CN1897962A - Glycopegylated erythropoietin - Google Patents

Abstract

The present invention provides conjugates between erythropoietin and PEG moieties. The conjugates are linked via an intact glycosyl linking group interposed between and covalently attached to the peptide and the modifying group. The conjugates are formed from glycosylated peptides by the action of a glycosyltransferase. The glycosyltransferase ligates a modified sugar moiety onto a glycosyl residue on the peptide. Also provided are methods for preparing the conjugates, methods for treating various disease conditions with the conjugates, and pharmaceutical formulations including the conjugates.

Description

Glycopegylated erythropoietin

The cross reference of related application

The application requires the U.S. Provisional Patent Application submitted on November 24th, 2003 number 60/524,989, the U.S. Provisional Patent Application of submitting on March 22nd, 2004 number 60/555,504, the U.S. Provisional Patent Application of submitting on July 23rd, 2004 number 60/590,573, the U.S. Provisional Patent Application of submitting on July 29th, 2004 number 60/592,744, the U.S. Provisional Patent Application submitted in 29th of JIUYUE in 2004 number 60/614,518 and U.S. Provisional Patent Application number 60/623,387 priority, every piece here is that all purpose integral body are incorporated by reference.

Background of invention

Erythropoietin (EPO) is the cytokine of being produced by the kidney regulating liver-QI, and it acts on hematopoietic stem cell, stimulates erythrocytic production.This albumen exists with two kinds of forms: a kind of is 165 amino acid peptides, and another kind is 166 amino acid peptides.166 amino acid peptides have the sequence identical with 165 amino acid peptides, except 166 amino acid peptides have the extra arginine at C-terminal position.Sophisticated 165 amino acid peptides are 34kD glycoproteins, and it comprises 3 N-glycosylation sites (Asn-24, Asn-38, and Asn-83), with 1 O-glycosylation site (Ser-126), and some variants are " high glycosylations ", comprise the glycosylation site that 5 N-connect.

Erythropoietin is synthetic is induced by the condition that can set up histanoxia effectively, for example reduces tremulous pulse O ₂The affinity for oxygen of tension force or increase blood.Under the usual conditions of stable state, hematocrit in the blood and hemoglobin concentration are kept constant in vivo, and by the macrophage in bone marrow, the spleen regulating liver-QI, erythropoiesis makes aged erythrocytic permanent destruction balance.Quantitatively, every day is the New Testament 1% red cell volume more, and it is about 2-3 * 10 ¹¹Erythrocyte.But, can set up effectively under the condition of histanoxia, for example to lose blood or be in high height above sea level, inducing of EPO can stimulate erythropoiesis to surpass 10 times or more times of normal levels.

Because EPO can stimulate erythropoiesis, it is the numerous disease relevant with the hematocrit that reduces and effective therapy of disease.Before about 20 years, reported first recover to suffer from the alternative medicine of the hematocrit among the patient of renal failure in latter stage with the recombinant human epo initial experiment (for example see Winearls, C.G.; Deng (1986) Lancet, 2,1175-1178, and Eschbach, J.W.; Deng (1987) N.Engl.J.Med., 316,73-78).This work provides the pathophysiology of further EPO and the power of pharmaceutical research (for example to see Jelkmann, W. and Gross, A. (1989) Erythropoietin; Springer, Berlin Heidelberg New York).

Because these early stage researchs, the recombinant human epo has been successfully used to treat many pathology diseases.For example, the recombinant human epo uses to the pharmacology of patient with operation, can reduce the seriousness and the persistent period of operation back anemia.Also verified, using of recombinant human epo is effective therapy of suffering from the patient of several non-kidney diseases, for example chronic inflammatory disease, malignant tumor and AIDS, wherein the relative shortage of endogenous EPO helps the development of anemia (for example to see, Means, R.T. and Krantz, S.B. (1992) Blood, 80,1639-1647, and Jelkmann, W. (1998) J.Interf.Cytokine Res., 18,555-559).In addition; report; EPO is that organization protection's agent (is for example seen in the damage of ischemic, traumatic, toxic and inflammatory; Brines M.; Deng (2004) PNAS USA 101:14907-14912 and Brines; M.L., wait (2000) .Proc.Natl.Acad.Sci.USA 97,10526-10531).

EPO is used for the treatment of anemia and the serviceability and the effectiveness of other disease of being caused by the reason of such broad variety, makes the recombinant human epo may become global best-selling medicine.In fact, estimate that annual sales volume surpasses 5,000,000,000 U.S. dollars.

Only a kind of recombinant human epo who produces in Chinese hamster ovary (CHO) cell line is widely used as therapeutic agent.Because mammal can both produce the polysaccharide of similar structures, Chinese hamster ovary (CHO), little hamster kidney (BHK), and human embryo kidney (HEK)-293 (HEK-293) is the preferred host cell that is used to produce the glycoprotein therapeutic agent.As known in the art, suitable glycosylation is the interior half-life of body and the immunogenic factor of fundamental influence treatment peptide.In fact, glycosylated relatively poorly albumen can be identified as " old " by liver, thereby, can more promptly remove than glycosylated albumen suitably from health.

Unfortunately, protein glycosylation that baffle and well-known aspect is the microheterogeneity phenomenon.Thereby, even preferably be used to produce the host cell of human therapy glycoprotein, EPO for example, the precision architecture that also can typically be created on polysaccharide comprises the peptide of the variation of certain limit.The scope of this inhomogeneity can be from glycosylation site to glycosylation site, from albumen to albumen with significantly different from cell type to cell type.Therefore, many glycosyl forms (wherein each all is different molecular speciess effectively) typically are present in the given arbitrarily glycoprotein goods.

Thereby the problem of microheterogeneity has proposed many problems of bigger industrial-scale production therapeutic glycoprotein.More specifically,, must carry out classification, with the single sugar form of purification needs to therapeutic glycoprotein goods because every kind of sugar form can be represented different molecular speciess.Other concurrent problem is derived from a fact, and promptly different production batch can there are differences aspect the percentage ratio of the target sugar form that constitutes glycoprotein therapeutic agent batch.Thereby, may must abandon every kind of goods bigger, be not always predictable part, so the whole productive rate of ideal sugar form can be lower.In a word, the problem of microheterogeneity means, cultivate the therapeutic glycopeptide of producing by mammalian cell and need higher production cost, this finally can convert the higher health expenditure more essential than possibility to, if can obtain the more effective method that is used to produce more persistent, more effective glycoprotein therapeutic agent.

Provide a solution of the problem of cost-effective glycopeptide therapeutic agent to be, for peptide provides the half-life in the longer body.For example, by synthetic polymer is connected on the peptide main chain, produced the glycopeptide therapeutic agent of pharmacokinetic property with raising.The exemplary polymer that has been conjugated on the peptide is Polyethylene Glycol (" PEG ").Verified, with the application of PEG derived peptide therapeutic agent, can reduce the immunogenicity of peptide.For example, U.S. Patent number 4,179,337 (Davis etc.) disclose the polypeptide that is coupled to the non-immunogenic on Polyethylene Glycol (PEG) or the polypropylene glycol, for example enzyme and peptide hormone.Except reducing immunogenicity, because the size of the increase of the PEG-conjugate of target polypeptides has prolonged the checkout time in circulation.

The derivant of PEG and it is that nonspecific combination by the peptide ammino acid residue (is for example seen U.S. Patent number 4,088,538, U.S. Patent number 4 to the bonded basic model of peptide, 496,689, U.S. Patent number 4,414,147, U.S. Patent number 4,055,635 and PCTWO 87/00056).The another kind of pattern that PEG is attached on the peptide is, by the nonspecific oxidation (see for example WO 94/05332) of glycosyl residue on glycopeptide.

In these nonspecific methods,, Polyethylene Glycol is added on the reactive residue of peptide main chain with at random, nonspecific mode.Certainly, adding at random of PEG molecule has its shortcoming, comprises that end-product lacks active probability biology or enzyme of homogeneity and minimizing peptide.Therefore, for the manufacture of therapeutic peptide, can form labelling specifically, easily characterization, the strategy of deriving of the product of homogeneous is superior basically.Developed such method.

By the effect of enzyme, can labelling, homogeneous the specifically peptide therapeutics of produced in vitro.Different with typical nonspecific method that synthetic polymer or other labelling are connected on the peptide, have regioselectivity and stereoselective advantage based on enzyme synthetic.2 primary categories of enzyme that are used for the peptide of complex sign are glycosyl transferase (for example, sialyltransferase, oligosaccharyl transferase, N-acetylglucosaminyl transferase), and glycosidase.These enzymes can be used for the specificity combination of sugar, and this sugar can be modified subsequently, to comprise treatment group (moiety).Perhaps, the glycosidase of glycosyl transferase and modification can be directly used in the sugar that will modify and be transferred to the peptide main chain and (for example see, United States Patent (USP) 6,399,336, with U.S. Patent Application Publication 20030040037,20040132640,20040137557,20040126838, with 20040142856, every piece incorporated by reference here).In conjunction with chemistry and the method for enzymatic synthin also is known (for example see, Carbohydr.Res.305:415-422 such as Yamamoto (1998) and U.S. Patent Application Publication 20040137557, it is incorporated by reference here).

Erythropoietin (EPO) is very valuable treatment peptide.Although now at the EPO that uses commercially available form, these peptides are lower than maximum effectiveness, and its reason is many factors, comprise the microheterogeneity that can increase the glucoprotein product of production cost, the relatively poor pharmacokinetics of the isolating glucoprotein product that obtains, or the combination of the two.Thereby this area still needs the EPO peptide of the pharmacokinetics of persistent effectiveness with raising and Geng Jia.And, for the individuality to maximum quantity is effectively, must have the dynamic (dynamical) EPO peptide of medicine of raising in industrial-scale production, it has predictable, the structure of homogeneous basically, and it can be easy to duplication of production again and again.

Fortunately, the EPO peptide and their method of preparation of treatment effectiveness have been had been found that now with raising.In fact, the invention provides the EPO peptide of pharmacokinetics with raising.The present invention also provides the industrial feasible and cost-effective method of the EPO peptide that is used to produce modification.EPO peptide of the present invention comprises for example PEG group of modification group, treatment group, biomolecule etc.Therefore, the present invention has satisfied the EPO peptide of pharmacokinetics of treatment effectiveness with raising and raising EPO can provide the disease of effective treatment and the needs in the disease treating wherein.

Summary of the invention

Have been found that now with controlled ground of one or more polyethylene groups modified erythropoietin (EPO), the EPO derivant of the new pharmacokinetic property with raising can be provided.And, have been found that and developed the reliable production method of the EPO peptide of cost-effective modification of the present invention.

In one aspect, the invention provides the erythropoietin peptide, it comprises group:

Wherein D is selected from-OH and R ¹The member of-L-HN-; G is selected from R ¹-L-and-C (O) (C ₁-C ₆) member of alkyl; R ¹It is the group that comprises the member who is selected from the group that contains straight chain or ramose Polyethylene Glycol residue; And L is a junctional complex, and it is to be selected from key, replacement or member unsubstituted alkyl and replacement or unsubstituted assorted alkyl, so when D was OH, G was R ¹-L-, and as G be-C (O) (C ₁-C ₆) during alkyl, D is R ¹-L-NH-.In one embodiment, R ¹-L has formula:

Wherein a is the integer of 0-20.In another embodiment, R ¹Have the following structures of being selected from:

Wherein e and f are the integers that is independently selected from 1-2500; And q is the integer of 1-20.In other embodiment, R ¹Have the following structures of being selected from:

E wherein, f and f ' they are the integers that is independently selected from 1-2500; And q and q ' are the integers that is independently selected from 1-20.

In another embodiment, the invention provides peptide, wherein R ¹Have the following structures of being selected from:

E wherein, f and f ' they are the integers that is independently selected from 1-2500; And q, q ' and q " be the integer that is independently selected from 1-20.In other embodiment, R ¹Have the following structures of being selected from:

Wherein e and f are the integers that is independently selected from 1-2500.

In yet another aspect, the invention provides peptide, it comprises the group with following formula:

In other embodiment, this group has formula:

[0021] in another exemplary embodiment, this peptide comprises the group according to following formula

Wherein AA is the amino acid residue of described peptide.In some embodiments, amino acid residue is the member who is selected from serine, threonine and tyrosine.In a preferred embodiment, amino acid residue is the serine at 126 places, position of SEQ.ID.NO:1.

In another exemplary embodiment, the invention provides the erythropoietin peptide, wherein this peptide comprises the group that at least one has following formula:

Wherein t equals 0 or 1 integer.Thereby in this embodiment, the sialic acids groups of modification can appear in any branch of two feeler structures.

In another related embodiment, the invention provides the erythropoietin peptide, wherein this peptide comprises the group that at least one has following formula:

In another embodiment, the invention provides the erythropoietin peptide, wherein this peptide comprises the group that at least one has following formula:

In this embodiment, the sialic acids groups of modification can appear in any or a plurality of branch of three feeler structures of any form.

In another embodiment, the invention provides the erythropoietin peptide, wherein this peptide comprises the group that at least one has following formula:

In this embodiment, the sialic acids groups of modification can appear in any or a plurality of branch of four feeler structures.

In yet another aspect, the invention provides the erythropoietin peptide, it is that bioactive erythropoietin peptide is arranged.In one embodiment, the erythropoietin peptide has the erythropoiesis activity.In another embodiment, the erythropoietin peptide does not have the erythropoiesis activity basically.In another embodiment, the erythropoietin peptide is a tissue protective.

In yet another aspect, the invention provides the method for the erythropoietin that comprises following radicals of preparation PEG-ization:

R wherein ¹It is the group that comprises straight chain or ramose Polyethylene Glycol residue; And L is a junctional complex, and it is to be selected from replacement or member unsubstituted alkyl and replacement or unsubstituted assorted alkyl.This method comprises, and under the condition that is suitable for shifting, makes to comprise glycosyl:

Substrate erythropoietin peptide contact have the PEG-sialic acid donor group of following formula:

And described PEG-sialic acid can be transferred to enzyme on the Gal of described glycosyl.In one embodiment, in suitable hosts, express the erythropoietin peptide.In one embodiment, the host is a mammalian cell, and in another embodiment, host cell is an insect cell.

In yet another aspect, the invention provides the method that treatment has the disease in this object that needs, wherein this disease is characterised in that, the impaired erythrocyte production in the object.This method comprises the step of using the erythropoietin peptide of the present invention of the amount that can improve the disease in the object effectively to object.

In yet another aspect, the invention provides the method for the erythrocyte production that strengthens in the mammal.This method comprises the erythropoietin peptide of the present invention that can strengthen the amount of the erythrocyte production in the mammal to administration effectively.

In yet another aspect, the invention provides treatment has the method for the tissue injury in this object that needs, described damage is characterised in that, by the damage that ischemia, wound, inflammation or contact toxicity material cause, described method comprises the step of using the erythropoietin peptide of the present invention of the amount that can improve the described tissue injury in the object effectively to object.

In yet another aspect, the invention provides pharmaceutical preparation, it comprises erythropoietin peptide of the present invention and pharmaceutically acceptable carrier.

In the erythropoietin conjugates that o-of the present invention connects, almost each amino acid residue of polymer combination has identical structure.For example, if peptide comprises the glycosyl residue that Ser connects, in the colony at least about 70%, 80%, 90%, 95%, 97%, 99%, 99.2%, 99.4%, 99.6%, or more preferably 99.8% peptide can have identical glycosyl residue, and it covalently is attached on the identical Ser residue.

From following detailed, those skilled in the art can understand other purpose of the present invention and advantage.

Description of drawings

Fig. 1. Fig. 1 has illustrated the ribotide that is used to realize exemplary modifications more of the present invention.

Fig. 2. Fig. 2 has illustrated the ribotide that is used to realize other exemplary modification of the present invention.

Fig. 3. Fig. 3 has illustrated the sialic acid nucleotide that is used to realize exemplary modification of the present invention.The structure of A:40 kilodalton cmp sialic acid-PEG.The structure of B:30 kilodalton cmp sialic acid-PEG.

Fig. 4. Fig. 4 has shown the exemplary Glycopegylated sketch map from the isolating EPO isotype of Chinese hamster ovary cell.The sugar form of the PEGization that 40 A. exemplary kilodalton O-connect.B: one of sugar form of the PEGization that several 30 kilodalton N-connect.The sialic acids groups that comprises the modification of PEG molecule can appear on any side chain of the glycosyl residue that one or more N-connect.And, this figure is exemplary, because that glycosylated arbitrarily EPO molecule can comprise is single-, two-, three-or any mixture of the glycosyl residue that connects of the N-of four-feeler, and any or a plurality of branch can also comprise the sialic acids groups of modification of the present invention.

Fig. 5. Fig. 5 has illustrated the deutero-EPO peptide of exemplary CHO-, it is non--Glycopegylated form.As Fig. 4 (above) legend in discussed, this figure is exemplary because that glycosylated arbitrarily EPO molecule can comprise is single-, two-, three-or any mixture of the glycosyl residue that connects of the N-of four-feeler.

Fig. 6. Fig. 6 shown contrast 2 kinds of CHO-deutero-non--experimental result of the pharmacokinetics of the deutero-Glycopegylated EPO form of Glycopegylated EPO form and two kinds of different CHO-.

Fig. 7. Fig. 7 has illustrated according to insecticide of the present invention-deutero-Glycopegylated EPO peptide.

Fig. 8. Fig. 8 shown contrast CHO-deutero-non--Glycopegylated EPO form, insecticide-deutero-is non--experimental result of the pharmacokinetics of Glycopegylated EPO form and the Glycopegylated form of their correspondence.

Fig. 9. Fig. 9 has shown two kinds of non--Glycopegylated EPO forms (A and B) and the relative activity of the EPO variant of two kinds of Glycopegylated variants (30 kilodaltons of Fig. 4 A and B and 40 kilodalton variants) and high glycosylation in the propagation that stimulates the TF1 cell of cultivating that carries the EPO receptor.

Figure 10. Figure 10 has shown the bonded inhibition to the chimeric EPO receptor of the EPO of isotope-labelling and reorganization of the EPO variant (A and B) of two kinds of non--PEGization of variable concentrations and two kinds of Glycopegylated variants (30 kilodaltons of Fig. 4 A and B and 40 kilodalton variants).

Detailed Description Of The Invention and embodiment preferred

Abbreviation

PEG, Polyethylene Glycol; PPG, polypropylene glycol; Ara, Arabic glycosyl; Fru, fructosyl; Fuc, fucosido; Gal, galactosyl; GalNAc, N-acetyl-amino galactosyl; Glc, glucityl; GlcNAc, N-acetyl-amino glucityl; Man, mannose group; ManAc, epichitosamine guanidine-acetic acid (mannosaminyl acetate); Xyl, xylosyl; And NeuAc, saliva acidic group (N-acetyl neuraminic acid); M6P, Man-6-P.

Definition

Unless otherwise defined, all technology used in this article and the scientific terminology implication that generally has those skilled in the art's common sense.Usually, term of Shi Yonging and the experimental technique in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization are well-known in the art and commonly used in this article.It is synthetic that standard techniques is used for nucleic acid and peptide.Usually, according to the conventional method of this area and the various general document that in presents, provides (usually referring to, Molecular Cloning:A LaboratoryManual such as Sambrook, 2d ed. (1989) Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., it is incorporated by reference here), carry out the operation of technology and method.The term of Shi Yonging and analytical chemistry that describes below and the experimental technique in the organic synthesis are well-known in the art and commonly used in this article.Standard techniques or its improvement can be used for chemosynthesis and chemical analysis.

All oligosaccharide as herein described be described as the title of non--reducing sugar or abbreviation (that is, and Gal), be thereafter glycosidic bond configuration (α or β), ring key (1 or 2), participate in the ring position (2 of the reducing sugar of bonding, 3,4,6 or 8), be then again the title of reducing sugar or abbreviation (that is, GlcNAc).Every kind of sugar is pyranose preferably.About the comment of the glycobiology term of standard, see that Essentials of Glycobiology Varki etc. compiles .CSHL Press (1999).

Think that oligosaccharide has reductive end and non-reducing end, no matter at the sugar of reduction end reducing sugar whether.According to the nomenclature of generally acknowledging, be that non-reduced end and right side are that reduction end is described oligosaccharide with the left side in this article.

Term " sialic acid " refers to any member of the acidifying sugared family of 9-carbon carboxylic.The modal member of sialic acid family is N-acetyl group-neuraminic acid (2-ketone-5-acetamido-3,5-dideoxy-D-glyceryl-D-galactononulopyranos-1-onic acid (often being 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 hydroxylated.The 3rd sialic acid family member is 2-ketone-3-deoxidation-nonulosonic acid (KDN) (Nadano etc. (1986) J.Biol.Chem.261:11550-11557; Kanamori etc., J.Biol.Chem.265:21811-21819 (1990)).Also comprise the sialic acid that 9-replaces, for example 9-O-C ₁-C ₆Acyl group-Neu5Ac resembles 9-O-lactyl-Neu5Ac or 9-O-acetyl group-Neu5Ac, 9-deoxidation-9-fluoro-Neu5Ac and 9-azido-9-'-deoxy-n eu5Ac.About the comment of sialic acid family, see, for example, Varki, Glycobiology 2:25-40 (1992); SialicAcids:Chemistry, Metabolism and Function, R.Schauer, Ed. (Springer-Verlag, New York (1992)).Synthetic and the application of sialylated compound in sialylated process is documented in the disclosed International Application No. WO 92/16640 on October 1st, 1992.

" peptide " refers to polymer, and wherein monomer is an aminoacid, and connects together by amido link, perhaps refers to polypeptide.In addition, also comprise non-natural aminoacid, for example, Beta-alanine, phenylglycine and homoarginine.The non-genomic amino acids coding also can be used for the present invention.And, be modified into the aminoacid that comprises reactive group, glycosylation site, polymer, treatment group, biomolecule etc. and also can be used for the present invention.All aminoacid of Shi Yonging can be D-or L-isomer in the present invention.The L-isomer is normally preferred.In addition, other peptide mimics (peptidomimetics) also can be used for the present invention.As used herein, " peptide " refers to glycosylated and nonglycosylated peptide.Also comprise by the glycosylated by halves peptide of system of energy expression of peptides.About general comment, see Spatola, A.F., in CHEMISTRY ANDBIOCHEMISTRY OF AMINO ACIDS, PEPTIDES AND PROTEINS, B.Weinstein, eds., Marcel Dekker, New York, p.267 (1983).

Term " peptide conjugate " refers to material of the present invention, and wherein peptide is attached on the sugar of modification as herein described.

Term " aminoacid " refers to natural generation and synthetic aminoacid and amino acid analogue and the amino acid analog thing of bringing into play function in the amino acid whose mode that is similar to natural generation.The aminoacid of natural generation is by those of genetic code coding, and those aminoacid of modifying later on, for example, and hydroxyproline, Gla and O-phosphoserine.Amino acid analogue refers to have the chemical compound of the basic chemical structure identical with the aminoacid of natural generation, promptly is attached to the α carbon on hydrogen, carboxyl, amino and the R group, for example, and homoserine, nor-leucine, methionine sulfoxide, methionine methyl sulfonium.Such analog has the R group (for example, nor-leucine) of modification or the peptide main chain of modifying, but has kept the basic chemical structure identical with the aminoacid of natural generation.The amino acid analog thing refers to have the structure different with amino acid whose general chemical constitution, still brings into play the chemical compound of function in the amino acid whose mode that is similar to natural generation.

As used herein, the carbohydrate that term " sugar of modification " refers to natively or non-natural ground produces, it is added on the aminoacid or glycosyl residue of peptide by enzymatic ground in the method for the invention.The sugar of modifying is selected from many zymolytes, include but not limited to, ribotide (single-, two-and three-phosphoric acid), the sugar of activatory sugar (for example, glycosyl halide, glycosyl methanesulfonates) and non-activated non-nucleotide." sugar of modification " is covalently functionalized with " modification group ".Useful modification group includes but not limited to, PEG group, treatment group, diagnosis group, biomolecule etc.Modification group preferably is not natural generation, or the carbohydrate of unmodified.Select to make it can not stop " sugar of modification " to be added on the peptide by enzymatic ground with the functionalized position of modification group.

Term " water miscible " but the water miscible group that refers to have some detection level.Detection and/or quantitative water miscible method are well-known in the art.Exemplary water miscible polymer comprises peptide, sugar, polyethers, polyamine, polycarboxylic acids etc.Peptide can be mixed sequence or be made up of single amino acids, for example, and polylysine.Exemplary polysaccharide is a Polysialic acid.Exemplary polyethers is a Polyethylene Glycol.Polyethyene diamine is exemplary polyamine, and polyacrylic acid is representational polycarboxylic acids.

The main polymer chain of water miscible polymer can be Polyethylene Glycol (being PEG).But, should be appreciated that other relevant polymer also is applicable to realization the present invention, and be intended to comprise rather than get rid of in the use of term PEG or Polyethylene Glycol aspect this.Term PEG comprises the Polyethylene Glycol of arbitrary form, comprise alkoxyl PEG, bifunctional PEG, the PEG of multi-arm, the PEG of bifurcated, ramose PEG, the PEG that dangles (promptly having the PEG of the functional group on one or more main polymer chains that dangle or relevant polymer), or wherein have the PEG of degradable linkage.

