sum of the opening on aromatic ring system; R ' wherein, R ", R and R " " preferentially are independently selected from hydrogen, replace or substituted alkyl not, replace or replace assorted alkyl, replacement or unsubstituting aromatic yl and replacement or substituted heteroaryl not.When compound of the present invention comprised more than one R group, for example, when having more than one these groups, each R group was selected independently, each R ', R ", R and R " " group is selected equally independently.In following route map, symbol X represents aforesaid " R ".

Two substituting groups on the adjacent atom of aryl or heteroaryl can be chosen wantonly by chemical formula-T-C (O)-(CRR ') _qThe substituting group of-U-replaces, and wherein T and U are-NR-independently ,-O-, and-CRR '-or singly-bound, and q is the integer of 0-3.Perhaps, two substituting groups on the adjacent atom of aryl or heteroaryl ring can be chosen (the CH by chemical formula-A-wantonly ₂) _rThe substituting group of-B-replaces, wherein A and B be independently-CRR '-,-O-,-NR-,-S-,-S (O)-,-S (O) ₂-,-S (O) ₂NR '-or singly-bound, and r is the integer of 1-4.One of singly-bound of the new ring of Xing Chenging can be chosen wantonly by two keys and replace like this.Perhaps, two substituting groups on the adjacent atom of aryl or heteroaryl ring can be chosen wantonly by chemical formula (CRR ') _s-X-(CR " R ) _d-substituting group replace, 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 '-.Substituent R, " and R preferably is independently selected from hydrogen or replacement or does not replace (C for R ', R ₁-C ₆) alkyl.

Term as used herein " hetero atom " comprises oxygen (O), nitrogen (N), sulphur (S), phosphorus (P) and silicon (Si).

Term " amino " or " amido " are meant group-NR ' R " (or-N ⁺RR ' R "), R wherein, R ' and R " are independently selected from hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, heteroaryl and substituted heteroaryl.The amine that replaces is that wherein R ' or R " are not the amidos of hydrogen.In primary amino radical, R ' and R " all being hydrogen, and in secondary amino group, R ' or R " any one (but not being two) is hydrogen.In addition, term " amine " and " amino " can comprise the protonated of nitrogen and season modification, comprise group-N ⁺RR ' R " and its biocompatibility anionic counter-ion.

Unless otherwise prescribed, itself or be meant fluorine, chlorine, bromine or iodine atom as term " halo " or " halogen " of another substituent part.Term comprises single haloalkyl and multi-haloalkyl such as " haloalkyl " intention in addition.For example, term " halo (C ₁-C ₄) alkyl is intended to including, but not limited to trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl etc.

Term as used herein " connects base " or " L " is meant single covalent bond or the stable covalent bond of series that has comprised 1-20 the non-hydrogen atom that is selected among C, N, O, S and the P, with covalent bond water-soluble polymer or branched water-soluble polymers are connected in another structure division such as chemically reactive group or put together material, comprise biology and abitotic substance.Exemplary coupling part comprises and containing-C (O) NH-,-C (O) O-,-NH-,-S-, the structure division of-O-etc." the connection base of cleavable " is the connection base with one or more cleavable groups that result or condition ruptured that can be reacted.Term " group of cleavable " is meant for example structure division sloughed from the remainder of conjugate of water-soluble polymer of a part that the key that connects the remainder of the structure division that will slough and conjugate by cracking makes conjugate.This cracking is a chemical property, or the enzyme mediation.Exemplary enzymatic lysis group comprises natural amino acid or with the peptide sequence of natural amino acid terminal.

Except the enzymatic lysis group, comprise one or more can be by the site of the effect of non-enzyme agent institute cracking.Exemplary non-enzymatic lysis agent is including, but not limited to acid, alkali, light (for example nitrobenzyl derivative, phenacyl, benzoin esters) and heat.Many cleavable groups are well known in the art.For example referring to people such as Jung, Biochem.Biophys.Acta, 761:152-162 (1983); People such as Joshi, J.Biol.Chem., 265:14518-14525 (1990); People such as Zarling, J.Immunol., 124:913-920 (1980); People such as Bouizar, Eur.J.Biochem., 155:141-147 (1986); People such as Park, J.Biol.Chem., 261:205-210 (1986); People such as Browning, J.Immunol., 143:1859-1867 (1989).And the difunctionality of multiple cleavable (with difunctionality and Heterobifunctional) spacerarm (spacer arm) can be commercial.

Exemplary cleavable group ester is to pass through for example cleavable group of sodium hydroxide cracking of reagent, has obtained to contain the fragment of carboxylate and the product of hydroxyl.

This connection base can be used for this compound is connected in another component of conjugate, such as guide frame part (for example antibody, part, non-covalent protein combination group etc.), analyte, biomolecule, medicine etc.

" non-covalent protein combination group " be with complete or sex change polypeptide with the interactional structure division of associated form.This interaction can be reversible or irreversible in biotic habitat." non-covalent protein combination group " be incorporated into provides the compound that has with non-covalent mode and the interactional ability of polypeptide in the fluorescent chemicals of the present invention.Hydrophobic-hydrophobic and electrostatic interaction that exemplary noncovalent interaction comprises.Exemplary " non-covalent protein combination group " comprises anionic group, for example phosphate radical, D2EHDTPA root, phosphonate radical, carboxylate radical, borate, sulfate radical, sulfone, thiosulfate anion and thiosulfonic acid root.

This paper employed " nucleic acid " is meant DNA, RNA, strand, hybridization motif double-stranded or that more highly assemble, and their any chemical variant.Variant is including, but not limited to those of chemical group that additional charge, polarizability, hydrogen bonding, electrostatic interaction and three-dimensional mutability (fluxionality) are incorporated into nucleic acid ligands main body or whole nucleic acid ligands are provided.This type of variant is including, but not limited to peptide nucleic acid (PNA), di-phosphate ester variant (for example thiophosphate, methyl phosphonate), 2 '-the sugared variant in position, 5-position pyrimidine variant, 8-position purine variant, encircle outer amine variant, the replacement of 4-thiouridine, the replacement of 5-bromine or 5-iodo-uracil; Backbone modification methylates, and improper base pairing is made up such as isobase, different cytidine and different guanidine etc.Nucleic acid can also comprise non-natural base, for example nitroindoline.Variant can also comprise 3 ' and 5 ' variant such as using quencher, fluorogen or other structure division end-blocking.

Term as used herein " reactive group " is meant and can reacts to form the group of covalent bond with other chemical group, that is, but under the reaction condition that is fit to the group of covalent reaction, and constituted the link position of other material usually.This reactive group be on the compound of the present invention can with functional group reactions on different compounds to form covalent bond, obtain the structure division of fluorescence or fluorescence labeling component, such as carboxylic acid or succimide ester.Reactive group generally includes nucleophile, close electric body and photoactivation group.

The exemplary reaction group comprises, but be not limited to alkene, alkynes, alcohol, phenol, ether, oxide, halide, aldehyde, ketone, carboxylic acid, ester, acid amides, cyanate, isocyanates, thiocyanates, isothiocyanates, amine, hydrazine, hydrazone, hydrazides, diazonium, diazol, nitro, nitrile, mercaptan, sulphide, disulphide, sulfoxide, sulfone, sulfonic acid, sulfinic acid, acetal, ketal, acid anhydrides, sulfuric ester, sulfenic acid, isonitrile, amidine, acid imide, imino-ester, nitrone, azanol, oxime, hydroxamic acid, sulfo-hydroxamic acid, the allene class, ortho esters, sulfite, enamine, ynamine, ureas, pseudo-urea class, semicarbazides, Carbodiimides, carbamate, imines, azide, azo-compound, azoxy compound, and nitroso compound.Reactive functional groups also comprises those that are used to prepare bioconjugates, N-maloyl imines ester for example, maleimide etc.Each the method for preparing these functional groups is known in the art.They are used for the application of specific purpose or the change made for specific purpose is (for example referring to Sandler and Karo in those skilled in the art's the limit of power, eds.ORGANIC FUNCTIONAL GROUP PREPARATIONS, Academic Press, SanDiego, 1989).

Term " target group " be meant (1) can be on one's own initiative with entity (for example fluorescence structure division) vector zone, for example cell that it connected; Or (2) preferred structure division that is adsorbed by the target area or in the target area, carry secretly passively.The target group can be little molecule, and expection comprises non-peptide and peptide.The target group can also be a macromolecule, and including, but not limited to carbohydrate, agglutinin, acceptor, the part of acceptor, albumen are such as BSA, and antibody gathers in (ether), dendritic macromole, poly-(amino acid) etc.

This paper employed " carrier molecule " is meant any molecule that connects compound of the present invention.Representative carrier molecule is not with and is restrictedly comprised albumen (for example enzyme, antibody), glycoprotein, peptide, sugar (for example monose, oligosaccharides and polysaccharide), hormone, acceptor, antigen, substrate, metabolite, transition state analog, co-factor, inhibitor, medicine, dyestuff, nutrients, growth factor etc." carrier molecule " also expresses possibility and is not considered to material in the classical range of definition of " molecule ", solid carrier (for example synthetic vectors, chromosorb, film) for example, virus and microorganism.

Preamble

The invention provides the conjugate of branched water-soluble polymers and branched water-soluble polymers.This conjugate is at branched water-soluble polymers of the present invention and comprise this branched water-soluble polymers and can put together between the material of reactive group thereon and form.The exemplary conjugate counter pair that is used for water-soluble polymer of the present invention comprises peptide, glycopeptide, lipid and glycolipid.Exemplary conjugate is that the modified sugars direct or indirect (for example by getting involved glycosyl residue) of wherein carrying branched water-soluble polymers of the present invention is connected on the glycosylation site of peptide.The method of producing conjugate of the present invention also is provided.

The method of conjugate of the present invention and formation conjugate illustrates with reference to peptide and glycopeptide conjugate here.The focus of discussing is in order to clearly demonstrate, should not to be considered to the purposes of branched water-soluble polymers disclosed herein is limited on the purposes that forms this conjugate.It will be understood by those skilled in the art that branched water-soluble polymers of the present invention can be used for forming various branched water-soluble polymers conjugates.

Described in part in front, the art-recognized chemical method dependence of covalency PEGization is chemically conjugated by the reactive group on amino acid or the carbohydrate.By careful design conjugate and reaction condition, used the strategy of puting together of chemistry mediation to prepare effective conjugate.The chemically conjugated major defect of polymer and albumen or glycoprotein is the optionally shortage of activated polymer, and this usually causes polymer influencing albumen or the glycoprotein biologically active connects on the site.Developed several method and solved site selectivity and put together chemistry, yet, only developed a kind of method in common that is suitable for various recombinant proteins.

Opposite with art-recognized method, the site-directed sugar of high selectivity that the invention provides branched water-soluble polymers is puted together, for example the novel means of sugar-PEGization.In exemplary of the present invention, the site-directed connection of branched water-soluble polymers is finished by the external enzymatic glycosylation of specific peptide sequence.Sugar puts together that can adopt can be with material branched water-soluble polymers-glycosyl, and for example the PEG-sialic acid is transferred to the glycosyl transferase of glycosylation site, and for example the sialic acid based transferase carries out (" sugar-PEGization ") by enzymatic method.

Branched water-soluble polymers

WSP-Y-R ^x

Wherein WSP is a water-soluble polymer.Symbol Y represents to connect base, key for example, or contain acid amides, carboxylate, urea alkane, mercaptan, replacement or the structure division of substituted alkyl etc. not.Exemplary connection base comprises: key, (CH ₂) _n, (CH ₂) _mC (O) O (CH ₂) _n, (CH ₂) _mC (O) NH (CH ₂) _n, (CH ₂) _mOC (O) NH (CH ₂) _n, (CH ₂) _mO (CH ₂) _n, (CH ₂) _mNH (CH ₂) _nAnd (CH ₂) _mS (CH ₂) _n, wherein m and n are the integers that is independently selected from 0-6.R ^xBe to be connected in the replacement of water-soluble polymer or substituted alkyl structure division not; Be connected in the amino acid or the amino acid whose dimer of water-soluble polymer; Perhaps be connected in the sugar or the ribotide of water-soluble polymer.WSP and R ^xWater-soluble copolymer component can be identical water-soluble polymer or different water-soluble polymers.

The exemplary water soluble polymer that is used for compound of the present invention comprises m-PEG, PEG, m-PPG, PPG, poly sialic acid, polyglutamic acid, poly-aspartate, polylysine, polymine, polyactide, polyglycerol ester and functionalized PEG, for example end-functionalization PEG.

Wherein X and Y are independently selected from OR ¹, NR ²R ³, SR ⁴, COOR ⁵, CONR ⁶R ⁷, OCONR ⁶R ⁷, replace and unsubstituted alkyl, and the member in replacement and the unsubstituted aryl.Z ¹Be to be selected from OR ¹', NR ²' R ³', SR ⁴', COOR ⁵', CONR ⁶' R ⁷', replace and substituted alkyl not, and the member in replacement and the unsubstituting aromatic yl.Symbol R ¹, R ⁴And R ⁵Represent water-soluble polymer.R ², R ³, R ⁶And R ⁷Be to be independently selected from H, replace and substituted alkyl not, replace and unsubstituting aromatic yl, replace and substituted heteroaryl not, replace and the unsubstituting heterocycle alkyl reactive functional groups, and member in the water-soluble polymer, prerequisite is to select these groups, and feasible compound according to general formula I comprises at least two water-soluble polymer structure divisions.Symbol R ¹', R ²', R ³', R ⁴', R ⁵', R ⁶' and R ⁷' expression is independently selected from H, replaces and substituted alkyl not, replaces and unsubstituting aromatic yl, replaces and substituted heteroaryl not, replaces and the unsubstituting heterocycle alkyl reactive functional groups, the group in carrier molecule and the water-soluble polymer.

Below provided exemplary compounds of the present invention according to general formula I:

With

R wherein ¹⁴Be OH or reactive functional groups.Exemplary reaction functional group is C (O) Q ', wherein selects Q ', makes that C (O) Q ' is a reactive functional groups.Q ' can also comprise carrier molecule (" part ").The exemplary material of Q ' comprises halogen, NHS, pentafluorophenyl group, HOBT, HOAt, and p-nitrophenyl.Subscript " m " and subscript " n " are independently to be selected from 1-20, the integer in 000.

[0098] above-claimed cpd and other compound of the present invention can easily be prepared by the initiation material such as following:

With

Below provided the exemplary approach that obtains compound of the present invention:

Part (ligand)=peptide, nucleotide sugar, lipid, glycolipid, sugar, DNA, RNA polymer

Below provided another the exemplary approach that obtains compound of the present invention:

With

R wherein ¹¹, R ^{11 '}, R ¹², R ^{12 '}, R ¹³And R ^{13 '}Be independently selected from H, replace or not substituted alkyl and water-soluble polymer, prerequisite is to select these groups, makes above-claimed cpd comprise at least two water-soluble polymer structure divisions.R ¹⁴Be to be selected from OH, reactive functional groups contains the group of sugared structure division or is connected in a kind of in the group of carrier molecule.A is selected from NH, a kind of among O and the S.The integer of subscript " s " expression 1-5.A is a member that is selected among NH, O and the S.

The chemical PEGization that can be used for other material (for example nucleic acid, peptide, sugar etc.) at each compound described in the above general formula.The method that forms the conjugate between the PEG (and contain PEG material) is well known in the art usually.For example, referring to Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; With people such as Feeney, MODIFICATION OF PROTEINS; Advances in ChemistrySeries, Vol.198, American Chemical Society, Washington, D.C., 1982.

Exemplary composition of the present invention comprises:

Such as:

Wherein " m ", " n " and " t " are independently selected from 1-20, the integer in 000; And R ¹⁴As mentioned above.

Other exemplary compounds comprises:

Such as:

In following table, provided other composition based on amino acid structure:

Among the figure that provides in last table, symbol a and b represent the numerical value of 1-10 independently.Symbol m and o represent 1-10 independently, 000 numerical value.Symbol X represents OH, H, and Q (activated group) and biological structure part, such as albumen, sugar, lipid, or nucleotide.

Exemplary compounds of the present invention has following general formula:

Sugar-O-(L-WSP) ₂

Wherein L is that connection base and WSP are water-soluble polymers.

Other exemplary compounds of the present invention has following general formula:

(C ₆H ₁₀O ₄)-(OC(O)-L-WSP) ₂

C wherein ₆H ₁₀O ₄Be a sugar core, wherein two sugared OH structure divisions are converted into OC (O)-connection base-WSP.

Another exemplary compounds of the present invention has following general formula:

Nucleotide-sugar-O-(L-WSP) ₂

Nu-O-(C ₆H ₉O ₃)-(OC(O)-L-WSP) ₂

Wherein Nu is a nucleotide.

R wherein ¹⁶And R ^{16 '}Be to be selected from H, the member in acetyl group and the general formula (I):

And R ¹⁷, R ¹⁸, R ¹⁹And R ¹⁹' be to be independently selected from H, OH, NH ₂, NHAc and according to the member in the structure division of general formula I.In general formula I, Z ²Be to be selected from O, S, CH ₂A kind of with among the S.R ¹¹As mentioned above, and the integer of subscript " a " expression 0-20, prerequisite is R ¹⁶, R ¹⁶', R ¹⁷, R ¹⁸, and R ¹⁹At least two structures that have according to general formula I.R ¹¹It can also be the key that is connected in the group of carrier molecule or is bonded in carrier molecule.R ¹⁵Be to be selected from H and activated group, a member in the phosphoric acid nucleoside acid for example.

In another exemplary, the connection base of general formula I has following structural formula:

Z wherein ³Be to be selected from NH, a member among O and the S.

In an exemplary, Z ²Be NH.

In another exemplary, the invention provides compound with following structure:

Wherein L connects base as herein defined.

