JP2011157365A - Use of galactose oxidase for selective chemical conjugation of protractor molecule to protein of therapeutic interest - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a conjugate of a glycoprotein having increased in vivo plasma half-life compared to the non-conjugated glycoprotein and comprising a glycoprotein having at least one terminal galactose or derivative thereof, and a protractor group covalently bonded thereto. <P>SOLUTION: There is provided a method comprising the steps of: (a) contacting a glycoprotein having at least one terminal galactose or derivative thereof with galactose oxidase to create a glycoprotein comprising an oxidized terminal galactose or derivative thereof having a reactive aldehyde functional group; and (b) contacting the glycoprotein product produced in the step (a) with a reactant X capable of reacting with an aldehyde group and comprising a protractor group to create a conjugate represented by the formula (glycoprotein)-(protractor group). <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

Field of Invention

  The present invention relates to the use of galactose oxidase in combination with terminal galactose-containing glycoproteins (eg, sialidase-treated glycoproteins (asialoglycoproteins), etc.) for selective chemical conjugation of retarder molecules. Continuous enzymatic treatment of natural glycoproteins with sialidase and galactose oxidase yields reactive aldehyde functional groups that can react chemically with nucleophilic conjugates, increasing circulation half-life or increasing distribution. Resulting in new modified glycoproteins with enhanced pharmacological properties such as

Background of the Invention

  Since proteins of biological origin often have high efficacy and high selectivity for their natural ligands, these proteins have great potential as therapeutic agents. Because these proteins are of biological origin, the organisms already have well-defined clearance mechanisms and metabolic pathways to process and excrete them, so these proteins are better than traditional small molecular drugs. It is non-toxic and is therefore safe to use. This makes the protein an ideal candidate drug along with the fact that it can now be produced by recombinant DNA technology in a variety of different expression systems capable of large-scale production. However, hormones, soluble receptors, cytokines, enzymes and most other proteins of therapeutic interest generally have a short circulating half-life in the body, which generally reduces their therapeutic utility .

  Therapeutic proteins can be removed from the circulation by many routes. For some pharmacologically active proteins, there are specific receptors that mediate removal from the circulatory system. Glycosylated proteins are cleared in the liver by lectin-like receptors that show specificity only for the sugar moieties of these molecules. Nonspecific clearance by the kidney of the following proteins and peptides (particularly non-glycosylated proteins and peptides) of about 50 KDa has also been described in the literature. It has been noted that asialoglycoproteins are cleared by the liver more rapidly than natural glycoproteins or proteins lacking glycosylation (Bocci (1990) Advanced Drug Delivery Reviews 4: 149).

  The problem solved by the present invention is the injection required to extend the circulating half-life of soluble glycoprotein derivatives and thus maintain a therapeutically effective level of circulating glycoprotein for treatment or prevention Reducing the amount of substance and the frequency of injections. The short in vivo plasma half-life of certain therapeutic glycoproteins is undesirable in terms of the frequency and amount of soluble protein required for treatment or prevention. The present invention provides a means of extending the circulating half-life of such glycoproteins while having conservative but more effective changes to the glycoprotein structure and substantially maintaining biological activity.

  More specifically, the present invention relates to general chemoenzymatic methods for modifying these to improve or enhance the pharmaceutical properties of glycoprotein glycans (particularly asparagine or N-linked glycans). I will provide a. The method involves optionally treating a glycoprotein with sialidase (neuraminidase) to remove any terminal sialic acid, and galactose oxidase in the presence of a hydrogen peroxide scavenger such as catalase or horseradish peroxidase. Oxidation of exposed galactose residues on the glycoprotein. When preformed under appropriate conditions, galactose oxidase treatment provides a glycoprotein with a reactive aldehyde functional group on the glycan end, which is then reacted chemically with a nucleophile A joined body can be produced. For glycoproteins with terminal galactose residues and high glycan content, any treatment with sialidase can be omitted.

Thus, in a first aspect, the present invention has an increased in vivo plasma half-life compared to non-conjugated glycoproteins and is covalently attached thereto with at least one terminal galactose or derivative thereof. A method for producing a glycoprotein conjugate comprising a glycoprotein having a retarding group comprising:
(A) contacting a glycoprotein having at least one terminal galactose or a derivative thereof with galactose oxidase to form an oxidized terminal galactose or a derivative thereof having a reactive aldehyde functional group;
(B) contacting the glycoprotein product produced in step (a) with a reactant X capable of reacting with an aldehyde group and containing a retarding group, represented by the formula (glycoprotein)-(retarding group) Forming a joined body;
A method comprising:

In a second aspect, the present invention provides an in vivo plasma half-life compared to non-conjugated glycoproteins and a delay group covalently attached thereto with at least one terminal galactose or derivative thereof. A method for producing a glycoprotein conjugate comprising a glycoprotein having:
(A) contacting a glycoprotein having at least one terminal galactose or a derivative thereof with galactose oxidase to form an oxidized terminal galactose or a derivative thereof having a reactive aldehyde functional group;
(B) Reactant X comprising a linking moiety that is capable of reacting the glycoprotein product produced in step (a) with an aldehyde group and further comprising a protected second reactive group. Contacting with a glycoprotein to form a conjugate of the linking moiety;
(C) contacting the product of step (b) with a retarding group capable of reacting with the second reactive group of the linking moiety, represented by the formula (glycoprotein)-(linking moiety)-(retarding group) Forming a joined body;
A method comprising:

In one embodiment of the present invention, the method further comprises steps (a1) and (a2) performed before step (a) or step (c), respectively:
(A1) Contacting a glycoprotein with one or more of sialidase, galacosidases, N-acetylhexosaminidase, fucosidases, mannosidase, endo-H, and endo-F3 to remove some of the glycan structures Forming a glycoprotein that is treated;
(A2) contacting the product of step (a1) with a galactosyltransferase and a galactose substrate to form a glycoprotein comprising at least one terminal galactose or derivative thereof.

In one embodiment of the invention, the method further comprises a first step (a3) performed before step (a):
(A3) contacting a glycoprotein having at least one terminal sialic acid residue with a sialidase or another reagent capable of removing sialic acid from glycans to form an asialoglycoprotein comprising at least one terminal galactose or derivative thereof Process.

  In a third aspect, the present invention provides a conjugate of a glycoprotein having an increased in vivo plasma half-life compared to a non-conjugated glycoprotein, optionally with at least one terminal galactose or derivative thereof, Provided is a conjugate comprising a glycoprotein having a retarder group covalently attached thereto via a linking moiety.

In a further aspect, the present invention provides a method for producing a glycoprotein conjugate having an increased in vivo plasma half-life compared to a non-conjugated glycoprotein, wherein the conjugate comprises at least one A glycoprotein having terminal galactose or a derivative thereof and a retarding group covalently bonded thereto,
A glycoprotein conjugate obtained by a method comprising the following steps is provided:
(A) contacting a glycoprotein having at least one terminal galactose or a derivative thereof with galactose oxidase to form an oxidized terminal galactose having a reactive aldehyde functional group or a derivative thereof; and (B) contacting the glycoprotein product produced in step (a) with a reactant X capable of reacting with an aldehyde group and containing a retarding group, represented by the formula (glycoprotein)-(retarding group) Forming a joined body.

In a further aspect, the present invention provides a method for producing a glycoprotein conjugate having an increased in vivo plasma half-life compared to a non-conjugated glycoprotein, wherein the conjugate comprises at least one A glycoprotein having a terminal galactose or a derivative thereof and a retarding group covalently bonded thereto via a linking moiety;
A glycoprotein conjugate obtained by a method comprising the following steps is provided:
(A) contacting a glycoprotein having at least one terminal galactose or a derivative thereof with galactose oxidase to form an oxidized terminal galactose having a reactive aldehyde functional group or a derivative thereof;
(B) Reactant X comprising a linking moiety that is capable of reacting the glycoprotein product produced in step (a) with an aldehyde group and further comprising a protected second reactive group. Contacting with a glycoprotein and a linking moiety to form a conjugate; and (c) contacting the product of step (b) with a retarding group capable of reacting with the second reactive group of the linking moiety. A step of forming a conjugate represented by the formula (glycoprotein)-(linking portion)-(retarding group).

  In a further aspect, the present invention provides a pharmaceutical composition comprising a glycoprotein conjugate according to the present invention.

FIG. 1 shows the amount of galactose residues exposed on FVIIa after treatment with neuraminidase (Vibro cholerae and Welsh bacterium on agarose) as determined by the galactose oxidase / amplex red assay from Molecular Probes (A-22179). Clostridium perfringens)). FIG. 2 shows an IEF-Gel analysis of FVIIa treated with neuramidase to produce FVIIa and Asialo-FVIIa. FIG. 3 shows MALDI-TOF spectra of FVIIa and Asialo-FVIIa. FIG. 4 shows IEF-gel analysis (Invitrogen method, pH 3-7) with the following settings: Lane 1: standard pI, Lane 2: CP FVIIa in MES buffer, Lane 3: CP FVIIa + agarose. Bound neuraminidase cholera, 16h, rt., Lane 4: CP FVIIa + agarose conjugated neuraminidase cholera, 36h, rt, lane 5: asialo CP FVIIa + galactose oxidase + aminoxyacetic acid, lane 6: MES buffer CP FVIIa, lane 7: CP FVIIa + agarose-binding neuraminidase Clostridium perfringens, 16h, rt, lane 8: CP FVIIa + agarose-binding neuraminidase Clostridium perfringens, 36h, rt, lane 9: standard pI, lane 10: CP FVIIa in MES buffer. FIG. 5 shows an SDS-PAGE gel (Invitrogen method) on two fractions of asialo FVIIa and PEG-5000 derivatized FVIIa obtained after subsequent reaction with galactose oxidase-catalase and PEG-5000 derivatized hydroxylamine. , Non-reducing conditions). FIG. 6 shows IEF-gel analysis (Invitrogen method, pH 3-7) with the following settings: Lane 1, standard pI; Lane 2: FVIIa; Lane 3: Asialo FVIIa; Lane 4-12, HiTrap ion Fraction of aminoxyacetic acid derivatized FVIIa after exchange purification. FIG. 7 shows the MALDI-TOF spectrum of derivatized N-glycans derivatized by the process with nitrobenzyloxyamine released by PNGase F treatment of FVIIa. FIG. 8 shows IEF gel analysis (Invitrogen method, pH 3-7) with the following settings: Lane 1: Standard pI, Lane 2: FVIIa, Lane 3: FVIIa after buffer exchange with MES buffer. Lane 4, asialo FVIIa (treated with Vibrio cholerae); lane 5: blank; lane 6: galactose oxidase / catalase / aminoxyacetic acid treatment performed with 30 mU galactose oxidase; lane 7: galactose oxidase / catalase / performed with 300 mU galactose oxidase Aminoxyacetic acid treatment. FIG. 9 shows the amino acid sequence of wild type human blood coagulation factor VII. FIG. 9 shows the amino acid sequence of wild type human blood coagulation factor VII. FIG. 10 illustrates a convergent solution phase synthesis of a first generation dendrimer capped with ethoxy 2- (2- [2-methoxyethoxy] ethoxy) acetic acid. FIG. 11 illustrates the solution phase synthesis of a second generation dendrimer capped at the center with a t-butyl protected carboxylic acid. FIG. 12 illustrates the solid phase synthesis of the second generation dendrimer. FIG. 13 illustrates a diverse solution phase synthesis of a second generation dendrimer with a free amino terminus in the center and a t-butyl protected carboxylic acid. FIG. 14 illustrates solution phase end capping of the generated second generation dendrimer as illustrated in FIG. 12 or 13. FIG. 15 illustrates end-capping of a second generation dendrimer using succinic acid mono tert-butyl ester and subsequent acid-mediated deprotection to make a polyanionic sugar mimetic polymer. FIG. 16 illustrates solution phase functionalization of a second generation dendrimer to create a handle that is compatible with the aldehyde functionality obtained by the present invention. SDS-PAGE electrophoresis gel (non-denaturing). Lane 1: Mark-12 Mw standard (Invitrogen); Lane 2: rFVIIa, Lane 3: Asialo rFVIIa, Lanes 4-10: 50 KDa band + fractions containing modified rFVIIa with several 1.7 KDa band additions .

Definition

  Natural peptides obtained from eukaryotic expression systems such as mammals, insects, or yeast cells are often isolated in these glycosylated forms. The glycosyl moiety is also referred to as the glycan moiety on such a peptide and can itself be used directly for conjugation purposes or, if appropriate, converted to a suitable attachment moiety for conjugation. It is a polyhydric alcohol that can be used. The glycan of interest is either an O-linked glycan, ie a glycoprotein in which the glycan is linked via the amino acid residue serine or threonine; or an N-glycan in which the glycan moiety is linked to the asparagine residue of the peptide. It is.