Main polymer chain can be linearity or ramose.Ramose main polymer chain is normally known in the art.Typically, ramose polymer has a center branch core group and many linear polymer chain that is connected on the center branch core.Usually use PEG with the branch form, it can prepare by oxirane being added on the various polyhydric alcohol (for example glycerol, tetramethylolmethane and sorbitol).The center branch group also can be derived from several seed amino acids, for example lysine.Ramose Polyethylene Glycol can be expressed as R (PEG-OH) _mGeneral type, wherein R represents the core group, for example glycerol or tetramethylolmethane, and m represents the number of arm.The PEG molecule of multi-arm, for example the whole U.S. Patent number incorporated by reference of this paper 5,932,462 described those, also can be used as main polymer chain.

Many other polymer also are applicable to the present invention.Non-peptide and water miscible have 2 main polymer chains to about 300 ends and be specially adapted to the present invention.The example of suitable polymers includes but not limited to; other poly alkylene glycol; polypropylene glycol (" PPG ") for example; the copolymer of ethylene glycol and propylene glycol etc.; poly-(the ethylating polyhydric alcohol of oxygen), polyalkenylalcohols, polyvinylpyrrolidone; poly-(hydroxypropyl methyl acrylamide); poly-(alpha-hydroxy acid), polyvinyl alcohol, polyphosphazene; poly- azoles quinoline; poly-(N-acryloyl morpholine), for example whole in this article U.S. Patent number incorporated by reference 5,629; 384 described and its copolymer, trimer and mixture.Although the molecular weight of each chain of main polymer chain can change, typically at about 100Da to about 100, in the scope of 000Da, often from about 6,000Da is to about 80,000Da.

As the peptide medicament administration being given in patient's the context employed, " area under a curve " or " AUC " is defined as the gross area under the curve of the drug level in patient's systemic circulation of describing 0 to the infinitely-great time of retinue and changing.

As the peptide medicament administration is given in patient's the context employed, term " half-life " or " t _1/2" be defined as the plasma drug level that makes the patient and reduce half required time.According to multiple removing mechanism well-known in the art, redistribution and other mechanism, can exist to surpass a kind of half-life relevant with the peptide medicine.Usually, definition α and β half-life, make the α stage relevant with redistribution, the β stage is relevant with removing.But,, can exist at least 2 kinds to remove the half-life for the protein drug that is limited to blood flow largely.For some glycosylated peptides, by the receptor on the macrophage, maybe can discern the endotheliocyte of terminal galactose, N-acetylamino galactosamine, N-acetyl glucosamine, mannose or fucose, can mediate 3 stages removing fast.By glomerular filtration and/or specific or nonspecific tissue of the molecule of effective radius＜2nm (approximately 68kD) are taken in and metabolism, the slower β stage can take place remove.Glycosyl PEGization can add medicated cap to the sugar (for example, galactose or N-acetyl group galactosamine) of end, thereby and stops by the stage of α fast that can discern these sugared receptors and remove.Also can give bigger effective radius, thereby reduce volume of distribution and tissue absorption, prolong the late β stage thus.Thereby as known in the art, glycosyl PEGization can change with size, glycosylation state and other parameter the accurate influence of α stage and β half-life in stage.About other explanation of " half-life ", see Pharmaceutical Biotechnology (1997, DFA Crommelin and RD Sindelar, eds., Harwood Publishers, Amsterdam, pp 101-120).

As used herein, term " sugar is puted together " refers to that the saccharide of the modification that enzymatic mediates is to the aminoacid of polypeptide (for example, erythropoietin peptide of the present invention) or puting together of glycosyl residue.The subgenus of " sugar is puted together " is " Glycopegylated ", wherein the modification group of the sugar of Xiu Shiing be Polyethylene Glycol and its alkyl derivative (for example, m-PEG) or reactive derivatives (for example, H2N-PEG, HOOC-PEG).

Term " on a large scale " and " " use interchangeably, refer to such reaction time, it can produce at least about 250mg after finishing single reaction time commercial scale, preferably at least about 500mg with more preferably at least about the 1g glycoconjugate.

As used herein, term " glycosyl linking group " refers to the covalently bonded glycosyl residue of modification group (for example, PEG group, treatment group, biomolecule); The glycosyl linking group can be connected to modification group the other parts of conjugate.In the method for the invention, " glycosyl linking group " covalently is attached on the glycosylated or not glycosylated peptide, thereby reagent is connected on the aminoacid and/or glycosyl residue of this peptide." glycosyl linking group " usually by " sugar of modification " enzymatic combination to the aminoacid and/or the glycosyl residue of peptide, and is derived from " sugar of modification ".The glycosyl linking group can be a sugar-deutero-structure, and it (for example, oxidation → Schiff's base formation → reduction be degraded in) the process, or the glycosyl linking group can be complete at the sugar bowl that forms modification group-modification." complete glycosyl linking group " refers to be derived from the linking group of glycosyl, and the sugar monomer that wherein modification group is connected to the other parts of conjugate is not degraded, for example, and oxidation, for example, by the sodium metaperiodate oxidation.By adding glycosyl units or removing one or more glycosyl units, can derive " complete glycosyl linking group " of the present invention from the oligosaccharide of natural generation from the sugared structure of female parent.

As used herein, term " targeting group " refers to optionally to be positioned at the particular organization of health or the material in the zone.The location is by mediations such as the molecular size of specific recognition, targeting agent or the conjugate of molecule decision base (determinant), ionic interaction, hydrophobic interactions.The known tissue that the reagent targeting is specific of those skilled in the art or other mechanism in zone.Exemplary targeting group comprises antibody, antibody fragment, transferrins, HS-glycoprotein, coagulation factors, serum albumin, β-glycoprotein, G-CSF, GM-CSF, M-CSF, EPO etc.

As used herein, " treatment group " refer to any reagent of being used for the treatment of include but not limited to antibiotic, anti-inflammatory agent, antineoplastic agent, cytotoxin and radioactive reagent." treatment group " comprises the prodrug of bioactive agents, and wherein the group of the treatment above is attached to the construct on the carrier, for example multivalence reagent.The treatment group also comprises albumen and comprises proteic construct.Exemplary albumen includes but not limited to, granulocyte colony-stimulating factor (GCSF), granulocyte macrophage colony stimulating factor (GMCSF), interferon (for example, interferon-' alpha ' ,-β ,-γ), interleukin (for example, interleukin I I), serum albumin (for example, factor VII, VIIa, VIII, IX, and X), human chorionic gonadotropin (HCG), follicle stimulating hormone (FSH) and lutropin (LH) and antibody fusion protein (for example Tumor Necrosis Factor Receptors ((TNFR)/Fc domain fusion protein)).

As used herein, " pharmaceutically acceptable carrier " comprises any material, and it can keep the activity of conjugate when making up with conjugate, and not with the immune system response of object.Example includes but not limited to, the pharmaceutical carrier of any standard, the saline solution of phosphate-buffered for example, water, emulsion, for example oil/aqueous emulsion and dissimilar wetting agent.Other carrier also can comprise aseptic solution, and tablet comprises coated tablet, and capsule.Typically, such carrier contains excipient, and for example starch is suckled sugar, the clay of some type, gelatin, stearic acid or its salt, magnesium stearate or calcium, Talcum, plant fat or oil, natural gum, ethylene glycol, or other known excipient.Such carrier also can comprise correctives and coloring agent or other composition.By well-known conventional method, can make the compositions that comprises such carrier.

As used herein, the dosage forms for oral administration of " using " point at objects, as using of suppository, local contact, intravenous, endoperitoneal, intramuscular, intralesional, intranasal or subcutaneous uses, or the implantation of delayed release device, for example, little-osmotic pumps.To use be by any approach, comprises (for example, per os, nose, vagina, the rectum, or transdermal) of parenteral and saturating mucosa.Parenteral using comprise, and be for example intravenous, intramuscular, in the small artery, Intradermal, subcutaneous, endoperitoneal, intraventricular and intracranial.In addition, when injection is when being used for the treatment of tumor, for example, cell death inducing can directly be administered to the tissue around tumor and/or the tumor.Other mode of movement includes but not limited to, uses Liposomal formulation, and intravenous is inculcated, transdermal patch, etc.

Term " improvement " refers to any successful sign in pathology or treatment of conditions, comprises any subjectivity or objective parameter, for example elimination of symptom, alleviation or minimizing, or the improvement of state patient's body or spirit.The improvement of symptom can be based on parameter subjectivity or objective; Comprise the result that physical examination and/or psychiatry are estimated.

Term " treatment " refers to " treatment " of disease or disease, comprise that prevent disease or disease take place in animal, this animal may easily suffer from this disease, but do not experience or show the symptom (prophylactic treatment) of this disease as yet, suppress disease (slow down or stop its development), remove the symptom or the side effect (comprising palliative treatment) of disease and alleviate disease (causing disappearing of disease).

Term " effective dose " or " can be effectively ... amount " or " treatment effective dose " or any grammer on the term that is equal to refer to, when being administered to animal and treating disease, be enough to realize the amount of this treatment of diseases.

Term " tissue protective " refers to that protective tissue avoids the effect of cell injury, and described damage is general experiences ischemia/anoxia, wound, toxicity and/or inflammation-related with tissue or organ.Cell injury may cause apoptosis and/or necrosis (that is toxic cell death).Thereby " tissue protective " interaction energy protective tissue avoids experiencing relevant with given traumatic, inflammatory, the toxic or ischemic damage usually apoptosis and/or the degree of toxic cell death.For example, EPO can reduce the infarct size of rodent model behind middle cerebral artery occlusion (Siren, A.L. etc. (2001) .Proc.Natl.Acad.Sci.U.S.A.98,4044-4049).Thereby under such condition, EPO can provide " tissue protective " effect by reducing effectively usually and relevant necrosis and/or the apoptosis of ischemic injury (for example, ischemic stroke)." tissue protective " refers to that also protective tissue avoids the effect of cell injury and the follow-up cell death relevant with degenerative disease (for example retinopathy, or neurodegenerative disease).

In fact or be substantially free of and be useful on the component of producing this material term " isolating " refers to a kind of material, and it.In fact or be substantially devoid of the component of the described material of following the mixture that is used for preparing this peptide conjugate usually for peptide conjugate of the present invention, term " isolating " refers to a kind of material, and it.Use interchangeably " isolating " and " pure ".Typically, isolating peptide conjugate of the present invention has certain purity level, preferably is expressed as certain scope.The lower limit of the purity range of peptide conjugate 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 also preferably was expressed as certain limit.The lower limit of purity range 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% purity.

By any analytical method well known in the art (for example, the band intensity on the silver-colored stained gel, polyacrylamide gel electrophoresis, HPLC, or similar approach), can detect purity.

As used herein, " each member in the colony basically " described the feature of peptide conjugate of the present invention colony, and wherein the sugar that adds the modification on the peptide to of particular percentile is added on a plurality of identical acceptor site of this peptide." each member in the colony basically " refers to be conjugated to " homogeneity " in the peptide site on the sugar of modification, and refers to conjugate of the present invention, and it is at least about 80%, preferably at least about 90% with more preferably at least about 95% homogeneous.

Structural integrity in the acceptor groups colony that the sugar that " homogeneity " refers to modify is puted together.Thereby in peptide conjugate of the present invention, wherein the glycosyl group of each modification is conjugated on the acceptor site of the identical structure of acceptor site that sugar with other modification with each puts together, claims that then peptide conjugate is about 100% homogeneous.Homogeneity is typically expressed and is certain scope.The lower limit of the homogeneity scope of peptide conjugate is about 60%, about 70% or about 80%, and the upper limit is about 70%, about 80%, about 90% or surpasses about 90%.

When peptide conjugate surpasses or equals about 90% all for the moment, their homogeneity also preferably is expressed as certain scope.The lower limit of homogeneity scope is about 90%, about 92%, about 94%, about 96% or about 98%.The upper limit is about 92%, about 94%, about 96%, about 98% or about 100% homogeneity.Typically, by one or more methods known to those skilled in the art, for example, liquid chromatography-mass spectrography (LC-MS), the auxiliary laser desorption flight time mass spectrum of substrate (MALDITOF), capillary electrophoresis etc., the purity of detection of peptides conjugate.

When referring to the glycopeptide material, " sugar form of homogeneous basically " or " glycosylation pattern of homogeneous basically " refers to the percentage ratio by the glycosylated acceptor groups of target glycosyl transferase (for example, fucosyltransferase).For example, under the situation of α 1,2 fucosyltransferase, if all basically (defining) Gal β 1 as following, by fucosylation, then there are the fucosylation pattern of homogeneous basically in 4-GlcNAc-R and its sialylated analog in peptide conjugate of the present invention.Those skilled in the art can recognize that raw material can contain glycosylated acceptor groups (for example, the Gal β 1 of fucosylation, 4-GlcNAc-R group).Thereby the glycosylation percentage ratio of calculating comprises by the glycosylated acceptor groups of method of the present invention, and glycosylated those acceptor groups in raw material.

In superincumbent " homogeneous basically " definition, term " basically " be often referred at least about 40%, at least about 70%, at least about 80%, or more preferably at least about 90% be glycosylated more preferably at least about the acceptor groups of 95% specific glycosyl transferase.

When with writing from left to right when representing substituent group with their conventional chemical formula, they comprise comparably writes the substituent group that chemically is equal to that structure obtains from right to left, for example, and-CH ₂O-also represents-OCH ₂-.

Except as otherwise noted, term " alkyl " self or as another substituent part, refer to straight chain or ramose chain, or cyclic alkyl, or its combination, it can be fully saturated, single-or polyunsaturated, and can comprise two-and polyvalent group, it has specified carbon atom number (is C ₁-C ₁₀Refer to 1-10 carbon).The example of saturated alkyl includes but not limited to that group is methyl for example, ethyl, n-pro-pyl, isopropyl, normal-butyl, the tert-butyl group, isobutyl group, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropyl methyl, its homologue and isomer, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, etc.Undersaturated alkyl is to have one or more pairs of keys or triple-linked alkyl.The example of undersaturated alkyl includes but not limited to, vinyl, 2-acrylic, cyclobutenyl, 2-isopentene group, 2-(butadienyl), 2,4-pentadienyl, 3-(1, the 4-pentadienyl), acetenyl, 1-and 3-propinyl, 3-butynyl and more high-grade homologue and isomer.Except as otherwise noted, term " alkyl " also is intended to comprise those alkyl derivatives of definition in more detail below, for example " assorted alkyl ".The alkyl that is limited to alkyl is called " homotype alkyl (homoalkyl) ".

Term " alkylidene " self or as another substituent part refers to be derived from the divalent group of alkane, as-CH ₂CH ₂CH ₂CH ₂-, but be not limited thereto, also comprise those groups that describe below to " assorted alkylidene ".Typically, alkyl (or alkylidene) group has the 1-24 carbon atom, have 10 or those groups of carbon atom still less be preferred in the present invention." low alkyl group " or " low-grade alkylidene " is to have 8 or the short-chain alkyl or the alkylidene of carbon atom still less usually.

Term " alkoxyl ", " alkyl amino " and " alkylthio group " (or thio alkoxy) use with their conventional sense, refer to respectively to be attached to those alkyl on the other parts of molecule by oxygen atom, amino or sulphur atom.

Except as otherwise noted, term " assorted alkyl " self or combined with another term, refer to stable straight chain or ramose chain, or cyclic hydrocarbon group, or its combination, its carbon atom by described number is selected from O with at least one, N, the hetero atom of Si and S is formed, and wherein nitrogen and sulphur atom can be randomly oxidized, and nitrogen heteroatom can randomly be quaternised.Hetero atom O, N and S and Si can be positioned at any interior location of assorted alkyl, or are positioned at the position on the other parts that alkyl is attached to this molecule.Example includes 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 ₃2 hetero atoms can be successive at the most, for example, and-CH ₂-NH-OCH ₃With-CH ₂-O-Si (CH ₃) ₃Similarly, term " assorted alkylidene " self or as another substituent part refers to be derived from the divalent group of assorted alkyl, as-CH ₂-CH ₂-S-CH ₂-CH ₂-and-CH ₂-S-CH ₂-CH ₂-NH-CH ₂-exemplified, but be not limited thereto.For assorted alkylidene, hetero atom also can occupy any or two chain ends (for example, alkylidene oxygen, alkylidene dioxygen, alkylidene amino, the alkylidene diaminourea, etc.).Further, for alkylidene and assorted alkylidene linking group, the direction of writing the linking group formula does not hint the direction of linking group.For example, formula-C (O) ₂R '-representative-C (O) ₂R '-and-R ' C (O) ₂-.

Except as otherwise noted, term " cycloalkyl " and " Heterocyclylalkyl " self or combined with other term represented respectively in " alkyl " and " assorted alkyl " of annular form.In addition, for Heterocyclylalkyl, hetero atom can occupy the position of the other parts of heterocycle binding molecule.The example of cycloalkyl includes but not limited to, cyclopenta, and cyclohexyl, the 1-cyclohexenyl group, the 3-cyclohexenyl group, suberyl, etc.The example of Heterocyclylalkyl includes but not limited to, 1-(1,2,5, the 6-tetrahydro pyridyl), piperidino, the 2-piperidyl, 3-piperidyl, 4-morpholinyl, morpholinyl, oxolane-2-base, oxolane-3-base, Tetramethylene sulfide-2-base, Tetramethylene sulfide-3-base, the 1-piperazinyl, the 2-piperazinyl, etc.

Except as otherwise noted, term " halo " or " halogen " self or as another substituent part refers to fluorine, chlorine, bromine, or iodine atom.In addition, term for example " haloalkyl " be intended to comprise single haloalkyl and multi-haloalkyl.For example, term " halo (C ₁-C ₄) alkyl " be intended to include but not limited to, trifluoromethyl, 2,2, the 2-trifluoroethyl, the 4-chlorobutyl, the 3-bromopropyl, etc.

Except as otherwise noted, term " aryl " refers to polyunsaturated aromatic substituents, and it can be monocyclic or polycyclic (preferably, 1-3 ring), and it is fused to together, or covalently connects.Term " heteroaryl " refers to contain 1-4 and is selected from N, the heteroatomic aryl of O and S (or ring), and wherein nitrogen and sulphur atom are randomly oxidized, and nitrogen-atoms is randomly by quaternized.Heteroaryl can be attached on the other parts of molecule by hetero atom.The non-limiting instance 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- azoles base, 4- azoles base, 2-phenyl-4- azoles base, 5- azoles base, the different  azoles of 3-base, the different  azoles of 4-base, the different  azoles of 5-base, 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-quinoline  quinoline base, 5-quinoline  quinoline base, the 3-quinolyl, tetrazole radical, benzo [b] furyl, benzo [b] thienyl, 2,3-dihydrobenzo [1,4] two  alkene-6-base, benzo [1,3] dioxole-5-base and 6-quinolyl.The substituent group of every kind of above-mentioned aryl and heteroaryl ring system is selected from following acceptable substituent group.

In brief, when making up with other term (for example, aryl oxide, aryl sulfo-oxygen, aryl alkyl), term " aryl " comprises aforesaid aryl and heteroaryl ring.Thereby, term " aryl alkyl " is intended to comprise that wherein aryl (for example is attached to alkyl, benzyl, phenethyl, pyridylmethyl etc.) on those groups, comprise that wherein carbon atom (for example, methylene) by alternate those alkyl of for example oxygen atom (for example, phenoxymethyl, 2-pyridine yloxymethyl, 3-(1-naphthyl oxygen) propyl group, etc.).

Each above-mentioned term (for example, " alkyl, " " assorted alkyl, " " aryl " and " heteroaryl ") is intended to comprise replacement and the unsubstituted form of indication group.The preferred substituted of every type group is provided below.

Alkyl and assorted alkyl (comprise often being called alkylidene alkenyl, assorted alkylidene, heterochain thiazolinyl, alkynyl, cycloalkyl, Heterocyclylalkyl, cycloalkenyl group, those groups with heterocycloalkenyl) substituent group usually is called " alkyl substituent ", and they can be to be selected from one or more in (but being not limited to) following numerous groups :-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, its quantity is 0 to (2m '+1), wherein m ' is the sum of the carbon atom in such group.R ', R ", R  and R " " preferably refer to hydrogen, assorted alkyl replacement or unsubstituted separately independently; aryl replacement or unsubstituted, for example, with the aryl of 1-3 halogen replacement; replacement or unsubstituted alkyl, alkoxyl or thio alkoxy, or aryl alkyl.For example, select each R group independently when chemical compound of the present invention comprises when surpassing a R group, resemble each R ' when existing, R above such group ", R  and R " " group.As R ' and R " when being attached to same nitrogen-atoms, they can form 5-with nitrogen-atoms, 6-, or 7-unit ring.For example ,-NR ' R " be intended to include but not limited to 1-pyrrolidinyl and 4-morpholinyl.From substituent group discussed above, those skilled in the art can understand that term " alkyl " is intended to comprise such group, and it comprises the carbon atom on the group that is attached to beyond the hydrogen group, for example 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 ₃, etc.).

With similar about the described substituent group of alkyl, the substituent group of aryl and heteroaryl usually is called " aryl substituent ".Substituent group is selected from, for example: halogen ,-OR ' ,=O ,=NR ' ,=N-OR ' ,-NR ' R " ,-SR ' ,-halogen ,-SiR ' R " R  ,-OC (O) R ' ,-C (O) R ' ,-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, fluoro (C ₁-C ₄) alkoxyl, and fluoro (C ₁-C ₄) alkyl, its quantity is 0 the sum of open valency to the aromatic rings system; And R ' wherein, R ", R  and R " " preferably be independently selected from hydrogen, replacement or unsubstituted alkyl, assorted alkyl replacement or unsubstituted, aryl replacement or unsubstituted and replacement or unsubstituted heteroaryl.When chemical compound of the present invention comprises when surpassing a R group, for example, select each R group independently, resemble each R ' when existing, R above such group ", R  and R " " group.In the scheme below, symbol X represents aforesaid " R ".

Aryl or heteroaryl ring close on 2 substituent groups on the atom can be randomly by formula-T-C (O)-(CRR ') _qThe substituent group of-U-substitutes, and wherein T and U are-NR-independently ,-O-, and-CRR '-or singly-bound, and q is the integer of 0-3.Perhaps, 2 substituent groups of closing on the atom of aryl or heteroaryl ring can be randomly by formula-A-(CH ₂) _rThe substituent group of-B-substitutes, 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 randomly be substituted by two keys like this.Perhaps, aryl or heteroaryl ring close on 2 substituent groups on the atom can be randomly by formula-(CRR ') _s-X-(CR " R ) _d-substituent group substitute, wherein s and d are the integer of 0-3 independently, and X is-O-,-NR '-,-S-,-S (O)-,-S (O) ₂-, or-S (O) ₂NR '-.Substituent R, R ', R " and R  preferably be independently selected from (C hydrogen or replacement or unsubstituted ₁-C ₆) alkyl.

As used herein, term " hetero atom " is intended to comprise oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).

Introduce

Erythropoietin (EPO) is a glycoprotein, and it plays the synthetic main regulator of erythrocyte.Erythropoietin is by kidney production, and by stimulating the precursor in the bone marrow to play a role, makes their divisions and be divided into sophisticated erythrocyte.EPO can be used as 165 or 166 aminoacid glycoproteins and exists.166 amino acid variant and the difference of 165 amino acid variant are extra arginine of the terminal existence of its C-.

For a period of time, the EPO that can obtain recombinating, the effective therapeutic agent as the various forms of anemias of treatment comprises the anemia relevant with chronic renal failure, the patient that the HIV of zidovidine treatment infects and the cancer patient of chemotherapy.Can use glycoprotein in the intestines and stomach other places, as intravenous (IV) or subcutaneous (SC) injection.

In order to improve the effectiveness of the recombinant erythropoietin that is used for the treatment of purpose, the invention provides conjugate glycosylated and not glycosylated erythropoietin peptide.By with different material (for example treat group, the diagnosis group, targeting group etc.) further put together, can further modify conjugate.

Be attached on the glycosylated or not glycosylated peptide by the carbohydrase that will modify shortly, can form conjugate of the present invention.Glycosylation site can provide modification group is conjugated to position on the peptide, for example, puts together by sugar.Exemplary modification group is water miscible polymer, Polyethylene Glycol for example, for example, methoxyl group-Polyethylene Glycol.The modification of EPO peptide can improve stability and the retention time of EPO in patient's circulation of reorganization, or reduces the antigenicity of the EPO of reorganization.

Method of the present invention makes the possibility that is assembled into of peptide with pattern of deriving of homogeneous basically and glycopeptide.The enzyme of Shi Yonging is optionally to the combination of specified amino acid residues, amino acid residue or the specific glycosyl residue of peptide usually in the present invention.This method also can be used to realize the peptide modified and the large-scale production of glycopeptide.Thereby method of the present invention provides and has been used for mass preparation and has the derive practical approach of glycopeptide of pattern of predetermined homogeneous.

The present invention also provides the glycosylated of treatment half-life with increase and the conjugate of glycosylated peptide not, the treatment half-life of described increase owing to, for example, the removing speed of minimizing, or the immunity that reduces or the absorption speed of reticuloendothelial system (RES).And, thereby method of the present invention provides the antigenic determinant on the shielding peptide to reduce or eliminate the immunoreactive method of host to this peptide.The selective binding of targeting agent, also can be used for peptide lead particular organization or to particular target to the specific cell surface receptor of agent.

Conjugate

Aspect first, the invention provides the modification group of selection and the conjugate between the EPO peptide.

Connection between peptide and the modification group comprises the glycosyl linking group between the group that places peptide and selection.As discussed herein, the group of selection is that can be incorporated on the sugar unit, form can be by the arbitrary substance of " sugar of modification " of suitable transferring enzyme (it can add the sugar of modifying on the peptide to) identification basically.When between the group that places peptide and selection, the saccharic composition of the sugar of modification for example can become " glycosyl linking group ", " complete glycosyl linking group ".The glycosyl linking group can by single arbitrarily-or widow-sugar form, the latter becomes and can add the sugar of modification to the aminoacid of peptide or the substrate of the enzyme on the glycosyl residue after with the modification group modification.