In yet another aspect, branched polymer is based on galactose or N-acetyl group galactose, and it has following general formula:

The exemplary arrangement for preparing branching sugar core water-soluble polymer of the present invention below is provided:

Below provided another exemplary arrangement for preparing sugared core branched water-soluble polymers of the present invention:

Single dispersion poly-(ethylene glycol)

The present invention also provides basic single method of disperseing colony of mono-disperse polymer amount PEG and poly-(ethylene glycol) molecule of preparation.This method comprises allows the PEG molecule difunctionality activated PEG that also has clear and definite molecular weight of PEG200 and at least 2 equivalents for example with clear and definite molecular weight, for example PEG200 contact, thus produce single dispersed sample of PEG, PEG600 for example:

G is a leaving group, such as sulphonic acid ester or tresylat e ester.Single dispersed sample of PEG600 can contact with difunctionality activated PEG 200 then, forms single PPEG100 of dispersion.Perhaps, single PEG600 that disperses can be converted into corresponding Bifunctionalized derivative, and the single dihydroxy-PEG600 that disperses with at least 2 equivalents reacts again, produces single PEG1800 of dispersion.Repeat method of the present invention, till the single PEG of dispersion that obtains required size.Can design this and synthesize, make the molecular weight differences permission between initiation material and product separate the material of any unreacted or partial reaction by SEC.

Therefore, in yet another aspect, the invention provides the activated water soluble polymer, especially the method for the substep of poly-(ethylene glycol) and analogue thereof assembling.This method provides the convenient way that obtains monofunctional and Bifunctionalized PEG molecule.

In step a, original glycol contacts with activated group (R-Y), the hydroxyl structure partial reaction of this activated group and glycol.Y is leaving group normally, and R is placed on one of the hydroxyl structure part of PEG molecule.In step b, the free hydroxyl group of gained adduct activates by the group that it is converted into sulfonic acid esters.The activation the PEG material with as initial PEG (" PEG _m") another PEG structure division contact of the identical or different degree of polymerization.In order to make and it is connected in another material, RO-PEG _(n+m)Choose wantonly in free hydroxyl group structural portion office and activate.

Single PEG that disperses of the present invention activates by art-recognized method easily, and the derivative of activation can be used for forming conjugate.Perhaps, this list dispersion PEG is introduced in and is used for forming among the branching PEG of the present invention of conjugate.

Water-soluble polymer

The hydrophily of the peptide of selecting is by puting together and improve such as containing amine, ester, hydroxyl and polyhydric molecule with polar molecule.Representative example is including, but not limited to polylysine, and polymine, and polyethers for example gather by (ethylene glycol), and m-gathers (ethylene glycol), poly-(propane diols), poly-(aklylene glycol) structure division of m-poly-(propane diols) and other O-alkyl.Preferred water-soluble polymer is non-fluorescence basically, perhaps sends the fluorescence of minimum and be not suitable for being used in tagged fluorescent agent in analysis.And usually preferred use does not belong to the polymer of naturally occurring sugar.This preferred exception is to use the naturally occurring sugar by covalently bound another entity (biomolecule is treated and used structure division for for example poly-(ethylene glycol), poly-(propane diols), and diagnosis grades with structural portion) modification.In another exemplary, treatment is puted together in linking arm with sugared structure division, and sugar-linking arm box (cassette) is puted together in peptide with method of the present invention subsequently.

Be used for the method and the chemical process of activated water soluble polymer and sugar and be used for sugar and polymer are puted together in the method for various materials and stated in the literature.The method of normally used activated polymer comprises functional group's cyanogen bromide, periodate, glutaraldehyde, di-epoxide, chloropropylene oxide, divinylsulfone, carbodiimides, sulfonic acid halide, the activation of three chlorotriazines etc. (referring to R.F.Taylor, (1991), PROTEIN IMMOBILISATION.FUNDAMENTALS ANDAPPLICATIONS, Marcel Dekker, N.Y.; S.S.Wong, (1992), CHEMISTRYOF PROTEIN CONJUGATION AND CROSSLINKING, CRC Press, Boca Raton; People such as G.T.Hermanson, (1993), IMMOBILIZED AFFINITY LIGANDTECHNIQUES, Academic Pres s, people such as N.Y., Eds.POLYMERIC DRUGS ANDDRUG DELIVERY SYSTEMS, ACS Symposium Series Vol.469, AmericanChemical Society, Washington, D.C.1991).

Many water-soluble polymers are that those skilled in the art is known, and can be used for implementing the present invention.The term water-soluble polymer comprises the material such as following: sugar (dextran for example, amylose, hyaluronic acid, poly-(sialic acid), heparan, heparin etc.; Poly-(amino acid); Nucleic acid; Synthetic polymer (for example poly-(acrylic acid), poly-(ether), for example poly-(ethylene glycol); Peptide, albumen etc.The present invention can implement with any water-soluble polymer, and unique restriction is the site that this polymer must comprise the remainder that can connect conjugate.

The method that is used for activated polymer can also be at WO 94/17039, US patent No.5,324,844, WO 94/18247, and WO 94/04193, US patent No.5,219,564, US patent No.5,122,614, WO 90/13540, US patent No.5,281,698 and WO93/15189 in find, and in order to put together activated polymer and peptide, blood coagulation factor VIII (WO 94/15625) for example, haemoglobin (WO 94/09027) is taken oxygen molecule (US patent No.4,412,989), ribalgilase and superoxide dismutase (people such as Veronese, App.Biochem.Biotech.11:141-45 (1985)).

Preferred water-soluble polymer be wherein in the sample of polymer most of polymer molecule have those of approximately identical molecular weight; These polymer are " single dispersions ".

The present invention further with reference to poly-(ethylene glycol) or mono methoxy-poly-(ethylene glycol) (m-PEG) conjugate illustrate.Can obtain to comment on and monograph with several pieces that put together about the functionalized of PEG.For example, referring to Harris, Macronol.Chem.Phys.C25:325-373 (1985); Scouten, Methods in Enzymology135:30-65 (1987); People such as Wong, Enzyme Microb.Technol.14:866-874 (1992); People such as Delgado, Critical Reviews in Therapeutic Drug Carrier Systems 9:249-304 (1992); Zalipsky, Bioconjugate Chem.6:150-165 (1995); With people such as Bhadra, Pharmazie, 57:5-29 (2002).

Poly-(ethylene glycol) that can be used for forming conjugate of the present invention is linearity or branching.Any molecular weight, 5Kd for example, 10Kd, the PEG structure division of 20Kd and 30Kd can be used for the present invention.

Reactive functional groups

The reactive derivatives of PEG (or other connects base) is used for one or more peptide structure divisions are connected in the basic purposes of this connection within the scope of the invention.The present invention is not limited by the identity of reactive PEG analog.Many activated derivatives of poly-(ethylene glycol) can be commercial, and state in the literature.Select and synthetic if desired to can be used to prepare the suitable activated PEG derivative that can be used for substrate of the present invention be in those skilled in the art's the limit of power fully.Referring to people such as Abuchowski, Cancer Biochem.Biophys., 7:175-186 (1984); People such as Abuchowski, J.Biol.Chem., 252:3582-3586 (1977); People such as Jackson, Anal.Biochem., 165:114-127 (1987); People such as Koide, Biochem Biophys.Res.Commun., 111:659-667 (1983)), tresylate (people such as Nilsson, Methods Enzymol., 104:56-69 (1984); People such as Delgado, Biotechnol.Appl.Biochem., 12:119-128 (1990)); The active ester that N-maloyl imines is derived (people such as Buckmann, Makromol.Chem., 182:1379-1384 (1981); People such as Joppich, Makromol.Chem., 180:1381-1384 (1979); People such as Abuchowski, Cancer Biochem.Biophys., 7:175-185 (1984); People such as Katre, Proc.Natl.Acad.Sci.U.S.A., 84:1487-1491 (1987); People such as Kitamura, Cancer Res., 51:4310-4315 (1991); People such as Boccu, Z.Naturforsch., 38C:94-99 (1983), carbonic ester (people such as Zalipsky, POLY (ETHYLENE GLYCOL) CHEMISTRY:BIOTECHNICAL AND BIOMEDICAL APPLICATIONS, Harris, Ed., Plenum Press, New York, 1992, pp.347-370; People such as Zalipsky, Biotechnol.Appl.Biochem., 15:100-114 (1992); People such as Veronese, Appl.Biochem.Biotech., 11:141-152 (1985)), formic acid imidazate (people such as Beauchamp, Anal.Biochem., 131:25-33 (1983)); People such as Berger, Blood, 71:1641-1647 (1988), 4-two sulfo-pyridines (people such as Woghiren, Bioconjugate Chem., 4:314-318 (1993)), isocyanates (people such as Byun, ASAIO Journal, M649-M-653 (1992)) and epoxides (US patent No.4,806,595, license to people such as Noishiki (1989).Other connects the basic urethane bond that is included between amino and the activated PEG.Referring to people such as Veronese, Appl.Biochem.Biotechnol., 11:141-152 (1985).

Can be used for implementing reactive group of the present invention and reaction type normally in the bioconjugates chemical field known those.The present favourable reaction type that obtains with the reactive sugars structure division is those that carry out under relative temperate condition.These are including, but not limited to nucleophilic displacement of fluorine (for example, the reaction of amine and alcohol and acyl halide, active ester), and parent's electricity replaces (for example enamine reaction) and the addition on carbon-to-carbon and carbon-hetero atom multiple bond (for example, Michael reaction, Diels-Alder addition).These and other useful reaction is for example at March, ADVANCEDORGANIC CHEMISTRY, 3 ^RdEd., John Wiley ﹠amp; Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; And people such as Feeney, MODIFICATION OF PROTEINS; Advances inChemistry Series, Vol.198, American Chemical Society, Washington, D.C. states in 1982.

The useful reactive functional groups of hanging from sugar nuclear or modification group upside including, but not limited to:

(a) carboxyl and various derivative thereof are including, but not limited to N-maloyl imines ester, N-hydroxybenzotriazole ester, carboxylic acid halides, acylimidazole, thioesters, p-nitrophenyl ester, alkyl, alkenyl, alkynyl and aromatic ester;

(b) hydroxyl, it can be converted into for example ester, ether, aldehyde etc.;

(c) haloalkyl, wherein halogen can be afterwards by nucleophilic group amine for example, carboxylate anion, the mercaptan anion, carboanion, or alkoxyl ion exchange, thus cause new group in the functional group of halogen atom, to connect with covalent bond;

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

(e) aldehydes or ketones group makes that follow-up derivatization can be by forming carbonyl derivative, imines for example, and hydrazone, semicarbazones or oximes, or undertaken by the mechanism such as Ge Liya addition or lithium alkylide addition;

(f) be used for reacting with amine subsequently, for example form the sulfonyl halide groups of sulfonamide;

(g) thiol group, it for example can be converted into disulphide or react with acyl halide;

(h) amine or sulfydryl, it for example can carry out acidylate, alkylation or oxidation;

(i) olefine, they for example can carry out cycloaddition, acidylate, Michael addition etc.; With

(j) epoxides, they for example can react with amine and hydroxy compounds.

Can choice reaction functional group, make them not participate in, perhaps disturb assembling reactive sugars nuclear or the necessary reaction of modification group.Perhaps, reactive functional groups can be protected by protecting group is provided, in order to avoid participate in reaction.Skilled in the art will recognize that how to protect the particular functional group, make it not interfere a group reaction condition of selection.About the example of effective protecting group, for example referring to people such as Greene, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, John Wiley ﹠amp; Sons, New York, 1991.

Peptide conjugate

Application of compound of the present invention illustrates by their purposes in the peptide conjugate that forms branched water-soluble polymers.The focus of discussing is in order to clearly demonstrate.The technical staff understands that this argumentation is relevant with the method that use branched water-soluble polymers of the present invention forms various conjugates.In exemplary, the chemical reactivity branched water-soluble polymers is puted together complementary interaction group on peptide by methods known in the art or its modification.

In another exemplary, branched water-soluble polymers comprises that perhaps, this branched water-soluble polymers is connected in sugar as a sugared structure division of type nuclear.Sugar is the substrate of glycosyl branched water-soluble polymers (or sugar-branched water-soluble polymers conjugate) being transferred to the enzyme of the amino acid of peptide or glycosyl residue.It will be apparent to those skilled in that said method is not limited to implement with peptide, but is widely used in other material, lipid for example, glycolipid, other treatment structure division of sugar nuclear.

The sugar of conjugate of the present invention by the branched water-soluble polymers modification is connected with the enzymatic of glycosylation or non-glycosylated peptide and forms.Modified sugars directly joins glycosylation position, or direct or indirect (for example by one or more glycosyl residues) are connected in the glycosyl residue of glycosylation position.

When inserting between the modification group of peptide (or glycosyl residue) and sugar, branched water-soluble polymers-modified sugars becomes this paper so-called " glycosyl connects base ".It can be " complete " that glycosyl connects base, and perhaps it can change in the connection procedure of branched water-soluble polymers and sugar, for example oxidation and reduction amination.Use the high selectivity of enzyme such as glycosyl transferase, the inventive method provides the peptide that carries branched water-soluble polymers at one or more ad-hoc locations.Therefore, according to the present invention, modified sugars is directly connected in the chosen position on the peptide chain, and perhaps, modified sugars is connected in the carbohydrate structure part of glycopeptide.Wherein the modified sugars peptide that is bonded in the glycopeptide carbohydrate and directly is bonded in the amino acid residue of peptide backbone also is within the scope of the invention.

Different with known chemistry with enzymatic peptide Processing Strategies, the invention provides and have basically uniformly the peptide of deriving mode-and glycopeptide-conjugate; The enzyme of Cai Yonging is generally selective to the combination of the particular amino acid residue of peptide or amino acid residue in the present invention.Conjugate of the present invention also can be with mass preparation.Therefore, the invention provides the practical approach that mass preparation has the glycopeptide of preselected even derivatization mode.These methods are particularly suitable for the modification of therapeutic peptide, including, but not limited at cell culture cell (mammalian cell for example, insect cell, plant cell, fungal cell, yeast cells or prokaryotic) or genetically modified plants or animal in incomplete in process of production glycosylated glycopeptide.

The branched water-soluble polymers conjugate of peptide generally is characterized as being to have the treatment half life period of increasing, for example because the clearance rate that lowers, or lower by the speed of immunity or reticuloendothelial system (RES) picked-up.And the antigenic determinant on the peptide composition of conjugate of the present invention shields with branched water-soluble polymers, has reduced or eliminated the immune response of host to this peptide.Use the directed agents of suitable modified sugars to be connected and can also be used to peptide guiding particular organization or the cell surface receptor special to specific directed agents with the selectivity of peptide.

Treatment with the half life period in the body of glycopeptide can also with comprise polyethylene glycol (PEG, m-PEG) and the enhancing of the branched water-soluble polymers of polypropylene glycol (PPG).For example, albumen has increased them with the chemical modification (PEGization, m-PEGization) of branching PEG molecular dimension and the surface that has reduced them-and functional group-accessibility, the latter is depended on the size of the PEG that is connected in albumen separately.Peptide generally is acknowledged as with the modification of water-soluble polymer and improves plasma half-life and proteolysis stability, and the promising strategy (people such as Chaffee, the J.Clin.Invest.89:1643-1651 (1992) that reduce immunogenicity and liver picked-up; People such as Pyatak, Res.Commun.Chem.Pathol Pharmacol.29:113-127 (1980)).The PEGization of having reported proleulzin has improved antitumor potential in its body (people such as Katre, Proc.Natl.Acad.Sci.USA.84:1487-1491 (1987)) and the PEGization of the F (ab ') 2 that obtains by monoclone antibody A7 improved its tumor-localizing (people such as Kitamura, Biochem.Biophys.Res.Commun.28:1387-1394 (1990)).Therefore, in a further preferred embodiment, improve with respect to the half life period in the body of non-derivative peptide with the half life period in the body of the peptide of water-soluble polymer derivatization by method of the present invention.

The increase of half life period is represented as the percentage scope that this amount increases best in the peptide body of conjugate of the present invention.The lower limit of the scope that percentage increases is about 40%, about 60%, about 80%, about 100%, about 150% or about 200%.The upper limit of this scope is about 60%, about 80%, about 100%, about 150% or surpasses about 250%.

In exemplary, the connection between peptide and choice structure part comprises that the intact glycosyl between peptide and water-soluble polymer connects base.As described herein, water-soluble polymer provides by " modified sugars " of suitable transferase identification with sugared structure division be connected (or use of a sugar type nuclear), and it is connected in peptide with modified sugars.When inserting between peptide and choice structure part, the saccharic composition of modified sugars becomes " glycosyl connects base ", for example " intact glycosyl connects base ".This glycosyl connects base and is formed by any monose or oligosaccharides, and after the modification of usefulness water-soluble polymer, it is the substrate of suitable transferase.

Conjugate of the present invention is usually corresponding to following general formula:

Wherein symbol a, b, c, d and s represent positive nonzero integer; And t is 0 or positive integer." reagent " is branched water-soluble polymers of the present invention.Perhaps, sugar-reagent provides by the branched water-soluble polymers that props up type nuclear based on sugar.This connection base can be hereinafter various any of base that connect.Perhaps, this connection base can be singly-bound or " zero level connects base ".The identity of peptide does not limit.

In exemplary, this water-soluble polymer is PEG, m-PEG, and PPG or m-PPG, and this branched water-soluble polymers is connected in peptide via complete glycosyl connection base with covalent bond.Glycosyl connects base is connected in peptide with covalent bond amino acid residue or glycosyl residue.Perhaps, this glycosyl connects one or more glycosyl units that base is connected in glycopeptide.The present invention also provides wherein, and glycosyl connects the conjugate that base (for example GalNAc) is connected in amino acid residue (for example Thr or Ser).

Except the basic conjugate that forms of intact glycosyl connection by the enzymatic addition is provided, the invention provides it and replace the uniform conjugate of mode height.Use method of the present invention, can form peptide conjugate, wherein all basically modified sugars structure divisions are connected in identical amino acid of structure or glycosyl residue in the colony of conjugate of the present invention.Therefore, aspect second, the invention provides the peptide conjugate with branched water-soluble polymers structure division colony, these structure divisions connect base by glycosyl, and for example intact glycosyl connection base is connected in peptide with covalent bond.In preferred conjugate of the present invention, each member basically of this colony connects the glycosyl residue that base key is connected to peptide via glycosyl, and each glycosyl residue of the basic peptide that is connected of glycosyl connection has identical structure.