  The term “glycoprotein” is intended to encompass peptides, oligopeptides, and polypeptides that contain one or more sugar residues (glycans) attached to one or more amino acid residues of a “backbone” amino acid sequence. The glycans may be N-linked or O-linked.

As used herein, the term “glycan” or interchangeably “oligosaccharide” refers to a complete oligosaccharide structure covalently linked to a single amino acid residue. Glycans are usually N-linked or O-linked, for example glycans are linked to asparagine residues (N-linked glycosylation) or serine or threonine residues (O-linked glycosylation). N-linked oligosaccharides may be multi-antenna types, such as two, three, or antenennary, and most often contain a core structure of GlcNAc-GlcNAc-Man ₃ .

  Some glycoproteins have glycan structures with terminal or “capping” sialic acid residues when produced in humans in situ. That is, the terminal sugar of each antenna is N-acetylneuraminic acid linked to galactose via an α2 → 3 or α2 → 6 bond. Other glycoproteins have glycans end-capped with other sugar residues. However, when produced in other situations, the glycoprotein may contain, for example, a lack of sialic acid residues; inclusion of N-glycolylneuraminic acid (Neu5Gc) residues in one or more of these antennas; galactose May contain oligosaccharides having different terminal structures, such as containing a terminal N-acetylgalactosamine (GalNAc) residue instead of N-linked and / or O-linked oligosaccharide patterns include, but are not limited to, high performance liquid chromatography (HPLC); capillary electrophoresis (CE); nuclear magnetic resonance (NMR); fast atom bombardment, electrospray, Or mass spectrometry (MS) using ionization techniques such as matrix-assisted laser desorption (MALDI); gas chromatography (GC); and anion exchange (AIE) HPLC, size exclusion chromatography (SEC), mass spectrometry (MS ), Gel electrophoresis (SDS-PAGE, CE-PAGE), isoelectric focusing gel, or treatment with exoglycosidase in combination with isoelectric focusing capillary electrophoresis (CE-IEF) It may be determined using any method known in the art. For example, Weber et al., Anal. Biochem. 225: 135 (1995); Klausen et al., J. Chromatog. 718: 195 (1995); Morris et al., In Mass Spectrometry of Biological Materials, McEwen et al. , eds., Marcel Dekker, (1990), pp 137-167; Conboy et al., Biol. Mass Spectrom. 21: 397, 1992; Hellerqvist, Meth. Enzymol. 193: 554 (1990); Sutton et al., Anal. Biohcem. 318: 34 (1994); Harvey et al., Organic Mass Spectrometry 29: 752 (1994).

  Thus, the term “terminal sialic acid” or interchangeably “terminal neuraminic acid” encompasses sialic acid residues linked as terminal sugar residues in a glycan or oligosaccharide chain, ie, each antenna. Is intended to be N-acetylneuraminic acid linked to galactose via a 2 → 3 or 2 → 6 linkage.

  The term “galactose or derivatives thereof” means galactose residues such as natural D-galactose or derivatives thereof, such as N-acetylgalactosamine residues. In one embodiment, the galactose derivative is N-acetylgalactosamine.

  The term “terminal galactose or derivatives thereof” means galactose or derivatives thereof linked as terminal sugar residues in a glycan or oligosaccharide chain, for example, the terminal sugar of each antenna is galactose or N-acetyl. Galactosamine.

  The term “asialoglycoprotein” means that at least one terminal sialic acid residue is derived from an underlying “layer” of galactose or N-acetylgalactosamine, for example by sialidase treatment or by chemical treatment. It is intended to include glycoproteins that expose galactose or N-acetylgalactosamine residues (“exposed galactose residues”).

  The term “oxidized asialoglycoprotein” is a reactive aldehyde function that is oxidized by galactose oxidase, optionally in combination with a hydrogen peroxide scavenger such as catalase or horseradish peroxidase, and thus located on the terminal galactose residue. It is intended to encompass asialoglycoproteins that give rise to groups or moieties.

  The term “hydrogen peroxide scavenger” is intended to include compounds or substances that can react, absorb or neutralize with hydrogen peroxide.

  The term “reactant X capable of reacting with an aldehyde group” means reacting with a nucleophile or nucleophile and an aldehyde group located at the oxidized terminal galactose residue of a glycoprotein and reacting with a terminal galactose residue. It means other drugs capable of forming a covalent bond with the substance (X). The reactant may include a polymeric group in some embodiments of the invention. Non-limiting examples of Reactant X include hydroxylamine derivatives, O-alkylated hydroxylamine derivatives, amines, stabilized carbanions, stabilized enolates, hydrazides, alkyl hydrazides, hydrazines, acyl hydrazines, and Ring-forming (eg thiazolidine-forming) nucleophiles such as thioethanamine, cysteine, or cysteine derivatives.

  As used herein, the term “retarding group” means a group that, when compared to an unmodified protein or peptide, increases the circulating half-life of the protein or peptide by conjugation to the protein or peptide. The specific principles behind the delayed effect are the increased size, the shielding of peptide sequences that can be recognized by peptidases or antibodies, or the glycan-specific receptors that are present in eg liver or macrophages It may occur by masking glycans in a way that is not recognized by the body to prevent or reduce clearance. The retarding effect of the retarding group can also be caused by binding to blood components such as albumin or nonspecific adhesion to vascular tissue. The conjugated glycoprotein should substantially preserve its biological activity.

  The term “electron withdrawing group” is used as defined in March, Advanced Organic Chemistry, 3rd edition, John Wiley & Sons, N.Y. 1985.

In one embodiment of the invention, the retarding group is selected from the group consisting of:
(A) Small molecule (15-1000 da) organic charged radicals that may include one or more carboxylic acids, amine sulfonic acids, phosphonic acids, or combinations thereof.

  (B) Low molecular weight (15-1000 da) neutral hydrophilic molecules such as cyclodextrins or optionally branched polyethylene chains.

  (C) Small molecules (15-1000 da) hydrophobic molecules such as fatty acids or cholic acids or their derivatives.

(D) polyethylene glycol with an average molecular weight of 2-40 kDa (e) a well-defined accurate such as a dendrimer with an accurate molecular weight ranging from 700 to 20.000 da, or more preferably between 700 and 10,000 da Polymer.

  (F) A substantially non-immunogenic polypeptide such as albumin or a portion of an antibody or optionally an antibody comprising an Fc-domain.

  (G) High molecular weight organic polymers such as dextran.

  The term “nucleophile” is interchangeable with “nucleophile” and “nucleophilic conjugate”.

  As used in this context, the term “covalent bond” refers to an oligosaccharide moiety (glycan) and reactant X that are covalently linked directly to each other or otherwise intervening moieties or bridges, spacers, or linkages. It is meant to include being indirectly covalently linked to each other via a moiety such as a moiety.

  The term “glycoconjugate” or interchangeably “conjugate” or “protein conjugate” refers to the sharing of one or more terminal galactose residues of a glycoprotein to one or more nucleophiles (reactant X). It is intended to indicate a heterogeneous molecule (in the sense of a complex or chimera) formed by binding. In this context, the term “protein” is intended to include peptides, oligopeptides and polypeptides having a sequence of at least 4 amino acid residues and preferably having between 4 and about 1000 residues.

  In one embodiment of the invention, the retarding group is a polymer molecule.

  As used herein, the term “polymer molecule” means a molecule formed by the covalent attachment of two or more monomers, neither of which is an amino acid residue.

  In one embodiment of the invention, the polymer molecule is a dendrimer, polyalkylene glycol (PAO) including polyalkylene glycol (PAG) such as polyethylene glycol (PEG) and polypropylene glycol (PPG), branched PEG, polyvinyl It is selected from the group consisting of alcohol (PVA), polycarboxylate, polyvinylpyrrolidone, polyethylene-co-maleic anhydride, polystyrene-co-maleic anhydride, and dextran including carboxymethyl dextran. In one embodiment of the invention, the polymer molecule is a PEG group. In one embodiment of the invention, the polymer molecule is a dendrimer. In one embodiment of the invention, the polymer molecule is a dendrimer having a molecular weight in the range of 700 to 10,000 Da.

  The polymer molecule is part of or attached to a reactant X that can react with the aldehyde group of the oxidized galactose residue located in the glycopeptide.

  Groups present on the polymer may be activated to yield reactant X prior to reaction with the oligosaccharide moiety or glycan, as described above. The activated group may be in the form of an activated leaving group, whether present in the oligosaccharide moiety or the polymer moiety. Methods and chemistries for polymer activation are described in the literature. Commonly used methods for polymer activation are cyanogen bromide, periodate, glutaraldehyde, biepoxides, epichlorohydrin, divinylsulfone, carbodiimide, sulfonyl halide, trichlorotriazine, etc. Activation of functional groups of (For example, Taylor (1991). Such electrophilic activated polymers can then be converted to reagents of type reagent X by reaction with eg binucleophilic linker moieties. Protein Immobilization, Fundamentals and Applications, Marcel Dekker, NY; Wong (1992), Chemistry of protein Conjugation and Crosslinking, CRC Press, Boca Raton; Hermanson et al., (1993), Immobilized Affinity Ligand Techniques, Academic Press, NY; Dunn et al., Eds. Polymeric Drugs and Drug Delivery Systems, ACS Symposium Series Vol. 469, See American Chemical Society, 1991).

  The terms “branched polymer” or interchangeably “dendritic polymer”, “dendrimer”, or “dendritic structure” are constructed by selection of monomeric building blocks, some of which contain branches. Means organic polymer.

  The term “generation” refers to a single uniform layer created by reacting one or more identical functional groups on an organic molecule with a particular monomer building block. For branched polymers made only from bifurcated monomers, the number of reactive groups in the generation is given by the formula (2 * (m-1)) 2 where m is 1,2 , 3 ... 8 and represents a specific generation). For branched polymers made from trifurcated monomers, the number of reactive groups is given by the formula (3 * (m-1)) 3 and is a multi-branched monomer with n or branches For branched polymers made from the number of reactive groups is given by (n * (m-1)) n. For branched polymers where different monomers are used for each individual generation, the number of reactive groups in a particular layer or generation is calculated to determine the density of each individual monomer layer in the dendritic structure. The position and the number of branches can be known recursively.

  In one embodiment of the invention, the retarding group is selected from the group consisting of albumin, fatty acid-like, C5-C24 fatty acids, serum protein binding ligands such as compounds that bind aliphatic diacids (eg C5-C24). The Other examples of retarding groups include moieties that change charge properties under physiological conditions such as carboxylic acids or amines, or prevent glycan-specific recognition of smaller alkyl substituents (eg, C1-C5 alkyl) Small organic molecules that contain functional substituents.

  In one embodiment of the invention, the retarding group is albumin. In one embodiment of the invention, the retarding group is selected from the group consisting of: dendrimer, polyalkylene oxide (PAO), polyalkylene glycol (PAG), polyethylene glycol (PEG), polypropylene glycol (PPG), Branched PEG, polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolidone, polyethylene-co-maleic anhydride, polystyrene-co-maleic anhydride, dextran, carboxymethyl dextran; fatty acid, C5-C24 fatty acid, fat Serum protein binding ligands, such as compounds that bind albumin, such as family diacids (eg C5-C24), structures that inhibit the binding of glycans to receptors (eg, asialoglycoprotein receptors and mannose receptors) Eg sialic acid derivatives or mimetics), carboxylic acids or Small organic molecules containing a moiety that alters charge properties under physiological conditions, such as min, or neutral substituents that prevent glycan-specific recognition, such as small alkyl substituents (eg, C1-C5 alkyl), one or more Low molecular weight organic charged radicals (eg C1-C25) that may contain carboxylic acids, sulfo group amines, phosphonic acids, or combinations thereof; low molecular weight neutral hydrophilic molecules (eg C1-C25), such as cyclodextrins; Or an optionally branched polyethylene chain; a polyethylene glycol having an average molecular weight of 2 to 40 KDa; a dendrimer having an accurate molecular weight in the range of 700 to 20.000 Da, or more preferably 700 to 10,000 Da, etc. A well-defined and precise polymer of; and substantially non-imunogenic, such as albumin or a portion of an antibody or optionally an antibody containing an Fc-domain Polypeptide.

  The term “activated leaving group” includes moieties that are readily displaced in organic or enzyme controlled substitution reactions. Activated leaving groups are known in the art, for example, Vocadlo et al., In Carbohydrate Chemistry and Biology, Vol 2, Wiley-VCH Verlag, Germany (2000); Kodama et al., Tetrahedron Letters 34: 6419 (1993); Lougheed et al., J. Biol. Chem. 274: 37717 (1999).