The glycosyl linking group can be, maybe can comprise, before adding modification group or the glycosyl group of the sex modification that is degraded in the process.For example, the glycosyl linking group can be derived from saccharide residue, and it generates by the oxidative degradation (for example, by the effect of metaperiodic acid salt) of complete sugar to the aldehyde of correspondence, and changes into Schiff's base with suitable amine subsequently, then it is reduced into corresponding amine.

Conjugate of the present invention typically corresponding universal architecture:

Wherein, symbol a, b, c, d and s represent non-0 positive integer; And t is 0 or positive integer." reagent " is therapeutic agent, bioactivator, detectable labelling, water miscible group (for example, PEG, m-PEG, PPG, and m-PPG) etc." reagent " can be peptide, for example, enzyme, antibody, antigen, etc.Junctional complex can be one of a large amount of linking groups hereinafter.Perhaps, junctional complex can be singly-bound or " zero level junctional complex ".

In exemplary embodiment, the modification group of selection is water miscible polymer, for example, and m-PEG.Water miscible polymer covalently is attached on the peptide by the glycosyl linking group.The glycosyl linking group covalently is attached on the amino acid residue or glycosyl residue of peptide.The present invention also provides conjugate, has wherein modified amino acid residue and glycosyl residue with the glycosyl linking group.

Exemplary water miscible polymer is a Polyethylene Glycol, for example, and methoxyl group-Polyethylene Glycol.The Polyethylene Glycol of Shi Yonging is not limited to any concrete form or molecular weight ranges in the present invention.For not ramose peg molecule, molecular weight preferably 500 to 100,000.Preferably use 2000-60,000 molecular weight, preferred about 5,000 to about 30,000.

In another embodiment, Polyethylene Glycol is ramose PEG, and it has the bonded PEG group of surpassing.The case history of ramose PEG is in U.S. Patent number 5,932,462; U.S. Patent number 5,342,940; U.S. Patent number 5,643,575; U.S. Patent number 5,919,455; U.S. Patent number 6,113,906; U.S. Patent number 5,183,660; WO 02/09766; KoderaY., Bioconjugate Chemistry 5:283-288 (1994); With Yamasaki etc., Agric.Biol.Chem., 52:2125-2127,1998.In a preferred embodiment, the molecular weight of each Polyethylene Glycol of ramose PEG is equal to or greater than about 40,000 dalton.

Except the conjugate that the glycosyl linking group formation of adding by enzymatic ground is provided, the invention provides their the highly homogeneous conjugate of substitute mode.Use method of the present invention, can form peptide conjugate, the glycosyl group of all basically modifications in the conjugate wherein of the present invention colony all is attached on the aminoacid or glycosyl residue identical on the structure of a plurality of copies.Thereby, aspect second, the invention provides peptide conjugate, it has a large amount of water miscible polymeric groups, and the latter covalently is attached on this peptide by complete glycosyl linking group.In preferred conjugate of the present invention, each member in the colony is attached on the glycosyl residue of this peptide by the glycosyl linking group basically, and the glycosyl linking group each glycosyl residue of bonded peptide have identical structure.

Peptide conjugate also is provided, and it has a large amount of glycosyl linking groups that pass through covalently in conjunction with thereon water miscible polymeric groups.In a preferred embodiment, each member basically of water miscible polymeric groups colony is attached on the amino acid residue of this peptide by the glycosyl linking group, and each amino acid residue that has in conjunction with thereon glycosyl linking group has identical structure.

The present invention also provides and those above-mentioned similar conjugates, and wherein this peptide is conjugated to the treatment group by complete glycosyl linking group, diagnosis group, targeting group, toxin group etc.Every kind of above-mentioned group can be a micromolecule, natural polymer (for example, polypeptide) or synthetic polymer.

Basically the peptide of erythropoietin arbitrarily that has arbitrary sequence can be as the component of conjugate of the present invention.In an exemplary embodiment, this peptide has sequence:

H ₂N-APPRLICDSR VLERYLLEAK EAENITTGCA EHCSLNENIT VPDTKVNFYA

WKRMEVGQQA VEVWQGLALL SEAVLRGQAL LVNSSQPWEP LQLHVDKAVS

GLRSLTTLLR ALGAQKEAIS PPDAASAAPL RTITADTFRK LFRVYSNFLR

GKLKLYTGEA CRTGD-COOH (SEQ ID NO：1).

In another exemplary embodiment, this peptide has sequence:

H ₂N-APPRLICDSR VLERYLLEAK EAENITTGCA EHCSLNENIT VPDTKVNFYA

WKRMEVGQQA VEVWQGLALL SEAVLRGQAL LVNSSQPWEP LQLHVDKAVS

GLRSLTTLLR ALGAQKEAIS PPDAASAAPL RTITADTFRK LFRVYSNFLR

GKLKLYTGEA CRTGDR-COOH (SEQ ID NO：2).

In above-mentioned sequence, there are two disulfide bond, one at C7-C161, and another is at C29-C33.Shown cysteine residues with bold Italic above.

Preferably, two ends are not deutero-.

Peptide of the present invention comprises glycosylation site that at least one N-connects or that O-connects, and it is glycosylated with the glycosyl residue that comprises the PEG group.PEG covalently is connected on the peptide by complete glycosyl linking group.The glycosyl linking group covalently is connected on the amino acid residue or glycosyl residue of peptide.Perhaps, the glycosyl linking group is attached on one or more glycosyl units of glycopeptide.The present invention also provides conjugate, and wherein the glycosyl linking group is attached on amino acid residue and the glycosyl residue.

The PEG group is directly or by non--glycosyl junctional complex, for example, replacement or unsubstituted alkyl, assorted alkyl replacement or unsubstituted is attached on the complete glycosyl junctional complex.

In a preferred embodiment, the erythropoietin peptide comprises the group shown in the formula I

Formula I

In formula I, D is selected from-OH and R ¹The member of-L-HN-; G is selected from R ¹-L-and-C (O) (C ₁-C ₆) member of alkyl; R ¹It is the group that comprises the member who is selected from the group that contains straight chain or ramose Polyethylene Glycol residue; And L is a junctional complex, and it is to be selected from key, replacement or member unsubstituted alkyl and replacement or unsubstituted assorted alkyl, so when D was OH, G was R ¹-L-, and as G be-C (O) (C ₁-C ₆) during alkyl, D is R ¹-L-NH-.

Compositions

As discussed above, the invention provides the sugar that carries modification group, the activatory analog of these materials, and the conjugate that forms between the materials such as sugar of peptide and lipid and modification of the present invention.

The sugar of modifying

The invention provides the sugar of modification, the conjugate of the ribotide of modification and the sugar of modification.In the sugar compounds of modification of the present invention, glycosyl group is sugar preferably, deoxidation-sugar, amino-sugar, or N-acyl group sugar.Term " sugar " and its equivalent word, " glycosyl " refers to monomer, dimer, oligomer and polymer.Glycosyl group also is functionalized with modification group.Modification group is conjugated in the glycosyl group, typically, by with sugar on the puting together of amine, sulfydryl or hydroxyl, for example, primary hydroxyl.In exemplary embodiment, by the amine groups on the sugar, for example, by amide, urethane or the urea that is formed by the reaction of the reactive derivatives of amine and modification group are in conjunction with modification group.

Any sugar can be as the sugared core of conjugate of the present invention.The exemplary sugared core that is used to form compositions of the present invention includes but not limited to, glucose, galactose, mannose, fucose, and sialic acid.Other useful steamed bun stuffed with sugar is drawn together amino sugar, glycosamine for example, galactosamine, mannosamine, sialic 5-amine analog etc.The sugar core can be the structure of natural discovery, maybe can modify it, to be provided for puting together the site of modification group.For example, in one embodiment, the invention provides sialic acid derivative, wherein the 9-hydroxyl is replaced by amine.With the activatory analog of the modification group of selecting, the amine of can easily deriving.

In the following discussion, with reference to the sialic derivant of selecting, explained the present invention.Those skilled in the art will appreciate that the purpose of discussion is for clarity, and described structure and compositions usually can be in glycosyl groups, the glycosyl group of modification is suitable between the conjugate of the glycosyl group of activatory modification and the glycosyl group of modifying.

In exemplary embodiment, the invention provides the osamine of modification with following formula:

Wherein G is a glycosyl, and L is key or junctional complex, and R ¹It is modification group.Exemplary key is reactive group (for example, the NH on glycosyl ₂, SH, or OH) and modification group on complementary reactive group between form those.Thereby exemplary key includes but not limited to, NHR ¹, OR ¹, SR ¹Deng.For example, work as R ¹When comprising hydroxy-acid group, this group can be activated, and uses NH ₂Group is connected on the glycosyl residue, forms to have structure NHC (O) R ¹Key.Similarly, OH and SH group can be changed into corresponding ether or sulfide derivative respectively.

Exemplary junctional complex comprises alkyl and assorted alkyl.Junctional complex comprises linking group, for example based on the linking group of acyl group, for example, and-C (O) NH-,-OC (O) NH-, etc.Linking group is the key that forms between the component of material of the present invention, for example, and between glycosyl and junctional complex (L), or at junctional complex and modification group (R ¹) between.Other linking group is an ether, thioether and amine.For example, in one embodiment, junctional complex is an amino acid residue, for example glycine residue.By with glycosyl residue on the reaction of amine, the hydroxy-acid group of glycine can be changed into corresponding amide, and by with the reaction of the carbonic ester of activatory carboxylic acid or modification group, the amine of glycine can be changed into corresponding amide or urethane.

Another kind of exemplary junctional complex is a PEG group or with the functionalized PEG group of amino acid residue.PEG is attached on the glycosyl by the amino acid residue a PEG end, and passes through other PEG end, is attached to R ¹Perhaps, amino acid residue is attached to R ¹On, and will not be attached on the glycosyl in conjunction with amino acid whose PEG end.

L-R ¹Exemplary material have formula-NH{C (O) (CH ₂) _aNH) _s{ C (O) (CH ₂) _b(OCH ₂CH ₂) _cO (CH ₂) _dNH} _tR ¹, wherein identifying s and t is 0 or 1 independently.Sign a, b and d are the integer of 0-20 independently, and c is the integer of 1-2500.Other similar junctional complex is based on such material, wherein-the NH group is replaced by, for example ,-S ,-O and-CH ₂

More specifically, the invention provides chemical compound, wherein L-R ¹Be: NHC (O) (CH ₂) _aNHC (O) (CH ₂) _b(OCH ₂CH ₂) _cO (CH ₂) _dNHR ¹, NHC (O) (CH ₂) _b(OCH ₂CH ₂) _cO (CH ₂) _dNHR ¹, NHC (O) O (CH ₂) _b(OCH ₂CH ₂) _cO (CH ₂) _dNHR ¹, NH (CH ₂) _aNHC (O) (CH ₂) _b(OCH ₂CH ₂) _cO (CH ₂) _dNHR ¹, NHC (O) (CH ₂) _aNHR ¹, NH (CH ₂) _aNHR ¹, and NHR ¹In these formulas, sign a, b and d are independently selected from the integer of 0-20, preferably 1-5.Sign c is the integer of 1-2500.

In an indicative embodiment, G is a sialic acid, and the chemical compound of selecting of the present invention has formula:

Can recognize that as those skilled in the art the sialic acids groups in the top exemplary chemical compound can be replaced by other amino-sugar arbitrarily, include but not limited to, glycosamine, galactosamine, mannosamine, their N-acetyl derivative, etc.

In another indicative embodiment, with modification group functionalized sugared primary hydroxyl.For example, sialic 9-hydroxyl can be changed into corresponding amine, and functionalized, to provide according to chemical compound of the present invention.Formula according to this embodiment comprises:

In another exemplary embodiment, the invention provides the sugar of modification, wherein the 6-hydroxy position is converted to corresponding amine groups, and it carries junctional complex-modification group box, for example above-mentioned those.Can comprise Gal as the exemplary glycosyl of the core of the sugar of these modifications, GalNAc, Glc, GlcNAc, Fuc, Xyl, Man, etc.Sugar according to the representational modification of this embodiment has formula:

R wherein ³-R ⁵And R ⁷Be to be independently selected from H, OH, C (O) CH ₃, NH, and NHC (O) CH ₃The member; R ⁶Be OR ¹, NHR ¹Or L-R ¹, it as mentioned above.

The conjugate of selecting of the present invention is based on mannose, galactose or glucose, or based on the stereochemical material with mannose, galactose or glucose.The general formula of these conjugates is:

In another exemplary embodiment, the invention provides aforesaid chemical compound, it is activated into corresponding nucleotide sugar.The exemplary ribotide that uses in the present invention with their modified forms comprise the nucleotide list-, two-or triphosphoric acid or its analog.In a preferred embodiment, the ribotide of modification is selected from the UDP-glucosides, CMP-glucosides, or GDP-glucosides.Even more preferably, the ribotide of the ribotide of modification partly is selected from the UDP-galactose, UDP-galactosamine, UDP-glucose, UDP-glycosamine, GDP-mannose, GDP-fucose, cmp sialic acid, or CMP-NeuAc.In exemplary embodiment, nucleotide phosphodiesterase is attached on the C-1.

Thereby, in an indicative embodiment (wherein glycosyl is a sialic acid), the invention provides chemical compound with following formula:

L-R wherein ¹As mentioned above, and L ¹-R ¹Representative is attached to the junctional complex on the modification group.About L, according to L ¹Exemplary junctional complex comprise key, alkyl or assorted alkyl.Ribotide chemical compound according to the exemplary modification of these embodiments is as illustrated in figs. 1 and 2.

In another exemplary embodiment, the invention provides sugar and substrate (for example, peptide in modification of the present invention, lipid, aglycon (aglycone), etc.) between the conjugate that forms, more specifically, between the glycosyl residue of the sugar of modifying and glycopeptide or glycolipid.In this embodiment, the glycosyl group of the sugar of modification can become the glycosyl linking group that places between substrate and the modification group.Exemplary glycosyl linking group is complete glycosyl linking group, and wherein the glycosyl group that maybe can form linking group is not degraded by the process (for example, oxidase) of (for example, the sodium metaperiodate) of chemistry or enzymatic.The conjugate of selecting of the present invention comprises the modification group on the amine groups that is attached to amino-sugar, and described amino sugar for example is a mannosamine, glycosamine, and galactosamine, sialic acid, etc.Exemplary modification group-complete glycosyl linking group box according to this motif is based on sialic acid structure, for example has following formula:

In the superincumbent formula, R ¹, L ¹And L ²As mentioned above.

In another exemplary embodiment, between the 1-position of substrate and glycosyl, form conjugate, wherein by at the junctional complex of the 6-of glycosyl carbon location in conjunction with modification group.Thereby, have following formula according to the illustrative conjugate of this embodiment:

Wherein group as mentioned above.The technical staff can recognize that the glycosyl of above-mentioned modification also can pass through 2,3,4, or 5 carbon atoms are conjugated on the substrate.

Illustrative chemical compound according to this embodiment comprises the chemical compound with following formula:

Wherein R group and sign are as mentioned above.

The present invention also provides at 6-carbon location L-R ¹The ribotide of modifying.Exemplary material according to this embodiment comprises:

Wherein R group, and L is represented aforesaid group.Sign " y " is 0,1 or 2.

Another kind of exemplary nucleotide sugar of the present invention is based on the stereochemical material with GDP mannose.

Exemplary material according to this embodiment has structure:

In another exemplary embodiment, the invention provides stereochemical conjugate based on the UDP galactose.Exemplary chemical compound according to this embodiment has structure:

In another exemplary embodiment, nucleotide sugar is based on the spatial chemistry of glucose.Exemplary material according to this embodiment has formula:

Modification group R ¹Be in many materials any, include but not limited to water miscible polymer, water-insoluble polymer, therapeutic agent, diagnostic agent etc.Discussed the character of exemplary modification group hereinafter in more detail.

Modification group

Water miscible polymer

Many water miscible polymer are known to those skilled in the art, and can be used to realize the present invention.The water miscible polymer of term comprises following substances, for example sugared (for example, glucosan, amylose, hyaluronic acid, Polysialic acid, heparinoid, heparin, etc.); Polyamino acid, for example, poly-aspartate and polyglutamic acid; Nucleic acid; Synthetic polymer (for example, polyacrylic acid, polyethers, for example, Polyethylene Glycol; Peptide, albumen, etc.Also can use water miscible arbitrarily polymer to realize the present invention, unique restriction is that this polymer must comprise the combinable point of other parts of conjugate.

The method of activated polymer also can be referring to WO 94/17039, U.S. Patent number 5,324,844, WO 94/18247, and WO 94/04193, U.S. Patent number 5,219,564, U.S. Patent number 5,122,614, WO 90/13540, U.S. Patent number 5,281,698, and WO93/15189, and puting together between activatory polymer and the peptide, for example blood coagulation factor VIII (WO94/15625), hemoglobin (WO 94/09027), oxygen delivery molecule (U.S. Patent number 4,412,989), ribonuclease and superoxide dismutase (Veronese etc., App.Biochem.Biotech.11:141-45 (1985)).

Preferred water miscible polymer is such, and wherein remarkable ratio of polymer molecule has approximately identical molecular weight in the polymer samples; Such polymer is " monodispersed (homodisperse) ".

By reference Polyethylene Glycol conjugate, further explained the present invention.Can obtain commenting on and monograph with several pieces that put together about the functionalized of PEG.See, for example, Harris, Macronol.Chem.Phys.C25:325-373 (1985); Scouten, Methods in Enzymology135:30-65 (1987); Wong etc., Enzyme Microb.Technol.14:866-874 (1992); Delgado etc., Critical Reviews in Therapeutic DrugCarrier Systems 9:249-304 (1992); Zalipsky, Bioconjugate Chem.6:150-165 (1995); And Bhadra, etc., Pharmazie, 57:5-29 (2002).Using the PEG molecule of reactive molecule preparation feedback and the approach of formation conjugate, is known in the art.For example, U.S. Patent number 5,672,662 disclose the water miscible and separable conjugate of active ester that is selected from linear or ramose poly-(alkylene oxide), poly-(the ethylating polyhydric alcohol of oxygen), poly-(enol) and gathers the polymeric acid of (acrylic morpholine).

U.S. Patent number 6,376,604 terminal hydroxyl and two (the 1-benzotriazole base) carbonic esters of having set forth by making polymer react in organic solvent, prepare the method for water miscible 1-benzotriazole base carbonic ester of the polymer of water miscible and non--peptide.Active ester is used for forming conjugate with bioactive reagent (for example albumen or peptide).

WO 99/45964 has described conjugate, it comprises bioactive reagent and activatory water miscible polymer, the latter comprises and has at least one is connected to the end on the main polymer chain by stable key main polymer chain, wherein at least one end comprises branch's group, the latter has the reactive group that is connected to the near-end on branch's group, and wherein bioactive reagent is connected on the reactive group of at least one near-end.Other ramose Polyethylene Glycol is documented among the WO96/21469, U.S. Patent number 5,932, and 462 have described the conjugate that forms with ramose PEG molecule, and this PEG molecule comprises ramose end, and the latter comprises reactive functional groups.Can obtain free reactive group,, form the conjugate between Polyethylene Glycol and the bioactive material to react with bioactive material (for example albumen or peptide).U.S. Patent number 5,446,090 has described bifunctional PEG junctional complex and it application in being formed on the conjugate that each PEG junctional complex end has peptide.

The conjugate that comprises degradable PEG connection is documented in WO 99/34833; And WO99/14259, and in the U.S. Patent number 6,348,558.Degradable connection like this is applicable to the present invention.

Above-mentioned polymer activation method well known in the art can be used to form ramose polymer as herein described in the context of the present invention, also can be used for these ramose polymer are conjugated to other material, for example, sugar, ribotide etc.

The exemplary peg molecule of Shi Yonging includes but not limited to have those of following formula in the present invention:

R wherein ⁸Be H, OH, NH ₂, replacement or unsubstituted alkyl, aryl replacement or unsubstituted, heteroaryl replacement or unsubstituted, Heterocyclylalkyl replacement or unsubstituted, assorted alkyl replacement or unsubstituted, for example, acetal, OHC-, H ₂N-(CH ₂) _q-, HS-(CH ₂) _q, or-(CH ₂) _qC (Y) Z ¹Sign " e " is represented the integer of 1-2500.Sign b, d and q represent the integer of 0-20 independently.Symbols Z and Z ¹Represent OH independently, NH ₂, leaving group, for example, imidazoles, p-nitrophenyl, HOBT, tetrazolium, halogenide, S-R ⁹, the alcohol moiety of activatory ester;-(CH ₂) _pC (Y ¹) V, or-(CH ₂) _pU (CH ₂) _sC (Y ¹) _vSymbol Y represents H (2) ,=O ,=S ,=N-R ¹⁰Symbol X, Y, Y ¹, A ¹And U represents group O, S, N-R independently ¹¹Symbol V represents OH, NH ₂, halogen, S-R ¹², the alkoxide component of activatory ester, the amine component of activatory amide, sugar-nucleotide, and albumen.Sign p, q, S and v are the integers that is independently selected from 0-20.Symbol R ⁹, R ¹⁰, R ¹¹And R ¹²Represent H independently, replacement or unsubstituted alkyl, assorted alkyl replacement or unsubstituted, aryl replacement or unsubstituted, Heterocyclylalkyl replacement or unsubstituted and replacement or unsubstituted heteroaryl.

In other exemplary embodiment, below peg molecule is selected from:

The Polyethylene Glycol that is used to form conjugate of the present invention is linearity or ramose.Be applicable to that ramose peg molecule of the present invention includes but not limited to, following formula described those:

R wherein ⁸And R ^{8 '}Be to be R above being independently selected from ⁸The member of the group of definition.A ¹And A ²Be to be A above being independently selected from ¹The member of the group of definition.Sign e, f, o and q are as mentioned above.Z and Y are as mentioned above.X ¹And X ^{1 '}Be to be independently selected from S, SC (O) NH, HNC (O) S, SC (O) O, O ,-NH, NHC (O), (O) CNH and NHC (O) O, the member of OC (O) NH.

In other exemplary embodiment, ramose PEG is based on cysteine, serine or two lysine cores.Thereby other exemplary ramose PEG comprises:

In another embodiment, ramose PEG group is based on three lysine peptides.Three lysines can be single-, two-, three-, or four-PEG-ization.Exemplary material according to this embodiment has formula:

E wherein, f and f ' are the integers of the 1-2500 that selects independently; And q, q ' and q " be the integer of the 1-20 that selects independently.

In exemplary embodiment of the present invention, PEG is m-PEG (5kD, 10kD, or 20kD).Exemplary ramose PEG material is a serine-(m-PEG) ₂Or cysteine-(m-PEG) ₂Wherein m-PEG is 20kD m-PEG.

Can understand that as the technical staff the ramose polymer of Shi Yonging comprises the variant of above-mentioned theme in the present invention.Two for example above-mentioned lysines-PEG conjugate can comprise three polymerization subunits, and the 3rd is attached on the α-amine of unmodified in the superincumbent structure.Similarly, with the use of three functionalized lysines of 3 or 4 polymerization subunits, also within the scope of the invention.

Specific embodiments according to the present invention comprises:

With the carbonic ester and the active ester of these materials, for example:

Be applicable to that other activation or leaving group linearity and ramose PEG that activation is used in the chemical compound as herein described in preparation includes but not limited to following substances:

In WO 04/083259, put down in writing with the activatory PEG molecule of these and other material and prepared the method for activatory PEG.

Those skilled in the art can recognize that one or more m-PEG arms of ramose polymer can replace with has different terminal PEG groups, for example, and OH, COOH, NH ₂, C ₂-C ₁₀-alkyl, etc.And, by (or removing carbon atom) between the functional group that the alkyl junctional complex is inserted alpha-carbon atom and side chain, can easily modify top structure.Thereby the homologue of " homologous " derivant and Geng Gao, and lower homologue are in the scope of the core that is used for ramose PEG of the present invention.

By for example following described method of scheme, can easily prepare ramose PEG material as herein described:

X wherein ^aBe O or S, and r is integer 1-5.Sign e and f are the integers of the 1-2500 that selects independently.

Thereby, according to this scheme, can make m-PEG derivant natural or non-natural aminoacid contact activation (being toluene fulfonate in this case), by alkylation side chain hetero atom X ^a, form 1.With reactive m-PEG derivant, the m-PEG aminoacid of monofunctional is carried out the N-acidylate, thereby assemble ramose m-PEG 2.Can recognize that as the technical staff toluenesulfonic acid leaving group can replace with suitable arbitrarily leaving group, for example, halogen, methanesulfonic acid, the trifluoromethane sulfonic acid ester, etc.Similarly, the reactive carbonic ester that is used for acylated amine can replace with active ester, for example, and N-hydroxy-succinamide, etc., or use dehydrant, dicyclohexylcarbodiimide for example, carbonyl dimidazoles etc. can in-situ activation acid.

In exemplary embodiment, modification group is the PEG group, still, by suitable junctional complex, can be with modification group arbitrarily, for example, and water miscible polymer, water-insoluble polymer, treatment groups etc. are integrated into glycosyl.By the method for enzymatic, chemical method, or its combination can form the sugar of modification, thereby generate the sugar of modifying.In exemplary embodiment, in the optional position that allows in conjunction with modification group, replace sugar, and still allow sugar to play the function of the substrate of enzyme with reactive amines, this enzyme can be connected to the sugar of modifying on the peptide.In exemplary embodiment, when galactosamine was modify sugared, amine groups was attached on the carbon atom of 6-position.