The present invention also provides to have by intact glycosyl and has connected the peptide conjugate of base with the colony of the branched water-soluble polymers structure division of covalent bond connection.In preferred embodiments, each member basically of the colony of branched water-soluble polymers structure division connects base key via complete glycosyl and is connected to the amino acid residue of peptide, and has each amino acid residue that connection intact glycosyl thereon connects base and have same structure.

The present invention also provides and above-mentioned those similar conjugates, and wherein this peptide is further puted together in the treatment structure division via intact glycosyl connection base, and structure division is used in diagnosis, target structure division, toxin structure part etc.The said structure part can be little molecule separately, natural polymer (for example, polypeptide) or synthetic polymer.

In another embodiment, the invention provides owing to the directed agents that exists as the component of conjugate, the selective fixed conjugate that is arranged in particular organization.In an exemplary, this directed agents is an albumen.Exemplary albumen comprises siderophillin (brain, blood pond), HS-glycoprotein (bone, brain; the blood pond), (brain has the tissue of antibody-specific antigen to antibody; the blood pond), labile factor-XII (damaged tissue, grumeleuse; cancer, the blood pond), haemocyanin; alpha-acid glycoprotein for example, myosin, α-fetus albumen (brain; the blood pond), beta 2-glycoprotein (liver, atherosclerotic plaque; brain, the blood pond), G-CSF; GM-CSF, M-CSF, and EPO (immunostimulation; cancer, the blood pond, erythrocyte excessively produces; neuroprotective), albumin (half life period increase), IL-2 and IFN-α.

In exemplary, this conjugate forms between branched water-soluble polymers and glycosylation or non-glycosylated peptide.This polymer, treatment connect base with structure division or biomolecule via intact glycosyl and put together in peptide, and this connects between base insertion peptide and the modification group (for example water-soluble polymer), and is connected in the two with covalent bond.This method comprises allows the peptide be that the mixture of the glycosyl transferase of its substrate contacts with containing modified sugars and modified sugars.This is reflected at is enough to carry out under the condition that forms covalent bond between modified sugars and the peptide.The sugared structure division of modified sugars is preferably from nucleotide sugar, activation sugar with not only selected non-nucleotide but also the non-activated sugar.

Acceptor peptide (glycosylation or non-glycosylated) is from the beginning synthetic usually, perhaps at prokaryotic (for example bacterial cell, such as E.coli) or eukaryotic such as recombinant expressed in mammal, yeast, insect, fungi or the plant cell.This peptide can be full-length proteins or fragment.And this peptide can be the peptide of wild type or sudden change.In exemplary, peptide comprises the sudden change that one or more total glycosylation sites is joined peptide sequence.

Method of the present invention also is provided for the modification of the incomplete glycosylated peptide of recombinant production.The glycopeptide of many recombinant production is by glycosylation by halves, exposed to have undesirable performance, for example immunogenicity, the carbohydrate residue discerned by RES.Use modified sugars in the method for the invention, peptide glycosylation simultaneously further and for example use derivatizations such as water-soluble polymer, therapeutic agent.The sugared structure division of modified sugars can be a residue of suitably puting together the acceptor in complete glycosylated peptide, perhaps has another glycosyl structure division of desired properties.

In table 1, provided the exemplary peptides component of conjugate of the present invention.

Table 1

Hormone and growth factor	Acceptor and Chimerical receptor
Hormone and growth factor	Acceptor and Chimerical receptor	·G-CSF	·CD4
·GM-CSF	TNF (THF) acceptor	·G-CSF	·CD4
·GM-CSF	TNF (THF) acceptor	·M-CSF	·α-CD20
·TPO	·MAb-CD20	·M-CSF	·α-CD20
·TPO	·MAb-CD20	·EPO	·MAb-α-CD3
The EPO variant	The MAb-TNF acceptor	·EPO	·MAb-α-CD3
The EPO variant	The MAb-TNF acceptor	·α-TNF	·MAb-CD4
Leptin	·PSGL-1	·α-TNF	·MAb-CD4
Leptin	·PSGL-1	Enzyme and inhibitor	·MAb-PSGL-1
·t-PA	Complement	Enzyme and inhibitor	·MAb-PSGL-1
·t-PA	Complement	The t-PA variant	GlyCAM or its chimera
Urokinase	N-CAM or its chimera	The t-PA variant	GlyCAM or its chimera
Urokinase	N-CAM or its chimera	Factor VII, VIII, IX, X	Monoclone antibody (immunoglobulin)
Deoxyribonuclease	MAb-resists-RSV	Factor VII, VIII, IX, X	Monoclone antibody (immunoglobulin)
Deoxyribonuclease	MAb-resists-RSV	Glucocerebrosidase
Hirudin	MAb-resists-the IL-2 acceptor	Glucocerebrosidase
Hirudin	MAb-resists-the IL-2 acceptor	α 1 anti-insulin	MAb-resists-CEA
Antithrombin III	MAb-antiplatelet IIb/IIIa acceptor	α 1 anti-insulin	MAb-resists-CEA
Antithrombin III	MAb-antiplatelet IIb/IIIa acceptor	The cell factor and the chimeric cell factor	MAb-resists-EGF
Il-1 (IL-1), 1B, 2,3,4	MAb-resists-the Her-2 acceptor	The cell factor and the chimeric cell factor	MAb-resists-EGF
Il-1 (IL-1), 1B, 2,3,4	MAb-resists-the Her-2 acceptor	Interferon-' alpha ' (IFN-α)	Cell
·IFN-α-2b	Erythrocyte	Interferon-' alpha ' (IFN-α)	Cell
·IFN-α-2b	Erythrocyte	·IFN-β	Leukocyte (for example T cell, B cell, dendritic cells, macrophage, NK cell, neutrophil leucocyte, monocyte etc.)
·IFN-γ	Stem cell	·IFN-β
·IFN-γ	Stem cell	Chimeric diptheria toxin I L-2

Other exemplary peptides component of conjugate of the present invention comprises member's (antibody for example of immunoglobulin family, MHC molecule, TXi Baoshouti etc.), iuntercellular acceptor (integrin for example, the acceptor of hormone or growth factor etc.) agglutinin, and cell factor (for example interleukin).Other example especially comprises tissue plasminogen activator (t-PA), feritin, and clotting factor is such as factor V-XII, bombesin, fibrin ferment, hemopoieticgrowth factor, colony stimulating factor, viral antigen, complement protein, alpha1-antitrypsin, erythropoietin(EPO), P-selects albumen glycopeptide part-1 (PSGL-1), granulocyte-macrophage colony stimulatory factor, Antithrombin III, interleukin, interferon, a-protein and C, fibrinogen, Trastuzumab (herceptin), Leptin (leptin), glycosidase, HS-glycoprotein, haemocyanin (for example, alpha-acid glycoprotein, myosin, α-fetus protein), beta 2-glycoprotein.This polypeptide list is exemplary, is not exclusiveness.The peptide composition of conjugate can also comprise fusion and chimeric protein, including, but not limited to containing by immunoglobulin such as the fragment of IgG or the immunoglobulin chimeric protein of FAb (Fc district) structure division of deriving for example.In addition, having provided in appendix 1 can be with the exemplary peptides of method modification of the present invention.Exemplary peptides provided herein is used to provide the selection that can be used for implementing peptide of the present invention; Therefore, they are not restrictive.The technical staff is clear, and the present invention can use all basically peptides that can use from any source to implement.

The peptide composition of conjugate of the present invention can be synthetic or wild type peptide, and perhaps they can be the peptides of sudden change, and the latter uses methods known in the art, produces such as directed mutagenesis.The glycosylation of peptide normally N-connect or O-connects.It is modified sugars and being connected of the side chain of asparagine residue that exemplary N-connects.Tripeptide sequence asparagine-X-serine is the recognition sequence that the carbohydrate structure part is connected with the enzyme process of asparagine side chain with asparagine-X-threonine (wherein X is any amino acid except proline).Therefore, any existence in polypeptide of these tripeptide sequences has produced potential glycosylation site.The glycosylation that O-connects is meant a sugar (N-acetyl group galactosamine for example, galactose, mannose; GlcNAc, glucose, fucose; wood sugar) and hydroxy-amino-acid, the connection of the hydroxyl side chain of preferred serine or threonine is though can also use 5-hydroxy-proline or 5-oxylysine.

And except peptide, the component of the conjugate of puting together with branched water-soluble polymers of the present invention can be the biological structure (for example glycolipid, lipid, sphingol, ceramide, whole cell etc.) except peptide.

Glycosylation site is joined peptide or other structure can make it contain one or more glycosylation sites and finish easily by changing amino acid sequence.This interpolation can be undertaken by the full chemosynthesis of sudden change or peptide.The peptide ammino acid sequence preference especially by make the dna mutation of this peptide of coding with preselected base, makes generation be translated as amino acid needed codon and changes by the change under dna level.Dna mutation preferably uses methods known in the art to carry out.

In exemplary, glycosylation site adds by reorganization (shuffling) polynucleotides.The polynucleotides of coding candidate's peptide can be reorganized operation with DNA and be regulated.DNA reorganization is the method for recurrence reorganization and sudden change, by random division related gene storehouse, re-assemblies fragment by the polymerase chain reaction class methods subsequently and carries out.For example, referring to Stemmer, Proc.Natl.Acad.Sci.USA 91:10747-10751 (1994); Stemmer, Nature370:389-391 (1994); With U.S. patent Nos.5,605,793,5,837,458,5,830,721 and 5,811,238.

Conjugate of the present invention can also comprise and added or removed one or more selection glycosyl residues, after this modified sugars puted together in the peptide of at least one of the selection glycosyl residue of peptide.For example, when hope was puted together modified sugars in the selection glycosyl residue that is not present on the peptide or do not exist with aequum, this embodiment was useful.Therefore, before modified sugars is coupled to peptide,, will select glycosyl residue to put together in peptide by enzymatic coupling or chemical coupling.In another embodiment, the glycosylation mode of glycopeptide was changing by remove the carbohydrate residue from glycopeptide before puting together modified sugars.For example, referring to WO 98/31826.

Increase or remove any carbohydrate structure part that is present on the glycopeptide and finish with chemical method or enzyme process.The chemistry deglycosylation is preferably finished by polypeptide variants contact compound trifluoromethanesulfonic acid or equivalent compound.This processing has caused great majority or all the sugared cracking except connecting sugar (N-acetyl glucosamine or N-acetyl group galactosamine), but keeps peptide complete.People such as Hakimuddin, people such as Arch.Biochem.Biophys.259:52 (1987) and Edge, Anal.Biochem.118:131 (1981) has described chemical deglycosylation.The enzymatic lysis of the carbohydrate structure part on polypeptide variants can be by use as by people such as Thotakura, and described various endoglycosidases of Meth.Enzymol.138:350 (1987) and exoglycosidase are finished.

The chemical addition of glycosyl structure division is undertaken by any technical generally acknowledged method.The modification of method as herein described is preferably used in the enzymatic addition of glycosyl structure division, carries out with the modified sugars that natural glycosyl units replaces using in the present invention.Other method that adds the saccharogenesis structure division is disclosed in US patent No.5, in 876,980,6,030,815,5,728,554 and 5,922,577.

The exemplary link position that is used to select glycosyl residue is including, but not limited to (a) the glycosylated total site of N-and O-; (b) belong to the terminal saccharide structure division of the acceptor of glycosyl transferase; (c) arginine, asparagine and histidine; (d) free carboxy; (e) free sulfhydryl groups is such as those of cysteine; (f) free hydroxyl group, such as serine, threonine, or those of hydroxy-proline; (g) aromatic moieties, such as phenylalanine, tyrosine, those of tryptophan; Or (h) amido of glutamine.Be used for illustrative methods of the present invention and be described in disclosed WO 87/05330 and Aplin and Wriston on September 11st, 1987, CRC, CRIT.REV.BIOCHEM. is among the pp.259-306 (1981).

In one embodiment, the invention provides by connecting the method that base connects two or more peptides.This connection base has any effective structure, can be selected from straight chain and branched structure.Preferably, each end that is connected in the connection base of peptide comprises modified sugars (that is, nascent complete glycosyl connect base).

In illustrative methods of the present invention, via comprising that branched water-soluble polymers connects basic syndeton part two peptides are connected together.This constructor is combined in the general formula shown in the above figure.As described herein, structure of the present invention comprises that two complete glycosyls connect base (being s+t=1).It is for purpose clearly that the PEG that concentrated description comprises two glycosyls connects base, should not be considered to limit the identity of the linking arm that is used for this embodiment of the present invention.

Modified sugars

Modification glycosyl donor material (" modified sugars ") is preferentially from modified sugars nucleotide, activation modification sugar and belong to not only non-nucleotide but the modified sugars of non-activated simple sugars in select.Any desirable carbohydrate structure can be incorporated in the conjugate of the present invention.Usually, this structure is a monose, but the invention is not restricted to use modification monose; Oligosaccharides and polypeptide also are useful.

The modification group is by the enzyme mode, chemical mode or their combination and be connected in sugared structure division, thus produce modified sugars.Sugar allow to connect the modified structure part, but still sugar is used from modified sugars is incorporated on any position of effect of substrate of enzyme of peptide and replace.In a preferred embodiment, when sialic acid was sugar, sialic acid was gone up or is normally replaced with the modification group on 5 of acetylizad amine structure part in sialic acid at 9 of pyruvoyl (pyruvyl) side chain.

In certain embodiments of the invention, utilize modified sugars nucleotide that modified sugars is added on the peptide.The exemplary ribotide that adopts in the present invention with its modified form comprises one, two or triphosphopyridine nucleotide or their analog.In preferred embodiments, modified sugars nucleotide is selected from the UDP-glucosides, CMP-glucosides, or GDP-glucosides.Also more preferably, modified sugars nucleotide is selected from the UDP-galactose, UDP-galactosamine, UDP-glucose, UDP-aminoglucose, GDP-mannose, GDP-fucose, cmp sialic acid, or CMP-NeuAc.The N-acetyl amine derivative of ribotide also can be used for method of the present invention.

The present invention also provides the use modified sugars, modification galactose for example, modification fucose, the method for modification GalNAc and modification sialic acid synthesis modification peptide.When adopting the modification sialic acid, sialic acid based transferase or trans sialidase (only for α 2, the sialic acid that 3-connects) can use in these methods.

In other embodiments, modified sugars is an activation sugar.Can be used for activation modification sugar of the present invention and normally comprise the glucosides that activates leaving group by synthetic changing into.Term as used herein " activation leaving group " is meant those structure divisions of being replaced easily in the nucleophilic substitution that enzyme is regulated.Many activation sugar have been known in the art.For example, referring to people such as Vocadlo, In CARBOHYDRATE CHEMISTRY AND BIOLOGY, Vol.2, people such as Ernst, Ed., Wiley-VCH Verlag:Weinheim, Germany, 2000; People such as Kodama, Tetrahedron Lett.34:6419 (1993); People such as Lougheed, J.Biol.Chem.274:37717 (1999).

The example of activated group (leaving group) comprises fluorine, chlorine, bromine, tosylate, methanesulfonates, triflate etc.Being used for preferred activation leaving group of the present invention is that not remarkable steric hindrance glucosides enzyme process is transferred to those on the acceptor.Therefore, the preferred embodiment of activation glycosides derivatives comprises glycosyl fluoride and glycosyl methanesulfonates, and wherein the glycosyl fluoride is particularly preferred.In the glycosyl fluoride, fluoridize α-galactose, fluoridize α-mannose; fluoridize phlorose, fluoridize α-fucose, fluoridize α-wood sugar; fluoridize α-sialic acid, fluoridize α-N-acetyl glucosamine, fluoridize α-N-acetyl group galactosamine; fluoridize beta galactose, fluoridize β-mannose, fluoridize β-glucose; fluoridize β-fucose; fluoridize β-wood sugar, fluoridize β-sialic acid, it is most preferred fluoridizing β-N-acetyl glucosamine and fluoridizing β-N-acetyl group galactosamine.

For instance, can be by at first with sugared acetylization with handle the cause free sugar with the HF/ pyridine then and prepare the glycosyl fluoride.This has produced the anomer of the most stable protection (acetylization) the glycosyl fluoride (that is alpha-glycosyl fluoride) of thermodynamics.Unsettled if desired anomer (that is, β-glycosyl fluoride), it can be by with HBr/HOAc or full acetylated sugar is converted into different bromide to HCl or chloride prepares.The acetylated glycosyl fluoride can be by reacting the protection of making a return journey with gentle (catalysis) alkali in methyl alcohol (for example NaOMe/MeOH).In addition, many glycosyl fluorides can be commercial.

Other activation glycosyl derivatives can use commonsense method well known by persons skilled in the art to prepare.For example, the methanesulfonic acid glycosyl ester can be by handling the sugar of complete benzyl hemiacetal form with mesyl chloride, and subsequent catalytic hydrogenation prepares to remove benzyl.

In another exemplary, modified sugars is the oligosaccharides with feeler structure.In a preferred embodiment, one or more ends of feeler carry the modified structure part.When more than one modified structure partly was connected in the oligosaccharides with feeler structure, this oligosaccharides can be used for " amplification " modified structure part; Put together in each oligosaccharides unit of peptide the modification group of a plurality of copies is connected in this peptide.Structure has comprised by adopting the feeler structure to prepare the multivalence material that conjugate of the present invention obtains as at the of the present invention typical conjugate as shown in the last figure.The sugared structure of many feelers is well known in the art, and the inventive method can be with restrictedly and implement with them.

Generally, sugared structure division and modification group connect together by using reactive group, and this reactive group is converted into new organo-functional group or unreacted matters by method of attachment usually.The sugar reactive functional groups is positioned on any position of sugared structure division.