  The reactive groups and classes of reactions useful in practicing the present invention are those that generally proceed under relatively mild conditions. These include, but are not limited to, nucleophilic substitution (eg, reaction of amines and alcohols with acyl halides, active esters). These and other useful reactions are described, for example, in arch, Advanced Organic Chemistry, 3rd edition, John Wiley & Sons, NY 1985; Hermanson, Bioconjugate Techniques, Academic Press, San Diego, 1996; Feeney et al, Modifications of Proteins , Advances in Chemistry Series, Vol. 198, American Chemical Society, 1982.

  The reactive functional groups can be selected such that they do not participate in or interfere with the reactions necessary to build the oligosaccharide and polymer moieties. Alternatively, the reactive functional group can be protected from participating in the reaction in the presence of a protecting group. For examples of useful protecting groups, see, for example, Greene et al., Protective groups in Organic Synthesis, John Wiley & Sons, N.Y., 1991.

  The term “naturally occurring glycosylation site in FVII” refers to positions Asn145 (N145), Asn322 (N322), Ser-52 (S52), and Ser-60 ( It is intended to indicate the glycosylation site of S60). In a similar manner, the term “naturally occurring in vivo O-glycosylation site” includes positions S52 and S60, while the term “naturally occurring in vivo N-glycosylation site” is represented by positions N145 and N322. including. The term “Figure: amino acid residues corresponding to amino acid residues S52, S60, N145, N322 of 3 (FVII wt.)” Is the sequence of wild-type factor VII when the sequences are aligned (Figure: 3 ) Is intended to indicate the Asn and Ser amino acid residues. Amino acid sequence homology / identity is conveniently suitable for sequence alignment such as the ClustalW program, version 1.8, 1999 (Thompson et al., 1994, Nucleic Acid Research, 22: 4673-4680). Determined from the aligned sequence using a computer program.

  The term “functional in vivo half-life” means in its usual meaning, ie the time during which 50% of the biological activity of the polypeptide or conjugate is still present in the body / target organ, or the activity of the polypeptide or conjugate. Is used at a time that is 50% of its initial value. As an alternative to determining functional in vivo half-life, “in vivo plasma half-life”, i.e., how long 50% of the polypeptide or conjugate molecule circulates in the plasma or bloodstream before it is cleared, can be determined. Good. Determination of plasma half-life is often simpler than determining functional half-life, and determination of plasma half-life magnitude is usually a good indicator of the magnitude of functional in vivo half-life. Alternative terms for plasma half-life include serum half-life, circulating half-life, circulating half-life, serum clearance, plasma clearance, and clearance half-life. The retained functionality is usually selected from a procoagulant, proteolytic, cofactor binding, receptor binding activity, or other type of biological activity associated with a particular protein.

  The term “increased” as used for functional in vivo half-life or plasma half-life is a reference such as a non-conjugated glycoprotein in which the relative half-life of the polypeptide or conjugate is determined under corresponding conditions. Used to show a statistically significant increase compared to that of the molecule. For example, the relative half-life may be increased to at least about 25%, such as at least about 50%, such as at least about 100%, 150%, 200%, 250%, or 500%. In some embodiments, a formulation of the invention has a half-life of at least about 0.25 h, preferably at least 0.5 h, more preferably at least about 1 h, and most preferably at least about 2 h compared to the half-life of the reference formulation. Increase.

  “Immunogenic” of a formulation refers to the ability of the formulation to elicit an adverse immune response when administered to a human, whether humoral, cellular, or both. There may be individuals in any human subpopulation that are sensitive to a particular administered protein. Immunogenicity may be measured by quantifying the presence of anti-glycoprotein antibodies and / or glycoprotein-responsive T cells in susceptible individuals using conventional methods known in the art. In some embodiments, the formulations of the invention are at least about 10%, preferably at least 25%, more preferably at least about 40%, and most preferably compared to the immunogenicity of the reference formulation against sensitive individuals. Indicates a decrease in immunogenicity in that individual of at least about 50%.

  “Linker moiety” or “L1” means any biocompatible molecule capable of reacting with an aldehyde group and functioning as a means of linking a retarding group to “Reactant X”. This is understood that in the final conjugate of the glycoprotein, the “linker moiety” links the glycoprotein to the retarding group via a chemical bond. It is well understood that the linker moiety may include covalent and non-covalent chemical bonds, or a mixture thereof.

Suitable linker moieties are peptides; polynucleotides; monosaccharides, disaccharides and oligosaccharides, cyclodextrins, and sugars including dextran; polyethylene glycol, polypropylene glycol, polyvinyl alcohol, hydrocarbons, polyacrylates, and amino, hydroxy Including, but not limited to, polymers including silicon, thio, or carboxy-functionalized polymers, other biocompatible material units; and combinations thereof. Such linker moiety materials described above are widely commercially available or generally available via synthetic organic methods known to those skilled in the art. The linker moiety may be selected, for example, from the following structures: linear or branched C _1-50 alkyl, linear or branched C _2-50 -alkenyl, linear or branched C _2-50 -alkynyl, 1-50 member straight or branched chain containing carbon in the chain and at least one N, O, or S atom, C _3-8 cycloalkyl, carbon in the ring and at least one N, 3- to 8-membered cyclic rings containing O or S atoms, aryl, heteroaryl, amino acids, structures optionally substituted with one or more of the following groups: H, hydroxy, phenyl, phenoxy, benzyl, thienyl , Oxo, amino, C _1-4 -alkyl, CONH ₂ , CSNH ₂ , C _1-4 monoalkylamino, C _1-4 dialkylamino, acylamino, sulfonyl, carboxy, carboxyamide, halogeno, C _1-6 alkoxy, C _1-6 alkylthio, trifluoromethyl alkoxy Alkoxycarbonyl, haloalkyl. The linker moiety may be linear or branched and may contain one or more double or triple bonds. The linker moiety may contain one or more heteroatom-like N, O, or S. It is well understood that the linker moiety can include multiple classes of groups as described above, as well as multiple members within a class. Where the linker moiety comprises multiple classes of groups, such linker moieties are preferably obtained by linking to different units via these functional groups. Methods for forming such bonds include standard organic synthesis and are well known to those skilled in the art.

  The term “alkyl” or “alkylene” refers to a C 1-6 alkyl or alkylene that represents a saturated, branched or straight hydrocarbon group having from 1 to 6 carbon atoms. Typically, C16 alkyl groups include methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, and the corresponding divalent radicals, including It is not limited.

  The term “alkenyl” or “alkenylene” refers to a C 2-6 alkenyl or alkenylene representing a branched or straight hydrocarbon group having from 2 to 6 carbon atoms and having at least one double bond. Typically, a C2-6 alkenyl group is ethenyl, 1-propenyl, 2-propenyl, isopropenyl, 1,3 butadienyl, 1-butenyl, 2-butenyl, 1-pentenyl, 2-pentenyl, 1-hexenyl, 2-hexenyl, 1-ethylprop-2-enyl, 1,1- (dimethyl) prop-2-enyl, 1-ethylbut-3-enyl, 1,1- (dimethyl) but-2-enyl, and the corresponding two Including, but not limited to, valent radicals.

  The term “alkynyl” or “alkynylene” refers to C 2-6 alkynyl or alkynylene, representing a branched or straight hydrocarbon group having from 2 to 6 carbon atoms and having at least one triple bond. Typically, alkynyl groups are vinyl, 1-propynyl, 2-propynyl, isopropynyl, 1,3-butazinyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 1-hexynyl, 2- Hexynyl, 1-ethylprop-2-ynyl, 1,1- (dimethyl) prop-2-ynyl, 1-ethylbut-3-ynyl, 1,1- (dimethyl) but-2-ynyl, and the corresponding divalent Including but not limited to radicals.

  The term “alkyleneoxy” or “alkoxy” refers to the radical —O—C 1-6 -alkyl or —O—C 1-6 -alkylene, where C 1-6 alkyl (ene) is as described above. Represents "C1-6-alkoxy" or alkyleneoxy. Representative examples are methoxy, ethoxy, n-propoxy, isopropoxy, butoxy, sec-butoxy, tert-butoxy, pentoxy, isopentoxy, hexoxy, isohexoxy and the like.

  The term “heteroaryl” as used herein is intended to include heterocyclic aromatic ring systems containing one or more heteroatoms selected from nitrogen, oxygen, and sulfur, such as: : Furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, isoxazolyl, isothiazolyl, 1,2,3-triazoyl, 1,2,4-triazoyl, pyranyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl 1,2,4-triazinyl, 1,3,5-triazinyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1 , 2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, tetrazolyl, thiadiazinyl, indolyl, isoindolyl, benzofuryl, benzo Thienyl, benzo-thiophenyl (thianaphthenyl), indazolyl, benzimidazolyl, benzthiazolyl, benzisothiazolyl, benzoxazolyl, benzisoxazolyl, purinyl, quinazolinyl, quinolidinyl, quinolinyl, isoquinolinyl, quinoxalinyl, naphthidyl Azepinyl, diazepinyl, acridinyl. Heteroaryl is also intended to include the partially hydrogenated derivatives of the heterocyclic systems listed above. Non-limiting examples of such partially hydrogenated derivatives are 2,3-dihydrobenzofuranyl, pyrrolinyl, pyrazolinyl, indolinyl, oxazolidinyl, oxazolinyl, oxazepinyl and the like.

Detailed Description of the Invention

  The purpose of the present invention is to stabilize the glycoprotein by cycling when protein glycosylation provides the primary determinant for clearance and when other clearance pathways not related to the glycan moiety are active. Provides a means for

  In the former case, with such glycoproteins, increased half-life is achieved by treatments that block or inhibit the removal of proteins by sugar-specific receptors such as galactose and mannose receptors in the liver. In particular, it describes the extension of soluble glycoprotein derivatives in the circulation. In the latter case, for example, an increase in half-life can be achieved by attaching a “protractor” moiety such as a serum protein binding ligand such as an albumin binding ligand or a large polymer such as PEG. In either case, increased circulating half-life is desirable in therapeutic proteins, since the frequency and / or size of doses can be reduced when the half-life is longer.

Galactose oxidase (GO) is a copper enzyme that catalyzes the two-electron oxidation of a number of primary alcohols to their corresponding aldehydes in combination with the reduction of diatomic oxygen to hydrogen peroxide: RCH ₂ OH + O ₂ → RCHO + H ₂ O ₂ . The enzyme exhibits broad specificity for a wide variety of primary alcohols that serve as effective substrates, including reduced forms and substituted benzyl alcohols. Galactose oxidase, along with catalase or horseradish peroxidase, provides a mild and highly selective method for introducing aldehyde functionality into glycoproteins, resulting in purified glycoproteins and lipid membranes of complete cell lines. It has been used extensively for the emission or fluorescence labeling of embedded glycoproteins (eg BBaumann H. and Doyle D., J. Biol. Chem. 1979, 254 (7) 2542-2550 and Wilchek M. Spiegel S. and Spiegel Y., Biochem. Biophys Res. Commun, 1980, 92 (4) 1215-1222). Galactose oxidase selectively oxidizes galactose (Gal) and acetylated galactosamine (Gal-NAc).

  Both neuraminidase and galactose oxidase were immobilized on beads to create a cost-effective storage and operationally stable enzyme reactor. See, for example, Bilkova Z. et. Al., J. Chromatography B. 2002, 770 / 1-2 (177-181). The application of such an immobilized enzyme to remove hydrogen peroxide by-products, optionally in conjunction with a similar immobilized enzyme-like catalase of horseradish peroxidase, is within the scope of the present invention. Since these are easily removed by simple filtration, the immobilized enzyme offers an unexpected advantage of increased stability in the subsequent purification process. Thus, in one embodiment of the invention, one or more of the enzymes involved in the process are immobilized on a support.

Sialidase treatment Some glycoproteins contain terminal sialic acid residues. In such cases, glycoproteins (which may be from natural sources or genetically modified mammals such as COS, BHK- or CHO cells) to expose at least one galactose residue from the inner layer of galactose residues. (Which can be obtained from animal cells or from reconstructed proteins obtainable from yeast cells) is first treated with sialidase. Some glycoproteins are not naturally terminal sialic acid added (does not include terminal sialic acid residues), and in such cases sialidase treatment is not used. Enzymatic desialidation can be achieved by using commercially available soluble sialidase, by using recombinant genetically modified sialidase, or by using sialidase coupled to a solid support, such as agarose. Either can be done. The progress of the reaction is conveniently carried out by IEF-gel monitoring as described in WO2003031464 (Neose) or by NK Klausen and T. Kornfelt, J. Chromatography A, 718, It can be monitored by capillary electrophoresis techniques such as those described in 195-202 (1995). If soluble sialidase is used for the reaction, a purification step may be necessary. Suitable purification techniques are known to those skilled in the art and can include, for example, ion exchange chromatography or other similar techniques.