The present invention also provides nucleotide sugar, has wherein modified glycosyl group.The ribotide of exemplary modification carries glycosyl group, and the latter is modified by the amine groups on the sugar.The ribotide of modifying, for example, the glycosyl-amine derivative of ribotide also can be used for method of the present invention.For example, can with glycosyl amine (not having modification group) enzymatic be conjugated on the peptide (or other material), and subsequently free glycosyl amine groups is conjugated on the modification group that needs.Perhaps, the ribotide of modification can play the function of the substrate of enzyme, and this enzyme can be transferred to the sugar of modifying on the glycosyl acceptor on the substrate, for example, peptide, glycopeptide, lipid, aglycon, glycolipid, etc.

In one embodiment, wherein sugared core is galactose or glucose, R ⁵Be NHC (O) Y.

In exemplary embodiment, the sugar of modification is based on 6-amino-N-acetyl group-glycosyl.About as shown in the N-acetyl group galactosamine,, can easily prepare 6-amino-glycosyl group as following by standard method.

In the superincumbent scheme, sign n represents the integer of 1-2500,10-1500 preferably, and 10-1200 more preferably.Symbol " A " is represented activated group, for example, and halogen, activatory ester () component for example, the N-hydroxy-succinamide ester, the component of carbonic ester (for example, p-nitrophenyl carbonic ester) etc.Those skilled in the art can recognize, by such and similar method, can easily prepare other PEG-amide nucleotide sugar.

In other exemplary embodiment, amide group is replaced by another group, for example urethane or urea.

In embodiment further, R ¹Be ramose PEG, for example, one of those materials recited above.Illustrative chemical compound according to this embodiment comprises:

X wherein ⁴Be key or O.

And, as discussed above, the invention provides with water miscible polymer-modified nucleotide sugar, described polymer is straight chain or ramose.For example, the chemical compound with following formula is within the scope of the invention:

X wherein ⁴Be O or key.

Similarly, the invention provides the nucleotide sugar of the saccharide of those modifications, wherein the carbon of 6-position has been modified:

X wherein ⁴Be key or O.

The conjugate of peptide and glycopeptide, lipid and glycolipid also is provided, and it comprises composition of the present invention.For example, the invention provides conjugate with following formula:

Water-insoluble polymer

In another embodiment, be similar to discussed above those, the steamed bun stuffed with sugar of modification contains water-insoluble polymer, rather than water miscible polymer.Conjugate of the present invention also can comprise one or more water-insoluble polymer.By using the conjugate conduct carrier of delivering therapeutic peptide in a controlled manner, explained this embodiment of the present invention.Polymeric delivery system is known in the art.See, for example, Dunn etc., compile POLYMERIC DRUGS and DRUGDELIVERY SYSTEMS, ACS Symposium Series Vol.469, American ChemicalSociety, Washington, D.C.1991. those skilled in the art can recognize that all known drug induction systems all are applicable to conjugate of the present invention basically.

Representational water-insoluble polymer includes but not limited to, polyphosphazine, poly-(vinyl alcohol), polyamide, Merlon, polyalkylene, polyacrylamide, poly alkylene glycol, polyalkylene oxide, the polyalkylene terephthalate, polyvingl ether, polyvinylesters, polyvinyl halide, polyvinylpyrrolidone, poly-Acetic acid, hydroxy-, bimol. cyclic ester, polysiloxanes, poly-urethane, poly-(methylmethacrylate), poly-(ethyl methacrylate), poly-(butyl isocrotonate), poly-(methacrylate isobutyl ester), poly-(the own ester of methacrylate), poly-(methacrylate isodecyl ester), poly-(methacrylate lauryl), poly-(methacrylate phenyl ester), poly-(acrylic acid methyl ester .), poly-(isopropyl acrylate), poly-(Isobutyl 2-propenoate), poly-(octadecyl acrylate) polyethylene, polypropylene, Polyethylene Glycol, poly-(oxirane), poly-(p-phthalic acid ethylidene ester), poly-(ethyl acetate), polrvinyl chloride, polystyrene, polyvinyl pyrrolidone, pluronics and polyvinylphenol and its copolymer.

The natural polymer of modifying synthetically that uses in conjugate of the present invention includes but not limited to alkylcellulose, hydroxy alkyl cellulose, cellulose ether, cellulose esters, and nitrocellulose.The particularly preferred member of the natural polymer of modifying synthetically of wide range of types includes but not limited to methylcellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl emthylcellulose, HBMC, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalic acid ester, carboxymethyl cellulose, primary cellulose acetate, the polymer of cellulose sulfate sodium salt and acrylate and methacrylate and alginic acid.

From commercial source, for example Sigma Chemical Co. (St.Louis, MO.), Polysciences (Warrenton, PA.), Aldrich (Milwaukee, WI.), Fluka (Ronkonkoma, NY), and BioRad (Richmond, CA), the polymer of these and other that can obtain easily that this paper discusses perhaps can use standard technique, and is synthetic from the monomer that is obtained by these suppliers.

The representational biodegradable polymer that uses in conjugate of the present invention includes but not limited to, polylactide, poly-Acetic acid, hydroxy-, bimol. cyclic ester and its copolymer, poly-(p-phthalic acid ethylidene ester), poly-butanoic acid, poly-valeric acid, poly-(lactide-altogether-caprolactone), poly-(lactide-co-glycolide), polyanhydride, poe, its mixture and copolymer.Use is especially, can form the compositions of gel, for example comprises those of collagen, pluronics etc.

The polymer of Shi Yonging comprises " heterozygosis " polymer in the present invention, and it comprises water-insoluble material, and it has in their part of structure at least can biological resorbent molecule.An example of such polymer is the polymer that comprises water-insoluble copolymer, and it has in every polymer chain can biological resorbent zone, hydrophilic region and a plurality of crosslinkable functional group.

For the purposes of the present invention, " water-insoluble material " comprises the material in the water insoluble basically or aqueous environment.Thereby although some zone of copolymer or fragment can be hydrophilic or water miscible, polymer molecule as a whole can not be soluble in water with any significance degree.

For the purposes of the present invention, comprise can be by the normal excretion pathway of health, the zone of metabolism or decomposition and absorption and/or elimination for term " can biological resorbent molecule ".Such metabolite or catabolite are preferably nontoxic to health basically.

Can biological resorbent zone can be hydrophobic or hydrophilic, as long as copolymer compositions is not water miscible as a whole.Thereby, keeping as a whole on the water-insoluble basis at polymer, selection can biological resorbent zone.Therefore, select the relative character of hydrophilic region, the type of the functional group that promptly comprises and relative scale that can biological resorbent zone are guaranteed useful can biological resorbent compositions keep water-insoluble.

Exemplary can comprise by resorbent polymer, for example, and the block copolymer of producing synthetically (seeing Cohn etc., U.S. Patent number 4,826,945) that can resorbently gather (Alpha-hydroxy-carboxylic acid)/poly-(oxyalkylene).These copolymers are not crosslinked, and are water miscible, so that health can be drained the block copolymer component of degraded.See Younes etc., J Biomed.Mater.Res.21:1301-1316 (1987); With Cohn etc., J Biomed.Mater.Res.22:993-1009 (1988).

At present, preferably can comprise that one or more are selected from following component by biological resorbent polymer: polyester, poly-hydroxy acid, polylactone, polyamide, poly-(ester-acid amide), polyamino acid, polyanhydride, poly-(ortho esters), poly-(carbonic ester), poly (phosphazine), poly-(phosphide), poly-(thioesters), polysaccharide and its mixture.More preferably, can comprise poly-(hydroxyl) acid constituents by biological resorbent polymer.In poly-(hydroxyl) acid, polylactic acid, polyglycolic acid, poly-caproic acid, poly-butanoic acid, poly-valeric acid and its copolymer and mixture are preferred.

But absorb in forming body the fragment of (" bio-absorbable "), preferred polymer coated also can formation of Shi Yonging can excretory and/or metabolizable fragment in the method for the invention.

More high-grade copolymer also can be used for the present invention.For example, Casey etc. disclose the triblock copolymer that is generated by the transesterification that gathers (aklylene glycol) that gathers (hydroxyacetic acid) and hydroxyl-ending at the U.S. Patent number 4,438,253 of mandate on March 20th, 1984.Disclosed such compositions can be used as can resorbent monofilament structure.By integrating aromatic orthocarbonic ester, for example four-p-methylphenyl orthocarbonic ester is integrated in the copolymer structure, can control the motility of such compositions.

Also can use other polymer based on lactic acid and/or hydroxyacetic acid.For example; the U.S. Patent number 5 that Spinu authorized on April 13rd, 1993; 202; 413 disclose biodegradable segmented copolymer; it has by the ring-opening polymerization of lactide on oligomeric diol or the diamidogen residue and/or Acetic acid, hydroxy-, bimol. cyclic ester, use bifunctional chemical compound then (vulcabond for example; diacyl chlorine or dichlorosilane) carry out chain extension, and the polylactide that generates and/or the orderly block thing of poly-Acetic acid, hydroxy-, bimol. cyclic ester.

Can be with the coating used in the present invention can biological resorbent zone design become hydrolyzable ground and/or the cutting of enzymatic ground.For the purposes of the present invention, " cutting of hydrolyzable ground " refer to the sensitivity of copolymer (especially can biological resorbent zone) to hydrolysis in water or aqueous environment.Similarly, as used herein, " but enzymatic ground cutting " refers to the sensitivity of copolymer (especially can biological resorbent zone) to the cutting of endogenic or ectogenic enzyme.

In the time of in placing body, hydrophilic region can be processed into can excretory and/or metabolizable fragment.Thereby hydrophilic region can comprise, for example, and polyethers, polyalkylene oxide, polyhydric alcohol, poly-(ethenyl pyrrolidone), poly-(vinyl alcohol), poly-(alkyl  azoles quinoline), polysaccharide, carbohydrate, peptide, albumen and its copolymer and mixture.And hydrophilic region also can be for example, to gather (alkylidene) oxide.Poly-(alkylidene) oxide like this can comprise that for example, poly-(ethylidene) oxide gathers (propylene) oxide and its mixture and copolymer.

Also can use polymer in the present invention as the component of hydrogel.Hydrogel is the polymeric material that can absorb a large amount of relatively water.The examples for compounds that can form hydrogel includes but not limited to, polyacrylic acid, and sodium carboxymethyl cellulose, polyvinyl alcohol, polyvinylpyrrolidone/, gelatin, carrageenin and other polysaccharide, hydroxy ethylene methacrylic acid (HEMA), and derivant, etc.Can produce stable, biodegradable and can biological resorbent hydrogel.And hydrogel composition can comprise the subunit that can show one or more such character.

Known can be by the biocompatible hydrogel composition of crosslinked its integrity of control, and be preferably used for now in the method for the present invention.For example, the U.S. Patent number 5 that authorize the April 25 nineteen ninety-five of Hubbell etc., 410,5 of mandate on June 25th, 016 and 1996,529,914 disclose water miscible system, and it is crosslinked block copolymer, and it has the water miscible central block fragment between the extension that is clipped in 2 hydrolytically unstables.Such copolymer end also has the acrylate-functional groups of photopolymerization.When crosslinked, these systems become hydrogel.The water miscible central block of such copolymer comprises Polyethylene Glycol; Yet the extension of hydrolytically unstable can be poly-(alpha-hydroxy acid), for example polyglycolic acid or polylactic acid.See Sawhney etc., Macromolecules 26:581-587 (1993).

In another preferred embodiment, gel is thermoreversible gel.The preferred thermoreversible gel of the present invention comprises component, pluronics for example, collagen, gelatin, hyaluronic acid, polysaccharide, poly-urethane hydrogel, poly-urethane-urea hydrogel and its combination.

In another preferred embodiment, conjugate of the present invention comprises the liposome component.According to method known to those skilled in the art, for example of the U.S. Patent number 4,522,811 that authorize the 11 days June in 1985 of Eppstein etc., can prepare liposome.For example, by with suitable lipid (stearoyl PHOSPHATIDYL ETHANOLAMINE for example, stearoyl phosphatidylcholine, Semen arachidis hypogaeae phosphatidyl choline, and cholesterol) be dissolved in the inorganic solvent evaporating solvent then, exsiccant membrane of lipoprotein is remained surface at container, can prepare Liposomal formulation.Then, the aqueous solution with reactive compound or its pharmaceutically acceptable salt imports this container.Then, manual stirred vessel gets off lipid material from the side release of container, disperses the lipid aggregation, thereby forms liposome suspension.

By the method for example, above-mentioned microgranule and the method for preparing microgranule is provided, described example is not intended to limit the scope of the microgranule that uses among the present invention.Those skilled in the art can understand, can use a series of microgranules of making by diverse ways among the present invention.

The version of discussing in the context of water miscible polymer (straight chain and branch) also is applicable to water-insoluble polymer usually above.Thereby, for example, can be with two functionalized cysteine of water-insoluble polymeric groups, serine, two lysines and three lysine branch cores.Be used to produce the method for these materials, usually very similar with the method that is used to produce water miscible polymer.

Method

Except conjugate discussed above, the invention provides the method for the conjugate for preparing these and other.And, the invention provides the object that is in the object in the danger that develops disease or suffers from disease by conjugate of the present invention is administered to, prevent, treat or improve the method for morbid state.

Thereby, the invention provides the method that between the group of selecting and peptide, glycolipid or aglycon (for example, ceramide or sphingol), forms covalent conjugates.For clarity, the conjugate that forms between the glycosyl with reference to the modification of the sugar of peptide and activatory modification of the present invention has been explained the present invention.The technical staff can recognize that the present invention similarly comprises the method that forms the conjugate of glycolipid and aglycon with the sugar of activatory modification of the present invention.

In exemplary embodiment, at water miscible polymer, the treatment group, targeting group or biomolecule, and between glycosylated or non--glycosylated peptide, formed conjugate.Polymer, treatment group or biomolecule are conjugated on the peptide by the glycosyl linking group, and described glycosyl linking group places between peptide and the modification group (for example, water miscible polymer), and covalently is connected on the two.This method comprises, makes the peptide contact contain the sugar of modification and the mixture of enzyme, and described enzyme for example is a glycosyl transferase, and it can be conjugated to the sugar of modifying on the substrate (for example, peptide, aglycon, glycolipid).Under the condition of sugar that is suitable for forming modification and the covalent bond between the peptide (or other substrate), carry out this reaction.Preferably, select the glycosyl group of the sugar of modification from nucleotide sugar.

Typically, synthesis of receptor peptide again, or express on reorganization ground in prokaryotic cell (for example, bacterial cell, for example escherichia coli) or eukaryotic cell (for example mammal, yeast, insecticide, fungus or 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 embodiment, this peptide comprises sudden change, and this sudden change can be added the glycosylation site of one or more N-or O-connection on the peptide sequence to.

Method of the present invention also provides the modification of the glycosylated by halves reorganization peptide that ground produces.The glycoprotein that many reorganization ground produces is glycosylated by halves, exposes the carbohydrate residue, and it may have unwanted character, and for example, immunogenicity is discerned by RES.Adopt the sugar of the modification in the method for the present invention, can use for example water miscible polymer, therapeutic agent etc., simultaneously further glycosylation and this peptide of deriving.The glycosyl group of the sugar of modifying can be the residue on the receptor that can suitably be conjugated in the complete glycosylated peptide, or has the another kind of glycosyl group of the character that needs.

The technical staff can recognize, uses basically the peptide or the glycopeptide in source arbitrarily, can realize the present invention.In WO 03/031464 and wherein said document, described and to be used to realize exemplary peptide of the present invention.

The peptide of modifying by method of the present invention can be synthetic or wild type peptide, perhaps they can be the peptides of the sudden change of producing by methods known in the art (for example rite-directed mutagenesis), the glycosylation of peptide typically N-connect or O-connects.It is the combination of the sugar of modification to the asparagine residue side chain that exemplary N-connects.Tripeptide sequence agedoite-X-serine and agedoite-X-threonine (wherein X is the arbitrary amino acid beyond the proline) is that carbohydrate group is to the bonded recognition sequence of the enzymatic of agedoite side chain.Thereby the existence of these tripeptide sequences in polypeptide can be set up potential glycosylation site.The glycosylation that O-connects (for example refers to a kind of sugar; N-acetyl group galactosamine; galactose; mannose, GlcNAc, glucose; fucose or xylose) to the combination of the hydroxyl side chain of hydroxy-amino-acid (preferably serine or threonine); although also can use uncommon or non-natural aminoacid, for example, 5-hydroxyproline or 5-oxylysine.

And, except peptide, can with other biological structure (for example, glycolipid, lipid, sphingol, ceramide, whole cell, etc., it contains glycosylation site), realize method of the present invention.

By changing aminoacid sequence, make it contain one or more glycosylation sites, can realize the interpolation of glycosylation site routinely to peptide or other structure.By one or more materials (for the glycosylation site of O-connection) of integrating existence-OH group in peptide sequence, preferably serine or threonine residues also can be added.By sudden change, or the complete chemosynthesis by peptide, can add.Preferably, by the variation of dna level, especially by the DNA of this peptide of encoding in predetermined base mutation, generation can be translated into the amino acid whose codon that needs, thereby can change the peptide ammino acid sequence.Preferably, use methods known in the art, carry out dna mutation.

In exemplary embodiment,, add glycosylation site by the reorganization polynucleotide.Utilize DNA reorganization method, can regulate and control the polynucleotide of the alternative peptide of encoding.DNA reorganization is the method for circulation reorganization and sudden change, and it is realized by the method assembling fragment that is similar to the polymerase chain reaction then by a large amount of relevant genes of random fragmentation.See, for example, Stemmer, Proc.Natl.Acad Sci.USA 91:10747-10751 (1994); Stemmer, Nature 370:389-391 (1994); With U.S. Patent number 5,605,793,5,837,458,5,830,721 and 5,811,238.

In WO03/031464 and relevant U.S. and PCT application, describe in detail and can be used to realize exemplary peptide of the present invention, add or remove glycosylation site and add or remove the method for glycosyl structure or substructure.

The present invention also provides the method for the glycosyl residue of one or more selections being added on the glycosyl residue of at least one selection that sugar that (or removal) will modify to peptide, then is conjugated to this peptide.For example, when on the glycosyl residue that the sugar of modifying need be conjugated to selection, can use the present embodiment, wherein said glycosyl residue is not present on the peptide, or does not exist with the amount of needs.Thereby, before the sugar that will modify is coupled on the peptide,, the glycosyl residue of selecting is conjugated on the peptide by coupling enzymatic or chemistry.In another embodiment, by removing the carbohydrate residue, before puting together the sugar of modification, change the glycosylation pattern of glycopeptide from glycopeptide.See that for example WO 98/31826.

Can be chemically or enzymatic ground add or remove any carbohydrate group that is present on the glycopeptide.Preferably, by polypeptide variants being exposed to the chemical compound trifluoromethanesulfonic acid, or the chemical compound that is equal to, come chemically deglycosylation.This processing can cause most of or all the sugared cuttings except connecting sugar (N-acetyl glucosamine or N-acetyl group galactosamine), keeps the integrity of peptide simultaneously.The deglycosylation of chemistry is documented in Hakimuddin etc., Arch.Biochem.Biophys.259:52 (1987) and Edge etc., Anal.Biochem.118:131 (1981).As Thotakura etc., Meth.Enzymol.138:350 (1987) is described, by using multiple inscribe-and circumscribed-glycosidase, can realize the enzymatic cutting of the carbohydrate group on the polypeptide variants.

By means commonly known in the art, can chemically add glycosyl.Preferably, use improving one's methods of method as herein described,, can realize that the enzymatic of glycosyl group adds with the sugar that natural glycosyl unit replaces the modification of using in the present invention.Add other method of glycosyl group, be disclosed in U.S. Patent number 5,876,980,6,030,815,5,728,554 and 5,922,577.

The exemplary binding site of the glycosyl residue of selecting includes but not limited to: (a) the glycosylated total site of N-connection, the glycosylated site that is connected with O-; (b) terminal saccharide, it is the receptor of glycosyl transferase; (c) arginine, agedoite and histidine; (d) free carboxyl; (e) free sulfydryl, for example those of cysteine; (f) free hydroxyl, for example those of serine, threonine or hydroxyproline; (g) aromatic residue, for example those of phenylalanine, tyrosine or tryptophan; Or (h) amide group of glutamine.The exemplary method of Shi Yonging in the present invention is documented in JIUYUE in 1987 disclosed WO 87/05330 on the 11st and Aplin and Wriston, CRC CRIT.REV.BIOCHEM., pp.259-306 (1981).

In one embodiment, the invention provides the method that connects 2 or a plurality of peptides by linking group.Linking group has useful arbitrarily structure, and can be selected from straight chain and structure side chain.Preferably, each end that is attached to the junctional complex on the peptide comprises the sugar (that is Xin Sheng complete glycosyl linking group) of modification.

In an exemplary method of the present invention, 2 peptides connect together by linking group, and described linking group comprises polymer (for example, PEG junctional complex).Construct is consistent with the described general structure of last figure.As described herein, construct of the present invention comprises 2 complete glycosyl linking groups (that is, s+t=1).To the concern of the PEG junctional complex that comprises 2 glycosyls is for purpose clearly, not should be understood to be limited in the character of the linking arm that uses in this embodiment of the present invention.

Thereby, with first kind of glycosyl units at first end-functionalization the PEG group, with second kind of glycosyl units at second end-functionalization.First kind with second kind of glycosyl units substrate of different transferring enzymes preferably, allow the quadrature combination of first kind and second kind of peptide respectively to first kind and second kind glycosyl units.In practice, make (glycosyl) ¹-PEG-(glycosyl) ²First kind of peptide of junctional complex contact and first kind of transferring enzyme, first kind of substrate that glycosyl units is this enzyme wherein, thus form (peptide) ¹-(glycosyl) ¹-PEG-(glycosyl) ²Then, randomly remove transferring enzyme and/or unreacted peptide from reactant mixture.Second kind of peptide and second kind of transferring enzyme (wherein second kind of substrate that glycosyl units is this enzyme) are added (peptide) ¹-(glycosyl) ¹-PEG-(glycosyl) ²Conjugate forms (peptide) ¹-(glycosyl) ¹-PEG-(glycosyl) ²-(peptide) ²At least one glycosyl residue is that directly or indirectly O-connects.Those skilled in the art can recognize that above-mentioned method also is applicable to and forms the conjugate that surpasses between 2 kinds of peptides, by for example using ramose PEG, dendritic (dendrimer), poly-(aminoacid), polysaccharide etc.

The preparation of the sugar of modifying

Usually, by using reactive group (it typically changes into new organo-functional group or nonreactive material by method of attachment), glycosyl group or glycosyl group-junctional complex box are connected together with PEG or PEG-junctional complex box group.The sugar reactive functional groups is positioned at the optional position of glycosyl group.In the present invention reactive group of Shi Yonging and reaction classification normally biology to put together chemical field well-known.The reaction classification that is fit to the present preference of reactive sugars group is such, and it can carry out under gentle relatively condition.They include but not limited to, nucleophilic displacement of fluorine (for example, the reaction of amine and alcohol and acyl halide, active ester), the interpolation of electrophilic substitution (for example, enamine reaction) and carbon-to-carbon and carbon-hetero atom multikey (for example, michael reaction, diels-alder addition).These and other useful reaction is documented in, for example, and March, ADVANCED ORGANICCHEMISTRY, 3rd Ed., John Wiley ﹠amp; Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; With Feeney etc., MODIFICATION OF PROTEINES; Advances in Chemistry Series, Vol.198, American Chemical Society, Washington, D.C., 1982.

Include but not limited to from the useful reactive functional groups that sugar is examined or modification group dangles:

(a) carboxyl and its various derivants include but not limited to N-hydroxy-succinamide ester, N-hydroxy-triazole ester, sour halogenide, acylimidazole, thioesters, p-nitrophenyl ester, alkyl, alkenyl, alkynyl and aromatic ester;

(b) hydroxyl, it can change into, for example, and ester, ether, aldehyde, etc.

(c) haloalkyl, wherein halogenide can replace with nucleophilic group later on, for example, and amine, the carboxylate anion, the mercaptan anion, carbanion, or alkoxide ion, thus cause the covalent bond of new group at the functional group place of halogen atom;

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

(e) aldehydes or ketones group, thus by forming carbonyl derivative, for example, imines, hydrazone, semicarbazones or oxime, or by such mechanism, as Grignard addition or lithium alkylide addition, deriving afterwards is possible;

(f) be used for subsequently sulfonyl halide group with the amine reaction, for example, to form sulfonamide;

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

(h) amine or sulfydryl, it can be, and is for example, acyl groupization, alkylating or oxidation;

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

(j) epoxide, it can react with for example amine and hydroxy compounds.

Can selective response functional group, they are not participated in or hinder assembly reaction sugar nuclear or the necessary reaction of modification group.Perhaps, by the existence of blocking group, can protective reaction functional group avoid participating in reaction.Those skilled in the art understands how to protect specific functional group, makes it not hinder a group reaction condition of selection.About the example of useful blocking group, see, for example, Greene etc., PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, JohnWiley ﹠amp; Sons, New York, 1991.

In the following discussion, many instantiations of the sugar that can be used to realize modification of the present invention have been set forth.In exemplary embodiment, sialic acid derivative is examined as the bonded sugar of modification group institute.To the concern of the discussion of sialic acid derivative, only be clear for what illustrate, not should be understood to limit the scope of the invention.Those skilled in the art can recognize, can be to be similar to the mode of using sialic acid to set forth as an example, activate and various other the glycosyl groups of deriving.For example, can obtain many methods, be used to modify galactose, glucose, N-acetyl group galactosamine and fucose (being several sugared substrates), it can easily be modified by means commonly known in the art.See, for example, Elhalabi etc., Curr.Med.Chem.6:93 (1999); With Schafer etc., J.Org.Chem.65:24 (2000)).