In the following discussion, the many object lessons that can be used for implementing modified sugars of the present invention have been illustrated.In exemplary, sialic acid is examined with the sugar that connects the modification group.It is in order to clearly demonstrate, should not to be considered to the restriction of scope of the present invention that argumentation concentrates on sialic acid derivative.It will be obvious to those skilled in the art that multiple other sugared structure division can be according to activating and derivatization with using the described as an example similar mode of sialic acid.For example, many methods can be used for the modification galactose, and glucose, N-acetyl group galactosamine and fucose provide several sugared substrates, and they come modification by art-recognized method easily.For example, referring to people such as Elhalabi, Curr.Med.Chem.6:93 (1999); With people such as Schafer, J.Org.Chem.65:24 (2000).

In exemplary, peptide by method modification of the present invention is at prokaryotic (for example E.coli.), the eukaryotic that comprises yeast and mammalian cell (for example, Chinese hamster ovary celI), or producing the glycopeptide that also therefore contains the oligonucleotide chain of N-and/or O-connection in the transgenic animal, their are by not exclusively sialylated.The oligonucleotide chain that lacks sialic acid but contain the glycopeptide of terminal galactose residues can carry out Glycopegylated, sugared PPGization or with the modification of modification sialic acid.

In scheme 2, the active ester of aminoglycoside 1 usefulness protection amino acid (for example glycine) derivative is handled, and the osamine residue is converted into corresponding protection amino acid amide adduct.This adduct is handled with aldolase, forms alpha-hydroxy carboxylic acid compounds salt 2.By the effect of CMP-SA synzyme, compound 2 is converted into corresponding C MP derivative, and this CMP derivative of subsequent catalytic hydrogenation forms compound 3.By allowing compound 3 and activation (m-) PEG or (m-) PPG derivative (for example PEG-C (O) NHS, PPG-C (O) NHS) reaction, form 4 or 5 respectively, via forming the site that amine that glycine adduct introduces connects as PEG or PPG.

Scheme 2

Wherein X-BWSP is an activation branched water-soluble polymers of the present invention, and BWSP is a branched water-soluble polymers.

Table 2 has provided the representative example with the sugared phosplate of PEG or PPG structure division derivatization.Some compound of table 2 prepares by the method for scheme 4.Other derivative prepares by art-recognized method.For example, referring to people such as Keppler, Glycobiology 11:11R (2001); With people such as Charter, Glycobiology 10:1049 (2000).Reactive PBG of other amine and PPG analog can be commercial, and perhaps they can prepare by the method that those skilled in the art is convenient to obtain.

Table 2

Wherein R is a branched water-soluble polymers of the present invention.

Can be used for implementing modified sugars phosphate of the present invention can be in other position and above-mentionedly be substituted on those.Below provided sialic preferred at present the replacement:

Wherein X connects base, preferentially be selected from-O-, and-N (H)-,-S, CH ₂-and-N (R) ₂, wherein each R is independently selected from R ¹-R ⁵In a member.Symbol Y, Z, A and B represent to be selected from the group in the described group of above identity for X separately.X, Y, Z, A and B can select independently of one another, and therefore, they can be identical or different.Symbol R ¹, R ², R ³, R ⁴And R ⁵Expression H, perhaps branched water-soluble polymers.In addition, these symbolic representations are bonded in the connection base of branched water-soluble polymers.

Crosslinked group

The preparation that is used for the modified sugars of method of the present invention comprises the modification group is connected in saccharide residue and forms stable adduct that the latter is the substrate of glycosyl transferase.Sugar and modification group can come coupling by zero level or senior crosslinking agent.Can be used for the modification group is connected in the exemplary difunctional compound of carbohydrate structure part including, but not limited to Bifunctionalized poly-(ethylene glycol), polyamide, polyethers, polyester etc.The conventional method that is used for carbohydrate is connected in other molecule is known in the literature.For example, referring to people such as Lee, Biochemistry28:1856 (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, WO92/18135.In the following discussion, the reactive group on the sugared structure division of newborn modified sugars carries out favourable processing.Concentrated argumentation is in order to clearly demonstrate.It will be apparent to those skilled in that this discusses relevant with reactive group on the modification group.

Exemplary policy comprises that use Heterobifunctional crosslinking aid S PDP (will protect sulfydryl to be incorporated into sugar and go up and then sulfydryl is gone protection, with another sulfydryl formation disulfide bond on the modification group by n-succimide base-3-(2-pyridine radicals two sulfo-s) propionic ester.

If SPDP has influenced the ability of modified sugars as the glycosyl transferase substrate nocuously, one of a series of other crosslinking agents are used for forming disulfide bond such as 2-imino group sulphur ring (2-iminothiolane) or N-succimide base S-acetyl group thiacetate (SATA).2-imino group sulphur ring and primary amine reaction are incorporated into unprotected sulfydryl in the molecule that contains amine immediately.SATA also with primary amine reaction, but introduced the sulfydryl of protection, it used azanol to come deacetylation afterwards, formed free sulfhydryl groups.In all cases, the sulfydryl of being introduced freely reacts such as SPDP with other sulfydryl or protection sulfydryl, forms required disulfide bond.

Above-mentioned strategy is to be used for the example that connects base of the present invention, is not restrictive.Can obtain other crosslinking agent, they can be used for modification group and the crosslinked Different Strategies of peptide.For example, (((S-(2-sulfo-pyridine radicals) sulfydryl-propionyl hydrazine) and the carbohydrate structure partial reaction of handling oxidation in advance by the periodate of gentleness, therefore the hydrazides at crosslinking agent partly and between the aldehyde of periodate generation has formed the hydrazone key to TPCH for S-(2-sulfo-pyridine radicals)-L-cysteine hydrazides and TPMPH.TPCH and TPMPH are incorporated into the sulfydryl of 2-pyridine radicals thioketones protection on the sugar, and it can go protection by enough DTT, is used to then put together, such as the disulfide bond that is formed between the component.

If find that disulfide bond is unsuitable for producing stable modified sugars, can use other crosslinking agent of having introduced the more stable key between the component.Heterobifunctional crosslinking agent GMBS (N-γ-dimaleoyl imino butyryl acyloxy) succimide) and SMCC (succimide base 4-(N-maleimide ylmethyl) cyclohexane) and primary amine reaction, therefore maleimide base group is incorporated on this component.Dimaleoyl imino can react with the sulfydryl on another component subsequently, and the latter can introduce by aforementioned crosslinking agent, therefore form the stable thioether bond between component.If the steric hindrance between component has been disturbed the activity of any component or the ability that modified sugars is used as the glycosyl transferase substrate, can use crosslinking agent, it between component, introduced long spacer arm and comprised aforementioned crosslinking agent some derivative (that is, SPDP).Therefore, has spendable a large amount of crosslinking agent that is fit to; They are produced the effect that is had according to it to best peptide conjugate and modified sugars separately and select.

Plurality of reagents can be used to the component of chemical cross-linking agent modification modified sugars in the molecule (about the commentary of crosslinking agent and crosslinked operation, 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, NewYork, 1981; Ji, T.H., Meth.Enzymol.91:580-609,1983; People such as Mattson, Mol.Biol.Rep.17:167-183,1993, they all are incorporated herein for reference).Preferred cross-linking agents is by various distances of zero mark, with difunctionality and the acquisition of Heterobifunctional crosslinking agent.The distance of zero mark crosslinking agent comprises directly puting together of two inherent chemical groups, does not introduce foreign material.The reagent that the catalysis disulfide bond forms belongs to this class.Another example is to bring out carboxyl and primary amino radical condensation, to form the reagent of amido link, and such as carbodiimides, ethyl chloroformate, Woodward reagent K (2-ethyl-5-phenyl-isoxazole azoles father-in-law-3 '-sulfonate), and carbonyl dimidazoles.Except these chemical reagent, enzyme-transglutaminase (glutamyl-peptide gamma-glutamyl based transferase; EC2.3.2.13) can be used as the distance of zero mark crosslinking agent.This enzymatic at the carboxamide groups place of protein bound glutaminyl residue usually with acyl group transfer reaction as the primary amino radical of substrate.Preferred with containing two identical or two different loci respectively with Heterobifunctional reagent, they can have reactivity to amino, sulfydryl, guanidine radicals, indoles or nonspecific group.

I. the preferred specific site in the crosslinking agent

1, amino-reactive group

In a preferred embodiment, the site on crosslinking agent is the amino-reactive group.The useful limiting examples of amino-reactive group comprises N-maloyl imines (NHS) ester, imino esters, isocyanates, acyl halide, aromatic yl azide, p-nitrophenyl ester, aldehyde, and sulfonic acid chloride.

The amino reaction primary (comprising aromatics) preferential and the modified sugars component of NHS ester.Known and the primary amine competitive reaction of the imidazole group of histidine, but product is unsettled and easy hydrolysis.This reaction relates to the nucleophillic attack of amine on the acid carboxyl of NHS, forms acid amides, discharges N-maloyl imines.Therefore, initial amino positive charge loses.

Imino esters is the most special acylating agent that is used for the reaction of the amido of modified sugars component.Under the pH of 7-10, imino esters only with primary amine reaction.Primary amine nucleophillic attack imino-ester is formed on the intermediate that resolves into amidine under the high pH or be decomposed into new imino-ester under low pH.This imidate can with another primary amine reaction, therefore make two amino crosslinked, that is, the situation of the monofunctional imino-ester of the difunctional reactant of inferring.With the primary product of primary amine reaction be the amidine that alkalescence is better than initial amine.Therefore initial amino positive charge is retained.

The primary amine reaction of isocyanates (and isothiocyanates) and modified sugars component forms stable key.They have obtained relative unsettled product with the reaction of sulfydryl, imidazoles and tyrosyl-.

Can also use acyl azide as amino specific reagent, wherein the nucleophilic amine of affinity component is under weak basic condition, and for example pH8.5 attacks acid carboxyl down.

Aryl halide is such as 1,5-two fluoro-2, and preferential amino and the reaction of tyrosine phenolic groups with the modified sugars component of 4-dinitro benzene, and react with sulfydryl and imidazole group.

The p-nitrophenyl ester of monocarboxylic acid and dicarboxylic acids also is useful amino-reactive group.Though this reagent specificity is not very high, α-and as if reaction the most apace of omega-amino-.

Aldehyde is such as the primary amine reaction of glutaraldehyde and modified sugars.Though formed unsettled Schiff alkali when the reaction of amino and aldehyde, glutaraldehyde can enough stable cross-linking modified this modified sugars.At pH6-8, cyclic polymer has experienced dehydration under the pH of typical crosslinked condition, forms α, the beta-unsaturated aldehyde polymer.Yet Schiff alkali is stable, when puting together in another pair key.The Resonant Interaction of two two keys has prevented the hydrolysis of Schiff key.In addition, the amine under high local concentrations can be attacked olefinic double bond, thereby forms stable Michael addition compound product.

Each site reaction of aromatics sulfonic acid chloride and modified sugars component, but with the reaction of amino be most important, formed stable sulfonamide key.

2, sulfydryl reactive group

In another preferred embodiment, this site is the sulfydryl reactive group.Useful, the limiting examples of sulfydryl reactive group comprise maleimide, alkyl halide, curing pyridine and sulfo-phthalimide.

Maleimide sulfydryl preferential and the modified sugars component reacts, thereby forms stable thioether bond.They also with slowly the primary amino radical and the imidazole group of many speed and histidine reacts.Yet under pH7, maleimide base group can be considered to sulfydryl specificity group, because under this pH, the reaction rate of simple mercaptan is higher 1000 times than the speed of corresponding amine.

Alkyl halide and sulfydryl, sulphide, imidazoles and amino reaction.Yet under alkalescent pH, the main and sulfydryl reaction of alkyl halide forms stable thioether bond in neutrality.Under higher pH, with the reaction of amino be preferential.

The curing pyridine reacts with free sulfhydryl groups via disulfide exchange, obtains mixed disulphide.As a result, the curing pyridine is the highest sulfydryl reactive group of specificity.

Sulfo-phthalimide and free sulfhydryl groups reaction form disulphide.

3, carboxyl-reactive residue

In another embodiment, the carbodiimides of water soluble and organic solvent is as carboxyl-reactive reagent.The reaction of these compounds and free carboxy forms pseudo-urea, and it can be coupled to obtainable amine then, has formed amido link, and this has instructed how to use carbodiimides modified carboxyl people such as (, Biochemistry 20:4836-4842,1981) Yamada.

Ii. the preferred non-specific site in cross-linking reagent

Except the reactive structure division of locus specificity, the present invention plans to use the nonspecific reaction group that sugar is connected in the modification group.

But the non-specific crosslinking agent of example comprises the group of photoactivation, is complete inertia in the dark, is converted into reactive materials when absorbing the photon of suitable energy.In a preferred embodiment, but the group of photoactivation is selected from the precursor in heating or the nitrene class that produces during the photodissociation azide.The sub-nitrene class of short of electricity reactive high, can with the various chemical bonding reactions that comprise N-H, O-H, C-H and C=C.Though can use three class azide (aryl, alkyl and acyl derivative), aromatic yl azide is preferred at present.Aromatic yl azide when photodissociation with reactivity N-H and O-H reactive good than with C-H.The sub-aryl nitrene of short of electricity class is encircled expansion apace, forms the dehydrogenation azatropylidene, and it tends to and the nucleophile reaction, inserts product but not form C-H.The reactivity of aromatic yl azide can improve such as nitro or hydroxyl by electron-withdrawing substituent is provided in ring.This type of substituting group is pushed to long wavelength with the absorption maximum of aromatic yl azide.Unsubstituted aromatic yl azide has the absorption maximum in the 260-280nm scope, and hydroxyl and nitro aromatic yl azide absorb a large amount of light that surpass 305nm.Therefore, hydroxyl and nitro aromatic yl azide are most preferred, because compare with unsubstituted aromatic yl azide, they allow to use not too harmful photodissociation condition to be used for affine component.

In a further preferred embodiment, but the photoactivation group is selected from the fluoro aryl azide.The photolytic product of fluoro aryl azide is an aryl nitrene class, and they all carry out the characteristic reactions of this group, comprises that c h bond inserts, and has high efficient people such as (, J.Org.Chem.55:3640-3647,1990) Keana.

In another embodiment, but the group of photoactivation is selected from the benzophenone residue.Benzophenone reagent has generally obtained higher cross linking yield than aromatic yl azide reagent.

In another embodiment, but the group of photoactivation is selected from diazonium compound, and it forms the sub-carbene of short of electricity when photodissociation.These carbene classes experience various reactions, comprise being inserted in the c h bond, add in two keys (comprising aromatic systems), and hydrogen attracts and is coordinated in nucleophilic center, obtains carbon ion.

In another embodiment, but the photoactivation group is selected from the diazonium pyruvate.For example, the p-nitrophenyl ester of diazonium pyruvic acid p-nitrophenyl ester reacts in aliphatic amine, obtains diazonium pyruvic acid acid amides, and the latter experiences ultraviolet photodissociation, forms aldehyde.The affine component of the diazonium pyruvate modification of photodissociation will be reacted as formaldehyde or glutaraldehyde, form crosslinked.

Iii. with difunctionality reagent

1, with the same bifunctional cross-linker of primary amine reaction

Synthetic, the performance of amine reactant cross-linker and application is a large amount of describes in the literature (for the commentary of crosslinked operation and reagent, referring to above).Many reagent can obtain (for example, PierceChemical Company, Rockford, I11.; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, Inc., Eugene, OR.).

Preferred limiting examples with difunctionality NHS ester comprises glutaric acid two succimide esters (DSG), suberic acid two succimide esters (DSS), suberic acid two (sulfo group succimide ester) (BS), tartaric acid two succimide esters (DST), tartaric acid two (sulfo group succimide) ester (sulfo group-DST), two-2-(succimide oxygen base carbonyl oxygen base) ethyl sulfone (BSOCOES), two-2-(sulfo group succimide oxygen base carbonyl oxygen base) ethyl sulfone (sulfo group-BSOCOES), ethylene glycol bis (succimide base succinate) (EGS), ethylene glycol bis (sulfo group succimide base succinate) (sulfo group-EGS), two thiobis (propionic acid succimide ester) (DSP), and two thiobis (propionic acid sulfo group succimide base ester) (sulfo group-DSP).Preferred with the difunctionality imino esters, limiting examples comprises malonyl imidic acid dimethyl ester (DMM), succinyl imidic acid dimethyl ester (DMSC), adipyl imidic acid dimethyl ester (DMA), heptanedioyl imidic acid dimethyl ester (DMP), suberoyl imidic acid dimethyl ester (DMS), 3,3 '-oxygen dipropyl imide dimethyl phthalate (DODP), 3,3 '-(methylene dioxy base) dipropyl imide dimethyl phthalate (DMDP), 3,3 '-(dimethylene dioxy base) dipropyl imide dimethyl phthalate (DDDP), 3,3 '-(tetramethylene dioxy base)-dipropyl imide dimethyl phthalate (DTDP), and 3,3 '-two thiobis propionyl imidic acid dimethyl ester (DTBP).

Preferred, limiting examples with the difunctionality isothiocyanates comprise: to phenylene diisothio-cyanate (DITC) and 4,4 '-two isothiocyanos-2,2 '-disulfonic acid stibene (DIDS).

Preferred limiting examples with the bifunctional isocyanate comprises XDI, Toluene-2,4-diisocyanate, the 4-vulcabond, Toluene-2,4-diisocyanate-isocyanates-4-isothiocyanates, 3-methoxyl group diphenyl methane-4,4 '-vulcabond, 2,2 '-dicarboxyl-4,4 '-the azobenzene vulcabond, and hexamethylene diisocyanate.

Preferred, limiting examples with the difunctionality aryl halide comprise 1,5-two fluoro-2, and 4-dinitro benzene (DFDNB) and 4,4 '-two fluoro-3,3 '-the dinitrophenyl sulfone.

Preferred, limiting examples with difunctionality aliphatic aldehydes reagent comprise glyoxal, malonaldehyde and glutaraldehyde.

The nitrobenzophenone ester that comprises dicarboxylic acids with preferred, the limiting examples of difunctionality acylating agent.

Preferred, limiting examples with difunctionality aromatics sulfonic acid chloride comprise phenol-2,4-disulfonic acid chloride, and alpha-Naphthol-2,4-disulfonic acid chloride.