The galactose oxidase glycoprotein or asialoglycoprotein obtained by sialidase treatment is then treated with galactose oxidase using molecular oxygen as the oxidant. Since the reaction by-product is hydrogen peroxide, some measurements can be made, for example, by adding a hydrogen peroxide scavenger to avoid its destructive power (eg, oxidation of methionine residues, etc.). Generally done. Catalase and horseradish peroxidase are both suitable, but other scavengers are known to those skilled in the art. The overall change in structure and function of the glycoprotein is made immediately during this reaction step, making it more difficult to monitor the reaction. Chemical conjugates with reporter molecules such as dansyl hydrazide are also an option. Alternatively, if horseradish peroxidase or catalase is excluded, the progress of the reaction can be estimated colorimetrically using a commercially available kit such as Amplex Red from Molecular Probes.

Chemical conjugation step In one embodiment of the invention, the glycoprotein for the chemical conjugation step is treated with sialidase to remove sufficient sialic acid to expose at least one galactose residue, Further examples are glycoproteins treated with galactose oxidase and horseradish peroxidase to generate reactive aldehyde functionality.

One reaction sequence is shown below using reactant X, which can react with aldehyde groups:
Reaction scheme 1

  In the formula, Sia means sialic acid linked to galactose or a galactose derivative (Gal) in either alpha-2,3- or alpha-2,6-configuration.

In one embodiment, Gal-OH represents galactose, in which case

In one embodiment, Gal-OH represents the galactose derivative N-acetylgalactosamine and galactose oxidase oxidizes acetylated galactosamine residues, in which case

  X is any type of molecule containing a chemical functionality capable of reacting covalently with an aldehyde to form a C-6 modified galactose or N-acetylgalactosamine residue (eg, a nucleophile, etc. ).

  L is a divalent organic radical linker that consists of natural monosaccharides such as fucose, mannose, N-acetylglycosamine, xylose, and arabinose, linked in any order and with many branches. Any organic diradical may be included, including those having one or more hydrocarbon moieties. L may also be a valence bond.

  Chemical conjugation may be performed in a number of ways depending on the particular reactant X involved.

  In one embodiment of the invention, Reactant X has the formula nuc-R, where nuc is a functional group that can react with an aldehyde to form a covalent bond, and R is a retarding group. It is a group.

  In one embodiment of the invention, reactant X has the formula nuc-L1-R, where nuc is a functional group capable of reacting with an aldehyde to form a covalent bond, and R is It is a delay group, and L1 is a linking moiety.

In one embodiment of the invention, Reactant X is H ₂ NR, HR1N-R, H ₂ NOR, HR1N-OR, H ₂ N-NH-COR, H ₂ N-CHR1-CHR-SH, H ₂ Selected from the group consisting of N—CHR—CHR1-SH, H ₂ N—NH—SO ₂ R, and Z′—CH ₂ —Z ″ —R; wherein R is a retarding group; Z ′ and Z ″ represent, for example, an electron withdrawing group such as COOEt, CN, NO ₂ , and one or both of the Z groups is connected to the R group Can do.

In one embodiment of the invention, Reactant X is H ₂ N-L1-R, HR1N-L1-R, H ₂ NO-L1-R, HR1N-O-L1-R, H ₂ N-NH— CO-L1-R, H ₂ N-CHR1-CH (L1-R) -SH, H ₂ N-CH (L1-R) -CHR1-SH, H ₂ N-NH-SO ₂ -L1-R, Z Selected from the group consisting of '-CH ₂ -Z''-L1-R; wherein L1 is a linking moiety, R is a delay group, and R1 is H or a second delay group Z ′ and Z ″ represent, for example, an electron withdrawing group such as COOEt, CN, NO ₂ , and one or both of the Z groups can be connected to the R group;

In one embodiment of the invention, Reactant X has the formula nuc-L1-Rn, _where nuc is a functional group capable of reacting with an aldehyde to form a covalent bond, and R Is a retarding group and L1 is a multifunctional linking moiety that connects one or more retarding groups (R) to a reactive functional group (nuc), and L1 may contribute to the delay, or It may not contribute; n represents an integer, 1-25 such as n = 1-10.

In certain embodiments, Reactant X is given by the following general formula:
nuc-R
In the formula, nuc is a group capable of reacting with an aldehyde group. Non-limiting examples for illustration include hydroxylamine, O-alkylated hydroxylamine, amine, stabilized carbanion, stabilized enolate, hydrazide, alkyl hydrazide, hydrazine, acyl hydrazine, and the like. Other embodiments include nucleophiles that form rings (eg, form thiazolidine) such as, for example, thioethanamine, cysteine, or cysteine derivatives, α-mercaptoacyl hydrazides.

In particular, nuc can be:
Hydrazine derivatives -NH-NH ₂ ,
Hydrazinecarboxylate derivatives -OC (O) -nH-NH ₂ ,
Semicarbazide derivative -NH-C (O) -nH-NH ₂ ,
Thiosemicarbazide derivatives -NH-C (S) -nH-NH ₂ ,
Carbonic dihydrazide derivatives -NHC (O) -NH-NH-C (O) -nH-NH ₂ ,
Carbazide derivatives -NH-NH-C (O) -nH-NH ₂ ,
Thiocarbazide derivatives -NH-NH-C (S) -nH-NH ₂ ,
Arylhydrazine derivatives -NH-C (O) -C ₆ H ₄ -nH-NH ₂ ,
Hydrazide derivative -C (O) -nH-NH2, and
Oxylamine derivatives such as —O—NH ₂ —, C (O) —O—NH ₂ , —NHC (O) —O—NH ₂ , and —NH—C (S) —O—NH ₂ .

R in the general formula nuc-R may be an organic radical selected from one of the following groups:
a) Linear, branched and / or cyclic C _1-30 alkyl, C _2-30 alkenyl, C _2-30 alkynyl, C _1-30 heteroalkyl, C _2-30 heteroalkenyl, C _2-30 heteroalkynyl, wherein one or more homocyclic aromatic or heterocyclic biradicals may be inserted, wherein the C _1-30 or C _2-30 radical is Substituted with one or more substituents selected from: —CO ₂ H, —SO ₃ H, —PO ₂ OH, —SO ₂ NH ₂ , —NH ₂ , —OH, —SH, halogen, or aryl. The aryl is optionally substituted with —CO ₂ H, —SO ₃ H, —PO ₂ OH, —SO ₂ NH ₂ , —NH ₂ , —OH, —SH, or halogen; Lipid radicals;
b) polysaccharide radicals (eg dextran); or cyclodextrins (polyamide radicals eg polyamino acid radicals); PVP radicals; PVA radicals; poly (1-3-dioxane); poly (1,3,6-trioxane); Maleic anhydride polymer;
c) Cibacron dyes such as Cibacron Blue 3GA and polyamide chains of specified lengths as disclosed in WO 00/12587, which is incorporated herein by reference.

  d) Albuminyl derivatives as described in Kan, SK et al in The Journal of Immunology 2001, 166 (2), 1320-1326 or in Stevenson, GT, The Journal of Immunology 1997, 158, 2242-2250 Blood components or substantially non-immunogenic protein residues such as antibodies or these domains such as Fc domains from human normal IgG1.

  e) Polyethylene glycol (PEG) or methoxypolyethylene glycol (MPEG) radicals and amino derivatives thereof having an average molecular weight between 500 and 100,000 Da, such as between 500 and 60,000 Da, such as between 1000 and 40,000 Da, etc. It may be between 5000 and 40,000 Da.

f) R is also (may represent R5) _3, each of R1, independently hydrogen addition _{-D - ((CH 2) -C} q O) r -OR6, -D-CH 2 -O _{- ((CH 2) q O} ) r -OR6, represent a moiety selected from among _{-DO ((CH 2) q O} ) r -OR6; wherein, q represents 1-6, r is 10 represents to 500, R6 is hydrogen or C ₁ -C ₆ - alkyl; and D represents a bond or a C _1-8 alkyl or C _1-8 heteroalkyl;
g) moieties known to bind to plasma proteins such as albumin (albumin binding properties are described in J. Med. Chem, 43, 2000, 1986-1992, which is incorporated herein by reference) Or an albumin binding moiety such as a peptide can be found in J. Biol Chem. 277, 38 (2002) 35035-35043, which is hereby incorporated by reference. Contains less than 40 amino acid residues such as the disclosed moieties.

_{h) C 1 -C 18 - C} 1 -C 20 , such as alkyl - alkyl. In particular it charged groups, polar groups, and / or may be substituted by halogen C ₁₄ -, C ₁₆ - and C ₁₈ - especially referring to alkyl. Examples of such substituents include —CO ₂ H and halogen. In certain embodiments, all hydrogens of C ₁ -C ₂₀ -alkyl are substituted with fluoro to form perfluoroalkyl.

Specific embodiments:
Specific embodiments of R-nuc, which is a PEG-2-40K derivative, are illustrated by way of example and not limitation:
In one embodiment of the invention, Reactant X has the formula nuc-R, where nuc is a functional group that can react with an aldehyde to form a covalent bond, and R is a PEG group. is there. In one embodiment of the invention, nuc-R is selected from the group consisting of:

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

Where mPEG has a molecular weight of 20 kDa,

In the formula, mPEG has a molecular weight of 20 kDa.

Where mPEG has a molecular weight of 10 kDa.

In the formula, mPEG has a molecular weight of 5 kDa.

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

Where mPEG has a molecular weight of 10 kDa,

  Where mPEG has a molecular weight of 10 kDa.

  In one embodiment of the invention, the retarding group is a compound that binds albumin.

  The phrase “compound that binds albumin” is used herein interchangeably with “albumin binder”.

In one embodiment of the invention, Reactant X has the formula nuc-R, where nuc is a functional group that can react with an aldehyde to form a covalent bond, and R binds to albumin. It is a compound. In one embodiment of the invention, nuc-R is selected from the group consisting of:

  Where k is an integer between 0 and 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

And structure

Where t is an integer between 10 and 24, v is an integer between 1 and 10 and v is an integer between 2 and 16 or an average molecular weight between 1 and 40 kDa, For example, the number of ranges describing polydisperse PEG polymers such as PEG ₃₄₀₀ , PEG ₅₀₀₀ , PEG ₁₀₀₀₀ , PEG ₂₀₀₀₀ and PEG ₄₀₀₀₀ .

  In one embodiment, Reactant X is a nucleophile, which can form a covalent bond upon dehydration. Non-limiting examples for illustration are hydroxylamine, O-alkylated hydroxylamine, amine, stabilized carbanion, stabilized enolate, hydrazide, alkyl hydrazide, hydrazine, acyl hydrazine, α-mercapto Includes acyl hydrazide and others. Other embodiments include ring-forming (eg, thiazolidine-forming) nucleophiles such as, for example, thioethanamine, cysteine, or cysteine derivatives.

In some cases (see below), the product of the reaction may be further reacted with a reducing agent (reduced form) to form a reduced product, as shown below:

  In such cases, and without limitation, examples of reducing agents (reducers) include sodium cyanoborohydride, pyridine borane, and sodium borohydride, and examples of x include hydrazide, primary, and secondary amines. Including.

In general, O-alkylated hydroxylamine derivatives form spontaneously stable oxime derivatives when reacted with aldehydes:

Although more reactive and in some cases directly harmful to the protein in question, alkyl hydrazine also reacts efficiently with aldehydes to form hydrazones. Hydrazones are stable in aqueous solution and can therefore be considered as a modification of hydroxylamine for derivatization:

Hydrazides, on the other hand, react spontaneously with aldehydes, but acyl hydrazone products are less stable in aqueous solution. Thus, when using hydrazide derivatized ligands, the resulting hydrazone is often reduced to N-alkyl hydrazides using mild reducing reagents such as sodium cyanoborohydride or pyridine borane. See, for example, Butler T. et al. Chembiochem. 2001, 2 (12) 884-894.

The formation of Schiff bases between amines and aldehydes provides another type of chemical conjugation method. As with hydrazide, in order to obtain a stable conjugate, it is often necessary to gently reduce the imine to produce an amine.

  Although mild reduction reagents are known, some problems can be foreseen in avoiding reduction of sulfide-sulfide (SS) bridges in proteins. In such cases, a bonding principle that avoids chemical reduction chemically is preferred.