In exemplary embodiment, the peptide of modifying by method of the present invention is a glycopeptide, and it is at mammalian cell (for example, Chinese hamster ovary celI) or produce in transgenic animal, and thereby contain the oligonucleotide chain that N-and/or O-connect, it is sialylated by halves.The oligonucleotide chain that lacks sialic acid and contain the glycopeptide of terminal galactose residues can be a PEGization, PPGization, or modify with the sialic acid of modifying.

In scheme 1, handle aminoglycoside 1 with the active ester of aminoacid (for example, the glycine) derivant of protecting, the osamine residue is changed into the amino acid amide adduct of corresponding protection.Handle adduct with aldolase, form alpha-hydroxycarboxylic ester 2.By the catalytic hydrogenation of the effect of CMP-SA synzyme and CMP derivant subsequently, chemical compound 2 is changed into corresponding CMP derivant, generate chemical compound 3.(for example, PEG-C (O) NHS, PEG-OC (O) O-p-nitrophenyl) reaction will be used as the bonded position of PEG by forming the amine that glycine adduct imports, and generate material for example 4 or 5 respectively by making chemical compound 3 and activatory PEG or PPG derivant.

Scheme 1

Table 1 has been set forth the representational example with the single phosphoric acid of the deutero-sugar of PEG group.By the method for scheme 1, prepared some chemical compound of table 1.By means commonly known in the art, the derivant that has prepared other.See, for example, Keppler etc., Glycobiology 11:11R (2001); With Charter etc., Glycobiology 10:1049 (2000)).Available other reactive PEG of amine and PPG analog on the market, or the method preparation that can easily obtain by those skilled in the art.

Table 1

Can be in other position and those positions recited above, replace the sugared phosphoric acid that is used to realize modification of the present invention.

Formula II has set forth present preferred sialic replacement;

Formula II

Wherein X is a linking group, and it preferably is selected from-O-,-N (H)-and ,-S, CH ₂-and-N (R) ₂, wherein each R is independently selected from R ¹-R ⁵Symbol Y, Z, A and B represent a group separately, and it is selected from the above-mentioned defined group of X that is.Select each X independently, Y, Z, A and B, therefore, they can be identical or different.Symbol R ¹, R ³, R ³, R ⁴And R ⁵Represent H, PEG group, treatment group, biomolecule or other group.Perhaps, these symbology junctional complexs, it is attached to the PEG group, and the treatment group is on biomolecule or other group.

The exemplary group that is attached on the conjugate disclosed herein includes but not limited to, PEG derivant (for example, acyl group-PEG; acyl group-alkyl-PEG, alkyl-acyl group-PEG carbamyl-PEG, aryl-PEG); PPG derivant (for example, acyl group-PPG, acyl group-alkyl-PPG; alkyl-acyl group-PPG carbamyl-PPG, aryl-PPG), the treatment group; the diagnosis group, Man-6-P, heparin; heparinoid, SLe _x, mannose, Man-6-P, saliva acidic group Lewis X, FGF, VFGF, albumen, chrondroitin, Keratin, dermatan, albumin, integrin, antenniferous oligosaccharide, peptide etc.Those skilled in the art can easily obtain various modification groups are conjugated to method (POLY (the ETHYLENE GLYCOLCHEMISTRY:BIOTECHNICAL AND BIOMEDICAL APPLICATIONS that glycosyl is rolled into a ball, J.MiltonHarris, Ed., Plenum Pub.Corp., 1992; POLY (ETHYLENE GLYCOL) CHEMICAL AND BIOLOGICAL APPLICATIONS, J.Milton Harris, Ed., ACSSymposium Series No.680, American Chemical Society, 1997; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; With Dunn etc., Eds.POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium Series Vol.469, American Chemical Society, Washington, D.C.1991).

Linking group (crosslinked group)

The preparation of the sugar of the modification of Shi Yonging in the method for the invention comprises the combination of PEG group to saccharide residue, and preferably, forms stable adduct, and the latter is the substrate of glycosyl transferase.Thereby, often preferably use junctional complex, for example, the junctional complex that the reaction by PEG and glycosyl group and cross-linking agent forms is to put together PEG and sugar.Can be used for the exemplary bifunctional chemical compound that modification group is attached on the carbohydrate group is included but not limited to bifunctional Polyethylene Glycol, polyamide, polyethers, polyester etc.From document, as can be known carbohydrate is connected to the conventional method on other molecule.See, for example, Lee etc., Biochemistry 28:1856 (1989); Bhatia etc., Anal.Biochem.178:408 (1989); Janda etc., J.Am.Chem.Soc.112:8886 (1990) and Bednarski etc., WO 92/18135.In the following discussion, the reactive group in the glycosyl group of the sugar of nascent modification is carried out gentleness processing.The purpose of discussing is clear for what illustrate.Those skilled in the art can recognize, the reactive group that also is applicable on the modification group is discussed.

Exemplary method comprises; use the isodigeranyl function cross-linking agent SPDP (n-succinimido-3-(2-pyridine radicals dithio) propionic ester is integrated into the sulfydryl of protection on the sugar, make sulfydryl go to protect then to be used for modification group on another sulfydryl form disulfide bond.

If SPDP can influence the ability that the sugar of modification plays the glycosyl transferase substrate-function unfriendly; then can use many other one of cross-linking agents; for example 2-imino group tiacyclopentane or N-succinimido S-acetyl group thiacetate (SATA) form disulfide bond.2-imino group tiacyclopentane energy and primary amine reaction are integrated into unprotected sulfydryl on the molecule that contains amine immediately.SATA also can with primary amine reaction, but integrate shielded sulfydryl, described sulfydryl uses the hydroxylamine deacetylateization later on, generates free sulfydryl.In each case, the sulfydryl of integration can freely react with other sulfydryl or the sulfydryl of protection (for example SPDP), forms the disulfide bond that needs.

Above-mentioned method is exemplary, but the junctional complex that is not limited to use in the present invention.Can obtain other cross-linking agent, it can be used for diverse ways, and modification group is linked on the peptide.For example, TPCH (S-(2-sulfo-pyridine radicals)-L-cysteine hydrazides and TPMPH ((S-(2-sulfo-pyridine radicals) sulfydryl-propionyl hydrazine) can be handled and the carbohydrate group reaction of oxidation by the periodate of gentleness with former, thus the hydrazone key between the aldehyde of the hydrazides part of formation cross-linking agent and periodate generation.TPCH and TPMPH can lead the sulfydryl of 2-pyridine radicals thioketone protection on the sugar, and it can go protection with DTT, is used to then put together, and for example forms the disulfide bond between the component.

Be not suitable for the sugar of producing stable modification if find disulfide bond, can use other cross-linking agent, it can integrate the more stable key between the component.The cross-linking agent GMBS of isodigeranyl function (N-γ-maleimide bytyry oxygen) butanimide) and SMCC (succinimido 4-(N-maleimide-methyl) cyclohexane extraction) can and primary amine reaction, thereby maleimide base group is imported on the component.Maleimide base group can be subsequently with other component on the sulfydryl reaction, described sulfydryl can import by aforementioned cross-linking agent, thereby forms the thioether bond between stable component.Play the ability of glycosyl transferase substrate as the sterically hindered sugar that can hinder the activity of component or modify between the fruit component, can use cross-linking agent, it can import the long spacer arm between the component, and comprises some aforementioned cross-linking agents (that is derivant SPDP).Thereby, there are many suitable useful cross-linking agents; To the effect that the sugar of the peptide conjugate of the best and modification is produced, select each according to it.

Using many reagent to modify the component of the sugar of the modification with intramolecularly chemical crosslinking (about the comment of cross-linking agent and cross-linking method, sees: 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; Mattson etc., Mol.Biol.Rep.17:167-183,1993, they are all incorporated by reference in this article).Preferred cross-linking agents be derived from various distance of zero mark degree, with cross-linking agent bifunctional and the isodigeranyl function.The cross-linking agent of distance of zero mark degree comprises directly puting together of two intrinsic chemical groups, does not import foreign substance.The reagent of the formation of energy catalysis disulfide bond belongs to the type.Another example is such reagent, and it can induce the condensation of carboxyl and primary amino radical, forms amido link, carbodiimide for example, ethyl chloroformate, Woodward reagent K (the 2-ethyl-different  azoles of 5-phenyl  (isoxazolium)-3 '-sulphonic acid ester), and carbonyl dimidazoles.Except these chemical reagent, enzyme transglutaminase (glutamyl-peptide gamma-glutamyl based transferase; EC 2.3.2.13) can be used as distance of zero mark degree cross-linking agent.The acyl group transfer reaction of this enzyme energy catalysis on the carboxylacyl amine group of protein bound glutaminyl residue uses primary amino radical as substrate usually.Preferred with-contain 2 identical or 2 different sites respectively with different-bifunctional reagent, its can with amino, sulfydryl, guanidine radicals, indole or nonspecific radical reaction.

I. the preferred specific site in the cross-linking agent

1. amino-reactive group

In a preferred embodiment, the site on the cross-linking agent is amino-reactive group.The useful non-limiting instance of amino-reactive group comprises N-hydroxy-succinamide (NHS) ester, imino-ester, isocyanates, acyl halide, aromatic yl azide, p-nitrophenyl ester, aldehyde, and sulfonic acid chloride.

Preferential primary (comprising aromatic series) of the amino reaction of NHS ester with the saccharic composition of modifying.The imidazole group of known group propylhomoserin can with the primary amine competitive reaction, but the product instability, and easily hydrolysis.React the nucleophillic attack of the amine on the sour carboxyl that comprises the NHS ester,, discharge N-hydroxy-succinamide to form amide.Thereby, can lose the positive charge of original amino.

Imino-ester is the most special catabolic acylating reagent of reaction to the amine groups of the saccharic composition of modifying.At pH 7-10, imino-ester only with primary amine reaction.Primary amine can be attacked imidic acid in nucleophilic ground, generates intermediate product, and the latter can be resolved into amidine at high pH, or resolves into new imidic acid at low pH.New imidic acid can with another kind of primary amine reaction, thereby crosslinked 2 amino, the situation that the unifunctional imidic acid of inferring reacts difunctionally.With the primary product of primary amine reaction be amidine, it is the alkali stronger than original amine.Therefore, the positive charge that has kept original amino.

Carbimide. (and isothiocyanic acid) can with the primary amine reaction of the saccharic composition of modifying, form stable key.The reaction of they and sulfydryl, imidazoles and tyrosyl group can produce unsettled relatively product.

Also as amino-specific reagent, wherein the nucleophilic amine of affinity component can be under weak basic condition for acyl azide, and for example pH 8.5, attack sour carboxyl.

Aryl halide for example 1,5-two fluoro-2, preferential amino and the reaction of tyrosine phenolic groups with the saccharic composition of modifying of 4-dinitro benzene, but also with sulfydryl and imidazole group reaction.

Single-and the p-nitrophenyl ester of dicarboxylic acids also be useful amino-reactive group.Although the specificity of this reagent is not very high, α-as if can the most promptly react with epsilon-amino.

Aldehyde for example glutaraldehyde can with the primary amine reaction of the sugar of modifying.Although unsettled Schiff's base forms by amino reaction with aldehyde, glutaraldehyde can be modified the sugar with stable crosslinked modification.At pH 6-8, i.e. the pH of typical crosslinked condition, cyclic polymer can experience dehydration, forms the undersaturated aldehyde polymer of alpha-beta.But in the time of on being conjugated to another pair key, Schiff's base is stable, and the Resonant Interaction of two two keys can stop the hydrolysis of Schiff key.And the amine of high local concentrations can be attacked the two keys of alkene, forms stable Michael addition adducts.

Aromatic sulfonic acid chloride can react with a plurality of sites of the saccharic composition of modifying, and still the reaction with amino is most important, can produce stable sulfonamide and connect.

2. sulfydryl-reactive group

In another preferred embodiment, the site is sulfydryl-reactive group.The useful non-limiting instance of sulfydryl-reactive group comprises maleimide, alkyl halide, pyridyl disulfide and thio phenyl dicarboximide.

Maleimide can react by preferential sulfydryl with the saccharic composition of modifying, and forms stable thioether bond.They also can be with the primary amino radical and the imidazole group reaction of lower speed and histidine.But, at pH 7, can think that maleimide base group is sulfydryl-specific group, because at this pH, the response speed of simple mercaptan is than the corresponding high 1000-of amine doubly.

Alkyl halide can react with sulfydryl, sulfide, imidazoles and amino.But to weakly alkaline pH, the main and sulfydryl reaction of alkyl halide forms stable thioether bond in neutrality.At higher pH, tend to and amino reaction.

Pyridyl disulfide can form blended disulphide by disulfide exchange and the reaction of free sulfydryl.As a result, pyridyl disulfide is the most specific sulfydryl-reactive group.

The thio phenyl dicarboximide can react with free sulfydryl, forms disulphide.

3. carboxyl-reactive residue

In another embodiment, the carbodiimide in water soluble and the organic solvent is used as carboxyl-reaction phenyl-diformyl reagent.These chemical compounds can with free carboxyl reaction, form pseudo-urea, the latter can be connected on the available amine, produces amide and connects, and instructs how to modify carboxyl (Yamada etc., Biochemistry 20:4836-4842,1981) with carbodiimide.

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

Except using site-specific reactive group, the present invention includes and use nonspecific reactive group, sugar is connected on the modification group.

But exemplary nonspecific cross-linking agent comprises the group of photoactivation, and it is inert fully in the dark, and behind the photon that absorbs suitable energy, it can change into reactive material.In a preferred embodiment, but the group of photoactivation is selected from the precursor of the nitrene that heating or photolysis azide produce.The nitrene that lacks electronics be have very much reactive, and can with many chemical bonding reactions, comprise N-H, O-H, C-H, and C=C.Although can use 3 class azide (aryl, alkyl, and acyl derivative), aromatic yl azide is preferred now.Aromatic yl azide is better with the reactivity of c h bond with the reactivity ratio of N-H and O-H after the photolysis.The aryl nitrene that lacks electronics can promptly encircle-enlarge, and forms dehydrogenation azepine , and it tends to and the nucleopilic reagent reaction, rather than forms C-H insertion product.By the existence of electron-withdrawing substituent, for example nitro or the hydroxyl in the ring can improve the reactivity of aromatic yl azide.Such substituent group is pushed into longer wavelength with the obtained the maximum absorption of aromatic yl azide.Unsubstituted aromatic yl azide has the obtained the maximum absorption of 260-280nm, but hydroxyl and nitro aromatic yl azide can significantly absorb the light above 305nm.Therefore, hydroxyl and nitro aromatic yl azide are most preferred, because their allow affinity component to adopt than the unsubstituted aromatic yl azide photolysis condition of harm still less.

In another preferred embodiment, but the group of photoactivation is selected from fluorizated aromatic yl azide.The photolysis products of fluorizated aromatic yl azide is the aryl nitrene, and they have all experienced the characteristic reaction of this group, comprises that high efficiency c h bond inserts (Keana etc., J.Org.Chem.55:3640-3647,1990).

In another embodiment, but the group of photoactivation is selected from benzo phenyl ketone residue.Benzo phenyl ketone reagent can produce the crosslinking rate higher than aromatic yl azide reagent usually.

In another embodiment, but the group of photoactivation is selected from diazonium compound, and it can form the carbene that lacks electronics after photolysis.These carbenes can experience many reactions, comprise the insertion in c h bond, add two keys (comprising aromatic system), and hydrogen attracts and to the coordination of nucleophilic center, produces carbon ion.

In another embodiment, but the group of photoactivation is selected from the diazo acetone acid.For example, the p-nitrophenyl ester of p-nitrophenyl diazo acetone acid can react with aliphatic amine, produces diazo acetone acid amide, and it can experience ultraviolet and decompose formation aldehyde.The affinity component energy weevil aldehyde or the glutaraldehyde of the diazo acetone acid-modification of photolysis equally react, and form crosslinked.

Iii. with bifunctional reagent

Can with the same bifunctional cross-linking agent of primary amine reaction

Synthetic, the character and the application (about the comment of cross-linking method and reagent, above seeing) of amine-reactive cross-linking agent have been described in the literature commercially.Can obtain many reagent (for example, Pierce Chemical Company, Rockford, Ill.; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, Inc., Eugene, OR.).

Preferred non-limiting instance with bifunctional NHS ester comprises two succinimido glutarates (DSG), two succinimido suberates (DSS), two (thiosuccimide base) suberates (BS), two succinimido tartrates (DST), dithiosuccinimide base tartrate (sulfo--DST), two-2-(succinimido oxygen ketonic oxygen) ethyl sulfone (BSOCOES), two-2-(thiosuccimide base oxygen-ketonic oxygen) ethyl sulfone (sulfo--BSOCOES), ethylene glycol bisthioglycolate (succinimido succinate) (EGS), ethylene glycol bisthioglycolate (thiosuccimide base succinate) (sulfo--EGS), dithio two (succinimido-propionic ester (DSP) and dithio two (thiosuccimide base propionic ester (sulfo--DSP).Preferred non-limiting instance with bifunctional imino-ester comprises dimethyl propylene two imide acid esters (DMM), dimethyl succinimide acid esters (DMSC), dimethyl adipyl imino-ester (DMA), dimethyl-g two imide acid esters (DMP), dimethyl-octa two imide acid esters (DMS), dimethyl-3,3 '-oxygen dipropyl imide acid esters (DODP), dimethyl-3,3 '-(methylene-dioxy) dipropyl imide acid esters (DMDP), dimethyl-, 3 '-(two methylene-dioxies) dipropyl imide acid esters (DDDP), dimethyl-3,3 '-(four methylene-dioxies)-dipropyl imide acid esters (DTDP), and dimethyl-3,3 '-dithio dipropyl imide acid esters (DTBP).

Preferred non-limiting instance with bifunctional isothiocyanic acid comprises: to phenylene diisothiocyanic acid (DITC) and 4,4 '-two isothiocyanos-2,2 '-disulfonic acid stilbene (DIDS).

Preferred non-limiting instance with bifunctional isocyanates comprises dimethylbenzene-vulcabond, Toluene-2,4-diisocyanate, the 4-vulcabond, Toluene-2,4-diisocyanate-Carbimide .-4-isothiocyanate, 3-methoxyl group diphenyl methane-4,4 '-vulcabond, 2,2 '-dicarboxyl-4,4 '-the azobenzene vulcabond, with 1,6-dihexyl vulcabond.

Preferred non-limiting instance with bifunctional aryl halide comprises 1,5-two fluoro-2, and 4-dinitro benzene (DFDNB) and 4,4 '-two fluoro-3,3 '-dinitrophenyl-sulfone.

Preferred non-limiting instance with bifunctional aliphatic aldehyde reagent comprises Biformyl, malonaldehyde, and glutaraldehyde.

The nitrobenzophenone ester that comprises dicarboxylic acids with the preferred non-limiting instance of bifunctional acylating reagent.

Preferred non-limiting instance with bifunctional aromatic sulfonic acid chloride comprises phenol-2,4-disulfonic acid chloride, and alpha-Naphthol-2,4-disulfonic acid chloride.

Other amino-reactive preferred non-limiting instance with bifunctional reagent comprises erithritol two carbonic esters, and itself and amine react, and produce diurethane.

Can with the same bifunctional cross-linking agent of free sulfydryl reaction

Synthetic, the character and the application (about the comment of cross-linking method and reagent, above seeing) of such reagent have been described in the literature.Available many described reagent (for example, PierceChemical Company, Rockford, Ill. on the market; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, Inc., Eugene, OR.).

Preferred non-limiting instance with bifunctional maleimide comprises dimaleimide hexane (BMH), N, N '-(1, the 3-phenylene) dimaleimide, N, N '-(1, the 2-phenylene) dimaleimide, azobenzene dimaleimide and two (N-maleimide methyl) ether.

Preferred non-limiting instance with bifunctional pyridyl disulfide comprises 1,4-23 '-(2 '-the pyridine radicals dithio) propionamido-butane (DPDPB).

With the preferred non-limiting instance of bifunctional alkyl halide comprise 2,2 '-dicarboxyl-4,4 '-diiodo-acetamido diphenyl diimide; α; α '-two iodo-xylol sulfonic acid, α, α '-two bromo-xylol 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 cross-linking agent of bifunctional photoactivation

Synthetic, the character and the application (about the comment of cross-linking method and reagent, above seeing) of such reagent have been described in the literature.More available reagent on the market (for example, PierceChemical Company, Rockford, Ill.; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, Inc., Eugene, OR.).

But the preferred non-limiting instance with the cross-linking agent of bifunctional photoactivation comprises 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 '-dithio diphenyl azide.

Iv. heterobifunctional agent

1. amino-reactive the heterobifunctional agent that has the pyridyl disulfide group

Synthetic, the character and the application (about the comment of cross-linking method and reagent, above seeing) of such reagent have been described in the literature.Available many described reagent (for example, PierceChemical Company, Rockford, Ill. on the market; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, Inc., Eugene, OR.).

Preferred non-limiting instance with heterobifunctional agent of pyridyl disulfide group and amino-reactive NHS ester comprises N-succinimido-3-(2-pyridine radicals dithio) propionic ester (SPDP), succinimido 6-3-(2-pyridine radicals dithio) propionamido-alkyl caproate (LC-SPDP), thiosuccimide base 6-3-(2-pyridine radicals dithio) propionamido-alkyl caproate (sulfo--LCSPDP), 4-succinimido oxygen carbonyl-Alpha-Methyl-α-(2-pyridine radicals dithio) toluene (SMPT) and thiosuccimide base 6-Alpha-Methyl-α-(2-pyridine radicals dithio) toluoyl amido alkyl caproate (sulfo--LC-SMPT).

2. amino-reactive the heterobifunctional agent that has maleimide base group

Synthetic, the character and the application of such reagent have been described in the literature.Preferred non-limiting instance with heterobifunctional agent of maleimide base group and amino-reactive NHS ester comprises succinimido dimaleoyl imino acetate (AMAS); succinimido 3-dimaleoyl imino propionic ester (BMPS); N-γ-maleimide bytyry oxygen succinimide ester (GMBS) N-γ-maleimide bytyry oxygen thiosuccimide ester (the succinimido 6-dimaleoyl imino alkyl caproate (EMCS) of sulfo--GMBS); succinimido 3-dimaleoyl imino benzoate (SMB); between maleimide benzoyl-N-hydroxysuccinimide eater (MBS); between maleimide benzoyl-N-hydroxy thiosuccinimide ester (sulfo--MBS); succinimido 4-(N-maleimide methyl)-cyclohexane extraction-1-formic acid esters (SMCC); thiosuccimide base 4-(N-maleimide methyl) cyclohexane extraction-1-formic acid esters (sulfo--SMCC); succinimido 4-(to maleimide phenyl) butyrate (SMPB) and thiosuccimide base 4-(to maleimide phenyl) butyrate (sulfo--SMPB).

3. amino-reactive the heterobifunctional agent that has the alkyl halide group

Synthetic, the character and the application of such reagent have been described in the literature.Preferred non-limiting instance with heterobifunctional agent of alkyl halide group and amino-reactive NHS ester comprises N-succinimido-(4-iodoacetyl) Aminobenzoate (SIAB); thiosuccimide base-(4-iodoacetyl) Aminobenzoate (sulfo--SIAB); succinimido-6-(iodoacetyl) aminocaproic acid ester (SIAX); succinimido-6-(6-((iodoacetyl)-amino) caproyl amino) alkyl caproate (SIAXX); succinimido-6-(((4-(iodoacetyl)-amino)-methyl)-cyclohexane extraction-1-carbonyl) aminocaproic acid ester (SIACX), and succinimido-4 ((iodoacetyl)-amino) hexahydrotoluene-1-formic acid esters (SIAC).

Preferred embodiment with heterobifunctional agent of amino-reactive NHS ester and alkyl dihalide group is a N-hydroxy-succinamide base 2,3-dibromo-propionic acid ester (SDBP).SDBP can be by puting together its amino, and intramolecular crosslinking is imported the affinity component.By reaction temperature, can control the reactivity (McKenzie etc., Protein Chem.7:581-592 (1988)) of dibromo propiono group and primary amine group.

Preferred non-limiting instance with heterobifunctional agent of alkyl halide group and amino-reactive p-nitrophenyl ester group comprises p-nitrophenyl iodoacetate (NPIA).

Other cross-linking agent is known to those skilled in the art.See, for example, Pomato etc., U.S. Patent number 5,965,106.Selection is used for the suitable crosslinking agent of special-purpose, in those skilled in the art's the limit of power.

V. the linking group that can cut

In another embodiment, for linking group provides a group that can be cut, to discharge modification group from saccharide residue.Many groups that cut are known in the art.See, for example, Jung etc., Biochem.Biophys.Acta 761:152-162 (1983); Joshi etc., J.Biol.Chem.265:14518-14525 (1990); Zarling etc., J.Immunol.124:913-920 (1980); Bouizar etc., Eur.J.Biochem.155:141-147 (1986); Park etc., J.Biol.Chem.261:205-210 (1986); Browning etc., J.Immunol.143:1859-1867 (1989).And, wide scope cut bifunctional (with-and different-bifunctional) linking group is can buy from supplier (for example Pierce) on the market.

Make use up, heat or reagent, mercaptan for example, hydroxylamine, alkali, periodates etc. can cut the exemplary group that cuts.And the endocytosis meeting causes cutting (for example, cis Aconitum carmichjaelii Debx. base in some preferred group body; See Shen etc., Biochem.Biophys.Res.Commun.102:1048 (1991)).Preferably the group that can cut comprises the following group that cuts, and it is to be selected from following member: disulphide, ester, imines, carbonic ester, nitrobenzyl, phenacyl and Benzoinum group.