Other amino-reactive comprises the erythrite double manganese ester with preferred, the limiting examples of difunctionality reagent, and it can react with amine, obtains double carbamate.

2. with the same bifunctional cross-linker of free sulfhydryl groups reaction

(about the commentary of crosslinked operation and reagent, referring to above) described in the literature in synthetic, the performance and the application of this type of reagent.Many reagent can commercial (for example, Pierce ChemicalCompany, Rockford, I11.; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, Inc., Eugene, OR.).

Preferred, limiting examples with the difunctionality maleimide comprise dimaleoyl imino hexane (BMH), N, N '-(1, the 3-phenylene) BMI, N, N '-(1, the 2-phenylene) BMI, azobenzene dimaleimide and two (N-maleimide ylmethyl) ether.

Preferred, limiting examples with difunctionality curing pyridine comprise 1,4-two-3 '-(2 '-the pyridine disulfide group) propionamido-butane (DPDPB).

With preferred, the limiting examples of difunctionality alkyl halide comprise 2,2 '-dicarboxyl-4,4 '-diiodo-acetamido azobenzene; α; α '-two iodo-paraxylene sulfonic acid, α, α '-two bromo-paraxylene sulfonic acid; N; N '-two (b-bromoethyl) benzyl amine, N, N '-two (acetyl bromide) phenyl hydrazine; with 1,2-two (acetyl bromide) amino-3-phenyl-propane.

3. but with the crosslinking agent of difunctionality photoactivation

Synthetic, the performance of this type of reagent and application is a large amount of describes in the literature (for the commentary of crosslinked operation and reagent, referring to above).Some of these reagent can commercial (for example, PierceChemical Company, Rockford, I11.; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, Inc., Eugene, OR).

But preferred, limiting examples with the crosslinking agent of difunctionality photoactivation comprise two-β-(4-azido salicyl amide groups) ethyl disulphide (BASED), two-N-(2-nitro-4-azido phenyl)-cystamine-S, S-dioxide (DNCO), with 4,4 '-two thiobis azidomethyl phenyl nitrogenize things.

Iv. Heterobifunctional reagent

1, the amino-reactive Heterobifunctional reagent that has curing pyridine radicals structure division

Synthetic, the performance of this type of reagent and application is a large amount of describes in the literature (for the commentary of crosslinked operation and reagent, referring to above).The many of these reagent can commercial (for example, PierceChemical Company, Rockford, I11.; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, Inc., Eugene, OR).

Have curing pyridine structure part and amino-reactive NHS ester Heterobifunctional reagent preferably, limiting examples comprises N-succimide base-3-(2-pyridine disulfide group) propionic ester (SPDP), succimide base 6-3-(2-pyridine radicals disulfide group) propionamido-capronate (LC-SPDP), sulfo group succimide base 6-3-(2-pyridine radicals disulfide group) propionamido-capronate (sulfo group-LCSPDP), 4-succimide base oxygen base carbonyl-Alpha-Methyl-α-(2-pyridine radicals disulfide group) toluene (SMPT) and sulfo group succimide base 6-Alpha-Methyl-α-(2-pyridine radicals disulfide group) toluoyl amido capronate (sulfo group-LC-SMPT).

2, the amino-reactive Heterobifunctional reagent that has the maleimide structure division

Synthetic, the performance of this type of reagent and application is a large amount of describes in the literature.Heterobifunctional reagent preferred with maleimide structure division and amino-reactive NHS ester; limiting examples comprises maleimide guanidine-acetic acid succimide ester (AMAS); 3-dimaleoyl imino propionic acid succimide ester (BMPS); N-γ-dimaleoyl imino butyryl acyloxy succimide ester (GMBS); N-γ-dimaleoyl imino butyryl acyloxy sulfo group succimide ester (sulfo group-GMBS); succimide base 6-dimaleoyl imino capronate (EMCS); succimide base 3-dimaleoyl imino benzoic ether (SMB); between-dimaleoyl imino benzoyl-N-maloyl imines ester (MBS); between-dimaleoyl imino benzoyl-N-hydroxyl sulfo group succimide ester (sulfo group-MBS); succimide base 4-(N-maleimide ylmethyl)-cyclohexane-1-carboxylate (SMCC); sulfo group succimide base 4-(N-maleimide ylmethyl)-cyclohexane-1-carboxylate (sulfo group-SMCC); succimide base 4-(to the dimaleoyl imino phenyl) butyrate (SMPB) and sulfo group succimide base 4-(to the dimaleoyl imino phenyl) butyrate (sulfo group-SMPB).

3, the amino-reactive Heterobifunctional reagent that has the alkyl halide structure division

Synthetic, the performance of this type of reagent and application is a large amount of describes in the literature.Heterobifunctional reagent preferred with alkyl halide structure division and amino-reactive NHS ester; limiting examples comprises N-succimide base-(4-iodoacetyl) Aminobenzoate (SIAB); sulfo group succimide base-(4-iodoacetyl) Aminobenzoate (sulfo group-SIAB); succimide base-6-(iodoacetyl) aminocaproic acid ester (SIAX); succimide base-6-(6-((iodoacetyl)-amino) caproyl amino) capronate (SIAXX); succimide base-6-(((4-(iodoacetyl)-amino)-methyl)-cyclohexane-1-carbonyl) aminocaproic acid ester (SIACX), and succimide base-4 (iodoacetyl)-amino) hexahydrotoluene-1-carboxylate (SIAC).

Preferred embodiment with Heterobifunctional reagent of amino-reactive NHS ester and alkyl dihalide structure division is a N-maloyl imido grpup 2,3-dibromo-propionic acid ester (SDBP).SDBP is incorporated into affine component by puting together its amido with intramolecular crosslinking.The reactivity of dibromo propiono structure division and primary amine groups is by controlling reaction temperature people such as (, Protein Chem.7:581-592 (1988)) McKenzie.

Preferred, limiting examples with Heterobifunctional reagent of alkyl halide structure division and amino-reactive p-nitrophenyl ester structure part comprise iodoacetic acid p-nitrophenyl ester (NPIA).

Other crosslinking agent is that those skilled in the art are known.For example, referring to people such as Pomato, U.S. patent No.5,965,106.Selecting suitable crosslinking agent to be used for application-specific is in those skilled in the art's limit of power.

V. the connection base of cleavable

In another embodiment, this connection base has can cracking, to discharge the group of modification group from saccharide residue.Many cleavable groups are well known in the art.For example, referring to people such as Jung, Biochem.Biophys.Acta 761:152-162 (1983); People such as Joshi, J.Biol.Chem.265:14518-14525 (1990); People such as Zarling, J.Immunol.124:913-920 (1980); People such as Bouizar, Eur.J.Biochem.155:141-147 (1986); People such as Park, J.Biol.Chem.261:205-210 (1986); People such as Browning, J.Immunol.143:1859-1867 (1989).And, various cleavables, difunctionality (with Heterobifunctional) being connected base can be commercial there such as Pierce from supplier.

Exemplary cleavable structure division can make and use up, heat or reagent is such as mercaptan, azanol, and alkali, periodate waits cracking.And, as response to endocytosis, some preferred group cracking in vivo (for example, cis-rhizome of Chinese monkshood base; Referring to people such as Shen, Biochem.Biophys.Res.Commun.102:1048 (1991)).The group of preferred cleavable comprises the structure division of cleavable, and it is to be selected from disulphide, ester, acid imide, carbonic ester, nitrobenzyl, the member in benzoyl and the benzoin group.

Method

The present invention also provides basic single method of disperseing colony of poly-(ethylene glycol) molecule of preparation.This method comprises allows the PEG molecule with clear and definite molecular weight, the difunctionality activated PEG that also has clear and definite molecular weight of PEG200 and at least 2 equivalents for example, and for example PEG200 contact, thus form single dispersed sample of PEG, PEG600 for example:

G is a leaving group, such as sulphonic acid ester and tresylate ester.Single dispersed sample of PEG600 can contact with difunctionality activated PEG 200 then, forms single PEG100 of dispersion.Perhaps, single PEG600 of dispersion can be converted into corresponding difunctionality derivative and disperse dihydroxy PEG600 reaction with at least 2 equivalents single, forms single PEG1800 of dispersion.Method of the present invention repeats to till the single PEG of dispersion that obtains required size.Can design this and synthesize, make the molecular weight differences permission between initiation material and product separate the material of any unreacted or partial reaction by SEC.

The activated PEG derivative

The present invention also provides the method for poly-(ethylene glycol) derivative of preparation.In scheme I, summarized this method:

Wherein, subscript m and n represent 1-100 independently, 000 integer.R is selected to replace or substituted alkyl not, replaces or replaces assorted alkyl, replaces or unsubstituting aromatic yl alkylamine, protection alkylamine, or the member in the activated group, trifluoromethanesulfonic acid root for example, tosylate etc.

R ' is selected from and replaces or substituted alkyl not, replaces or replaces assorted alkyl, replaces or unsubstituting aromatic yl, replaces or unsubstituting heterocycle alkyl and replacement or substituted heteroaryl not.When not comprising, R is not used to activate the CH that it connects ₂During the leaving group of-O structure division, R ' generally is a leaving group, or comprises leaving group.

In exemplary, R is a low alkyl group, such as methyl.In another exemplary, R ' is a substituted alkyl, such as chloro-carbonic acid p-nitrophenyl ester.

In step a, original glycol contacts with activated group (R-Y), the hydroxyl structure partial reaction of this activated group and glycol.Y is leaving group normally, and R is placed on one of the hydroxyl structure part of PEG molecule.In step b, the free hydroxyl group of gained adduct by its be converted into such as halogen for example the group chlorine or the sulphonic acid ester activate.The activation the PEG material with as initial PEG (" PEG _m") another PEG structure division contact of the identical or different degree of polymerization.In order to make it be connected in another material, RO-PEG _(n+m)Choose wantonly in free hydroxyl group structural portion office and activate.

Generally, the R group is connected in the PEG structure division via the material that comprises reactive functional groups.And two poly-(ethylene glycol) fragments connect together by using reactive functional groups, and they are converted into new organo-functional group or unreacted matters by method of attachment.Any position on reactive functional groups is positioned at poly-(ethylene glycol) structure division, but preferably one of endways.

Puting together of the sugar of branched polymer modification and peptide

Modified sugars uses suitable enzyme to put together in glycosylation or non-glycosylated peptide, puts together to reconcile this.Preferably, select modification to give the concentration of body sugar, enzyme and acceptor peptide, till making glycosylation proceed to acceptor to be consumed.Though illustrate with regard to the sialic acid based transferase, following consideration generally is applicable to other glycosyl transferase reaction.

It is known using many methods of the synthetic required oligosaccharide structure of glycosyl transferase, and generally is applicable to the present invention.Illustrative methods for example is described in people such as WO96/32491, Ito, Pure Appl.Chem.65:753 (1993), and U.S.Pat.Nos.5,352,670,5,374,541 and 5,545,553.

The present invention uses the bond of single glycosyl transferase or glycosyl transferase to implement.For example, can use the bond of sialic acid based transferase and galactosyltransferase.In these embodiments of the enzyme that uses more than one, enzyme and substrate preferably merge in initial reaction mixture, and perhaps in case first enzymatic reaction finishes or almost finishes, the enzyme and the reagent that will be used for second enzymatic reaction join reaction medium.By carry out two kinds of enzymatic reactions according to the order of sequence in single container, total recovery is than the wherein operation improvement of separation of intermediates material.And, reduced the removing and the processing of added solvent and accessory substance.

In preferred embodiments, each glycosyl transferase naturally of first and second kinds of enzymes.In another preferred embodiment, a kind of enzyme is interior glycosidase.In other embodiment preferred, use two or more enzymes to assemble modified proteins of the present invention.Before modified sugars is added to peptide and afterwards, use enzyme to change the sugared structure of any position on the peptide.

The glycosyl structure division that the O-of conjugate of the present invention connects is general assigns to produce with the GalNAc structural portion that is connected in peptide.Any member of the family of GalNAc transferase can be used for the GalNAc structure division is incorporated into peptide (Hassan H, Bennett EP, Mandel U, Hollingsworth MA, and Clausen H (2000)).Mucus type O-glycosylation: the control of O-glycan occupation rate is instructed (Eds.Ernst, Hart, and Sinay) by the substrate specificity of polypeptide GalNAc-transferase.Wiley-VCH chapter″Carbohydrates inChemistry and Biology-a Comprehension Handbook″，273-292)。GalNAc structure division itself can be that complete glycosyl connects base.In addition, glycosyl residue can use the glycosyl substrate of one or more enzymes and one or more suitable enzymes to increase and build, and modified sugars is added into and increases on the glycosyl structure division of building.

In another embodiment, this method is utilized one or more outer or interior glycosidases.Glycosidase generally is a mutant, and it is formed glycosyl bond by through engineering approaches, but not cracking they.The mutant dextranase generally comprises the amino acid residue that replaces the avtive spot acidic amino acid residue.For example, when interior dextranase be interior-during H, replace the avtive spot residue and generally be Asp, at 132 Glu, or their combination at 130.Amino acid is generally used serine, alanine, and asparagine or glutamine replace.

Mutant enzyme is usually by coming this reaction of catalysis with the similar synthesis step of the reverse reaction of interior dextranase hydrolysing step.In these embodiments, glycosyl donor molecule (for example, required oligosaccharides or monose structure) contains leaving group, and this reaction is undertaken by donor molecule being added on the GlcNAc residue on the albumen.For example, leaving group can be a halogen, such as fluorine.In other embodiments, leaving group is Asn, or Asn-peptide structure division.In other embodiments, the GlcNAc residue on the glycosyl donor molecule is modified.For example, the GlcNAc residue can comprise 1,2-oxazoline structure division.

In preferred embodiments, the various enzymes that are used to produce conjugate of the present invention exist with catalytic amount.The catalytic amount of certain enzyme changes such as temperature, time and pH value according to the concentration and the reaction condition of the substrate of enzyme.The mode of measuring the catalytic amount of set enzyme under preselected concentration of substrate and reaction condition is as well known to those skilled in the art.

The temperature of carrying out above method can be just the temperature more than freezing to the temperature of the most responsive enzyme denaturation.Preferred temperature range is about 0 to about 55 ℃, and is more preferably about 20 to about 30 ℃.In another exemplary, one or more components of the inventive method at high temperature use Zimadzhunt L 340 to handle.

Reactant mixture keeps acceptor to be enough to by glycosylation, thereby forms the time of required conjugate.Some of conjugate usually can detect after several hours, and recyclable amount reclaims in 24 hours or shorter time usually.Those skilled in the art will recognize that reaction rate depends on the value (for example enzyme concentration is given bulk concentration, acceptor density, temperature, solvent volume) of variable factor, these values can be optimized for selective system.

The present invention also provides the industrial-scale production method of modification peptide.Commercial scale used herein has generally produced at least approximately 250mg, preferred at least approximately 500mg and more preferably finished product, the purification conjugate of about at least 1g, preferably after finishing a reaction time, that is, this conjugate is not the bond from the product of the synthesis cycle of identical, continuous repetition.

In the following discussion, the present invention illustrates with puting together of glycosylated peptide by modification sialic acid structure part.Exemplary modification sialic acid (m-) PEG mark.Below discussing the use that concentrates on PEG modification sialic acid and glycosylated peptide is in order to clearly demonstrate, and is not the conjugate that hint the present invention is limited to these two kinds of counter pairs.The technical staff understands, and this argumentation generally is applicable to the addition of the modified sugars based structures part except sialic acid.And this argumentation is equally applicable to the modification that glycosyl units is used the reagent (comprising other water-soluble polymer, treatment structure division and biomolecule) except PEG.

Enzymatic method can be used for the carbohydrate selectivity of branched polymer modification is incorporated into peptide or glycopeptide.This method adopts the modified sugars that contains branched water-soluble polymers, and combines with suitable glycosyl transferase or sugared synzyme.Glycosyl transferase by select obtaining required carbohydrate key and adopt modified sugars as giving the body substrate, this branched water-soluble polymers can be introduced directly on the peptide backbone, on the existing saccharide residue of glycopeptide or added on the saccharide residue on the peptide.

The acceptor of sialic acid based transferase is as naturally occurring structure, perhaps as reorganization, the structure of enzyme process or chemical method setting is present in will be by on the method modification peptide of the present invention, and the acceptor that is fit to for example comprises the galactosyl acceptor, such as GalNAc, Gal β 1,4GlcNAc, Gal β 1,4GalNAc, Gal β 1,3GalNAc, LNT, Gal β 1,3GlcNAc, Gal β 1,3Ara, Gal β 1,6GlcNAc, Gal β 1,4Glc (lactose), and other acceptor well known by persons skilled in the art (for example referring to people such as Paulson, J.Biol.Chem.253:5617-5624 (1978)).

In one embodiment, the acceptor of sialic acid based transferase is present on the glycopeptide of modification when synthesizing in the body of glycopeptide.It is sialylated that this type of glycopeptide can use desired method, and do not answer preliminary election to change the glycosylation mode of glycopeptide.Perhaps, method of the present invention can be used for the sialylated peptide that does not comprise suitable acceptor; People at first can pass through this peptide of method known to those skilled in the art modification, to introduce acceptor.

In exemplary, the galactosyl acceptor is by being connected in galactose residue the suitable acceptor that links to each other with peptide, and for example GalNAc assembles.This method comprises the peptide of giving the reactant mixture hatching modification of wanting of body (for example UDP-galactose) with the galactosyl that contains an amount of galactosyltransferase (for example Gal β 1,3 or Gal β 1,4) and be fit to.It is complete basically that this reaction is proceeded to, perhaps, when having added the galactose residue of scheduled volume, cessation reaction.It is that those skilled in the art is conspicuous that other method of saccharide acceptor is selected in assembling.

In another embodiment, at first overall or oligosaccharides that part " prunings " glycopeptide connects with the acceptor of exposure sialic acid based transferase, perhaps can add the structure division of one or more suitable residues with the acceptor that obtains to be fit to.Enzyme can be used for connecting and pruning reaction such as galactosyltransferase and endoglycosidase (for example referring to US patent No.5,716,812).