C6-oxidized galactose residues also react efficiently with amino thiols such as cysteine or cysteine derivatives or aminoethane thiol to form thiazolidine, as shown below:

Also, the same type of modification that yields a cyclic product includes α-mercaptoacyl hydrazide:

Also, C6-oxidized galactose residues can be reacted with carbanion organophosphorus reagents in the Horner-Wadsworth-Emmons reaction. This reaction forms alkenes as shown below. The strength of the nucleophile can be altered by using different organophosphorus reagents, such as those used in the Wittig reaction.

Alternatively, the C6 oxidized galactose residue can be reacted with a carbanion nucleophile. This example can be an aldol-type reaction, as illustrated below. Z 'and Z''groups represent electron withdrawing groups such as COOEt, CN, NO2 (see March, Advanced Organic Chemistry, 3rd edition, John Wiley & Sons, NY 1985), which is the acidity of the methylene proton Increase the degree. In the present invention, it will also be possible to connect one or both of the Z groups to the R group (protractor), thereby improving the properties of the glycoprotein.

  The examples listed above for modifying oxidised galactose at the C6-position serve as non-limiting examples of the invention. Chemical procedures for modifying aldehyde function, such as those present in other nucleophiles and C6-oxidized galactose, are known to those skilled in the art (March, Advanced Organic Chemistry, 3rd edition, John Wiley & (See Sons, NY 1985).

The modification of the linker molecule or oxidized (asialo) glycoprotein may proceed in multiple steps before reaching the final product. Thus, in one embodiment, a C6 oxidized galactose residue is first reacted with a linker molecule that has specificity for the aldehyde moiety. The linker molecule, which itself contains additional chemical handles (bifunctional), is then reacted by attaching yet another molecule (eg, a retarding group) to give the final product:
Glycoprotein-CHO → Glycoprotein-Linker-X → Glycoprotein-Linker-R
Suitable bifunctional linkers are well known to those skilled in the art or can be readily considered. Examples include, but are not limited to, bifunctional linkers including hydroxylamines, amines, or hydrazides in combination with malimides, succimidyl esters, thiol hydroxylamines, amines, hydrazides, and the like.

  Although described as a stepwise reaction in Reaction Scheme 1 above, in some cases, a nucleophile is added directly to the reaction mixture when oxidation is performed using a galactose oxidase-catalase or galactose oxidase horseradish peroxidase enzyme pair. It is preferable. Such a one-pot condition can prevent any protein-protein reaction between any molecule of the aldehyde functional group on one protein and the amino group on the other (eg, epsilon amine in a lysine residue). . Intramolecular and intermolecular Schiff base (imine) formation between proteins can result in incomplete reaction with nucleophiles or precipitation of the protein in question. Also, since the aldehyde functionality can be readily reacted with nucleophiles present in the reaction medium, the one-pot condition prevents the possibility of any overoxidation mediated by galactose oxidase. The concentration ratio of nucleophile to protein can depend on the protein in question and the type of nucleophile selected for conjugation (eg, hydroxylamine, hydrazide, amine, etc.). Optimum conditions may be found by experiment, for example, a variation in the concentration ratio of the nucleophile to the protein, or a variation in the overall concentration of the protein, such as in solution.

  Although galactose or N-acetylgalactosamine residues are generally exposed after treatment with sialadase, the present invention can be used to covalently attach a retarding group to any terminal galactose moiety. Can do. One example is the use of galactosyltransferase to add terminal galactose residues to glycans, and such terminal galactose residues can be modified by the techniques described by the present invention.

Suspension principle The method described in the present invention allows the selection of a wide range of known clearance mechanisms, particularly for proteins and glycoproteins. Clearance methods for glycoproteins include glomerular filtration and renal clearance, proteolytic degradation by proteases present in plasma, such as recognition by glycan-specific receptors in the liver, and antibody-mediated immunoneutralization However, it is not limited to these.

Glomerular filtration and renal clearance:
Glomerular filtration is an important clearance pathway for proteins. Glomerular filtration is generally thought to be related to protein size and overall charge characteristics. The basement membrane of the glomerular snare wall is an ionic filter composed of negatively charged proteoglycans, which are neutral blood components, i.e. water and urea, and charged blood components such as salts, peptides and proteins Can be distinguished.

  The charge on the protein surface can interact with the immobilized, negatively charged heparin sulfate proteoglycans of the glomerular basement membrane. Proteins with a negative surface charge tend to be rejected by the immobilized negatively charged heparin sulfate proteoglycans that make up the glomerular basement membrane and are therefore less likely to pass into the urine. For example, the microclearance of negatively charged horseradish peroxidase was estimated to be 0.007 compared to the microclearance of neutral horseradish peroxidase estimated to be 0.061 (HG Rennke, Y. Patel, and MA Venkatachalam, Kidney Int. 13 1978 278-288).

  Thus, changes in surface charge can have a significant effect of directing protein sensitivity towards glomerular filtration. Thus, in one embodiment, in accordance with the present invention, a protein is treated with neuraminidase and galactose oxidase and then reacted with a molecular ligand containing one or more positively or negatively charged moieties to have altered surface charge properties. Protein is produced.

  As mentioned, the size of the protein also determines its ability to pass through the glomerular snare wall. Larger proteins (eg, proteins with a molecular weight of 50 KDa or more) are less prone to renal clearance than smaller proteins. Thus, increasing the given protein size may increase the circulating half-life by minimizing renal clearance.

  Thus, in another embodiment, in accordance with the present invention, the protein is treated with neuraminidase and galactose oxidase to prevent renal clearance and then contains a retarding group such as reactant X, eg, PEG or one or more dendrimers, For example, reacting with a nucleophile form produces a protein of significantly increased size.

Dendrimer:
The following sections consist of the exact number of monomer building blocks, using appropriate monomer protection and activation strategies, and oligomerized in any order, either on a solid support or in solution. A new class of branched polymers is described.

General structure of monomers Dendrimer monomers are generally linear, branched, 2, 3, or 4 branches of general structure A-L2-C- (L3-B) _n (general formula I) Where C serves as an attachment moiety for A-L2 and as a molecular moiety for n number of L3-B, where L2 and L3 are both The linker part is:

  A and B are both functional groups selected in such a method, and together they can form a covalent bond under appropriate conditions. The nature of the newly formed covalent bond depends on the choice of A and B, including but not limited to: amide bond, carbamate bond, ester bond, phosphate ester bond, thiophosphate ester Ester bonds, phosphoramidates, ethers, and thioether bonds.

A is selected from (but not limited to): COOH, COOR, OCOOR, OP (NR2) OR, OP (OR) 2, COCl, COBr, OCOCl, OCOBr, CHO, Br, Cl, I, OTs , OMs, alkynes,
Where R is alkyl, aryl, or substituted aryl;
Preferably, moiety A of general formula I represents an activated moiety capable of reacting with either nucleophiles on the peptide or of type B. Preferably A is selected from the following groups:
Functional groups that can react with amino and hydroxy groups such as:
a) a carbonate such as p-nitrophenyl, or succinimidyl;
b) carbonylimidazole or carbonyl chloride;
c) Carboxylic acids activated in situ;
d) activated esters such as carbonyl halides, N-hydroxysuccinimide esters, N-hydroxybenzotriazole esters, esters of 1,2,3-benzotriazin-4 (3H) -one,
e) phosphoramidite and H-phosphonate, or
f) Isocyanates or isothiocyanates.

B may be selected from NH ₂ , OH, N ₃ , NHR ′, OR ′, O—NH ₂ , O—NHR ′,
Where R ′ is a protection group that includes, but is not limited to:

  Other examples of suitable protecting groups are known to those skilled in the art and can be found in Green & Wuts “Protection groups in organic synthesis”, 3.ed. Wiley-interscience.

Preferably, moiety B of general formula I represents a protected nucleophile moiety that is preferably capable of reacting with an electrophile of type A. Preferably B is selected from the following group:
a) Fmoc protected amino group,
b) a free amino group,
c) an azide that can be reduced to an amino group,
d) an azide which may be added together with an alkyne to form a triazole,
e) O-substituted hydroxylamine,
f) hydroxyl group,
g) DMT, MMT, or trityl protected hydroxyl groups.

  In other embodiments of the invention, the definitions of A and B may be exchanged to facilitate branched polymer construction by a convergent approach, as described in the references below.

  C is preferably either a hydrophilic, linear (divalent organic radical) or branched (multivalent branched organic radical) linker. This preferably includes various functionalized alkyl groups containing up to 18, more preferably between 1 and 10 carbon atoms. Some heteroatoms such as nitrogen, oxygen, or sulfur may be included in the alkyl chain. The alkyl chain may be branched by carbon or nitrogen atoms. In one embodiment of the invention, C is a single nitrogen atom.

Examples of C include, but are not limited to: divalent organic radicals such as ethylene, arylene, propylene, ethyleneoxy,
Or propane-1,2,3-triyl, benzene-1,3,4,5-tetrayl, 1,1,1-nitrogen triyl, or polyvalent cyclic carbons including but not limited to Multivalent organic radicals of:

In one embodiment, C is

It is.

C may be separated from A or B by linkers L2 and L3, which is preferably hydrophilic. Examples of such linkers include, but are not limited to:
1,2-ethanediyl, 1,3-propanediyl, 1,4-butanediyl, 1,5-pentanediyl, 1,6-hexanediyl, (CH ₂ CH ₂ O-) _n , where n is 0-10 Is an integer between
-(CR1R2-CR3R4-O) _n- , where n is an integer between 0 and 0 and R1, R2, R3, and R4 are independently H, Me, Et, Pr it can.

((CH ₂ ) _m O) _n- , where m is ..2, 3, 4, 5, 6 and n is an integer between 0 and 10.

  However, C need not be symetrically.

  Also, L2, T3, or both can be valence bonds.

Preferably L2 and T3 are selected from water-soluble organic divalent radicals. Thus, in one embodiment, L2 or L3, or both are divalent organic radicals containing about 1-5 PEG (—CH ₂ CH ₂ O—) groups.

In one embodiment, L2 is - oxy - or - oxymethyl, and L3 is a _{(CH 2 CH 2 O-) 2} :

  Thus, in one embodiment, A is a carboxyl group and B is a protected amino group that, after deprotection, can be attached to a new monomer of the same type via the carboxy group. Good is an amide.

  In another embodiment, A is a phosphoramidite and B is a suitable protected hydroxyl group, which, by deprotection, binds to other monomers of the same type to form triphosphite. Esters can be formed and subsequently oxidized to form stable phosphate triesters or thiophosphate triesters.

  In yet another embodiment, A is a reactive carbonate such as nitrophenyl carbamate and B is preferably its protected amino group.

  In yet another embodiment, A is an acyl halide such as COCl or COBr and B is preferably its protected amino group.

In certain embodiments, A-L2-C- (L3-B) _n is

It is.

In certain embodiments, A-L2-C- (L3-B) _n is

It is.

In certain embodiments, A-L2-C- (L3-B) _n is

It is.

In certain embodiments, A-L2-C- (L3-B) _n is

It is.

Synthetic branched polymers of polymers with dendritic structures are generally derived from the monomers described above using one of two fundamentally different oligomerization strategies called the divergence approach and the convergence approach. Can be built.

Construction of branched polymers by divergence approach:
In one embodiment, the branched polymer is constructed by an iterative process of synthesis cycles, where each cycle has a functional end group that can itself further extend (ie, polymer growth). Use appropriately activated reactive 2, 3, or multiple branched monomers. Functional end groups usually need to be protected to prevent self-polymerization, in which case a deprotection step is required to generate the functional end groups necessary for further extension. . In an iterative process, generation is completed by a cycle of addition of such activated (reactive) monomers and subsequent deprotection. The divergence approach is illustrated in solution phase chemistry using FIG. 13 and in solid phase chemistry using FIG.

Convergence construction of branched polymers:
However, when achieving higher product in such an iterative process, a high packing density of functional end groups often appears, which prevents further regular growth and leads to incomplete production. . In fact, in all systems that require the reaction of a large number of surface functional groups for growth, it is difficult to ensure that everything reacts at each growth step. Since unreacted functional end groups can result in failure sequences (cleavages) or pseudoreactivity in later stages of the step-growth sequence, this is regularly monodispersed and highly organized. This presents a significant problem in the synthesis of branched structures.

  Thus, in one embodiment of the present invention, the branched (dendritic) polymer is constructed by the convergent approach described in US Pat. No. 5,041,516. A convergent approach to building macromolecules involves building the final molecule around it, rather than starting with its core, as with the divergent approach. This avoids problems such as incomplete covalent bond formation, typically associated with reactions at cumulatively multiple sites.