Sugar the puting together of modifying to peptide

Use suitable enzyme to mediate and put together, the sugar that PEG modifies can be conjugated on glycosylated or non--glycosylated peptide.Preferably, select the concentration of donor sugar, enzyme and the receptor peptide of modification, exhaust thereby make glycosylation proceed to receptor.Although the viewpoint of discussing below is to set forth, also be applicable to other glycosyl transferase reaction usually in the context of sialyltransferase.

Known many use glycosyl transferases synthesize the method for the oligosaccharide structure that needs, and are applicable among the present invention usually.Exemplary method is documented in, and for example, WO 96/32491, Ito etc., Pure Appl.Chem.65:753 (1993), U.S. Patent number 5,352,670,5,374,541,5,545,553, with the U.S. Patent number of owning together 6,399,336 and 6,440,703, it is incorporated by reference in this article.

Use the independent glycosyl transferase or the combination of glycosyl transferase, can realize the present invention.For example, can use the combination of sialyltransferase and galactosyltransferase.In these use to surpass a kind of embodiment of enzyme, enzyme and substrate preferably were combined in the initial action mixture, perhaps in case finish or near finishing first enzymatic reaction, the enzyme and the reagent that will be used for second enzymatic reaction add reaction medium.By in a jar, carrying out 2 enzymatic reactions successively,, can improve gross production rate with respect to the method for separation of intermediates wherein.And, reduced the removing and the processing of unnecessary solvent and by-product.

In a preferred embodiment, first kind and second kind of enzyme all are glycosyl transferases.In another preferred embodiment, a kind of enzyme is an endoglycosidase.In another preferred embodiment, use is assembled the glycoprotein of modification of the present invention above 2 kinds of enzymes.Before or after adding to the sugar of modifying on the peptide, use enzyme to change sugared structure on the arbitrfary point of peptide.

In another embodiment, this method is used one or more exoglycosidases or endoglycosidase.Glycosidase is mutant typically, and it forming glycosyl bond, rather than is destroyed them by through engineering approaches.The dextranase of sudden change comprises that typically the amino acid residue of avtive spot acidic amino acid residue replaces.For example, when the polysaccharide restriction endonuclease was endo-H, the avtive spot residue of replacement is 130 the Asp in the position typically, 132 the Glu in the position, or its combination.Usually, use serine, alanine, agedoite, or glutamine substitutes this aminoacid.

The enzyme of sudden change can catalytic reaction, normally the synthesis step of the back reaction by being similar to inscribe dextranase hydrolysing step.In these embodiments, and the glycosyl donor molecule (for example, the widow who needs-or list-sugared structure) containing leaving group, reaction is proceeded, and donor molecule is added on the proteic GlcNAc residue.For example, leaving group can be halogen, for example fluoride.In other embodiment, leaving group is Asn, or Asn-peptide group.In other embodiment, the GlcNAc residue on the glycosyl donor molecule is modified.For example, the GlcNAc residue can comprise 1,2  azoles quinoline group.

In a preferred embodiment, the every kind of enzyme that is used to produce conjugate of the present invention exists with catalytic amount.The catalytic amount of certain enzyme is with the concentration of the substrate of enzyme and reaction condition (for example, temperature, time and pH value) and change.The method of the catalytic amount of the given enzyme of detection under predetermined concentration of substrate and reaction condition is that those skilled in the art is well-known.

Can carry out the temperature range of said method, can be the temperature during to the most responsive enzyme denaturation just above freezing point.Preferred temperature range is about 0 ℃ to about 55 ℃, more preferably about 20 ℃ to about 30 ℃.In another exemplary embodiment, use thermophilic enzyme, at elevated temperatures, carry out one or more component of the present invention.

Reactant mixture kept is enough to make the glycosylated time of receptor, thereby forms the conjugate that needs.After a few hours, often can detect some conjugates, usually in 24 hours or the shorter time, obtain callable amount.Those skilled in the art can understand that response speed depends on many variable factors (for example, enzyme concentration, donor concentration, acceptor density, temperature, solvent volume), and they are optimized at the system of selecting.

The present invention also provides the industrial-scale production of the peptide of modifying.As used herein, commercial scale can be produced the conjugate of the final purification of 1g at least usually.

In the following discussion, by the sialic acids groups of modifying to the puting together of glycosylated peptide, illustration the present invention.With the PEG labelling sialic acid of exemplary modification.It is in order to get across, to be not intended to hint that the present invention is limited to puting together of these two gametophytes that the sialic acid of PEG-modification and the purpose of glycosylated peptide are used in following discussion.The technical staff can understand that this discussion is applicable to the glycosyl that adds sialic acid modification in addition usually.And this discussion is equally applicable to use PEG reagent in addition that glycosyl units is modified, and comprises other PEG group, treatment group, and biomolecule.

Can use the method for enzymatic, carbohydrate PEGization or PPGization is optionally imported on peptide or the glycopeptide.This method is used the sugar of modifying, and it contains PEG, PPG, or the reactive functionality of covering, the sugar of this modification and suitable glycosyl transferase or the combination of glycosyl synthase.By select to produce the carbohydrate that needs connects and the sugar of utilization modification as the glycosyl transferase of donor substrate, PEG or PPG directly can be imported on the peptide main chain, on the existing saccharide residue of glycopeptide, or added on the saccharide residue on the peptide.

The receptor of sialyltransferase is present on the peptide that will modify by method of the present invention, as the structure of natural generation, or reorganization ground, enzymatic ground or the structure of generation chemically.Suitable receptor comprises, for example, the galactosyl receptor is Gal β 1 for example, 4GlcNAc, Gal β 1,4GalNAc, Gal β 1,3GalNAc, lactose-N-tetrose, Gal β 1,3GlcNAc, Gal β 1,3Ara, Gal β 1,6GlcNAc, Gal β 1,4Glc (lactose) and other receptor known to those skilled in the art (are seen, for example, Paulson etc., J.Biol.Chem.253:5617-5624 (1978)).

In one embodiment, the receptor of sialyltransferase is present in the body of glycopeptide on the glycopeptide to be finished of synthetic back.Use described method, need not to modify in advance the glycosylation pattern of glycopeptide, can sialylated such glycopeptide.Perhaps, can use the sialylated peptide that does not comprise suitable receptor of method of the present invention; By method known to those skilled in the art, at first modified peptides makes it comprise receptor.In exemplary embodiment,, add the GalNAc residue by the effect of GalNAc transferring enzyme.

In exemplary embodiment,, galactose residue is connected peptide (for example, GlcNAc) the suitable receptor on, assembling galactosyl receptor by being attached to.This method comprises, hatches the reactant mixture of peptide to be finished and the galactosyltransferase (for example, gal β 1,3 or gal β 1,4) that contains appropriate amount and suitable galactosyl donor (for example, UDP-galactose).Make and carry out in the reaction substantially fully, perhaps, when adding the galactose residue of scheduled volume, cessation reaction.Those skilled in the art can understand other method of the saccharide acceptor that assembling is selected.

In another embodiment, the oligosaccharide of glycopeptide-connection is " trimmed (trimmed) " at first whole or in part, maybe will add the group of one or more suitable residues with the receptor that exposes sialyltransferase, obtains suitable receptor.Enzyme, for example glycosyl transferase and endoglycosidase (seeing that for example U.S. Patent number 5,716,812) can be used for combination and finishing reaction.

In the following discussion, the sugar of use modifying (it has the PEG group of the face of being connected thereto), illustration method of the present invention.The purpose of discussing is clear for what illustrate.The technical staff can recognize that the sugar that this discussion is equally applicable to wherein modify carries those embodiments of treatment group, biomolecule etc.

In exemplary embodiment of the present invention (wherein before adding the sugar of modifying, " finishing " carbohydrate residue), high mannose is trimmed to the first generation pair feeler structures.The sugar that carries the modification of PEG group is conjugated on the one or more saccharide residues that expose by " finishing ".In one embodiment, by being conjugated to the GlcNAc group on the PEG group, add the PEG group.The GlcNAc that modifies is attached on one or two terminal mannose residue of two feeler structures.Perhaps, the GlcNAc of unmodified can be added on one or two end of ramose material.

In another exemplary embodiment, the sugar (it is conjugated on the GlcNAc residue that adds terminal mannose residue to) of the modification by having galactose residue adds the PEG group on one or two terminal mannose residue of two feeler structures.Perhaps, the Gal of unmodified can be added on one or two terminal GlcNAc residue.

In another embodiment, use the sialic acid of modifying, the PEG group is added on the Gal residue.

In another exemplary embodiment, high mannose (high mannose) structure " is trimmed to " mannose of two feeler structure bifurcation.In one embodiment, by with polymer-modified GlcNAc, add the PEG group.Perhaps, adding the GlcNAc of unmodified to mannose, is thereafter the Gal with bonded PEG group.In another embodiment, adding the GlcNAc and the Gal residue of unmodified to mannose successively, is thereafter the sialic acids groups with the PEG base group modification.

In another exemplary embodiment, high mannose " is trimmed to " the bonded GlcNAc of first mannose.GlcNAc is conjugated on the Gal residue that carries the PEG group.Perhaps, the Gal of unmodified is added on the GlcNAc, add with water miscible sugar-modified sialic acid then.In another embodiment, put together terminal GlcNAc, use the fucose fucosylation GlcNAc of the modification of carrying the PEG group subsequently with Gal.

Also high mannose can be trimmed to first GlcNAc on the Asn that is attached to peptide.In one embodiment, the GlcNAc that puts together GlcNAc-(Fuc) a residue with the GlcNAc that carries water miscible polymer.In another embodiment, modified the GlcNAc of GlcNAc-(Fuc) a residue with the Gal that carries water miscible polymer.In another embodiment, modified GlcNAc, be conjugated to subsequently on the sialic Gal with the PEG base group modification with Gal.

Other exemplary embodiment is documented in the U.S. Patent Application Publication of owning together: 20040132640; 20040063911; 20040137557; Application No.: 10/369,979; 10/410,913; 10/360,770; 10/410,945 and PCT/US02/32263 in, wherein every piece is incorporated by reference here.

The above embodiments provide the strong explanation of method as herein described.Use method as herein described, can " repair " and set up the carbohydrate residue of all structures that need basically.Can add the sugar of modifying the end of aforesaid carbohydrate group to, perhaps it can be between the end of peptide core and carbohydrate.

In exemplary embodiment, use sialidase, remove existing sialic acid from glycopeptide, thereby expose all or most following galactosyl residue.Perhaps, use galactose residue, or with the oligosaccharide residue of galactose units ending, labelling peptide or glycopeptide.After exposing or adding galactose residue, use suitable sialyltransferase to add the sialic acid of modification.This method summary is in scheme 2.

Scheme 2

[0293] in the another kind of method of summing up in scheme 3, the reactive functionality of covering is present on the sialic acid.The sialic acid that the reactive group that covers preferably is used for modifying is attached to the condition effect on the erythropoietin.After covalently being attached to the sialic acid of modifying on the peptide, removal is sheltered, with reagent for example PEG put together peptide.By it with the saccharide residue of modifying on the reaction of uncovered reactive group, in specific mode reagent is conjugated on the peptide.

Scheme 3

Terminal sugar according to the oligosaccharide side chain of glycopeptide can use the sugar of modifying arbitrarily (table 2) with its suitable glycosyl transferase.As discussed above, can be in the expression process, import the terminal sugared of the required glycopeptide of the structure of PEGization natively, perhaps use suitable glycosidase, the mixture of glycosyl transferase or glycosidase and glycosyl transferase generates after expression.

Table 2

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, junctional complex-M, M. M=PEG, for example, m-PEG

In another exemplary embodiment, make UDP-galactose-PEG and Lac Bovis seu Bubali β 1, the reaction of 4-galactosyltransferase, thus the galactose of modifying is transferred to suitable terminal N-acetyl glucosamine structure.In the expression process, can produce the terminal GlcNAc residue on the glycopeptide, as can occurring in the expression systems such as mammal, insecticide, plant or fungus, but also can be as required, by producing with sialidase and/or glycosidase and/or glycosyl transferase processing glycopeptide.

In another exemplary embodiment, use the GlcNAc transferring enzyme, GNT1-5 for example transfers to terminal mannose residue on the glycopeptide with the GlcN of PEGization.In another exemplary embodiment, remove the glycan structures that N-and/or O-connect from glycopeptide enzymatic ground, to expose aminoacid or terminal saccharide residue, it is puted together with the sugar of modifying subsequently.For example, the structure that the N-that uses the inscribe dextranase to remove glycopeptide connects is to expose the terminal GlcNAc as the GlcNAc-connection-Asn on the glycopeptide.Use UDP-Gal-PEG and suitable galactosyltransferase that PEG-galactose functional group is imported on the GlcNAc of exposure.

In an alternative embodiment, use the known glycosyl transferase that saccharide residue can be transferred on the peptide main chain, the sugar of modifying is directly added on the peptide main chain.This exemplary embodiment is as described in the scheme 4.Be used to realize that exemplary glycosyl transferase of the present invention includes but not limited to, GalNAc transferring enzyme (GalNAc T1-14), GlcNAc transferring enzyme, fucosyltransferase, glucosyltransferase, xylosyltransferase, mannose transferase etc.Use this method, the sugar of modifying directly can be added on the peptide that lacks any carbohydrate, perhaps, on the existing glycopeptide.In both cases, the interpolation of the sugar of modification occurs on the specific position of peptide main chain, and the substrate specificity of this glycosyl transferase defines, rather than the random fashion that takes place in the process with the peptide main chain that uses the chemical method modified protein.By suitable aminoacid sequence is made up into polypeptide chain, many reagent can be imported albumen or the glycopeptide that lacks glycosyl transferase peptide substrate sequence.

Scheme 4

In each above-mentioned exemplary embodiment, after the sugar that will modify is conjugated on the peptide, can use one or more other modification steps chemistry or enzymatic.In exemplary embodiment, use enzyme (for example, fucosyltransferase) that glycosyl units (for example, fucose) is added on the sugar of the modification that is attached to the end on the peptide.In another embodiment, the site of using enzymatic reaction can not put together the sugar of modifying " adds medicated cap ".Perhaps, use chemical reaction to change the structure of the sugar of the modification of puting together.For example, sugar that makes the modification of puting together and the reagent reacting that is connected that can stablize or go stable and peptide composition, described peptide composition combines the sugar of modification.In another embodiment, after being conjugated on the peptide, the component of the sugar of modification is gone protection.The technical staff can recognize, has many enzymatics and method chemistry, and it can be used in the stage of the sugar that will modify after being conjugated on the peptide in the method for the invention.The further modification of sugar-peptide conjugate of modifying, within the scope of the invention.

I. enzyme

Sugar shifts

Except the top enzyme of in the context of the conjugate that forms acyl group-connections, discussing, by using the method for other enzyme, can refinement, the glycosylation pattern of finishing or modification conjugate and initial substrate (for example, peptide, lipid).In the WO03/031464 A2 of disclosed DeFrees on April 17th, 2003, the enzyme reconstruct peptide that saccharide donor can be transferred on the receptor and the method for lipid used has been discussed very at length.Following simplified summary the enzyme of the selection used in the method.

Glycosyl transferase

The sugar (donor NDP-sugar) of glycosyl transferase energy catalytic activation adds on albumen, glycopeptide, lipid or the glycolipid in mode progressively, or the non--reduction end of the oligosaccharide of growth.Oligosaccharide donor Dol-PP-NAG by transferring enzyme and lipid-be connected ₂Glc ₃Man ₉Can synthesize the glycopeptide that N-connects in the mode (enblock transfer) that module shifts, repair core subsequently.In this case, " core " sugar character a little with subsequently in conjunction with different.Very a large amount of glycosyl transferases is known in the art.

The glycosyl transferase of Shi Yonging can be arbitrarily in the present invention, as long as it can utilize the sugar of modification as saccharide donor.The example of such enzyme comprises Leloir approach glycosyl transferase; galactosyltransferase for example; N-acetyl-amino glucosyltransferase; N-acetyl-amino galactosyltransferase; fucosyltransferase, sialyltransferase, mannose transferase; xylosyltransferase, glucuronyl transferase etc.

Sugar for the enzymatic that relates to the glycosyl transferase reaction is synthetic, can clone glycosyl transferase, or from source separation arbitrarily.Many clones' glycosyl transferase is known, and their polynucleotide sequence also is like this.See, for example, " The WWW Guide To ClonedGlycosyltransferases, " (http://www.vei.co.uk/TGN/gt_guide.htm).The glycosyl transferase aminoacid sequence and can from its infer aminoacid sequence can encoding glycosyl the nucleotide sequence of transferring enzyme also be documented in many data bases that can openly obtain, comprise GenBank, Swiss-Prot, EMBL, and other.

Can include but not limited to glycosyl transferase in the method for the invention; galactosyltransferase, fucosyltransferase, glucosyltransferase; N-acetyl-amino galactosyltransferase; the N-acetylglucosaminyl transferase, glucuronyl transferase, sialyltransferase; mannose transferase; glucuronyl transferase, galacturonic acid transferring enzyme, and oligosaccharyl transferase.Suitable glycosyl transferase comprises those that obtain from eukaryote and prokaryote.

By chemosynthesis, by reverse transcription thing from suitable cell or cloned culture screening mRNA, by from suitable cell screening genomic library, or the combination by these methods, can obtain can the encoding glycosyl transferring enzyme DNA.Oligonucleotide probe with producing from the glycosyltransferase gene sequence can screen mRNA or genomic DNA.According to known and be used in method in the conventional cross experiment, can be with detectable group for example fluorophor, radioactive atom or chemiluminescent group label probe.In replacement scheme, by using polymerase chain reaction (PCR) method, utilize the PCR oligonucleotide primers that generates from the glycosyltransferase gene sequence, can obtain the glycosyltransferase gene sequence.See the U.S. Patent number 4,683,195 of Mullis etc. and the U.S. Patent number 4,683,202 of Mullis.

In carrier transformed host cells, can synthesize glycosyl transferase with the DNA that contains energy encoding glycosyl transferring enzyme.Use carrier increase can the encoding glycosyl transferring enzyme DNA, and/or express can the encoding glycosyl transferring enzyme DNA.Expression vector is reproducible DNA construct, and DNA sequence that wherein can the encoding glycosyl transferring enzyme is operably connected on the suitable control sequence, and the latter can realize the expression of glycosyl transferase in suitable hosts.To the needs of such control sequence, different with the method for transformation of host who selects and selection.Usually, control sequence comprises transcripting promoter, the operator sequence that optional control is transcribed, the sequence of the mRNA ribosome binding site that can encode suitable and can control the sequence of transcribing with translation termination.Amplification vector does not need to express control domain.The ability (this is given by origin of replication usually) that only need in the host, duplicate and select gene (with the identification of promotion transformant).

In exemplary embodiment, the present invention uses the prokaryote enzyme.Such glycosyl transferase comprises the enzyme that participates in synthetic fat oligosaccharide (LOS), and it generates (Preston etc., Critical Reviews in Microbiology 23 (3): 139-180 (1996)) by many gram negative bacteria.Such enzyme includes but not limited to, the albumen of the rfa operon of escherichia coli and Salmonella typhimurium (Salmonell atyphimurium), it comprises β 1,6 galactosyltransferases and β 1,3 galactosyltransferases (are seen, for example, EMBL registration number M80599 and M86935 (escherichia coli); EMBL registration number S56361 (Salmonella typhimurium); glucosyltransferase (Swiss-Prot registration number P25740 (escherichia coli); β 1; 2-glucosyltransferase (rfaJ) (Swiss-Prot registration number P27129 (escherichia coli) and Swiss-Prot registration number P19817 (Salmonella typhimurium); with β 1,2-N-acetyl-amino glucosyltransferase (rfaK) (EMBL registration number U00039 (escherichia coli).The glycosyl transferase of other of known its aminoacid sequence comprises by operon those of rfaB coding for example, it characterizes in biology, Klebsiella pneumonia (Klebsiella pneumoniae) for example, escherichia coli, Salmonella typhimurium, intestinal Salmonella (Salmonella enterica), yersinia enterocolitica (Yersinia enterocolitica), Mycobacteriumleprosum also comprises the rh1 operon of Pseudomonas aeruginosa (Pseudomonas aeruginosa).

Also be applicable to and among the present invention be; participate in generating and contain lacto-N-neotetraose; D-galactosyl-β-1; 4-N-acetyl group-D-aminoglucose base-β-1; 3-D-galactosyl-β-1; the 4-D-glucose; with Pk blood group trisaccharide sequence D-galactosyl-α-1; 4-D-galactosyl-β-1; the glycosyl transferase of the structure of 4-D-glucose (it identifies in the LOS of mucosal disease substance Diplococcus gonorrhoeae (Neisseriagonnorhoeae) and Neisseria meningitidis (N.meningitidis)) (Scholten etc., J.Med.Microbiol.41:236-243 (1994)).From Neisseria meningitidis immunologic pattern L3 and L1 (Jennings etc., Mol.Microbiol.18:729-740 (1995)) and Diplococcus gonorrhoeae mutant F62 (Gotshlich, J.Exp.Med.180:2181-2190 (1994)) in, the Neisseria meningitidis of the biosynthetic glycosyl transferase that having identified to encode participates in these structures and the gene of Diplococcus gonorrhoeae.In Neisseria meningitidis, by 3 gene lgtA, the locus that lgtB and lgE the form required glycosyl transferase (Wakarchuk etc., J.Biol.Chem.271:19166-73 (1996)) of last 3 sugar in the interpolation lacto-N-neotetraose chain of encoding.Recently, confirmed the enzymatic activity of lgtB and lgtA gene outcome, for their the glycosyl transferase function of supposition provides first hand positive evidence (Wakarchuk etc., J.Biol.Chem.271 (45): 28271-276 (1996)).In Diplococcus gonorrhoeae, there is 2 other genes: lgtD, it can add β-D-GalNAc to 3 positions of the terminal galactose of lacto-N-neotetraose structure, and lgtC, it can add terminal α-D-Gal to the lactose element of the LOS of truncate, thereby set up Pk blood group antigen structure (Gotshlich (1994), the same).In Neisseria meningitidis, independent immunologic pattern L1 also can express the Pk blood group antigen, and has been proved and carries lgtC gene (Jennings etc., (1995), the same).The Neisseria gonorrhoeae glycosyl transferase also is documented in USPN5 with relevant gene, among 545,553 (Gotschlich).Also characterized α 1,2-fucosyltransferase and α 1, the gene of 3-fucosyltransferase (Martin etc., J.Biol.Chem.272:21349-21356 (1997)) from helicobacter pylori (Helicobacter pylori).Also can use in the present invention campylobacter jejuni jejunum subspecies (Campylobacter jejuni) glycosyl transferase (see, for example, http://afmb.cnrs-mrs.fr/～pedro/CAZY/gtf_42.html).

Fucosyltransferase

In some embodiments, the glycosyl transferase that uses in the method for the invention is a fucosyltransferase.Fucosyltransferase is known to those skilled in the art.Exemplary fucosyltransferase comprises the enzyme that the L-fucose can be transferred to the hydroxy position of acceptor saccharide from the GDP-fucose.Non-nucleoside acid sugar can be transferred to the fucosyltransferase on the receptor, also can be with in the present invention.

In some embodiments, acceptor saccharide is, for example, and the GlcNAc in the Gal β in the oligosaccharide glucosides (1 → 3,4) the GlcNAc beta-yl group.The suitable fucosyltransferase that is used for this reaction comprises Gal β (1 → 3,4) GlcNAc β 1-α (1 → 3,4) fucosyltransferase (FTIII E.C.No.2.4.1.65), it characterizes from human milk first (sees, Palcic, etc., Carbohydrate Res.190:1-11 (1989); Prieels, etc., J.Biol.Chem.256:10456-10463 (1981); And Nunez, etc., Can.J.Chem.59:2086-2095 (1981)) and Gal β (1 → 4) GlcNAc β-α fucosyltransferase (FTVI), it is found in human serum for FTIV, FTV.Also characterized FTVII (E.C.No.2.4.1.65), i.e. saliva acidic group α (2 → 3) Gal β ((1 → 3) GlcNAc β fucosyltransferase.Also characterized the Gal β (1 → 3 of recombinant forms, 4) GlcNAc β-α (1 → 3,4) fucosyltransferase (is seen, Dumas, Deng, Bioorg.Med.Letters 1:425-428 (1991) and Kukowska-Latallo, etc., Genes and Development 4:1288-1303 (1990)).Other exemplary fucosyltransferase comprises, for example, and α 1,2 fucosyltransferase (E.C.No.2.4.1.69).By Mollicone,, can carry out the fucosylation of enzymatic etc., the method described in Eur.J.Biochem.191:169-176 (1990) or the U.S. Patent number 5,374,655.Be used to produce the cell of fucosyltransferase, also comprise the enzymatic system that is used for synthetic GDP-fucose.

Galactosyltransferase

In another group embodiment, glycosyl transferase is a galactosyltransferase.Exemplary galactosyltransferase comprises α (1,3) galactosyltransferase (E.C.No.2.4.1.151, see, for example, Dabkowski etc., Nature 345:229-233 (1990) such as Transplant Proc.25:2921 (1993) and Yamamoto, cattle (GenBank j04989, Joziasse etc., J.Biol.Chem.264:14290-14297 (1989)), Mus (GenBank m26925; Larsen etc., Proc.Nat ' l.Acad.Sci.USA 86:8227-8231 (1989)), pig (GenBank L36152; Strahan etc., Immunogenetics 41:101-105 (1995)).Another kind of suitable α 1,3 galactosyltransferase is to participate in synthetic blood group B antigenic (EC 2.4.1.37, Yamamoto etc., J.Biol.Chem.265:1146-1151 (1990) (mankind)).Another kind of exemplary galactosyltransferase is core Gal-T1.