In exemplary, the carbohydrate residue is before adding modified sugars " pruning ".For example, the GalNAc-Gal residue is trimmed to GalNAc again.Carry the modified sugars of water-soluble polymer and put together the one or more saccharide residues that expose in by " pruning ".In an example, " pruning " glycopeptide, via the glycosyl structure division of puting together in water-soluble polymer, Sia for example, Gal or GalNAc structure division add to water-soluble polymer on gained O-side chain amino acid or the glycopeptide glycan.The modified sugars based structures partly is connected on the acceptor site on the glycopeptide of " pruning ".Perhaps, unmodified glycosyl structure division, for example Gal can be in the terminal connection of the glycan that O-connects.

In another exemplary,, branched water-soluble polymers is added on the GalNAc residue via modified sugars with galactose residue.Perhaps, unmodified Gal can be added to terminal GalNAc residue.

In another example, use the modification sialic acid that branched water-soluble polymers is added on the Gal residue.

Above-mentioned exemplary provides the explanation of the ability of method as herein described.Use method of the present invention, can " cut down " and the carbohydrate residue of " increase " any basically structure that caters to the need.Modified sugars can add to the end of aforesaid carbohydrate structure part, and perhaps it can be between peptide nuclear and carbohydrate end.

In exemplary, use the sialic acid of polymer modification that branched water-soluble polymers is added on the terminal Gal residue.Use suitable sialic acid based transferase to come the addition modification sialic acid.This method is summed up in scheme 3.

Scheme 3:

In another method of summing up in scheme 4, the reactive functional groups of sealing is present in the sialic acid.This capping group preferably is not used for the modification sialic acid is connected in the condition effect of peptide.After the modification sialic acid is covalently attached to peptide, remove to cover and close thing, use such as PEG, PPG, treatment reagent or other reagent with structure division, biomolecule and put together peptide.This reagent by the not capping group on itself and the modified sugars residue reaction and put together in peptide with ad hoc fashion.

Scheme 4

Depend on the terminal sugar (table 3) of the oligosaccharides side chain of glycopeptide, any modified sugars can be used with its suitable glycosyl transferase.As mentioned above, introducing the terminal sugar of the required glycopeptide of branched water-soluble polymers structure can introduce in the expression process naturally, perhaps it can be by the suitable glycosidase of use, glycosyl transferase, and perhaps the mixture of glycosidase and glycosyl transferase generates after expression.

Table 3

X＝O，NH，S，CH ₂，N-(R _1-5) ₂. Y＝X；Z＝X；A＝X；B＝X. Q＝H ₂，O，S，NH，N-R. R，R _1-4=H connects base-M, M M=branched water-soluble peptide

In alternative embodiment, use and known glycosyl residue is transferred to glycosyl transferase on the glycosylation site that the O-of peptide backbone connects, modified sugars is directly added on the peptide backbone.This exemplary is set forth in scheme 5.Can be used for implementing exemplary glycosyl transferase of the present invention including, but not limited to GalNAc transferase (GalNAc T1-20), GlcNAc transferase, fucosyltransferase, glucosyltransferase, xylosyltransferase, mannose transferase etc.Use this method modified sugars directly can be added to the peptide that lacks any carbohydrate, on the perhaps existing glycopeptide.In both cases, the addition of modified sugars occurs in as by on the ad-hoc location on the peptide backbone of the substrate specificity defined of glycosyl transferase, rather than resembles the random fashion that takes place in the peptide backbone process of using the chemical method modified protein.By with suitable amino acid sequence through engineering approaches in polypeptide chain, reagents series can be incorporated in the albumen or glycopeptide that lacks glycosyl transferase peptide substrate sequence.

Scheme 5

In above-mentioned each exemplary, modified sugars is being puted together after peptide, can adopt one or more additional chemical or enzyme modification step.In exemplary, use enzyme (for example fucosyltransferase) that glycosyl units (for example fucose) is connected in the terminal-modified sugar that links to each other with peptide.In another example, the site of using enzymatic reaction to come " end-blocking " (for example sialylated) modified sugars to fail to put together.Perhaps, adopt chemical reaction to change the structure of the modified sugars of puting together.Modified sugars of for example, puting together and the reagent reacting of stablizing or go to stablize the key of its peptide composition that is connected with modified sugars.In another example, put together after peptide, the component of modified sugars is gone protection at it.The technical staff is clear, in the stage of modified sugars being puted together after peptide, has the serial enzymes that the can be used for method of the present invention chemical process of urging to become reconciled.The further details of modified sugars-peptide conjugate is within the scope of the invention.

In another exemplary, glycopeptide is puted together in directed agents, siderophillin (be used to carry peptide to pass blood-brain barrier, and arrive in endosome) for example, carnitine (is used for peptide is transported to muscle cell; For example referring to people such as LeBorgne, Biochem.Pharmacol.59:1357-63 (2000), and phosphonate ester, for example bisphosphonate (is used for peptide is directed to bone and other calcic tissue; For example referring to Modern Drug Discovery, in August, 2002, the 10th page).Other reagent that can be used for leading is that those skilled in the art institute is conspicuous.For example, glucose, glutamine and IGF also can be used for being directed to muscle.

Guide frame part and therapeutic peptide are puted together by any method as herein described or other method known in the art.The technical staff is clear, also can derivatization as described herein except above-mentioned peptide those.The unexamined of submitting in October 10 calendar year 2001, the US temporary patent application No.60/328 that owns together have set forth exemplary peptides in the 523 appended appendix.

In exemplary, directed agents and therapeutic peptide are via being connected the coupling of based structures part.In this embodiment, the method according to this invention, at least a being coupled to via complete glycosyl connection base of therapeutic peptide or directed agents connects the based structures part.In exemplary, connect based structures and comprise that partly poly-(ether) is such as poly-(ethylene glycol).In another exemplary, connect based structures and partly be included in conjugate is transported to after the destination organization or position of human body, degradation in vivo discharges at least one key of therapeutic peptide from directed agents.

In another exemplary, distributing in the body of therapeutic peptide partly changes by changing the sugared shape of treatment with structure division, therapeutic peptide need not being puted together in guide frame.For example, therapeutic peptide can be by avoiding being absorbed by reticuloendothelial system with the terminal galactose structure division of sialic acid (or derivatives thereof) end-blocking glycosyl.

I. enzyme

1, glycosyl transferase

Glycosyl transferase with substep mode catalytic activation sugar (give body NDP-sugar) on albumen, glycopeptide, lipid or the glycolipid or the addition on the non-reduced end of growth oligosaccharides.The glycopeptide that N-connects is given body Dol-PP-NAG via the oligosaccharides that transferase is connected with lipid ₂Glc ₃Man ₉Synthetic in the mode that global transfer and pruning are subsequently examined.In this case, the character of " nuclear " sugar is different from follow-up connection a bit.Known in the art have a large amount of glycosyl transferases.

It can be any being used for glycosyl transferase of the present invention, as long as it can use modified sugars to give body as sugar.The example of this fermentoid comprises Leloir approach glycosyl transferase, such as galactosyltransferase, N-acetylamino glucosyl transferase, N-acetylamino galactosamine based transferase, fucosyltransferase, sialic acid based transferase, mannose transferase, xylosyltransferase, glucuronyl transferase etc.

Synthetic for the enzymatic sugar that relates to the glycosyl transferase reaction, glycosyl transferase can be cloned, and perhaps separates from any source.Many clones' glycosyl transferase is known, and their polynucleotide sequence also is known.For example referring to " The WWW Guide To ClonedGlycosyltransferases ", ( Http:// www.vei.co.uk/TGN/gt_guide.htm).The glycosyl transferase amino acid sequence also can find in the obtainable database of the various public with the nucleotide sequence that can reason out the encoding glycosyl transferase of amino acid sequence, comprises GenBank, Swiss-Prot, EMBL etc.

The glycosyl transferase that can use in the method for the invention is including, but not limited to galactosyltransferase, fucosyltransferase, glucosyltransferase, N-acetylamino galactosamine based transferase, N-acetylamino glucosyl transferase, glucuronyl transferase, the sialic acid based transferase, mannose transferase, glucuronyl transferase, galacturonic acid transferase and oligosaccharyl transferase.The glycosyl transferase that is fit to comprises those that are obtained by eukaryotic and prokaryotes.

The glycosyl transferase of dna encoding can be by chemosynthesis, from the reverse transcription product of suitable cell or cloned culture screening mRNA, by the gene pool of screening from suitable cell, perhaps by obtaining in conjunction with these operations.The screening of mRNA or genomic DNA can be carried out with the oligonucleotide probe that is produced by the glycosyltransferase gene sequence.Probe can be according to operation known and that in common cross experiment, use with detectable group such as fluorophor, radioactive atom or chemiluminescent groups are come mark.In the scheme that substitutes, the glycosyltransferase gene sequence can obtain by using polymerase chain reaction (PCR) operation, and wherein the PCR Oligonucleolide primers is produced by the glycosyltransferase gene sequence.Referring to US patent No.4,683,195 (people such as Mullis) and US patent No.4,683,202 (Mullis).

Glycosyl transferase can be synthetic in the carrier transformed host cells of the DNA that contains the encoding glycosyl transferase.Carrier is used for the DNA of amplification coding glycosyl transferase and/or expresses the DNA of encoding glycosyl transferase.Expression vector is reproducible dna structure, and wherein the dna sequence dna of encoding glycosyl transferase is operably connected to and can expresses the suitable control sequence (control sequence) of this glycosyl transferase in being fit to the host.Demand for this control sequence will change according to selected host and selected method for transformation.Generally, control sequence comprises transcripting promoter, and the manipulation group sequence of transcribing is controlled in optional being used to, the sequence of the mRNA ribosome binding site that coding is fit to, and the sequence of controlling the termination of transcribing and translating.Amplification vector does not need to express the control zone.Required whole are the abilities of in the host, duplicating, given by starting point of duplicating and the selection gene that helps the identification of transformant usually.

In exemplary, the present invention adopts former ribozyme.This glycosyl transferase is included in the enzyme that relates in fat oligosaccharides (LOS) synthetic, and they produce people such as (, Critical Reviews in Microbiology23 (3): 139-180 (1996)) Preston by many Gram-negative bacterias.This fermentoid is including, but not limited to the albumen of species such as the rfa operon of E.coli and salmonella typhimurium, they comprise β-1,6-galactosyltransferase and β-1, and the 3-galactosyltransferase is (for example, referring to EMBL Accession Nos.M80599 and M86935 (E.coli); EMBL Accession No.S56361 (S.typhimurium)), glycosyl transferase (Swiss-Prot Accession No.P25740 (E.coli), β 1,2-glucosyltransferase (rfaJ) (Swiss-Prot Accession No.P27129 (E.coli) and Swiss-Prot Accession No.P 19817 (S.typhimurium)), with β 1,2-N-acetylamino glucosyl transferase (rfaK) (EMBL Accession No.U00039 (E.coli)).Other glucosyltransferase that amino acid sequence is known comprise by operon such as rfaB (they in microorganism such as klebsiella pneumoniae, Escherichia coli, bacillus typhi murium, intestines salmonella (Salmonella enteric), YE, identify in the Mycobacterium leprae (Mycobacterium leprosum)), and those of the rhl operon of pseudomonas aeruginosa coding.

Also being applicable to of the present invention is to contain Lacto-N-neo-tetraose (lacto-N-neotetraose) in production, D-galactosyl-β-1,4-N-acetyl group-D-glucose amido-β-1,3-D-galactosyl-β-1,4-D-glucose, and P ^kBlood group three glycosylation sequences, D-galactosyl-α-1,4-D-galactosyl-β-1, the glycosyl transferase that relates in the structure of 4-D-glucose, their identify in the LOS of mucosal disease substance Diplococcus gonorrhoeae (Neisseria gonnorhoeae) and Neisseria meningitidis (N.meningitidis) people such as (, J.Med.Microbiol.41:236-243 (1994)) Scholten.The Neisseria meningitidis of the encoding glycosyl transferase that relates in the biosynthesis of these structures and the gene of Diplococcus gonorrhoeae are by Neisseria meningitidis immunologic pattern (immunotypes) L3 and L1 (people such as Jennings, Mol.Microbiol.18:729-740 (1995)) and Diplococcus gonorrhoeae mutant F62 (Gotshlich, J.Exp.Med.180:2181-2190 (1994)) identification.In Neisseria meningitidis, by three gene lgtA, the locus that lgtB and lgE the form required glycosyl transferase of in Lacto-N-neo-tetraose (lacto-N-neotetraose) last three sugar of interpolation people such as (, J.Biol.Chem.271 (45): 19166-73 (1996)) Wakarchuk of having encoded.Recently, the enzymatic activity of lgtB and IgtA gene outcome is proved, and their first positive evidence (people such as Wakarchuk, J.Biol.Chem.271 (45): 28271-276 (1996)) of glycosyl transferase function that is proposed is provided.In Diplococcus gonorrhoeae, have two other genes, β-D-GalNAc is added to the lgtD on 3 of terminal galactose of Lacto-N-neo-tetraose (lacto-N-neotetraose) structure and terminal α-D-Gal added to the lactose construction unit that blocks LOS, therefore produced P ^kThe lgtC of blood group antigens structure (Gotshlich (1994) above).In Neisseria meningitidis, independently immunologic pattern L1 has also expressed P ^kBlood group antigens, and shown carry the lgtC gene (people such as Jennings, (1995), above).Neisseria glycosyl transferase and related gene also are described in USPN 5,545, in 553 (Gotschlich).From the α 1 of helicobacter pylori, 2-fucosyltransferase and α 1, the gene of 3-fucosyltransferase are also identified people such as (, J.Biol.Chem.272:21349-21356 (1997)) Martin.Also can be used for of the present invention is that the glycosyl transferase of campylobacter jejuni is (for example referring to http://afmb.cnrs-mrs.fr/～pedro/CAZY/gtf_42.html).

A) fucosyltransferase

In certain embodiments, the glycosyl transferase that is used for method of the present invention is a fucosyltransferase.Fucosyltransferase is known to those skilled in the art.Exemplary fucosyltransferase comprises the enzyme of the L-fucose being transferred to the hydroxy position of acceptor saccharide from the GDP-fucose.The fucosyltransferase of non-nucleotide sugar being transferred to acceptor also can be used for the present invention.

In certain embodiments, acceptor saccharide for example is the GlcNAc in Gal β (1 → 3,4) the GlcNAc beta-yl group in the oligosaccharides glucosides.The fucosyltransferase that is applicable to this reaction comprises Gal β (1 → 3,4) GlcNAc β 1-α (1 → 3,4) fucosyltransferase (FTIIIE.C.No.2.4.1.65), and their at first quilt evaluations from human milk (referring to people such as Palcic, Carbohydrate Res.190:1-11 (1989); People such as Prieels, J.Biol.Chem.256:10456-10463 (1981); And people such as Nunez, Can.J.Chem.59:2086-2095 (1981)), and Gal β (1 → 4) GlcNAc β-α fucosyltransferase (FTVI), they are found in human serum for FTIV, FTV.FTVII (E.C.No.2.4.1.65), saliva acidic group α (2 → 3) Gal β (also identified by (1 → 3) GlcNAc β fucosyltransferase.Gal β (1 → 3,4) GlcNAc β-α (1 → 3,4) recombinant forms of fucosyltransferase is also identified (referring to people such as Dumas, people such as Bioorg.Med.Letters 1:425-428 (1991) and Kukowska-Latallo, Genes andDevelopment 4:1288-1303 (1990)).Other exemplary fucosyltransferase for example comprises α 1,2-fucosyltransferase (E.C.No.2.4.1.69).The enzymatic fucosylation can pass through people such as Mollicone, Eur.J Biochem.191:169-176 (1990) or U.S. patent No.5, and the method described in 374,655 is carried out.The cell that is used to produce fucosyltransferase also comprises the enzymatic system that is used for synthetic GDP-fucose.

B) galactosyltransferase

In another group embodiment, the galactosyltransferase of glycosyl transferase.Exemplary galactosyltransferase comprises α (1,3) galactosyltransferase (E.C.No.2.4.1.151, for example referring to people such as Dabkowski, people such as Transplant Proc.25:2921 (1993) and Yamamoto, Nature 345:229-233 (1990), ox (GenBank j04989, people such as Joziasse, J.Biol.Chem.264:14290-14297 (1989)), murine (GenBank m26925; People such as Larsen, Proc.Nat ' l.Acad.Sci.USA86:8227-8231 (1989)), pig (GenBank L36152; People such as Strahan, Immunogenetics 41:101-105 (1995)).Another α 1,3 galactosyltransferase that is fit to is the sort of (EC 2.4.1.37, people such as Yamamoto, the J.Biol.Chem.265:1146-1151 (1990) (people)) that relates in blood group B antigen synthetic.Another exemplary galactosyltransferase is nuclear Gal-T1.

That also be applicable to method of the present invention is β (1,4) galactosyltransferase, for example comprise EC2.4.1.90 (LacNac synzyme) and EC2.4.1.22 (lactose synthetase) (ox (people such as D ' Agostaro, Eur.J.Biochem.183:211-217 (1989)), people (people such as Masri, Biochem.Biophys.Res.Commun.157:657-663 (1988)), mouse (people such as Nakazawa, J.Biochem.104:165-168 (1988)), and E.C.2.4.1.38 and ceramide galactosyltransferase (EC 2.4.1.45, people such as Stahl, J.Neurosci.Res.38:234-242 (1994)).Other galactosyltransferase that is fit to for example comprises α 1,2 galactosyltransferase (for example from Schizosaccharomycespombe, people such as Chapell, Mol.Biol.Cell 5:519-528 (1994)).