  A convergent approach for construction 2 generation dendritic material is illustrated in FIG. 10 and FIG. 11 using an embodiment using one of the monomers of the present invention.

It is important to note that the variation final branched polymer in individual layers may consist of different types of monomer building blocks in each of the layers or generations, if desired. Branched polymers with custom properties can be made using different monomers in each layer. By that method, the overall properties of the polymer and polymer-peptide conjugate can be controlled.

  For example, one can be interested in controlling the overall stiffness of the branched polymer. Select a branched monomer in the first layer, followed by a linear monomer in one or several layers to create a polymer structure with a few branches and an overall flexible structure can do. On the other hand, in each layer, repeatedly use highly branched monomers, such as 3 or 4 branched, while removing any linear, less branched monomers, A highly branched polymer having a high density and an overall dense structure can be obtained. Rigidity can also be controlled by the design of the particular monomer, for example by using a strong core structure (C) or by using a strong linker moiety (L2, L3). Then, it can be obtained by using a strong monomer in one or more specific layers mixed with a monomer having a flexible property by fine adjustment or adjustment of rigidity. It may also be of interest to fine tune the overall hydrophilic nature of the polymer. This can be achieved by selecting monomers with more hydrophobic core structure (C) or more hydrophobic linker moieties (L2 and L3) in one or more dendritic layers.

Also, it is often desirable to use different monomers in the outer layer of the branched polymer where the final peptide conjugate is exposed to the surrounding environment. Some monomers described in the present invention have a protected amine function as the end group (B) and after the deprotection step and at physiological conditions, i.e. neutral physiologically buffered pH. Under about 7.4, it is protonated to produce a polycationically charged overall structure. Such polycationic structures have proven to be toxic in animal studies and, although they are generally cleared rapidly from the blood circulatory system, they are avoided in any pharmaceutical context It should be. By carefully choosing the monomers used to make the final layer, polycation structures can be avoided. One example as illustrated in FIG. 14 uses Me (Peg) 2 CH ₂ COOH acid to coat the final layer of the dendritic structure, otherwise terminated with an amine.

  On the other hand, polyanionic biopolymers are well known, for example in naturally occurring glycoproteins, which generally have a plurality of anionic charged sialic as termination groups on the antenna structure of these N-glucans. Have acid. In addition, such glucans can be mimicked with respect to their polyanionic nature, according to the present invention and by appropriate selection of the monomers used to make the final layer. One such example is shown in FIG. 15, where the dendritic layer is coated with succinic acid mono tert butyl ester (estes) and is negatively protected under physiological conditions by acid deprotection. Provides a charged polymer surface.

Construction of solid phase oligomer-forming monomers into polymers can be done on solid supports as described by NJ Wells, A. Basso and M. Bradley in Biopolymers 47, 381-396 or US Pat. No. 5,041,516. As described by Frechet et al. In classical organic solution phase chemistry, either in a suitable organic solvent.

  Thus, in one embodiment of the invention, the branched polymer is constructed on a solid support derivatized with appropriate linkages in a repetitive divergence process, as illustrated above and illustrated in FIG. For monomers designed with Fmoc or Boc protected amino groups (B) and acylation moieties (A) of reactive functional groups, conveniently adapt solid phase protocols useful for conventional peptide synthesis be able to. A suitable standard solid phase method (see Fields, ed., Solid phase peptide synthesis, in Meth Enzymol 289), such as those described in the literature, with a suitable programmable device (eg ABI 430A) or similar This can be done either by using conventional home build machines or using standard filtration techniques for manual support separation and washing.

For monomers with, for example, DMT-protected alcohol groups (B) and, for example, reactive phosphoramidites (A), it is convenient to use solid oligonucleotides used for standard oligonucleotide synthesis such as Applied Biosystems Expidite 8909. Phase equipment and conditions such as those recently described by M. Dubber and JMJ Frechet in Bioconjugate chem. 2003, 14 , 239-246 can be applied. Such solid phase synthesis of phosphodiesters according to the conventional phosphoramidite method requires that the intermediate phosphite triester be oxidized to phosphotriester. This type of solid support oxidation is conveniently accomplished using iodine in an inert organic solvent and requires that the monomer be resistant to iodine oxidation with or without protecting groups. is there. In addition, the phosphoramidite method is based on the simple substitution of iodine in organic thiolating reagent solutions such as elemental sulfur pyridine or 3H-1,2-benzodithiol-3-one-1,1-dioxide. A convenient synthesis is possible (see for example M. Dubber and JMJ Frechet in Bioconjugate chem. 2003, 14 , 239-246).

  When complete, the branched polymer attached to the resin can be isolated from the resin under suitable conditions. A linker capable of cleaving between a growing polymer and a solid support is an individual monomer oligomerization process that includes any deprotection, oxidation, or reduction steps used in individual synthesis cycles. It is important to select in such a way that the final branched polymer can be cut intact and spoken off under appropriate conditions when desired. One skilled in the art can make an appropriate selection of linkers and supports, and reaction conditions for the oligomer formation process, deprotection process, and optionally an oxidation process, depending on the monomer in question. Resins derivatized with suitable functional groups to which monomeric units can be attached and subsequently serve as cleavable moieties are commercially available (see, for example, Bachem and NovoBiochem catalogs). See).

  In other embodiments of the present invention, the branched polymer is supplied by cleavage, as described below, with functional groups that can act directly on the peptide as attachment groups in subsequent solution phase conjugation processes. The branched polymer product is produced or alternatively synthesized on a resin with an appropriate linker that can be converted to such an attachment group by appropriate chemical means.

Solution Phase Oligomer Formation In some cases, it may be advantageous to synthesize a dendritic polymer of constant size and composition using classical solution phase techniques.

Thus, in another embodiment of the invention, the branched polymer is constructed in a suitable solvent by adding the appropriate activated monomer over time to the growing polymer. . After each addition, a deprotection step may be necessary before the construction of the next layer or generation can begin. In order to achieve a complete reaction, it is desirable to use an excess of monomer. In one embodiment of the invention, removal of excess monomer takes advantage of the fact that hydrophilic polymers are poorly soluble in diethyl ether or the same type of solvent. Thus, the growing polymer is precipitated, leaving excess monomer, coupling reagent, biproducts, etc. in solution. The phase separation can then be carried out by simple decantation, more preferably by decantation following centrifugation. The polymer may also be subjected to normal or reverse phase conditions, eg, by conventional chromatographic techniques on silica gel or as described in PR Ashton et al. J. Org. Chem. 1998, 63, 3429-3437. It can be separated from the biproduct by using an HPLC or MPLC system under either. Alternatively, considerrbly large polymers can be obtained using size exclusion chromatography, optionally as described in ERGillies and JMJ Frechet in J. Am. Chem. Soc. 2002, 124 , 14137-14146. In combination with dialysis, it can be separated from low molecular components such as excess monomer and biproduct.

  In contrast to the solid phase method, this may also be used in the solution phase for the construction of branched polymers as outlined above and further in SMGrayson and JMJ Frechet, Chem. Rev. 2001, 101, 3819-3867. Can be used for convergence approach. In this approach, it is desirable to initiate the synthesis with monomers, in which case the initially protected functional end group (B) is ultimately converted to a moiety present on the outer surface of the final branched polymer. The Thus, the functional moiety (A) of general formula I in most cases requires appropriate protection that allows stepwise chemical manipulation of the end group (B). The protecting group for the functional moiety (A) depends on the actual functional group. For example, if A in general formula I is a carboxyl group, a tert-butyl ester derivative that can be removed by TFA would be a suitable choice. Suitable protecting groups are known to those skilled in the art and other examples can be found in Green & Wuts “Protection groups in organic synthesis”, 3.ed. Wiley-interscience. The convergent construction of branched polymers is illustrated in FIGS. 10 and 11. In step (i) of FIG. 10, the tert-butyl ester functionality (A) is prepared by reaction of t-butyl α-bromoacetic acid with a suitable precursor. In step (ii), the end groups (B) are manipulated in such a way that they can be acylated in step (iii) with carboxylic acids and converted to acyl halides in step (iv). In step (v), the t-butyl ester functionality (A) is removed to produce a terminal (B) capped monomer. This end-capped monomer serves as a starting material for preparing the product of the second generation of FIG. 11, in which two equivalents are combined with the product of step (ii) of FIG. Used for acylation reactions. The product of this reaction is a new t-butyl ester that, after deprotection, can be returned to the initial step of FIG. 11 in an iterative manner to yield a higher generation material.

Conjugation to peptides and proteins
In order to covalently attach the polymer molecule to the peptide in the attachment moiety solution to the branched polymer , the polymer must be provided with a reactive handle, i.e. a reactive functional group (examples of which are , Primary amino groups, hydrazides, hydrazides, β and γ amino thiols, or hydroxylamine, by way of example and not limitation).

  Suitable attachment moieties on the branched polymer, such as those described above, may be made after the polymer is constructed using conventional solution phase chemistry or solid phase chemistry. A non-limiting example of how to make a nucleophilic attachment moiety on a branched polymer containing a carboxylic acid group is listed in FIG.

One or more of the activated branched polymers can be attached to the biologically active polypeptide by standard chemical reactions according to the present invention. The conjugate is represented by the general formula II:
(Branched polymer) _z- L ⁴ (Polypeptide)
(Formula II)
Where (branched polymer) is a water-soluble, non-antigenic polymer consisting essentially of monomers according to general formula I, and L ⁴ is essentially L of general formula I Linking moieties defined with respect to ² and L ³ , (z) represents the number of branched polymers conjugated to a biologically active polypeptide with an integer ≧ 1. The upper limit of (z) is determined by the number of available attachment points on the polypeptide and the extent of polymer attachment investigated by those skilled in the art.

  The degree of conjugation can be modified by changing the reaction stoichiometry using well-known techniques, as described above. A plurality of branched polymers conjugated to a polypeptide can be obtained by reacting a stoichiometric excess of activated polymer with the polypeptide.

  Biologically active polypeptides can be reacted with the activated branched polymer in an aqueous reaction medium that can be buffered, depending on the pH requirements of the polypeptide. The optimum pH for the reaction is generally between about 6.5 and about 8, and preferably about 7.4 for most polypeptides.

  Optimal reaction conditions for polypeptide stability, reaction efficiency, etc. are within the level of ordinary skill in the art. A preferred temperature range is between 4 ° C and 37 ° C. The temperature of the reaction medium cannot exceed the temperature at which the polypeptide may denature or degrade. The polypeptide is preferably reacted with an excess of activated branched polymer. Following the reaction, the conjugate was recovered and purified by diafiltration, column chromatography including size exclusion chromatography, ion exchange chromatography, affinity chromatography, electrophoresis, or combinations thereof.

Specific examples of suitable protected monomers included in the present invention are:
General formula Ia-linear monomer (A-L2-C-L3-B):

General Formula Ib-Dichodron Monomer (A-L2-C- (L3-B) ₂ ):

General formula Ic-Trifurcated monomer (A-L2-C- (L3-B) ₃ ):

Proteolytic shielding:
A significant problem when using proteins and peptides as therapeutic agents is that they are sensitive to proteolytic degradation by proteases present in plasma. Degradation not only plays a clear role in the removal of damaging or abnormal polypeptides, but also affects the half-life of normal proteins and peptides, and an individual's turnover rate depends on the peptide sequence, structure, and protein In this case, it can strongly depend on the surface characteristics.

  The shielding effect on proteolysis depends not only on structural or temporal factors but also on overall biophysical factors such as overall hydrophobicity or overall charge characteristics.

  Thus, even slight changes in the overall charge characteristics of a protein can have an effect on protease-mediated proteolysis in serum. For example, such changes in the glycan portion of a glycoprotein by incorporating a portion that is charged under physiological conditions are also included in the present invention.

  Non-limiting examples of moieties that are negatively charged under physiological conditions include carboxylic acids, sulfonic acids, phosphonic acids, phosphoric acids, phosphoramidates, and the like. Non-limiting examples of moieties that are positively charged under physiological conditions include primary, secondary, tertiary, and quaternary amino groups, guanidine, and heterocycles such as pyridine, imidazole, quinoline, and others.

  Accordingly, in one embodiment of the present invention, glycoproteins treated with sialidase (optionally) and galactose oxidase according to the present invention to produce modified glycoproteins with enhanced stability against serum proteases. Is reacted with a nucleophile containing a positively or negatively charged moiety under physiological conditions.