Also be fit to use β (1 in the method for the invention, 4) galactosyltransferase, it comprises, for example, EC 2.4.1.90 (LacNAc synzyme) and EC 2.4.1.22 (lactose synthetase) (cattle (D ' Agostaro etc., Eur.J.Biochem.183:211-217 (1989)), people (Masri etc., Biochem.Biophys.Res.Commun.157:657-663 (1988)), Mus (Nakazawa etc., J.Biochem.104:165-168 (1988)), and E.C.2.4.1.38 and ceramide galactosyltransferase (EC 2.4.1.45, Stahl etc., J.Neurosci.Res.38:234-242 (1994)).Other suitable galactosyltransferase comprises, for example, and α 1,2 galactosyltransferase (from for example, foxtail millet wine fragmentation sugar yeast (Schizosaccharomyces pombe), Chapell etc., Mol.Biol.Cell 5:519-528 (1994)).

Sialyltransferase

Sialyltransferase is the glycosyl transferase of another kind of type, cell that it can be used for recombinating and reactant mixture of the present invention.Can produce the cell of the sialyltransferase of reorganization and also can produce cmp sialic acid, the latter is the sialic acid donor of sialyltransferase.The example that is applicable to sialyltransferase of the present invention (for example comprises ST3Gal III, rat or people ST3Gal III), ST3Gal IV, ST3Gal I, ST6Gal I, ST3Gal V, ST6Gal II, ST6GalNAcI, ST6GalNAc II, with ST6GalNAc III (sialyltransferase nomenclature of Shi Yonging such as Tsuji etc., Glycobiology 6:v-xiv (1996) is described) in this article.Exemplary α (2, the 3) sialyltransferase that is called α (2,3) sialyltransferase (EC 2.4.99.6) can be transferred to sialic acid the non-reduced terminal Gal of Gal β 1 → 3Glc disaccharide or glucosides.See Van denEijnden etc., J.Biol.Chem.256:3159 (1981), Weinstein etc., J.Biol.Chem.257:13845 (1982) and Wen etc., J.Biol.Chem.267:21011 (1992).Another kind of exemplary α 2,3-sialyltransferase (EC 2.4.99.4) can be transferred to sialic acid the non-reduced terminal Gal of disaccharide or glucosides.See Rearick etc., J.Biol.Chem.254:4444 (1979) and Gillespie etc., J.Biol.Chem.267:21004 (1992).Other exemplary enzyme comprises Gal-β-1,4-GlcNAc α-2,6 sialyltransferase (seeing Eur.J.Biochem.219:375-381 such as Kurosawa (1994)).

Preferably, glycosylation for the carbohydrate of glycopeptide, sialyltransferase can be transferred to sialic acid sequence Gal β 1,4GlcNAc-(it is the modal penult sequence below the terminal sialic acid on the fully sialylated carbohydrate structure) (seeing table 2).

Table 2: use Gal β 1, the 4GlcNAc sequence is as the sialyltransferase of receptor substrate

Sialyltransferase	The source	The sequence that forms	List of references
				ST6Gal I	Mammal	NeuAc 2，6Galβ1，4GlCNAc-	1
ST3Gal III	Mammal	NeuAc 2，3Galβ1，4GlCNAc- NeuAc 2，3Galβ1，3GlCNAc-	1
				ST3Gal IV	Mammal	NeuAc 2，3Galβ1，4GlCNAc- NeuAc 2，3Galβ1，3GlCNAc-	1
ST6Gal II	Mammal	NeuAc 2，6Galβ1，4GlCNA
				ST6Gal II	Photobacteria	NeuAc 2，6Galβ1，4GlCNAc-	2
ST3Gal V	The Neisseria meningitidis Diplococcus gonorrhoeae	NeuAc 2，3Galβ1，4GlCNAc-	3

1) Goochee etc., Bio/Technology 9:1347-1355 (1991)

2) Yamamoto etc., J.Biochem.120:104-110 (1996)

3) Gilbert etc., J.Biol.Chem.271:28271-28276 (1996)

The example of the sialyltransferase that uses in described method is ST3Gal III, and it is also referred to as α (2,3) sialyltransferase (EC 2.4.99.6).This enzyme can catalysis sialic acid to Gal β 1,3GlcNAc or Gal β 1, the transfer of the Gal of 4GlcNAc glucosides (see, for example, Wen etc., J.Biol.Chem.267:21011 (1992); Van den Eijnden etc., J.Biol.Chem.256:3159 (1991)), and the oligosaccharide of the agedoite-connection in the responsible glycopeptide is sialylated.Sialic acid is connected to Gal, forms the α-connection between 2 sugar.Bonding between the sugar (connection) is between the 3-position of the 2-position of NeuAc and Gal.Can separate this specific enzyme from following: rat liver (Weinstein etc., J.Biol.Chem.257:13845 (1982)); People cDNA (Sasaki etc. (1993) J.Biol.Chem.268:22782-22787; Kitagawa ﹠amp; Paulson (1994) J.Biol.Chem.269:1394-1401) and genome (Kitagawa etc. (1996) J.Biol.Chem.271:931-938) DNA sequence be known, help by this enzyme of recombinant expressed production.In a preferred embodiment, described sialylated method is used rat ST3Gal III.

In the present invention the exemplary sialyltransferase of other of Shi Yonging comprise from campylobacter jejuni jejunum subspecies isolating those, comprise α (2,3).See, for example, WO99/49051.

Sialyltransferase those that list except table 2 also can be used for commercially important glycopeptide sialylated economy and effective large-scale methods.Simple experiment as the effectiveness of finding these other enzyme makes every kind of not commensurability enzyme (1-100mU/mg albumen) and takes off sialic acid-α ₁AGP (1-10mg/ml) reaction, with contrast target sialyltransferase and cattle ST6Gal I, ST3Gal III or two kinds of sialyltransferases are compared the ability of sialylated glycopeptide.Perhaps, other glycopeptide, or the oligosaccharide of the N-that discharges from peptide main chain enzymatic ground connection can be used for substituting the sialic acid-α that takes off that is used for this evaluation ₁AGP.Can be used to the large-scale methods that realizes that peptide is sialylated than the ST6Gal I sialyltransferase of the oligosaccharide that connects of the N-of sialylated glycopeptide more effectively.

The GalNAc transferring enzyme

N-acetyl-amino galactosyltransferase can be used to realize the present invention, on the aminoacid of the glycosylation site that connects especially for the O-that the GalNAc group is attached to peptide.Suitable N-acetyl-amino galactosyltransferase includes but not limited to; α (1; 3) N-acetyl-amino galactosyltransferase; β (1; 4) N-acetyl-amino galactosyltransferase (Nagata etc.; J.Biol.Chem.267:12082-12089 (1992) and Smith etc., J.Biol Chem.269:15162 (1994)) and polypeptide N-acetyl-amino galactosyltransferase (Homa etc., J.Biol.Chem.268:12609 (1993)).

Produce for example enzyme GalNAcT of albumen by genetic engineering from cloned genes as everyone knows _1-XXSee, for example, U.S. Patent number 4,761,371.A kind of method comprises collects enough samples, determines the aminoacid sequence of enzyme then by the N-end sequencing.Then, use this information to come the cDNA clone of separation energy coding total length (membrane-bound) transferring enzyme, this cDNA is cloned among the insect cell line Sf9 and expresses, and causes the synthetic of complete active enzyme.Then, use the known glycosylation site amino acid whose semi-quantitative analysis on every side in 16 kinds of different albumen, study the glycosylation of synthetic peptide subsequently externally, determine the receptor-specific of enzyme.This work is verified, and some amino acid residue is excessive existence in glycosylated fragments of peptides, and the residue in the ad-hoc location around glycosylated serine and the threonine residues may have than other amino acid group receptor efficient remarkable influence more.

Cell-bonded glycosyl transferase

In another embodiment, the enzyme that uses in the method for the invention is cell-bonded glycosyl transferase.Although known many soluble glycosyl transferases (see, for example, U.S. Patent number 5,032,519), when following cell, glycosyl transferase is film-bonded form normally.Think that the many films-bonded enzyme of research so far is an albumen in the film; In other words, they are discharged from film, and need detergent to carry out solubilising by supersound process.On the surface of vertebrates and invertebral zooblast, identified surperficial glycosyl transferase, have realized that also these surperficial transferring enzymes can keep catalytic activity under physiological condition.But the more generally acknowledged function of cell surface glycosyl transferase is intercellular identification (Roth, MOLECULAR APPROACHESto SUPRACELLULAR PHENOMENA, 1990).

Developed the method that changes the glycosyl transferase of cellular expression.For example, Larsen etc., Proc.Natl.Acad.Sci.USA 86:8227-8231 (1989) has reported a kind of genetic method, with the cDNA sequence of separating clone, the latter can determine the expression of cell surface oligosaccharide structure and their associated sugars based transferase.Will be by expressing the UDP-galactose from known:. β .-D-galactosyl-1,4-N-acetyl group-D-glucosaminide α-1, the COS-1 cell is advanced in the cDNA library transfection that the isolating mRNA of the mouse cell line of 3-galactosyltransferase produces.Then, cultivate cells transfected, and measure α 1-3 galactosyltransferasactivity activity.

Francisco etc., Proc.Natl.Acad.Sci.USA 89:2713-2717 (1992) discloses a kind of method that beta-lactamase is anchored to colibacillary outer surface.Produced signal sequence by (i) outer membrane protein, (ii) outer membrane protein stride triple fusants that membrane portions and (iii) ripe completely beta-lactamase sequence are formed, produced the beta-lactamase molecule of activated surface combination.But the Francisco method only only limits to the prokaryotic cell system, and recognizes as the author, and suitable function needs complete triple fusants.

Sulfotransferase

The present invention also provides the method for producing peptide, and described peptide comprises Sulfated molecule, comprises, for example Sulfated polysaccharide is heparin for example, heparinoid, and carrageenin is with relevant chemical compound.Suitable sulfotransferase comprises, for example, and chrondroitin-6-sulfotransferase (Fukuta etc., the described chicken cDNA of J.Biol.Chem.270:18575-18580 (1995); GenBank registration number D49915, glycosaminoglycans N-acetyl glucosamine N-deacetylase/N-sulfotransferase 1 (Dixon etc., Genomics 26:239-241 (1995); UL18918), with glycosaminoglycans N-acetyl glucosamine N-deacetylase/N-sulfotransferase 2 (Orellana etc., J.Biol.Chem.269:2270-2276 (1994) and Eriksson etc., the described Mus cDNA of J.Biol.Chem.269:10438-10443 (1994); The described people cDNA of GenBank registration number U2304).

Glycosidase

The present invention also comprises use wild type and saltant glycosidase.Verified, the saltant beta galactosidase can be by being coupled to the alpha-glycosyl fluoride on the galactosyl acceptor molecule formation of catalysis disaccharide (Withers, U.S. Patent number 6,284,494; The mandate on the 4th of calendar year 2001 JIUYUE).Other glycosidase of Shi Yonging comprises in the present invention, for example, and β-Pu Tangganmei; beta galactosidase, beta-Mannosidase, β-acetyl-amino glucosidase; β-N-acetyl-amino tilactase, xylobiase, β-fucosidase; cellulase, xylanase, Galactanase; mannase; hemicellulase, amylase, glucoamylase; alpha-Glucosidase; alpha-galactosidase, alpha-Mannosidase, α-N-acetyl-amino glucosidase; α-N-acetyl-amino tilactase; α-xylosidase, Alpha-Fucosidase, and neuraminidase/sialidase.

Immobilized enzyme

The present invention also provides the application of the enzyme of immobilization on solid and/or the soluble carrier.In exemplary embodiment, glycosyl transferase is provided, its method according to this invention, the glycosyl junctional complex by complete is conjugated on the PEG.PEG-junctional complex-enzyme conjugate randomly is attached on the solid carrier.The enzyme application in the method for the invention that solid is supported can be simplified the foundation of reactant mixture and the purification of product, also can easily reclaim enzyme.The glycosyl transferase conjugate can be used in the method for the invention.Those skilled in the art can understand other combination of enzyme and support.

Fusion rotein

In other exemplary embodiment, method of the present invention is used to have and is surpassed a kind of fusion rotein that participates in the enzymatic activity of the synthetic glycopeptide conjugate that needs.Fused polypeptide can be made up of the catalytic activity territory of for example glycosyl transferase and the catalytic activity territory of connected auxiliary enzymes.The auxiliary enzymes catalytic domain can, for example, catalysis forms the step of nucleotide sugar (it is the donor of glycosyl transferase), or catalysis relates to the reaction of glycosyl transferase circulation.For example, the polynucleotide of energy encoding glycosyl transferring enzyme can be connected on the polynucleotide by reading frame, the latter can encode and participate in the synthetic enzyme of nucleotide sugar.Then, what the fusion rotein that obtains not only can the catalysis nucleotide sugar is synthetic, and can catalysis glycosyl group to the transfer of acceptor molecule.Fusion rotein can be two or more cyclophorases, and it connects into an effable nucleotide sequence.In other embodiment, fusion rotein comprises the catalytic activity territory of two or more glycosyl transferases.See, for example, 5,641,668.Can easily use various suitable fusion rotein designs and produce the glycopeptide (seeing that for example, PCT patent application PCT/CA98/01180, it is disclosed as WO 99/31224 on June 24th, 1999) of modification of the present invention.

The purification of erythropoietin conjugates

Can use the product of producing by said method without further purification.But, preferably reclaim product usually.Can use the technology of the glycosylated sugar of well-known recovery of standard, for example thin layer or thick layer chromatography, column chromatography, ion exchange chromatography or membrane filtration.Preferably use membrane filtration, more preferably use reverse osmosis membrane, or one or more column chromatography technology that are used to reclaim, as what discuss in the document of hereinafter quoting with this paper.For example, can use membrane filtration to remove for example glycosyl transferase of albumen, wherein film has about 3000 to about 10,000 weight shutoff value.Then, can use nanofiltration or reverse osmosis make a return journey desalination and/or purified product sugar (seeing that for example, WO 98/15581).Nanofiltration membrane is a class reverse osmosis membrane, and it can pass through monovalent salt, but can keep multivalent salts and greater than about 100 to about 2,000 daltonian uncharged solutes, depend on the film of use.Thereby in typical application, the sugar for preparing by method of the present invention can be retained in the film, and the salt that pollutes can pass through.

If the glycoprotein of modifying is to produce in the cell, as the first step, remove granular debris, no matter be host cell or cracked fragment, for example, by centrifugal or ultrafiltration; Randomly, can be with commercially available albumen thickening filtration device protein concentrate, then by being selected from following one or more steps, isolated polypeptide variant from other impurity: immunoaffinity chromatography, ion exchange column (for example separates, diethylamino ethyl (DEAE) contain carboxymethyl or the substrate of sulfo-propyl group in), at Blue-Sepharose, CM Blue-Sepharose, MONO-Q, MONO-S, lens culinaris agglutinin-Sepharose, WGA-Sepharose, Con A-Sepharose, Ether Toyopearl, Butyl Toyopearl, chromatograph on Phenyl Toyopearl or the protein A Sepharose, the SDS-PAGE chromatograph, the silicon dioxide chromatograph, chromatofocusing, reversed-phase HPLC (for example, silicon gel with additional aliphatic group), use for example Sephadex molecular sieve or the stratographic gel filtration of size exclusion, can be optionally in conjunction with the chromatograph on the post of polypeptide and ethanol or ammonium sulfate precipitation.

Usually, by preliminary extraction cell, enzyme etc., then by one or morely concentrate, saltout, aqueous ion exchange or size exclusion chromatographic step, be separated in the glycopeptide of the modification of producing in the culture.In addition, by affinity chromatography, the glycoprotein of can purification modifying.At last, can use HPLC to carry out last purification step.

Can with protease inhibitor for example Fumette (PMSF) be included in any aforesaid step, with the Profilin hydrolysis, and can comprise antibiotic, with the prevention external contaminant growth.

In another embodiment, use commercially available albumen thickening filtration device, for example, Amicon or Millipore Pellicon ultra filtration unit at first concentrate the supernatant from the system of the glycopeptide of producing modification of the present invention.Behind concentration step, concentrate can be applied on the suitable purification substrate.For example, suitable affinity substrate can comprise the part that is attached to the peptide on the suitable carriers, agglutinin or antibody molecule.Perhaps, can use anion exchange resin, for example, have the substrate or the substrate of the DEAE group of side extension.Suitable substrate comprises acrylamide, agarose, glucosan, cellulose, or other type of generally using in protein purification.Perhaps, can use cation-exchange step.Suitable cationite comprises various insoluble substrate, comprises sulfo-propyl group or carboxymethyl.The sulfo-propyl group is particularly preferred.

At last, can use one or more RP-HPLC steps (adopt hydrophobic RP-HPLC medium, for example, have the methyl of side extension or the silicon gel of other aliphatic group), to be further purified the polypeptide variants compositions.Also can use some or all of aforesaid purification steps, the glycoprotein of the modification of homogeneous is provided with different combinations.

By being similar to Urdal etc., J.Chromatog.296:171 (1984) disclosed method, the glycopeptide of the modification of the present invention that can purification obtains by large scale fermentation.The document has been described 2 successive RP-HPLC steps that are used for purification of Recombinant human IL-2 on preparation scale HPLC post.Perhaps, can use technology such as affinity chromatography, the glycoprotein that purification is modified.

Pharmaceutical composition

In yet another aspect, the invention provides pharmaceutical composition.This pharmaceutical composition comprises the covalent conjugates between PEG group, treatment group or biomolecule and the glycosylated or non--glycosylated peptide that pharmaceutically acceptable diluent and non-natural ground produces.Polymer, treatment group or biomolecule are conjugated on the peptide by placing between peptide and polymer, treatment group or the biomolecule and covalently connecting thereon complete glycosyl linking group.

Pharmaceutical composition of the present invention is applicable to many delivery systems.The appropriate formulation of Shi Yonging in the present invention is referring to Remington ' s Pharmaceutical Sciences, MacePublishing Company, Philadelphia, PA, 17th ed. (1985).About the simple comment of medicament delivery method, see Langer, Science 249:1527-1533 (1990).

Pharmaceutical composition can be mixed with and be used for any suitable administering mode, for example comprise, partial, per os, nose, intravenous, intracranial, endoperitoneal, subcutaneous or intramuscular administration.For parenteral, subcutaneous injection for example, carrier preferably comprises water, saline, alcohol, fat, wax or buffer agent.For oral administration, can use any above-mentioned carrier or solid carrier, mannitol for example, lactose, starch, magnesium stearate, saccharin sodium, Talcum, cellulose, glucose, sucrose, and magnesium carbonate.Also can be with the carrier of biodegradable microsphere (for example, poly-lactic acid ester polyglycolic acid ester) as pharmaceutical composition of the present invention.Suitable biodegradable microsphere is documented in, for example, and U.S. Patent number 4,897,268 and 5,075,109.

Usually, the intestines and stomach other places drug administration compositions, for example, intravenous ground.Thereby, the invention provides the compositions that is used for parenteral administration, it comprises the chemical compound that is dissolved or suspended in the suitable carriers, preferred aqueous carrier, for example, water, buffered water, saline, PBS etc.For near physiological condition, compositions can contain pharmaceutically acceptable auxiliary substance, for example pH regulator agent and buffer agent, tension regulator, wetting agent, detergent etc.

By the sterilization technology of routine, these compositionss can be sterilized, or can filtration sterilization.The aqueous solution former state that obtains can be packaged spare, or lyophilizing, before using, freeze dried goods are mixed mutually with aseptic aqueous carrier.The pH of goods is 3-11 typically, more preferably 5-9, most preferably 7-8.

In some embodiments, glycopeptide of the present invention can be integrated in the liposome that the lipid that can form vesicle by standard forms.Can obtain many methods that prepare liposome, for example, Szoka etc., Ann.Rev.Biophys.Bioeng.9:467 (1980), U.S. Patent number 4,235,871,4,501,728 and 4,837,028 is described.Using many targeting agent (for example, saliva acidic group galactoside of the present invention) guiding liposome, is (see, for example, U.S. Patent number 4,957,773 and 4,603,044) well-known in the art.

Can use the standard method that the targeting agent is coupled to liposome.These methods comprise usually lipidic component (for example PHOSPHATIDYL ETHANOLAMINE) are integrated in the liposome, and it can be activated, and is used in conjunction with targeting agent or deutero-lipophilic chemical compound, lipid for example of the present invention-deutero-glycopeptide.

The common requirement of targeting mechanism, the targeting agent can be positioned at the surface of liposome with the interactional mode of target (for example, cell surface receptor) with the target group.Use method known to those skilled in the art (for example, using the hydroxyl on chain alkyl halogenide or fatty acid alkanisation or the acidylate carbohydrate respectively), can before forming liposome, carbohydrate of the present invention be attached on the lipid molecule.Perhaps, can construct liposome in the following manner, the coupling part at first is integrated in the film when forming film.The coupling part must have lipophilic portion, its implant securely with anchor in film.It also must have reactive moieties, and it can be on the aqueous surface of liposome and participate in chemical reaction.The selective response part chemically is applicable to the targeting agent or the carbohydrate that add later on it and forms stable chemical bond.In some cases, target reagent directly can be attached on the link molecule, but in most of the cases, more suitably be to use the third molecule as chemical bridge, thereby the link molecule in the film is connected with targeting agent or carbohydrate, and the latter extends the vesicle surface three-dimensionally.

Chemical compound by method preparation of the present invention also can be used as diagnostic reagent.For example, the chemical compound of labelling can be used for locating the zone or the neoplasm metastasis of the inflammation of suspecting the patient who suffers from inflammation.For this purposes, can use ¹²⁵I, ¹⁴C, or tritiated compound.

The active component that uses in pharmaceutical composition of the present invention is that Glycopegylated erythropoietin has the derivant that makes medullary cell increase production skein cell and erythrocytic biological property with it.Liposome disperse system of the present invention can be as the parenteral administration of treatment hematologic effects, described hematologic effects is characterised in that low or defective erythrocyte production, for example various forms of anemias, comprise the anemia relevant with chronic renal failure, the patient that the HIV of zidovidine treatment infects and the cancer patient of chemotherapy.It also can be used for the treatment of the not normal state of various disease states, obstacle and hematology, drepanocytosis for example, and β-thalassemia, cystic fibrosis, pregnancy and menoxenia, the early stage anemia of prematureness, spinal cord injury, space flight, acute bleeding, aging etc.Preferably, the intestines and stomach other places (for example IV, IM, SC or IP) uses EPO compositions of the present invention.Effective dose can change greatly according to the disease of treatment and route of administration, but scope should be the active substance of about 0.1 (about 7U) to 100 (about 7000U) μ g/kg body weight.The preferred dosage that is used for the treatment of the anemia disease is about 50 to about 300 units/kg, 3 times weekly.Because the invention provides erythropoietin, when using compositions of the present invention, can randomly reduce described dosage with time of staying in the enhanced body.

Provide the following examples to explain conjugate of the present invention and method, but can requirement for restriction the invention of protection.

Embodiment

Embodiment 1

The preparation of UDP-GalNAc-6 '-CHO

(200mg 0.30mmol) is dissolved in 1mM CuSO with UDP-GalNAc ₄Solution (20mL) and 25mM NaH ₂PO ₄(pH 6.0 for solution; 20mL).Then, add beta-Galactose oxidase (240U; 240 μ L) and catalase (13000U; 130 μ L), response system has assembled the balloon of filling oxygen, and stirring at room 7 days.Then, filter reaction mixture (concentrator bowl; MWCO 5K), (about 40mL) is deposited in 4 ℃ with filtrate, up to needs.TLC (silicon dioxide; EtOH/ water (7/2); R _f=0.77; Visual with the anisaldehyde dyestuff).

Embodiment 2

UDP-GalNAc-6 '-NH ₂Preparation:

At 0 ℃, with ammonium acetate (15mg, 0.194mmol) and NaBH ₃CN (1M THF solution; 0.17mL, 0.17mmol) add the UDP-GalNAc-6 '-CHO solution (2mL or about 20mg) that obtains from above, be heated to ambient temperature overnight.Through G-10 post filtering reaction thing, collect water and product.The corresponding fraction of lyophilization, and freezing.TLC (silicon dioxide; Ethanol/water (7/2); R _f=0.72; Visual with ninhydrin reagent).

Embodiment 3

UDP-GalNAc-6-NHCO (CH ₂) ₂The preparation of-O-PEG-OMe (1KDa)

With galactosaminyl-1-phosphoric acid-2-NHCO (CH ₂) ₂(58mg 0.045mmol) is dissolved in DMF (6mL) and the pyridine (1.2mL)-O-PEG-OMe (1KDa).Add then UMP-morpholidate (60mg, 0.15mmol), the mixture 48h that obtains 70 ℃ of stirrings.By with nitrogen bubbling in reactant mixture, remove solvent, by reversed phase chromatography (C-18 silicon dioxide, the stagewise gradient of 10-80%, methanol) purification residue.Collect the fraction that needs, and drying under reduced pressure, 50mg (70%) white solid produced.TLC (silicon dioxide, propanol/H ₂O/NH ₄OH, (30/20/2), R _f=0.54).MS (MALDI): observed value, 1485,1529,1618,1706.