C) sialic acid based transferase

The sialic acid based transferase is the another kind of glycosyl transferase that can be used for recombinant cell and reactant mixture of the present invention.The cell of producing reorganization sialic acid based transferase also produces cmp sialic acid, and it is that the sialic acid of sialic acid based transferase is given body.The example that is applicable to sialic acid based transferase of the present invention comprises ST3Gal III (for example mouse or people ST3Gal III), ST3Gal IV, ST3GalI, ST6Gal I, ST3Gal V, ST6Gal II, ST6GalNAc I, ST6GalNAc II and ST6GalNAc III (in sialic acid based transferase nomenclature used herein as people such as Tsuji, described in the Glycobiology 6:v-xiv (1996)).Exemplary α (2,3) the sialic acid based transferase that is called α (2,3) sialic acid based transferase (EC2.4.99.6) is transferred to sialic acid on the non-reduced terminal Gal of Gal β 1 → 3Glc disaccharides or glucosides.Referring to people such as Van denEijnden, 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 exemplary α 2,3-sialic acid based transferase (EC2.4.99.4) is transferred to sialic acid on the non-reduced terminal Gal of 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).Other exemplary enzyme comprises Gal-β-1,4-GlcNAc α-2,6 sialic acid based transferase (referring to people such as Kurosawa, Eur.J.Biochem.219:375-381 (1994)).

Preferably, glycosylation for the carbohydrate of glycopeptide, the sialic acid based transferase can be transferred to sialic acid sequence Gal β 1, on the 4GlcNAc-, be positioned at the sequence modal second from the bottom (referring to table 4) below the terminal sialic acid on the complete sialylated carbohydrate structure.

Table 4: use Gal β 1, the 4GlcNAc sequence is as the sialic acid based transferase of receptor substrate

The sialic acid based transferase	The source	Formed sequence	List of references
The sialic acid based transferase	The source	Formed sequence	List of references	ST6Gal I	Mammal	NeuAcI2，6Galβ1，4GlCNAc-	1
ST3Gal III	Mammal	NeuAcI2，3Galβ1，4GlCNAc- NeuAcI2，3Galβ1，3GlCNAc-	1	ST6Gal I	Mammal	NeuAcI2，6Galβ1，4GlCNAc-	1
ST3Gal III	Mammal	NeuAcI2，3Galβ1，4GlCNAc- NeuAcI2，3Galβ1，3GlCNAc-	1	ST3Gal IV	Mammal	NeuAcI2，3Galβ1，4GlCNAc- NeuAcI2，3Galβ1，3GlCNAc-	1
ST6Gal II	Mammal	NeuAcI2，6Galβ1，4GlCNAc-		ST3Gal IV	Mammal	NeuAcI2，3Galβ1，4GlCNAc- NeuAcI2，3Galβ1，3GlCNAc-	1
ST6Gal II	Mammal	NeuAcI2，6Galβ1，4GlCNAc-		ST6Gal II	Luminous bacteria	NeuAcI2，6Galβ1，4GlCNAc-	2
ST3Gal V	The Neisseria meningitidis Diplococcus gonorrhoeae	NeuAcI2，3Galβ1，4GlCNAc-	3	ST6Gal II	Luminous bacteria	NeuAcI2，6Galβ1，4GlCNAc-	2

1) people such as Goochee, Bio/Technology 9:1347-1355 (1991)

2) people such as Yamamoto, J.Biochem.120:104-110 (1996)

3) people such as Gilbert, J.Biol Chem.271:28271-28276 (1996)

The example that can be used for the sialic acid based transferase of desired method is ST3Gal III, also is called α (2,3) sialic acid based transferase (EC2.4.99.6).This enzymatic sialic acid is transferred to Gal β 1,3GlcNAc or Gal β 1, the Gal of 4GlcNAc (for example referring to people such as Wen, J.Biol.Chem.267:21011 (1992); And cause the sialylated of oligosaccharides that the asparagine in glycopeptide connects people such as Van den Eijnden, J.Biol.Chem.256:3159 (1991)).This sialic acid is connected in Gal, has formed the α key between two sugar.Between the keyed jointing between the sugar (connection) is at 2 of NeuAc and Gal 3.This specific endonuclease capable separates (people such as Weinstein, J.Biol.Chem.257:13845 (1982)) from the mouse liver; People cDNA (people (1993) such as Sasaki, J.Biol.Chem.268:22782-22787; Kitagawa﹠amp; Paulson (1994), J.Biol.Chem.269:1394-1401) and genome (people such as Kitagawa, (1996) J.Biol.Chem.271:931-938) dna sequence dna be known, help by recombinant expressed this enzyme of producing.In a preferred embodiment, desired sialylated method is used mouse ST3Gal III.

Be used for other exemplary sialic acid based transferase of the present invention and comprise those that separate from campylobacter jejuni, comprise α (2,3).For example referring to WO99/49051.

Except enumerate in the table 5 those the sialic acid based transferase also can be used for the economy of sialylated industrial important glycopeptide and effective large-scale methods.As the simple experiment of the purposes of finding these other enzymes, each enzyme (1-100mU/mg albumen) of different amounts with take off sialic acid-α ₁AGP (1-10mg/ml) reaction is with sialic acid based transferase and the ox ST6Gal I that is relatively studied, the ability of the sialylated glycopeptide of ST3GalIII or these two kinds of glycosyl transferases.Perhaps, the oligosaccharides that connects of other glycopeptide that discharges from peptide backbone with enzyme process or N-can replace taking off sialic acid-α ₁AGP is used for this evaluation.Have than ST6Gal I more effectively the sialic acid based transferase of the ability of the oligosaccharides that connects of the N-of sialylated glycopeptide can be used for the sialylated practical large-scale method of peptide (as illustrated to ST3Gal III in the disclosure).

D) GalNAc transferase

N-acetyl group galactosamine based transferase can be used for implementing the present invention, is particularly useful for the GalNAc structure division is incorporated into the amino acid of the glycosylation site that the O-of peptide connects.The N-acetyl group galactosamine based transferase that is fit to is including, but not limited to α (1; 3) N-acetyl group galactosyltransferase; β (1; 4) N-acetyl group galactosamine based 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-acetyl group galactosamine based transferase (people such as Homa, J.Biol.Chem.268:12609 (1993)).

Produce albumen such as enzyme GalNAcT by gene engineering by cloned genes _I-XXMethod be known.For example referring to US patent No.4,761,371.A kind of method comprises collects enough samples, measures the amino acid sequence of enzyme then by the N-end sequencing.Use this information to separate the cDNA clone of coding total length (film combination) transferase then, synthesized complete organized enzyme when it is expressed in insect cell line Sf9.Use the amino acid whose semi-quantitative analysis around the known glycosylation site of 16 kinds of different albumen then, synthesize the external glycosylation of peptide subsequently and study the receptor-specific of measuring enzyme.This research work is verified, some amino acid residue is excessively performance in the glycosylated peptide segment, and the residue in the ad-hoc location around glycosylation serine and the threonine residues can have more appreciable impact to acceptor efficient than other amino acid structure part.

2, sulfotransferase

The present invention also provides to produce and has comprised sulfating numerator, and for example the sulphation polypeptide is such as heparin, Heparan sulfate, the method for the peptide of carrageenan (carragnen) and related compound.The sulfotransferase that is fit to for example comprise chondroitin-6-sulfotransferase (by people such as Fukuta, the described chicken cDNA of J.Biol.Chem.270:18575-18580 (1995); GenBank Accession No.D49915), glycosaminoglycan N-n acetylglucosamine n N-deacetylase/N-sulfotransferase 1 (people such as Dixon, Genomics 26:239-241 (1995); UL 18918), and glycosaminoglycan N-n acetylglucosamine n N-deacetylase/N-sulfotransferase 2 is (people such as Orellana, people such as J.Biol.Chem.269:2270-2276 (1994) and Eriksson, the murine cDNA described in the J.Biol.Chem.269:10438-10443 (1994); At the people cDNA described in the GenBankAccession No.U2304).

3, the glycosyl transferase of cell combination

In another embodiment, the enzyme that adopts in the method for the invention is the glycosyl transferase of cell combination.Though many soluble sugar based transferases are known (for example referring to US patent No.5,032,519), when getting in touch with cell, glycosyl transferase exists with film combining form usually.Many membrane bound enzymes of being studied are considered to intrinsic protein so far; That is, they can not disengage from film by ultrasonic processing, need be used for the washing agent of solubilising.The surface glycosyl transferase is identified on the surface of vertebra and invertebral zooblast, has realized that also these surperficial transferases have kept catalytic activity under physiological condition.Yet the function of more generally acknowledged cell surface glycosyl transferase is iuntercellular identification (Roth, MOLECULAR APPROACHES toSUPRACELLULAR PHENOMENA, 1990).

The method of having developed changes the glycosyl transferase by cellular expression.For example, people such as Larsen, Proc.Natl.Acad.Sci.USA 86:8227-8231 (1989) has reported the genetic method of cDNA sequence of the separating clone of the expression of measuring cell surface oligosaccharide structure and their related glycosyl transferase.Will be by known expression UDP-galactase: β-D-galactosyl-1,4-N-acetyl group-D-glucosaminide α-1, the generation cDNA library transfection of the mRNA that the murine cell-line of 3-galactosyltransferase is separated is to the COS-1 cell.Cultivate cells transfected then, and analyze α-1,3-galactosyltransferasactivity activity.

People such as Francisco, Proc.Natl.Acad.Sci.USA 89:2713-2717 (1992) discloses the method that beta-lactamase is anchored in colibacillary outer surface.Production is by the burst of (i) outer membrane protein, (ii) outer membrane protein stride three fusions that film section and (iii) full ripe beta-lactamase sequence are formed, obtained the beta-lactamase molecule of active surface combination.Yet the Francisco method only limits to the prokaryotic system, and according to author's understanding, suitable playing a role needs complete three to merge.

4, fusion

In other exemplary, method of the present invention adopts the fusion that has with the enzymic activity of synthetic relevant more than one of desired sugars peptide conjugate.Fused polypeptide can be for example be made up of the catalytic activity zone of the glycosyl transferase in the catalytic activity zone that is connected in auxiliary enzymes.The auxiliary enzymes catalysis region for example can catalysis belongs to the step in the formation of the nucleotide sugar of giving body of glycosyl transferase, the perhaps reaction that relates in the circulation of catalysis glycosyl transferase.For example, the polynucleotides of encoding glycosyl transferase can be connected in the polynucleotides that are coded in the enzyme that nucleotide sugar relates in synthetic in frame.The gained fusion then not only can the catalysis nucleotide sugar synthetic, and catalysis sugar structure division is transferred on the acceptor molecule.This fusion can be the two or many cyclophorases that are connected in an effable nucleotide sequence.In other embodiments, fusion comprises the catalytic activity zone of two or more glycosyl transferases.For example referring to 5,641,668.Modification glycopeptide of the present invention can easily utilize various suitable fusions to design and produce (for example, referring to PCT patent application PCT/CA98/01180, it is open on June 24th, 1999 as WO99/31224).

5, immobilized enzyme

Except the enzyme of cell combination, the present invention also provides the purposes that is fixed on the enzyme on solid and/or the soluble carrier.In exemplary, provide the method according to this invention to connect the basic glycosyl transferase of puting together in PEG via intact glycosyl.PEG-connects the optional solid carrier that is connected in of base-enzyme conjugate.The post processing of reactant mixture and the purification of product have been simplified in the enzyme that solid supports application in the method for the invention, can also reclaim enzyme easily.The glycosyl transferase conjugate adopts in the method for the invention.Other combination of enzyme and carrier is that those skilled in the art institute is conspicuous.

The purification of peptide conjugate

The product of producing in order to last method can be purified and be used.Yet, the preferred usually product that reclaims.Can adopt the standard that is used to reclaim glycosylation sugar, technique known such as thin layer or thick layer chromatography method, column chromatography, ion exchange chromatography or membrane filtration.The preferred membrane filter method that uses more preferably adopts reverse osmosis membrane, perhaps as hereinafter with one or more column chromatography technology that are used to reclaim described in this paper citing document.For example, wherein the membrane filtration of film with weight shutoff of about 3000 to about 10,000 can be used for removing deproteinized such as glycosyl transferase.Can use nanofiltration or reverse osmosis to remove then desalts and/or purified product sugar (for example referring to WO 98/15581).Nano-filtration membrane is by monovalent salt, but keeps the class reverse osmosis membrane greater than about 100 to about 2,000 daltonian multivalent salts and uncharged solute, and this depends on employed film.Therefore, in the typical case used, the sugar for preparing with method of the present invention was retained in the film, but the salt that pollutes will pass through.

If modified proteins produces, as the first step, for example remove granular debris, i.e. host cell or dissolving fragment by centrifugal or ultrafiltration in molecule; Randomly, this albumen can concentrate with commercial albumen thickening filtration device, subsequently by being selected from one or more step isolated polypeptide variants and other impurity among following: immunoaffinity chromatography, ion exchange column classification (for example use diethylamino ethyl (DEAE) or contain carboxymethyl or the matrix of sulfo group propyl group), use Blue-Sepharose, CM Blue-Sepharose, MONO-Q, MONO-S, LCA (lentil lectin)-Sepharose, WGA-Sepharose, Con A-Sepharose, Ether Toyopearl, Butyl Toyopearl, Phenyl Toyopearl, SP-Sepharose, or protein A Sepharose chromatography, the SDS-PAGE chromatography, silica gel chromatography, chromatofocusing, reversed-phase HPLC (silica gel that for example has additional aliphatic group), for example use the gel filtration of Sephadex molecular sieve or SEC, use the chromatography of the pillar of selective binding polypeptide, and ethanol or ammonium sulfate precipitation.

The modification glycopeptide of producing in culture is usually by initially extraction from cell, enzyme etc., and subsequently one or more concentrate, saltout, water-based ion exchange or size exclusion chromatography step (for example SP Sepharose) are separated.In addition, modified proteins can be purified by affinity chromatography.HPLC can also be used for one or more purification step.

Protease inhibitors, for example methanesulfonyl fluoride (PMSF) can in officely how be gone up in the step and comprise, with the Profilin hydrolysis, and can comprise antibiotic, to prevent the growth of external contaminant.

In another embodiment, at first use commercial albumen thickening filtration device from the supernatant of the system that produces modification glycopeptide of the present invention, for example Amicon or MilliporePellicon ultra filtration unit concentrate.After concentration step, this concentrate can be put on suitable purifying matrix.For example, the affinity substrate of Shi Heing can comprise the part of the peptide, agglutinin or the antibody molecule that are incorporated into suitable carrier.Perhaps, can use anion exchange resin, for example have matrix or substrate that side is hung the DEAE group.The matrix that is fit to comprises acrylamide, agarose, dextran, cellulose, or in albumen is purified normally used other type.Perhaps, can use cation-exchange step.The cation-exchanger that is fit to comprises the various insoluble matrix that contains sulfo group propyl group or carboxymethyl.The sulfo group propyl group is particularly preferred.

At last, adopt hydrophobic RP-HPLC medium, for example have side and hang one or more RP-HPLC steps of the silica gel of methyl or other aliphatic group and can be used for the polypeptide variants composition of further purifying.Some or all of the above-mentioned purification step of various combinations can also be used to providing the glycoprotein of even modification.

The modification glycopeptide of the present invention that is obtained by large scale fermentation can be by in by people such as Urdal, and disclosed those the similar methods of J.Chromatog.296:171 (1984) are purified.This list of references has been described with two of preparation HPLC pillar purification recombinant human il-2 RP-HPLC steps according to the order of sequence.In addition, the technology such as affinity chromatography can be used to the modified proteins of purifying.

Except above-mentioned conjugate, the invention provides the method for these and other conjugate of preparation.And, the invention provides by conjugate of the present invention is delivered medicine to and have the method that the individuality or the patient that suffer from disease risks prevented, treat or improved morbid state.

Pharmaceutical composition

Put together in the treatment of branched water-soluble polymers of the present invention and have various medicinal applications with structure division.For example modification hematopoietin (EPO) can be used for the treatment of general anemia, alpastic anemia, the damage of chemotherapy induction (such as the damage to marrow), chronic renal failure, ephritis, and thalassemia.Modification EPO can be further used for treating nerve problems such as brain/spinal cord injury, multiple sclerosis and Alzheimer disease.

Second example is interferon-alpha (IFN-α), it can be used for treating AIDS and the hepatitis B or third liver, by various viruses such as human papilloma virus (HBV), coronavirus, human immunodeficiency virus (HIV), herpes simplex virus (HSV), and the virus infections that causes of varicella virus (VZV), cancer is such as hairy cell leukemia, AIDS correlation Kaposi sarcoma, malignant mela noma, the non-hodgkin lymphoma of folliculus, Philadelphia chromosome (Ph) positive, chronic granulocytic leukemia (CML), kidney, myeloma, chronic myelogenous leukemia, head and neck neoplasm, osteocarcinoma, and cervical atypism hyperplasia and central nervous system (CNS) disease are such as multiple sclerosis.In addition, the IFN-α of the method according to this invention modification can be used for treating various other diseases and state such as dry syndrome (autoimmunity disease), behcet's disease (autoimmunity inflammatory disease), fibromyalgia (flesh and skeleton pain/fatigue diseases), aphthous ulcer (canker sore), chronic fatigue syndrome, and pulmonary fibrosis.

Another example is a beta-interferon, can be used for treating the CNS obstacle such as multiple sclerosis (recurrence type/mitigation type or chronic progressive external), AIDS and hepatitis B or third liver, by various viruses such as human papilloma virus (HBV), human immunodeficiency virus (HIV), herpes simplex virus (HSV), and the virus infections that causes of varicella virus (VZV), ear infection, flesh and infection of bone, and cancer, comprise breast cancer, the cancer of the brain, colorectal cancer, non-small cell lung cancer, Head and Neck cancer, basal-cell carcinoma, the cervical atypism hyperplasia, melanoma, cutaneum carcinoma, and liver cancer.The IFN-β of the method according to this invention modification also is used for the treatment of other disease and state such as graft rejection (for example bone-marrow transplantation), Huntington's chorea, colitis, encephalitis, pulmonary fibrosis, macular degeneration, cirrhosis, and keratoconjunctivitis.