  Other variations that can increase stability against proteolytic degradation include attachment of hydrophobic side chains such as long chain alkanes and polyaromates or large hydrophilic polymers such as polyethylene glycol (PEG) Including adhesion.

An albumin binding agent or a ligand that binds a serum protein such as albumin may be conjugated to the glycan portion of the therapeutic glycoprotein using the procedures described in the present invention. This allows glycoproteins to form non-covalent complexes, eg, specific for albumin, when modified in this way, which escapes glomerular filtration depending on the overall size. be able to. Thus, non-covalent complexes can reduce the biological activity of the glycoprotein counterpart; to retain biological activity, the affinity of the ligand can be tailored to the particular serum protein selected. desirable.

  Thus, in another embodiment of the present invention, a galactose oxidase-treated glycoprotein according to the present invention can be coupled with human serum albumin or diacid or lithocholic acid long chain fatty acid residues (eg C12, C14, C16 or C18) is reacted with reactant X (for example in the form of a nucleophile).

  In other embodiments of the present invention, a glycoprotein treated with galactose oxidase according to the present invention is a reactant X comprising a portion of a protein having a long circulating half-life, such as an immunoglobulin (eg in the form of a nucleophile) React with. In certain embodiments, the glycoprotein is treated with reactant X (eg, in the form of a nucleophile) comprising the Fc domain of IgG.

Epitope masking—Sugar-specific recognition process inhibition:
Mammalian glycoproteins often have N-acetylneuraminic acid (sialic acid) as the external (terminal) residue of the oligosaccharide chain, which may be N-linked or O-linked (See Osawa and Tsuji (1987) Ann. Rev. Biochem. 56:21). If the nature of the oligosaccharide is the primary determinant of clearance from circulation, in general, glycoproteins with terminal sialic acid residues removed (asialoglycoproteins) are more likely than their untreated counterparts. Cleared rapidly. Circulating glycoproteins are exposed to sialidase (or neuraminidase) that can remove terminal sialic acid residues. Typically, removal of sialic acid exposes galactose residues that are recognized and bound by hepatocyte galactose-specific receptors (Ashwell and Harford (1982) Ann. Rev. Biochem. 51: 531). The liver also contains other sugar-specific receptors that mediate the removal of glycoproteins from the circulation. Such receptor specificities also include N-acetylglucosamine, mannose, fucose, and phosphomannose. Glycoproteins cleared by hepatocyte galactose receptors undergo substantial degradation and then enter bile; glycoproteins cleared by stellate mannose receptors enter the reticuloendothelial system (Ashwell and Harford 10 (1982) Ann. Rev. Biochem. 51:53). The studies with asialo-ceruloplasmin and derivatives are extended compared to asialo-ceruloplasmin, where galactose residues are oxidized by treatment with galactose oxidase and horseradish peroxidase and asialogalacto-ceruloplasmin. Showed a circulatory half-life (Morell et al. (1968) J. Biol. Chem. 243: 155). Efficient removal by the galactose receptor appears to be necessary with at least two exposed galactose residues. Glycoproteins where sialic acid addition is an important determinant of clearance from the circulation, and glycoproteins where sialic acid addition does not share a role for clearance or oligosaccharides play a relatively minor role in clearance From the above cited reference, it can be concluded that most of the apparent mechanisms for the fate and clearance of a particular circulating glycoprotein are empirically determined.

  Modifications that result in increased half-life include those by exposing galactose residues, followed by oxidation or derivatization of galactose, such that the binding of the modified glycoprotein to the galactose receptor is inhibited or blocked. It is not limited. Thus, in one embodiment of the present invention, a galactose oxidase treated glycoprotein according to the present invention is reacted with a nucleophile comprising a steric moiety that prevents glycan specific receptor recognition.

  In one embodiment, the terminal sialic acid residue of the oligosaccharide side chain of the soluble protein or soluble protein derivative is removed by neuraminidase treatment, and then the exposed galactose residue is converted to galactose oxidase (optionally horseradish peroxidase treatment). ) Is oxidized. Oxidation of exposed galactose residues has the effect of preventing rapid clearance of the modified glycoprotein from circulation by liver specific galactose receptors. Other structural modifications of terminal galactose residues (including addition of functional groups or small molecules, or mild oxidation treatments that have a blocking effect, including inhibiting or preventing recognition of terminal galactose residues without destroying biological activity) But is not limited thereto) is understood to be functionally equivalent.

  Modified glycoproteins made in accordance with the present invention can block or inhibit clearance through liver sugar-specific receptors, reduce renal clearance, or minimize proteolytic degradation This includes structural changes (modifications) in the oligosaccharide portion of the glycoprotein that result in an increase in circulation half-life. Modifications that substantially reduce the biological activity of the glycoprotein are avoided.

  Preferably, the glycoprotein is synthesized in a recombinant mammalian host. Chemical and enzymatic treatments that result in structural changes in the oligosaccharide should not substantially alter the glycoprotein binding reaction to its biological target protein. The circulating half-life of the modified glycoproteins of the invention is 4 to 48 hours, such as at least 24 hours in humans (or at least about 2 to 12 hours such as at least about 6 hours as measured in a rat animal model). Preferably there is. Most preferably, the structural modification of the oligosaccharide to extend the circulation half-life is the most conservative structural change that achieves the purpose. A variety of chemical derivatization procedures or chemical and / or enzymatic procedures that are understood in the art may be used to produce the modified glycoproteins of the invention.

  Most desirably, any modified glycoprotein made in accordance with the present invention does not induce an immune response in a human patient exposed to the modified glycoprotein. In addition, biological activity should be such that its activity is significantly reduced and does not have a detrimental effect on the therapeutic function of glycoproteins due to structural modifications used to confer prolonged circulation. . It is also most desirable that any structural modification of the glycoprotein does not cause toxicity in the patient to whom the modified glycoprotein is administered. Obviously, modified glycoproteins for therapeutic use should minimize addictive, irritating, or other side effects from administration to humans. Therefore, strategies for extending the circulation of specific glycoproteins must be assessed individually.

  Chemical modification or derivatization of the C6 position of galactose maximizes half-life, minimizes clearance without significantly affecting biological function and inducing negative physiological responses Preferred; minimal and gentle treatment is preferred. Functional groups or other structurally modified molecules that may be added to the oligosaccharide portion of a glycoprotein according to the present invention can be any chemistry of a size from a single methyl group to a larger polymeric group such as, for example, polyethylene glycol. Contains groups. Modifications that substantially reduce the biological activity of the glycoprotein are avoided.

  In general, the most evaluated mechanism for clearance, or strategy for slowing or avoiding clearance, must consider maintaining the desired biological activity or function, potential toxicity, potential immunogenicity, and cost. I must.

  As long as the desired therapeutic effect is achieved by these molecules, and clearance by clearance that mediates glycospecific receptors by proteolysis or by renal clearance is inhibited by modifications made to the glycoprotein, Alternatively, as long as it is prevented, the uniformly modified glycoprotein may be incorporated into a therapeutic composition, or a mixture of modified glycoproteins may be formulated in such a composition. Thus, the methodology is generally applicable to therapeutic glycoproteins that are cleared from the circulation by renal clearance, glycospecific receptors, or proteases present in serum. Those skilled in the art will be able to obtain assay results other than those specifically described herein to obtain the same or equivalent results and to achieve the goals described herein, It will be readily apparent that reagents, procedures, and techniques can be used. For example, chemical means of oxidation by removal of sialic acid can be easily replaced with specifically described enzymatic means. All such variations are encompassed within the spirit and scope of the present invention.

  In one embodiment of the invention, a modified soluble glycoprotein derivative is produced that has an increased plasma half-life compared to the unmodified derivative. Increased circulating half-life of glycoproteins is generally due to removal of glycoproteins by galactose, mannose, or other sugar-specific receptors, or by means of inhibiting renal clearance, proteolytic degradation, or immunological neutralization Achieved by means of blocking or inhibiting.

  Accordingly, in another embodiment, a protein comprising an antenennary N-glucan is reacted with sialidase, galactose oxidase and hydrogen peroxide scavenger (eg, catalase) according to the present invention to produce the resulting protein product O -Treatment with pegylated hydroxylamine produces a glycol-sugar conjugate with reduced renal clearance.

  In another embodiment, a galactose-oxidized glycoprotein prepared according to the present invention is reacted with O-carboxymethyl hydroxylamine, without any sialidase labile neuraminic acid, Glycoproteins with similar isoelectric characteristics are produced.

  In yet another embodiment, a galactose-oxidized glycoprotein prepared according to the present invention is reacted with O-diethylaminoethylhydroxylamine to produce a glycoprotein with altered binding properties to liver lectin receptors. Generated.

  In one embodiment, the glycoprotein is reacted with sialidase followed by optionally galactose oxidase and a nucleophile to produce a glycoconjugate with reduced renal clearance compared to unmodified glycoprotein. In one of these embodiments, the produced glycoconjugate has at least a 50% decrease in renal clearance compared to the unmodified glycoprotein. In another of these embodiments, the glycoconjugate produced has a decrease in renal clearance of at least 100% compared to the unmodified glycoprotein.

  In another embodiment, a glycoprotein is reacted with sialidase, followed by galactose oxidase and optionally a nucleophile to produce a glycoconjugate with reduced binding to an asialoglycoprotein receptor compared to an unmodified glycoprotein. Generated.

  In another embodiment, the glycoprotein is reacted with sialidase, optionally followed by galactose oxidase and a nucleophile, to produce a glycoconjugate with reduced binding to the mannose receptor compared to the unmodified glycoprotein. Is done.

  In another embodiment, the glycoprotein is reacted with sialidase, followed by galactose oxidase and optionally a nucleophile to produce a glycoconjugate with reduced clearance in the liver compared to unmodified glycoprotein. . In one of these embodiments, the produced glycoconjugate has at least a 50% reduction in liver clearance compared to the unmodified glycoprotein. In another of these embodiments, the produced glycoconjugate has a reduction in liver clearance of at least 100% compared to an unmodified glycoprotein.

  In another embodiment, the glycoprotein is optionally reacted with a sialidase followed by galactose oxidase and a nucleophile to produce a glycoconjugate with increased serum stability compared to the unmodified glycoprotein. Wake up.

  In another embodiment, glycoconjugates are reacted with sialidase and then optionally with galactose oxidase and nucleophiles to increase the circulating half-life in animal models compared to unmodified glycoproteins. Is generated. In one of these embodiments, the glycoconjugate produced has an increased circulating half-life of 100% in an animal model compared to an unmodified glycoprotein.

In one series of embodiments, a galactose oxidized glycoprotein prepared according to the present invention is reacted with a nucleophile connected to a fatty acid (eg, C ₅ -C ₂₄ ). The fatty acid can be connected to the nucleophile via a linker moiety. The linker moiety may be a simple structure designed to connect a fatty acid to a nucleophile or it may contain functional groups that enhance the in vivo properties of this embodiment (eg, carboxylic acid , Amines, alcohol, etc.).

In another series of embodiments, a galactose oxidized glycoprotein prepared according to the present invention is reacted with a nucleophile connected to an aliphatic diacid (eg, C ₅ -C ₂₄ ). The aliphatic diacid can be connected to the nucleophile via a linker moiety. The linker moiety may be a simple structure designed to connect an aliphatic diacid to a nucleophile or it may contain functional groups that enhance the in vivo properties of this embodiment (e.g. Carboxylic acids, amines, alcohols, etc.).

  In another set of embodiments, a galactose oxidized glycoprotein prepared according to the present invention is reacted with reactant X (eg, in the form of a nucleophile), which binds to an albumin-like serum protein. Connected to things. Buildings that bind to serum proteins can be connected to the nucleophile via a linker moiety. The linker moiety may be a simple structure designed to connect an aliphatic diacid to a nucleophile or it may contain functional groups that enhance the in vivo properties of this embodiment (e.g. Carboxylic acids, amines, alcohols, etc.).

  In another series of embodiments, a galactose oxidized glycoprotein prepared in accordance with the present invention is reacted with reactant X (eg, in the form of a nucleophile), which accepts the glycoprotein from the circulation. Connected to structures (eg, sialic acid derivatives) that inhibit glycans from binding to the body (eg, asialoglycoprotein receptor and mannose receptor).

  In another series of embodiments, the glycoprotein is first treated sequentially with a series of enzymes such as neuramidases, galactosidase, mannosidase, endo-H, and endo-F3, or directly with endo-H or endo-F3. Thus, part of the glycan structure is removed (see for example K. Witte et al. J. Am. Chem. Soc., 119, 2114 (1997)). The galactose moiety can then be added to the new terminus by using a galactosyltransferase and an appropriate galactose substrate, or a series of transferases with various sugar substrates can be used before using the galactosyltransferase. May be. The new glycan structure can then be oxidized and reacted with the nucleophile described by the invention, thus a glycoprotein having an alternative glycan structure and a galactose modification that improves its therapeutic properties. Arise.