Embodiment 4

Cysteine-PEG ₂(2) preparation

4.1 chemical compound 1 is synthetic

Under argon, potassium hydroxide (84.2mg, 1.5mmol is as powder) is added L-cysteine (93.7mg, 0.75mmol) solution in absolute methanol (20L).In stirring at room mixture 30 minutes, then,, divide several parts to add the mPEG-O-toluenesulfonic acid (Ts of molecular weight 20 kilodaltons through 2 hours; 1.0g, 0.05mmol).Mixture stirring at room 5 days, is concentrated by rotary evaporation.Water (30mL) dilution residue, and, destroy all 20 unnecessary kilodalton mPEG-O-toluenesulfonic acids stirring at room 2 hours.Then, use the acetic acid neutralization solution, pH regulator to pH 5.0, and is loaded on reversed phase chromatography (C-18 silicon dioxide) post.With the gradient elution post (at about 70% methanol-eluted fractions product) of methanol, monitor the product eluting by the steam light scattering, and collect corresponding fraction, water (500mL) dilution.This solution is carried out chromatograph (ion exchange, XK 50Q, BIG pearl, 300ml, hydroxide form; Water is to the gradient of water/acetic acid-0.75N), with acetic acid the pH of corresponding fraction is reduced to 6.0.Then, on reversed-phase column (C-18 silicon dioxide), catch this solution, and with aforesaid methanol gradient elution.Merge the product fraction, concentrate, soluble in water again, and lyophilization, obtain 453mg (44%) white solid (1).The structured data of this chemical compound is as follows: ¹H-NMR (500MHz; D ₂O) δ 2.83 (t, 2H, O-C-CH ₂-S), 3.05 (q, 1H, S-CHH-CHN), 3.18 (q, 1H, (q, 1H, S-CHH-CHN), 3.38 (s, 3H, CH ₃O), 3.7 (t, OCH ₂CH ₂O), 3.95 (q, 1H, CHN).By SDS PAGE, confirmed degree of purity of production.

4.2 chemical compound 2 (cysteine-PEG ₂) synthetic

Triethylamine (about 0.5mL) is dropwise added chemical compound 1 (440mg, 22 μ mol) be dissolved in anhydrous CH ₂Cl ₂In the solution (30mL), be alkalescence up to solution.Through 1 hour,, divide several parts to add 20 kilodalton mPEG-O-p-nitrophenyl carbonic esters (660mg, 33 μ mol) and N-hydroxy-succinamide (3.6mg, 30.8 μ mol) at CH in room temperature ₂Cl ₂Solution (20mL).Stirring at room reactant mixture 24 hours.Then, remove solvent,, pH is transferred to 9.5 with 1.0N NaOH with residue water-soluble (100mL) by rotary evaporation.Stirring at room alkaline solution 2 hours, be neutralized to pH 7.0 with acetic acid then.Then, with sample on the solution to reversed phase chromatography (C-18 silicon dioxide) post.With the gradient elution post (at about 70% methanol-eluted fractions product) of methanol, monitor the product eluting by the steam light scattering, and collect corresponding fraction, water (500mL) dilution.This solution is carried out chromatograph (ion exchange, XK 50Q, BIG pearl, 300ml, hydroxide form; Water is to the gradient of water/acetic acid-0.75N), with acetic acid the pH of corresponding fraction is reduced to 6.0.Then, on reversed-phase column (C-18 silicon dioxide), catch this solution, and with aforesaid methanol gradient elution.Merge the product fraction, concentrate, soluble in water again, and lyophilization, obtain 575mg (70%) white solid (2).The structured data of this chemical compound is as follows: ¹H-NMR (500MHz; D ₂O) δ 2.83 (t, 2H, O-C-CH ₂-S), 2.95 (t, 2H, O-C-CH ₂-S), 3.12 (q, 1H, S-CHH-CHN), 3.39 (s, 3H CH ₃O), 3.71 (t, OCH ₂CH ₂O).By SDS PAGE, confirmed degree of purity of production.

Embodiment 5

UDP-GalNAc-6-NHCO (CH ₂) ₂The preparation of-O-PEG-OMe (1KDa)

With galactosaminyl-1-phosphoric acid-2-NHCO (CH ₂) ₂(58mg 0.045mmol) is dissolved in DMF (6mL) and the pyridine (1.2mL)-O-PEG-OMe (1 kilodalton).Then, add UMP-morpholidate (60mg, 0.15mmol), and the mixture that obtains 70 ℃ of stirrings 48 hours.By with nitrogen bubbling in reactant mixture, remove solvent, by reversed phase chromatography (C-18 silicon dioxide, the stagewise gradient of 10-80%, methanol) purification residue.Collect the fraction that needs, and drying under reduced pressure, 50mg (70%) white solid produced.TLC (silicon dioxide, propanol/H ₂O/NH ₄OH, (30/20/2), R _f=0.54).MS (MALDI): observation, 1485,1529,1618,1706.

Embodiment 6

GnT1 in single jar and GalT1 reaction

6.1 the reaction in single jar

By at 32 ℃, containing 150mM NaCl, 5mM UDP-GlcNAc, 5mM UDP-Gal, 5mM MnCl ₂0.02% Hydrazoic acid,sodium salt, hatch EPO (1mg/mL) 16h among the 100mM Tris HCl pH 7.5 of the GlcNAc transferring enzyme-1 of 30mU/mL purification and the galactosyl transferase-1 of 200mU/mL purification or the MESpH 6.5, GlcNAc transferring enzyme-1 in carrying out single jar and galactosyl transferase-1 reaction.

6.2 purification EPO on Superdex75

With the flow velocity of 5mL/min, with 100mM MES pH of buffer 6.5 balance Superdex 75 posts that contain 150mM NaCl.To be loaded on the post from the product EPO of step 6.1 (top), and use the level pad eluting.By absorbance and electrical conductivity, monitor eluate at 280nm.Use SDS-PAGE to determine that the peak fraction of which collection contains EPO, and be used for other experiment.

6.3 ST3Gal-III reaction

By at 32 ℃; containing 150mM NaCl; 0.5mM CMP-N-acetyl group-neuraminic acid-20 kilodaltons-PEG; 0.02% Hydrazoic acid,sodium salt; with hatch 1mg/mLEPO-Gal (from top step 6.2) 16h among the 100mM Tris HCl pH 7.5 of the ST3Gal-III of 200mU/mL purification or the MES pH 6.5, carry out ST3Gal III reaction.

Embodiment 7

GnT1 in single jar, GalT1 and ST3Gal-III (using CMP-NAN-20KPEG) reaction

In 100mM MES pH of buffer 6.5; with 30mU/mL GlcNAc transferring enzyme-1; 200mU/mL galactosyl transferase-1 and 500mU/mL ST3GalIII, ribotide and CMP-N-acetyl group-neuraminic acid-20Kd PEG is hatched EPO (1mg/mL), and uses SDS-PAGE to analyze.Similar with the result who in the enzyme reconstruct reaction of two steps, obtains, in single jar three enzyme preparation, observed the EPO of the PEGization of 3 bands.

Embodiment 8

The production of the PEG-EPO of two feelers

8.1 GlcNAc is to the interpolation of rEPO

At 32 ℃, use 3mM USP-GlcNAc, 50mU/mg GlcNAc transferring enzyme-1 and 50mU/mg GlcNAc transferring enzyme-II are incubated in 0.1M Tris, 0.15M NaCl, 5mMMnCl ₂With the EPO of the reorganization of in baculovirus (1mg/mL), expressing among the 0.02% Hydrazoic acid,sodium salt pH 7.2 24 hours.

8.2 the interpolation of galactose

In the peptide of the GlcNAc-labelling of step 8.1 (above), add 3mM UDP-Gal and 0.2U/mg galactosyl transferase-1.Mixture was hatched 36 hours at 32 ℃.By the gel filtration chromatography on Superdex 75 posts, in the Tris-buffered saline, separate the product of galactosylation.The product of purification is concentrated into 1mg/mL.

8.3 the interpolation of sialic acid or sialic acid PEG

At 32 ℃, (wherein PEG has molecular weight 5 kilodaltons with 200mU/mg ST3GalIII and 0.5mM cmp sialic acid or cmp sialic acid-PEG, 10 kilodaltons or 20 kilodaltons), at 0.1M Tris, hatch among the 0.1M NaCl pH 7.2 from the product (1mg/mL) of the galactosylation of step 8.2 (top) 24 hours.

Embodiment 9

Carry out the 30K PEGization that N-connects by CST-II

(5mg, 0.166 μ mol is 5ml) also with tris buffer (50mM Tris, 0.15M NaCl, 0.001M CaCl to be concentrated in the glycosylated EPO that expresses in CHO (Chinese hamster ovary) cell ₂+ 0.005%NaN3) buffering exchanges to the final volume of 5ml.Then, add cmp sialic acid-PEG (30 kilodaltons, 25mg, 0.833 μ mol; The structure of 30K dalton cmp sialic acid-PEG is seen Fig. 3 B), 0.25mL 100mM MnCl ₂0.25ml and from the bifunctional sialyltransferase CST-II of campylobacter jejuni jejunum subspecies (1.4U/mL, 0.5ml, 0.7U).The mixture that obtains was shaken 48 hours at 32 ℃.

When reaction finished, to the 1mL final volume, enriched mixture used 25mM NaOAc+0.005% Tween-80 (pH 6.0) buffering to exchange to 2.5ml then by ultrafiltration.Use 25mM NaOAc+2M NaCl+0.005% Tween-80 (pH 6.0) as eluant, carry out Q-Sepharose IEX chromatograph.Collect peak 2, and by ultrafiltration and concentration to 1.5ml, use 1XPBS (pH 5.5+0.005% Tween80) as eluant then, carry out the superdex-200 purification (post: Superdex 200,16/60 GL, Amersham).Collect peak 2, and be concentrated into 1.5ml.With the material aseptic filtration that obtains, and use 10mM NaOAc (0.75%NaCl, pH 5.5), prepare final volume to 2.5mL.Protein concentration 264 μ g/ml; 660 μ g albumen (BCA mensuration) have been obtained.

Embodiment 10

The following examples have illustrated the method for using ST3GalIII to prepare the EPO that 40 kilodalton PEG that O-connects connect.

10.1 asialoglycoproteinization

In this step, the EPO asialoglycoproteinization that will in Chinese hamster ovary cell (Chinese hamster ovary celI), grow.GlcNAc-Gal connects as the receptor that shifts the sialic acid PEG that modifies in the step 10.2 below.

With Tris buffer (20mM Tris, 50mM NaCl, 5mM CaCl ₂, 0.02% NaN ₃, pH 7.2) and cushion the glycosylated EPO solution 10ml (10mg, 0.33 μ mol) that exchange is expressed in CHO (Chinese hamster ovary) cell, obtain the 10ml final volume.Then, the 750mU 2,3,6 of self-produced urea arthrobacterium in future (Arthrobacter Ureafaciens), 8-neuraminidase (neuramidase) adds solution.At 32 ℃, shook the mixture that obtains 48 hours.The product of this step is directly used in the next procedure (face as follows) of this method.

10.2 the 40K PEGization that O-connects

In this step, use the O-sialyltransferase with sialic acid-PEG group transfer of modifying to EPO from the asialoglycoproteinization of top step 10.1.

With cmp sialic acid-PEG (40 kilodaltons, 33mg, 0.825 μ mol; The structure of 40 kilodalton CMP-SA-PEG is seen Fig. 3 A), and the O-sialyltransferase (1.4U/ml, 300mU) and 0.25mL 100mM MnCl ₂Add the mixture above half.At 32 ℃, shook this mixture 48 hours.After 48 hours,, reactant mixture is concentrated into 2.8ml, uses 25mM NaOAc+0.001% Tween-80 (pH 6.0) buffering to exchange to the 3ml final volume then by ultrafiltration (MWCO 5K).Go up the ion-exchange purification end-product 3 times at SP (5mL) (3 injections, each 1ml).Collect the EPO (peak 2) of PEGization, to the 2ml final volume, be used for the SEC purification by ultrafiltration and concentration.Purification on superdex 200, the final step that can provide the parsing of target protein: EPO-GlcNAc-Gal-SA-PEG (40K) to be used to react.

10.3 the complete end of CHO-EPO-GalNAc-Gal-SA-PEG (40K) is sialylated

In this step of method, sialic acid is added to the end of the glycosyl structure of the sialic acid residues that does not carry modification.

EPO (about 2mg is from the reaction among the top step b) by the concentrated PEGization that merges of ultrafiltration (MWCO 5K) uses tris buffer (0.05M Tris, 0.15M NaCl, 0.001M CaCl then ₂+ 0.005% NaN ₃) buffering exchanges to the 2mL final volume.Then, add CMP-N-acetyl neuraminic acid (CMP-NANA; 1.5mg, 2.4 μ mol), (8.9U/mL, 10 μ l are 0.089U) with 50 μ l 100mM MnCl for ST3GalIII ₂The mixture that obtains was shaken 24 hours at 32 ℃, be concentrated into the 1ml final volume then.Use 1XPBS (pH5.5+0.005% Tween 80) as eluant, this solution is directly carried out Superdex 200 purification.Collect peak 1, and be diluted to 10ml.Protein concentration 52.8ug/ml (BCA).Totally 528 μ g albumen have been obtained.The sketch map of final peptide prod is presented among Fig. 4 A.

Embodiment 11

In this embodiment, use ELISA to measure, contrasted the pharmacokinetic characteristic of the Glycopegylated variant of deutero-EPO of CHO-(sketch map is presented at Fig. 5) that intravenous uses and the deutero-EPO of CHO-.

After giving rat single 30 μ g/kg intravenous administrations, by ELISA, the pharmacokinetics of the deutero-erythropoietin of CHO-(Fig. 4 A) of the deutero-erythropoietin of CHO-(Fig. 4 B) of the 30KPEGization of contrasted the deutero-erythropoietin of CHO-of 2 batches of non--PEGization, producing by method of the present invention and the 40K PEGization by method production of the present invention.

11.1 preparation ELISA flat board

With 100 μ L/ holes, will distribute at the capture antibody of people EPO the institute of 96-hole flat board into porose in.Cover flat board with dull and stereotyped fluid sealant, and hatched 2 hours at 37 ℃.By with Tris-buffered saline (TBST) washing that contains 0.2%Tween-20 2 times, remove capture antibody from flat board.After the washing, 3% milk lock solution (TBST+3% milk) is added flat board for the third time, cover flat board with dull and stereotyped fluid sealant, and 4 ℃ of overnight incubation.

In the morning, by with TBST washing 3 times, remove lock solution.Suitably dilute rat plasma sample and standard protein with rat plasma, and be assigned in the hole with 100 μ L/ holes.Cover flat board with dull and stereotyped fluid sealant, and 4 ℃ of overnight incubation.

Morning next day, use standard protein, produce the linear regression of standard for every kind of epo protein testing its pharmacokinetic property.Carry out the rp-hplc analysis of standard protein, and by area under the corresponding peak of albumen that calculates and test, detectable concentration.

11.2 preparation and interpolation sample

The dilution per sample (p.s.), and, the sample that dilutes is distributed into ELISA flat board with 100 μ L/ holes.Then, cover flat board with dull and stereotyped fluid sealant, and 4 ℃ of overnight incubation.

11.3 measure the europium counting

In the morning, remove the rat blood serum sample, and wash dull and stereotyped 3 times with TBST.To be that mice Anti-Human EPO is applied on the ELISA flat board with the europium labelling and through the detection antibody of gel filtration column purification in advance.Under 100rpm stirs, dull and stereotyped 1 hour of incubated at room.

By with dull and stereotyped 6 times of TBST washing, remove and detect antibody.With 200 μ L/ holes, will strengthen solution add dull and stereotyped, dull and stereotyped 20 minutes of incubated at room.Use the europium counting procedure, read fluorescence with the Wallac plate reader.

11.4 result

11.4a the linear regression of the standard of generation

Use produces the linear regression curve and the equation of standard from the europium counting of each dull and stereotyped standard protein.Use this equation, the europium counting is changed into the corresponding EPO amount of each sample well.

11.4b pharmacokinetics result

The result as shown in Figure 6.The detectable limit of the EPO of non--PEGization is about 0.4ng/mL, and the detectable limit of the EPO of 30 kilodaltons and 40 kilodalton PEGization is about 0.8ng/mL.

The deutero-EPO of the CHO-of 30 kilodalton PEGization compares with the homologue of their non--PEGization with the deutero-EPO of the CHO-of 40 kilodalton PEGization, clearly shows very superior intravenous and removes parameter.As can be seen from Fig., the order of various EPO isotypes is: the deutero-EPO of CHO-of the deutero-EPO of the CHO-of 40 kilodalton PEGization～30 kilodalton PEGization〉〉〉homologue of non--PEGization.

Embodiment 12

In this embodiment, use ELISA to measure, detected the pharmacokinetic characteristic of the deutero-Glycopegylated EPO of non--Glycopegylated EPO, the Glycopegylated EPO of insect cell growth of the deutero-erythropoietin of CHO-(EPO), high glycosylation of subcutaneous administration and Chinese hamster ovary celI.

Use 10 μ g/kg subcutaneous dosages of single to rat after, pass through ELISA, contrasted the insect cell-deutero-erythropoietin (sketch map is seen Fig. 7) of the PEGization that the deutero-EPO of CHO of the high glycosylation of non--Glycopegylated deutero-EPO of CHO-, non--PEGization, the Glycopegylated deutero-EPO of insect cell, 10K N-connect, the pharmacokinetics of the deutero-erythropoietin of CHO-(seeing Fig. 4 A) of the PEGization that is connected with 40 kilodalton O-.

Prepared the ELISA flat board, and as sealing as described in the embodiment 10.Also prepare standard protein, and measured the europium counting as mentioned above.

12.1 preparation and interpolation rat sample

After subcutaneous (S.C.) injection, to compare with the intravenous injection of equivalent, the amount of the EPO in the circulation reduces.In injection back 30 minutes to 48 hours, the plasma concentration of the epo protein that general energy measurement is injected to S.C..

12.2 pharmacokinetics result

These result of experiment as shown in Figure 8.Fig. 8 shown for each EPO variant group, different time point behind inject time=0 hour, average magnitude and standard deviation in the EPO of ng/mL in the rat blood serum sample.The detectable limit of the EPO of non--PEGization and the EPO of PEGization is about 0.3ng/mL.

Under the situation of the EPO of the 10K PEGization of growing in insect cell and the CHO-EPO of 40 kilodalton PEGization, absorb seemingly gradually, set up such situation, wherein many such EPO variants keep being absorbed considerably beyond peak serum levels (C _Max).

The EPO variant of the 10K PEGization of growing in insect cell time range of 24-36 hour after injection reaches C _MaxBut the CHO-EPO variant of 40 kilodalton PEGization reached C in 40-60 hour after injection _MaxIn addition, after the current injected dose of injection 96 hours, there is the variant of the PEGization of considerable level.

Serum ordering t _1/2As follows: the CHO-EPO of 40 kilodalton PEGization〉the EPO variant of the 10K PEGization of in insect cell, growing〉CHO-EPO of high glycosylation〉CHO-EPO. of non--PEGization

Embodiment 13

The EPO variant (A and B) of 2 kinds of non--PEGization and the PEG of two kinds of Glycopegylated variants (30 kilodaltons and 40 kilodalton PEG) and high glycosylation are contrasted at the relative activity aspect the TF1 cell proliferation that carries the EPO receptor that stimulates cultivation.The Glycopegylated activity of EPO peptide in this mensuration is similar to the EPO variant of high glycosylation.

Embodiment 14

EPO of the not PEGization of various concentration (A and B) and Glycopegylated 30 kilodaltons and 40 kilodalton PEG variants are to the EPO of the isotope-labelling bonded inhibition to the chimeric EPO receptor of reorganization.With regard to receptor affinity (Ki), the EPO of PEGization and Glycopegylated variant are not similar.

Be to be understood that, embodiment as herein described and embodiment only are used for illustrative purpose, those skilled in the art can propose various improvement or variation with reference to them, and described improvement and variation are also included within the scope of the spirit and scope of this application and appended claims.All publications, patent and the patent application of quoting in this article all here are that all purpose integral body are incorporated by reference.

Claims (34)

1. erythropoietin peptide, it comprises group:

Wherein

D is selected from-OH and R ¹The member of-L-HN-;

G is selected from R ¹-L-and-C (O) (C ₁-C ₆) member of alkyl;

R ¹It is the group that comprises the member who is selected from the group that contains straight chain or ramose Polyethylene Glycol residue; With

L is a junctional complex, and it is to be selected from key, replacement or member unsubstituted alkyl and replacement or unsubstituted assorted alkyl,

Thereby when D was OH, G was R ¹-L-, and as G be-C (O) (C ₁-C ₆) during alkyl, D is R ¹-L-NH-.

2. according to the peptide of claim 1, L-R wherein ¹Have formula:

Wherein

A is the integer of 0-20.

3. according to the peptide of claim 1, R wherein ¹Have the following structures of being selected from:

Wherein

E and f are the integers that is independently selected from 1-2500; And

Q is the integer of 0-20.

4. according to the peptide of claim 1, R wherein ¹Have the following structures of being selected from:

Wherein

E, f and f ' are the integers that is independently selected from 1-2500; And

Q and q ' are the integers that is independently selected from 1-20.

5. according to the peptide of claim 1, R wherein ¹Have the following structures of being selected from:

Wherein

E, f and f ' are the integers that is independently selected from 1-2500; And

Q, q ' and q " be the integer that is independently selected from 1-20.

6. according to the peptide of claim 1 R wherein ¹Have the following structures of being selected from:

Wherein

E and f are the integers that is independently selected from 1-2500.

7. according to the peptide of claim 1, wherein said group has formula:

8. according to the peptide of claim 1, wherein said group has formula:

9. according to the peptide of claim 1, wherein said group has formula:

Wherein AA is the amino acid residue of described peptide.

10. according to the peptide of claim 9, wherein said amino acid residue is the member who is selected from serine or threonine.

11. according to the peptide of claim 10, wherein said peptide has the aminoacid sequence of SEQ.ID.NO:1.

12. according to the peptide of claim 11, wherein said amino acid residue is the serine at 126 places, position of SEQ.ID.NO:1.

13. according to the peptide of claim 1, wherein said peptide comprises at least one described group according to following formula:

Wherein AA is the amino acid residue of described peptide, and t equals 0 or 1 integer.

14. according to the peptide of claim 13, wherein said amino acid residue is an asparagine residue.

15. according to the peptide of claim 14, wherein said peptide has the aminoacid sequence of SEQ ID NO:1, and wherein said amino acid residue is asparagine residue, it is to be selected from N24, N38, the member of N83 and its combination.

16. according to the peptide of claim 1, wherein said peptide comprises at least one described group according to following formula:

Wherein AA is the amino acid residue of described peptide, and t equals 0 or 1 integer.

17. according to the peptide of claim 16, wherein said amino acid residue is an arginine residues.

18. according to the peptide of claim 17, wherein said peptide has the aminoacid sequence of SEQ ID NO:1, and wherein said amino acid residue is asparagine residue, it is to be selected from N24, N38, the member of N83 and its combination.

19. the peptide of claim 1, wherein said peptide comprise at least one described group according to following formula:

Wherein AA is the amino acid residue of described peptide, and t equals 0 or 1 integer.

20. according to the peptide of claim 1, wherein said peptide comprises at least one described group according to following formula:

Wherein AA is the amino acid residue of described peptide, and t equals 0 or 1 integer.

21. according to the peptide of claim 20, wherein said amino acid residue is an asparagine residue.

22. according to the peptide of claim 21, wherein said peptide has the aminoacid sequence of SEQ ID NO:1, and wherein said amino acid residue is asparagine residue, it is to be selected from N24, N38, the member of N83 and its combination.

23. according to the peptide of claim 1, wherein said peptide is bioactive erythropoietin peptide.

24. according to the peptide of claim 23, wherein said peptide has promoting erythrocyte and generates active.

25. according to the peptide of claim 24, wherein said peptide does not have promoting erythrocyte basically and generates active.

26. according to the peptide of claim 25, wherein said peptide is a tissue protective.

27. the method for the erythropoietin that comprises following radicals of preparation PEG-ization:

Wherein

R ¹It is the group that comprises straight chain or ramose Polyethylene Glycol residue; And

L is a junctional complex, and it is to be selected from replacement or member unsubstituted alkyl and replacement or unsubstituted assorted alkyl,

Described method comprises:

(a) under the condition that is suitable for shifting, make the substrate erythropoietin peptide that comprises following glycosyl:

Contact has the PEG-sialic acid donor group of following formula:

With the enzyme on the Ga1 that described PEG-sialic acid can be transferred to described glycosyl.

28. the method for claim 27 also comprises, in step (a) before:

(b) in suitable hosts, express described substrate erythropoietin peptide.

29. the method for claim 28, wherein said host is selected from insect cell and mammalian cell.

30. the method for claim 29, wherein said insect cell are fall army worm (Spodoptera frugiperda) cell line.

31. treatment has the method for the disease in this object that needs, described disease is characterised in that erythrocyte production impaired in the described object, and described method comprises the step according to the peptide of claim 1 of using the amount that can improve the described disease in this object effectively to object.

32. strengthen the method that the erythrocyte in the mammal is produced, described method comprises to the peptide of described administration according to claim 1.

33. treatment has the method for the tissue injury in this object that needs, described damage is characterised in that, by the infringement that ischemia, wound, inflammation or contact toxicity material cause, described method comprises the step according to the erythropoietin peptide of claim 1 of using the amount that can improve the infringement relevant with tissue injury in this object effectively to object.

34. pharmaceutical preparation, it comprises according to the erythropoietin peptide of claim 1 and pharmaceutically acceptable carrier.

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