Granulocyte colony stimulating factor (G-CSF) is another example.The G-CSF of the method according to this invention modification can be used as auxiliary agent in the chemotherapy of treatment cancer, and is used to prevent or alleviate the state of an illness relevant with some medical procedure or complication, for example bone marrow injury of chemotherapy induction; Leukocyte reduces (generality); The heat generation neutrophil leucocyte of chemotherapy induction reduces; The neutrophil leucocyte relevant with bone-marrow transplantation reduces; And heavy chronic neutrophil leucocyte reduces.Modification G-CSF also can be used for transplanting; Peripheral blood cells is mobilized; Accepting the mobilization that marrow melts the peripheral blood CFU-GM that is used to collect among the patient of (myeloablative) or bone marrow suppression chemotherapy; And the neutrophil leucocyte after the inducing of acute myelogenous leukemia/after treatment reduces, fever, and antibiotic uses, the minimizing of the duration of hospitalization.Other state of an illness or obstacle can comprise asthma and allergic rhinitis with modification G-CSF treatment.

As an other example, the human growth hormone (HGH) of the method according to this invention modification (hGH) can be used for treating with the relevant disease of growing such as nanism, children and adult's is of short and small stature, cachexia/muscle consumption, general amyotrophy, and sex chromosomal abnormality (for example Turner's synodrome).Can comprise with other disease of modification hGH treatment: short bowel syndrome, LD, osteoporosis, uremia (uraemaia), burn, female sterility, bone regeneration, general diabetes, type ii diabetes, osteoarthritis, chronic obstructive pulmonary disease (COPD), and insomnia (insomia).And modification hGH can also be used to promote various processes, for example general regeneration, and osteanagenesis, and wound healing are perhaps as vaccine adjuvant.

Therefore, in yet another aspect, the invention provides pharmaceutical composition.This pharmaceutical composition comprises that medicine acceptable diluent and non-natural exist, water-soluble polymer, treats the covalent conjugates with structure division or biomolecule and glycosylation or non-glycosylated peptide.This polymer, treatment connect base with structure division or biomolecule via intact glycosyl and put together in peptide, this intact glycosyl connect base between and with covalent bond be connected in peptide with polymer, treat with structure division or biomolecule.

Pharmaceutical composition of the present invention is applicable to various drug delivery systems.Be applicable to that preparation of the present invention can be at Remington ' s Pharmaceutical Sciences, Mace PublishingCompany, Philadelphia, PA, 17th ed. finds in (1985).For the summary of the method that is used for the medicine conveying, referring to Langer, Science249:1527-1533 (1990).

This pharmaceutical composition can be prepared and be used for any suitable administering mode, for example comprises the part, mouthful, nose, intravenous, encephalic, in the peritonaeum, subcutaneous or intramuscular administration.For parenteral, such as hypodermic injection, carrier preferably includes water, salt solution, alcohol, fat, wax or buffer.For oral administration, any or solid carrier that can use above carrier is such as mannitol, lactose, starch, dolomol, sodium sugar, talcum, cellulose, glucose, sucrose, and magnesium carbonate.Can also use biodegradable matrix, such as the carrier of microballoon (for example polylactic acid poly glycolic) as pharmaceutical composition of the present invention.The biodegradable microballoon that is fit to for example is disclosed in US patent Nos.4, in 897,268 and 5,075,109.

Usually, the subcutaneous or parenteral of pharmaceutical composition, for example intravenous administration.Therefore, the invention provides to comprise and be dissolved in or be suspended in acceptable carrier, preferred aqueous carrier, water for example, buffered water, salt solution, the composition that is used for parenteral of the compound of PBS etc.Composition can also contain washing agent such as Tween20 and Tween80; Stabilizing agent is such as mannitol, sorbierite, sucrose and trehalose; With preservative such as EDTA and metacresol.Composition can contain the required medicine acceptable assistant of simulation physiological condition, such as pH regulator and buffer, and opening property conditioning agent, wetting agent, washing agent etc.

These compositions can be sterilized by common sterilization technology, perhaps can aseptic filtration.Obtained aqueous solution can be packed, and is used for direct use, perhaps freeze-drying.Lyophilized formulations merged with aseptic aqueous carrier before administration.The pH of said preparation generally is 3-11, more preferably 5-9, most preferably 7-8.

In some embodiments, glycopeptide of the present invention can be incorporated into by the standard vesica and form in the formed liposome of lipid.Several different methods is available for preparing liposome, for example is described in people such as Szoka, Ann.Rev.Biophys.Bioeng.9:467 (1980), and U.S.Pat.Nos.4 is in 235,871,4,501,728 and 4,837,028.Use being directed at of liposome (referring to for example US patent Nos.4,957,773 and 4,603,044) known in the art of various directed agents (saliva acidic group galactoside for example of the present invention).

Can adopt the standard method that is used for directed agents is coupled to liposome.These methods generally comprise and can activate the lipidic component that is used to connect directed agents, and such as phosphatidyl-ethanolamine, perhaps the derivatization lipophilic compound is incorporated into liposome such as lipid derivatization glycopeptide of the present invention.

Guiding mechanism generally requires directed agents to be positioned on the surface of liposome, make object construction part can with object, for example cell surface receptor interacts.Carbohydrate of the present invention can be connected in lipid molecule (alkylation or the acidylate that for example, are present in the hydroxyl on the carbohydrate that has chain alkyl halide respectively or have fatty acid) before using method known to those skilled in the art formation liposome.Perhaps, liposome can be made by the mode that when forming film the coupling part at first is incorporated in the film.This coupling part must have lipophilic portion, and it embeds and anchoring in film securely.It also must have reactive part, and it can utilize by chemistry on the aqueous surface of liposome.The choice reaction part makes it form stable chemical bond at the directed agents or the carbohydrate that chemically are suitable for adding afterwards.In some cases, can directly directed agents be connected in the connection molecule, but in most of the cases, be more suitable for using the 3rd molecule to come therefore the connection molecule in the film to be connected with directed agents of stretching out from the vesica surface three-dimensional or carbohydrate as chemical bridge.

Compound with method preparation of the present invention can also be used as diagnostic reagent.For example, the compound of mark can be used for locating the inflammation among the patient who is suspected to have inflammation or the zone of metastases.For this reason, compound can be used ¹²⁵I, ¹⁴C or tritiated.

Following examples provide and are used for illustrating compound of the present invention and method, but do not limit desired invention.

Embodiment

In embodiment 1-3, use following symbol.Main PEG subunit is made up of four glycol monomethyl units.This molecule has 194 molecular weight, is rounded to 200 in the drawings.Following symbol is used for representing main PEG subunit:

The molecular weight that rounds up shown in the bond of main PEG subunit alternatively is used between the functionalized end is as shown below represented.

Following symbol is used for representing monofunctional methoxyl group PEG subunit:

Embodiment 1

Single preparation that disperses PEG and their activated form

List disperses or unimodal molecular weight PEG is as follows prepares.By regulating the size of the fragment that is produced, prepared the PEG of any yardstick.Then by the end end-blocking of alkylation with glycol, and activation, be used to put together in the biological structure part such as albumen, sugar, lipid or nucleotide.

Leaving group can be connected in main PEG subunit, so that produce the main PEG of activation shown below.In this reaction, Q can be any leaving group compatible with chemical process of the present invention.Exemplary leaving group comprises halogen, tresylate, tosylate and methanesulfonate.

After producing the main PEG of activation, as follows, this compound and the reaction of main PEG subunit.This product is a first generation PEG continuation.

Second generation PEG continuation is according to producing with the same mode of the first generation.

The generation as follows of third generation PEG continuation.

The generation as follows of the 4th generation PEG continuation.

PEG prolongs process by allowing the reaction of one of monofunctional structure division and difunctional compound stop.In this reaction, the monofunctional structure division is any group compatible with chemical process of the present invention.Exemplary end-capping group comprises alkoxyl-PEG and alkyl.

In following exemplary, leaving group is added on methoxyl group-PEG subunit.This molecule reacts with the 4th generation PEG continuation then.

In another exemplary, the methyl subunit is added on the 4th generation PEG continuation.

After end of end-blocking, the other end of PEG continuation is as follows to be activated and is used for biology and puts together.In this reaction, X is any leaving group that can form ester.Symbol X is independently selected from imidazole radicals, HOBt, HOAt, NHS and p-nitrophenyl ester.

At last, PEG prolongs that molecule is as follows to be puted together in the biological structure part.

Embodiment 2

Do not require that the single PEG of dispersion of activation must have and PEG subunit with the prolongation molecule similar number of its reaction.In exemplary, activation single disperses PEG to have than with the prolongation molecule of its reaction PEG subunit of big figure more.In another exemplary shown below, the single PEG of dispersion of activation has the PEG subunit than the prolongation molecule lesser number of reacting with its.

The end blocking method of these molecules is with similar in the method described in the embodiment 1.

Embodiment 3

Can add excessive activated PEG subunit, so that produce single PEG of dispersion shown below:

N is 1-100 or 1-20,000, depend on the source

By changing the ratio of reactant, employed alkali, temperature, solvent and concentration can conditioned reactions, obtain required main molecules amount (n).

This method provides simple, quick, the effective means of the single PEG of dispersion for preparing any size.Purification is simplified by this method, and there is difference in (with therefore physicochemical characteristics) because single molecular weight that disperses PEG.This purification technique that allow to use simple, standard is such as silica gel, anti-phase cellulose, membrane filtration (nanofiltration and ultrafiltration).The PEG glycol of purifying is derived then and is turned to required any functionalized form.

Embodiment 4

The production of alkoxyl PEG

Conventional method shown below is used for preparing alkoxyl PEG or other monofunctional PEG.

In first embodiment, activate the generation as follows of Bifunctionalized PEG molecule:

Wherein symbol n represents 1-100,000 numerical value.Symbol Q represents any leaving group compatible with chemical process of the present invention.Exemplary leaving group comprises halogen, tresylate, tosylate and methanesulfonate.Symbol X represents any counter ion counterionsl gegenions compatible with leaving group.

This activates the Bifunctionalized PEG molecule length that is used to prolong the PEG molecule as follows:

Wherein symbol m represents 1-100,000 numerical value.

In second embodiment, as follows, prolong monofunctional PEG, activation is used for partly puting together with biological structure then.

In the first step, monofunctional PEG carries out tosylation.

Wherein symbol n represents 1-100,000 numerical value.

In second step, prolong monofunctional PEG:

Wherein symbol m represents 1-100,000 numerical value.

In last step, as follows, the monofunctional PEG compound activating of prolongation is used for partly puting together with biological structure.

Embodiment 5

Be used to prepare other composition and the method for two feeler polymer

Other pair feeler structure of the present invention has following general formula:

Wherein symbol X represents OH, H, and Q (activated group) and biological structure part, such as albumen, sugar, lipid, or nucleotide.Symbol n represents the numerical value of 1-10.Term " polymer " " can be PEG, mPEG (methoxy poly (ethylene glycol)), PPG (polypropylene glycol), mPPG, polyglutamic acid, poly-aspartate, PLA, and poly sialic acid.

In exemplary, two feeler structures have following general formula:

Wherein symbol m and n represent 1-10 independently, 000 numerical value.Symbol X represents OH, H, and Q (activated group) and biological structure part, such as albumen, sugar, lipid or nucleotide.

In another exemplary, two feeler structures have following formula:

Wherein symbol a and b represent the numerical value of 1-24 independently.Symbol m and o represent 1-10 independently, 000 numerical value.Symbol X represents OH, H, and Q (activated group), and biological structure part are such as albumen, sugar, lipid or nucleotide.

Though disclose the present invention with reference to specific embodiments, apparent, under the situation that does not depart from true spirit of the present invention and scope, others skilled in the art can design other embodiment of the present invention and modification.

All patents of enumerating in this application, patent application and other publication are introduced for reference comprehensively.

Claims

1, peptide, it has following general formula:

In the formula:

R ¹¹, R ^{11 '}, R ¹², R ^{12 '}, R ¹³And R ^{13 '}Be independently selected from H, replace or not substituted alkyl and water-soluble polymer, prerequisite is R ¹¹, R ^{11 '}, R ¹², R ^{12 '}, R ¹³And R ^{13 '}At least two be the water-soluble polymer structure division; And R ¹⁴Be selected from OH, reactive functional groups contains the group of sugared structure division or is connected in a kind of in the group of carrier molecule.

2, according to the peptide of claim 1, wherein said water-soluble polymer structure division comprises poly-(ethylene glycol).

3, according to the peptide of claim 2, it has following general formula:

4, according to the peptide of claim 2, it has following general formula:

Wherein:

M, n and t are independently selected from integer 1-20,000.

5, according to the peptide of claim 1, R wherein ¹⁴Comprise sugared structure division.

6, according to the peptide of claim 5, wherein said sugared structure division is a nucleotide sugar.

7, according to the peptide of claim 5, wherein said sugared structure division is puted together a kind of in being selected from second kind of peptide and lipid.

8, according to the peptide of claim 5, wherein said sugared structure division is puted together a kind of in amino acid that is selected from described peptide and glycosyl residue.

9, peptide according to Claim 8, wherein said sugared structure division are that the glycosyl between described peptide and described second kind of peptide connects base.

10, according to the peptide of claim 9, wherein said sugared structure division is that the intact glycosyl between described peptide and described second kind of peptide connects base.

11, comprise according to the peptide of claim 1 and the pharmaceutical preparation of medicine acceptable carrier, wherein R ¹⁴Comprise carrier molecule, it is selected from a kind of with in the structure division of treatment.

12, amino acid, it has following general formula:

In the formula:

R ¹¹, R ^{11 '}And R ¹²Be independently selected from H, replace or not substituted alkyl and water-soluble polymer, prerequisite is R ¹¹, R ^{11 '}And R ¹²At least two be the water-soluble polymer structure division; With

R ¹⁴Be selected from OH, reactive functional groups contains the group of sugared structure division or is connected in a kind of in the group of carrier molecule.

13, according to the amino acid of claim 12, wherein said water-soluble polymer structure division comprises poly-(ethylene glycol).

14, according to the amino acid of claim 12, wherein said water-soluble polymer structure division has following general formula:

15, according to the amino acid of claim 14, it has following general formula:

16, according to the amino acid of claim 12, R wherein ¹⁴Comprise sugared structure division.

17, according to the amino acid of claim 16, wherein said sugared structure division is a nucleotide sugar.

18, according to the amino acid of claim 16, wherein said sugared structure division is puted together a kind of in being selected from second kind of peptide and lipid.

19, according to the amino acid of claim 16, wherein said sugared structure division is puted together a kind of in amino acid that is selected from described peptide and glycosyl residue.

20, according to the amino acid of claim 19, wherein said sugared structure division is that the glycosyl between described peptide and described second kind of peptide connects base.

21, according to the amino acid of claim 20, wherein said sugared structure division is that the intact glycosyl between described peptide and described second kind of peptide connects base.

22, comprise according to the amino acid of claim 12 and the pharmaceutical preparation of medicine acceptable carrier, wherein R ¹⁴Comprise carrier molecule, it is selected from a kind of with in the structure division of treatment.

23, branched water-soluble polymers, it has the general formula that is selected among following:

With

In the formula:

Q is selected from H, comprises carrier molecule and activated group, makes that C (O) Q ' is a kind of of reactive functional groups; And m and n be independently selected from 1-20, the integer in 000.

24, according to the branched water-soluble polymers of claim 23, wherein Q ' is selected from halogen, perfluorophenyl, HOBT, a kind of in HOAt and the p-nitrophenol.

25, according to the branched water-soluble polymers of claim 23, wherein Q ' comprises sugared structure division.

26, according to the branched water-soluble polymers of claim 25, wherein said sugared structure division is a nucleotide sugar.

27, according to the branched water-soluble polymers of claim 25, wherein said sugared structure division is puted together a kind of in being selected from second kind of peptide and lipid.

28, according to the branched water-soluble polymers of claim 25, wherein said sugared structure division is puted together a kind of in amino acid that is selected from described peptide and glycosyl residue.

29, according to the branched water-soluble polymers of claim 28, wherein said sugared structure division is that the glycosyl between described peptide and described second kind of peptide connects base.

30, according to the branched water-soluble polymers of claim 29, wherein said sugared structure division is that the intact glycosyl between described peptide and described second kind of peptide connects base.

31, comprise according to the amino acid of claim 23 and the pharmaceutical preparation of medicine acceptable carrier, wherein Q ' comprises carrier molecule, and it is selected from a kind of with in the structure division of treatment.

32, branched water-soluble polymers, it has following general formula:

R wherein ¹⁶, R ^{16 '}, R ¹⁷, R ¹⁸And R ¹⁹Be selected from H, OH, NH ₂, NHAc and general formula (I):

In the formula

Z ²Be selected from O, S, CH ₂A kind of with among the S,

R ¹¹Be water-soluble polymer and

The integer of subscript " a " expression 0-20,

Prerequisite is R ¹⁶, R ^{16 '}, R ¹⁷, R ¹⁸, and R ¹⁹At least two structures that have according to general formula I; With

R ¹⁵Be selected from H, a kind of in the key of nucleotide sugar and connection carrier molecule.

33, according to the branched water-soluble polymers of claim 32, wherein said water-soluble polymer comprises poly-(ethylene glycol).

34, according to the branched water-soluble polymers of claim 32, wherein said carrier molecule is selected from a kind of in peptide and the lipid.

35, according to the branched water-soluble polymers of claim 32, it has following general formula:

36, branched water-soluble polymers, it has following general formula:

R wherein ¹⁶, R ¹⁷, R ¹⁸And R ¹⁹Be selected from H, OH, NH ₂, NHAc and general formula (I):

In the formula

Z ²Be selected from O, S, CH ₂A kind of with among the S,

R ¹¹Be water-soluble polymer and

The integer of subscript " a " expression 0-20,

R ¹⁵Be to be selected from H, a kind of in the key of nucleotide sugar and connection carrier molecule.

37, according to the branched water-soluble polymers of claim 36, wherein said water-soluble polymer comprises poly-(ethylene glycol).

38, according to the branched water-soluble polymers of claim 36, wherein said carrier molecule is selected from a kind of in peptide and the lipid.