Peptides conjugated with glycoprotein retarding groups suitable for conjugation according to the present invention have been described as being “biologically active”. However, the term is not limited to being physiologically or pharmacologically active. For example, several polymer conjugates of the present invention comprising proteins such as immunoglobulins, enzymes with proteolytic activity are also useful as laboratory diagnostics, ie for in vivo studies and the like. An important feature of all conjugates is that at least the same part of the activity associated with the unmodified bioactive peptide is maintained.

  Thus, the conjugate is biologically active and has numerous therapeutic applications. A human in need of treatment containing a bioactive peptide will be able to be treated by administering an effective amount of a branched polymer conjugate containing the desired bioactive peptide. For example, humans in need of enzyme replacement therapy or blood factors can be given branched polymer conjugates containing the desired peptide.

  Bioactive peptides of interest of the present invention include, but are not limited to proteins, peptides, peptides, and enzymes. Enzymes of interest include sugar chain specific enzymes, proteolytic enzymes, oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases. Examples of enzymes of interest, but not limited to specific enzymes, include asparaginase, arginase, arginine deaminase, adenosine deaminase, superoxide dismutase, endotoxinases, cataiases, chymotrypsin, lipase, uricase, adenosine Includes diphosphatase, tyrasinases, and bilirubin oxidase. Sugar chain specific enzymes of interest include glucose oxidase, glycosidase, glucocerebrosidase, glucouronidases and the like.

  Glycosyltransferases using glycosyltransferases as described in Li Shao et all. Glycobiology 12 (11) 762-770 (2002) or enzymatically, eg, standard peptide chemistry and N-galactosylated asparagine Peptides and proteins that do not contain a glycan moiety that are chemically synthesized by using derivatized amino acid components can be glycosylated.

  Alternatively, glycosylation sites may be designed into proteins or peptides that are normally generated in vivo in these unglycosylated forms. For example, the insertion of a consensus sequence Cys-XXX-Ser-XXX-Pro-Cys within the EGF repeat allows selective serine O-glycosylation using UDP-glucose and glucosyltransferase. all. Glycobiology 12 (11) 762-770 (2002), while consensus sequence Asn-XXX-Ser / Thr allows N-glycosylation RA Dwek, Chem. Rev. 1996, 96, 683-720 . Also, peptide sequences containing threonine or serine are subject to glycosylation in a sequence-dependent manner in the presence of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase and UDP-GalNAc (eg BC O'Connell FKHagen and LA Tabak in J. Biol. Chem. 267 (35), 25010-25018 (1992)). Alternatively, to introduce galactose or galactose containing sugar structures via mixed disulfide formation as described by DP Gamblin et al. In Angew. Chem. Int. Ed., 43, 828 (2004). Alternatively, site directed mutagenesis introducing cysteine mutations can be used. In addition, peptides and proteins containing galactose or N-acetylgalactosamine include non-biogenic treatments such as the method described by PG Schultz in J. Am. Chem. Soc, 125, 1702 (2003). It can be made by conjugation to a protein or peptide or non-specifically by direct glycosylation of the peptide using a glycosyl donor substrate such as trichloroacetamidyl galactoside. Also, glycosidase inhibition to fermentation culture as described in US 4925796A / US 5272066A1 to obtain proteins containing galactose or N-acetylgalactosamine and for enzymatic modification of glutamine residues using TGase A glycoprotein having a truncated glycan structure can be produced by adding an agent (see, for example, M. Sato et al. Angew. Chem. Int. Ed. 43, 1516-1520, (2004)). Wanna)

  Production of N-glycosylated proteins is not limited to the use of mammalian host cells such as CHO or BHK cells, but is described by M. Wacker et al. In Science, 298, 1790-1793 (2002) It can also be performed using cells.

  Proteins and peptides of interest are hemoglobin, factor VII, FX, FII, factor FV, protein C, protein S, tPA, PAI-1, tissue factor, FXI, FXII, and FXIII, and sequences FVIII, FIX, these Serum proteins such as blood factors including mutants; cytokines such as immunoglobulins, interleukins, α-, β-, and γ-interferons, colony-stimulating factors including granulocyte colony-stimulating factor, platelet-derived growth factor, and phospholipase activity Including but not limited to activated protein (PUP). Other proteins and peptides of general biological and therapeutic interest are plant proteins such as insulin, lectin and lysine, tumor necrosis factor and related alleles, soluble forms of tumor necrosis factor receptor, interleukins Soluble forms of receptors and interleukin receptors, growth factors such as TGFa or TGFps, and tissue growth factors such as epidermal growth factor, hormones, somatomedin, erythropoietin, pigment hormone, hypothalamic release factor, antidiuretic hormone, prolactin, villi Includes gonadal stimulating hormone, follicle stimulating hormone, thyroid stimulating hormone, tissue plasminogen activator, etc. The immunoglobulins of interest include IgG, IgE, IgM, IgA, IgD, and fragments thereof.

In one embodiment of the invention, the glycoprotein is selected from the group consisting of: aprotinin, tissue factor pathway inhibitor, or other protease inhibitor, insulin or insulin precursor, human or bovine growth hormone, inter Leukin, glucagon, oxyntomodulin, GLP-1, GLP-2, IGF-I, IGF-II, tissue plasminogen activator, transforming growth factor γ or β, platelet-derived growth factor, GRF (growth hormone) Release factors), human growth factors, immunoglobulins, EPO, TPA, protein C, FVII, FVIII, FIX, FX, FII, FV and other blood clotting factors, protein C, protein S, PAI-1, tissue factor, FXI, FXII and FXIII, exendin-3, exendin-4, and enzymes or functional analogues thereof. In this context, the term “functional analogue” is meant to indicate a protein that has a function similar to that of the native protein. A protein may be structurally similar to a natural protein, with the addition of one or more amino acids to either or both of the C and N termini of the natural protein, one or more at one or many different sites of the natural amino acid. Amino acid substitutions, natural protein either or both, or deletion of one or more amino acids at one or several sites in the amino acid sequence, or at one or more sites in the natural amino acid sequence It may be derived from a natural protein by insertion of one or more amino acids. Further, the protein may be acylated at one or more positions, including WO 98/08871 (which discloses acylation of GLP-1 and analogs thereof) and WO 98/08872. (This discloses the acylation of GLP-2 and analogs thereof). An example of an acylated GLP-1 derivative is Lys26 (N ^epsilon -tetradecanoyl)-, which is GLP-1 (7-37) in which the epsilon-amino group of the Lys residue at position 26 is tetradecanoylated. GLP-1 (7-37).

  In one embodiment of the invention, the glycoprotein is selected from the group consisting of: FVII, FVIII, FIX, FX, FII, FV, protein C, protein S, tPA, PAI-1, tissue factor, FXI , FXII, FXIII, and sequence variants thereof; immunoglobulins, cytokines such as interleukins, α-, β-, and γ-interferons, colony-stimulating factors including granulocyte colony-stimulating factor, platelet-derived growth factor, and phospholipase Activated protein (PUP). Other proteins and peptides of general biological and therapeutic interest are plant proteins such as insulin, lectin and lysine, tumor necrosis factor and related alleles, soluble forms of tumor necrosis factor receptor, interleukins Soluble forms of receptors and interleukin receptors, growth factors such as TGFa or TGFps, and tissue growth factors such as epidermal growth factor, hormones, somatomedin, erythropoietin, pigment hormone, hypothalamic release factor, antidiuretic hormone, prolactin, villi Includes gonadal stimulating hormone, follicle stimulating hormone, thyroid stimulating hormone, tissue plasminogen activator, etc. The immunoglobulins of interest include IgG, IgE, IgM, IgA, IgD, and fragments thereof.

  In one embodiment of the invention, the glycoprotein is FVII. In one embodiment of the invention, the glycoprotein is FVIII. In one embodiment of the invention, the glycoprotein is FIX. In one embodiment of the invention, the glycoprotein is FXIII.

  Proteins or portions thereof can be prepared or isolated using techniques known to those skilled in the art, such as by tissue culture, extraction from animal sources, or by recombinant DNA methods. Also contemplated are transgenic sources such as proteins, peptides, amino acid sequences. Such materials are obtained from transgenic animals, ie mice, pigs, cows, etc., in which the protein is expressed in milk, blood or tissue. Transgenic insect and baculovirus expression systems are also envisioned as sources. Furthermore, mutant versions of proteins such as mutant TNF and / or mutant interferon are also within the scope of the invention. Other proteins of interest are allergenic proteins such as ragweed, antigen E, bee venom, mite allergen, and the like.

  The foregoing is illustrative of bioactive peptides suitable for conjugates with a retarder group according to the present invention. Although not specifically mentioned, it is understood that biologically active substances with suitable peptides are also contemplated and within the scope of the present invention.

  In one embodiment of the invention, the protein is not a CD4 protein; in another embodiment, the protein is not a soluble CD4 protein.

  In one embodiment, the glycoprotein is FVII having the amino acid sequence of wild type factor VII (FIG. 9). In one embodiment, the polypeptide is wild type Factor VIIa.

  In one embodiment of the invention, the glycoprotein is a Factor VII polypeptide.

  In one embodiment, the Factor VII polypeptide is a Factor VII variant having substantially the same biological activity as wild-type Factor VII, including: S52A-FVIIa (S60A-FVIIa (Lino et al. , Arch. Biochem. Biophys. 352: 182-192, 1998); FVIIa variants exhibiting increased proteolytic stability as disclosed in US Pat. No. 5,580,560; between residues 290 and 291 or Factor VIIa cleaved by proteolysis between residues 315 and 316 (Mollerup et al., Biotechnol. Bioeng. 48: 501-505, 1995); oxidized form of factor VIIa (Kornfelt et al., Arch Biochem. Biophys. 363: 43-54, 1999); FVII variants as disclosed in PCT / DK02 / 00189; and as disclosed in WO 02/38162 (Scripps Institute), FVII variants with increased proteolytic stability; modified as disclosed in WO 99/20767 (University of Minnesota) FVII mutants having a Gla-domain and exhibiting enhanced membrane binding; and FVII mutations as disclosed in WO 01/58935 (Maxygen ApS) and WO 04/029091 (Maxygen ApS) body.

  In one embodiment, the Factor VII polypeptide is WO 01/83725, WO 02/22776, WO 02/077218, PCT / DK02 / 00635, Danish Patent Application No. PA 2002 01423. , FVII variants disclosed in Danish patent application PA 2001 01627; WO 02/38162 (Scripps Institute); and JP 2001061479 (Chemo-Sero-Therapeutic Res Inst.) FVII variants with increased biological activity compared to wild type FVIIa, including FVIIa variants with enhanced activity.

In one embodiment, the Factor VII polypeptide is selected from the group consisting of: L305V-FVII, L305V / M306D / D309S-FVII, L305I-FVII, L305T-FVII, F374P-FVII, V158T / M298Q- FVII, V158D / E296V / M298Q-FVII, K337A-FVII, M298Q-FVII, V158D / M298Q-FVII, L305V / K337A-FVII, V158D / E296V / M298Q / L305V-FVII, V158D / E296V / M298Q / K337A-FVII, V158D / E296V / M298Q / L305V / K337A-FVII, K157A-FVII, E296V-FVII, E296V / M298Q-FVII, V158D / E296V-FVII, V158D / M298K-FVII, S336G-FVII; positions 290 and / or 291, preferably A factor VII sequence variant with 290 amino acid residues substituted; and a factor VII sequence variant with positions 315 and / or 316, preferably 31 amino acid residues substituted. In another embodiment, the Factor VII polypeptide is selected from the list consisting of: WO 01/83725, WO 02/22776, WO 02/77218, WO Factor VII variants with increased biological activity compared to wild type FVIIa, as disclosed in 03/27147 and WO 03/37932; L305V / K337A-FVII, L305V / V158D-FVII, L305V / E296V-FVII, L305V / M298Q-FVII, L305V / V158T-FVII, L305V / K337A / V158T-FVII, L305V / K337A / M298Q-FVII, L305V / K337A / E296V-FVII, L305V / K337A / V158D-FVII, L305V / V158D / M298Q-FVII, L305V / V158D / E296V-FVII, L305V / V158T / M298Q-FVII, L305V / V158T / E296V-FVII, L305V / E296V / M298Q-FVII, L305V / V158D / E296V / M298Q-FVII, L305V / V158T / E296V / M298Q-FVII, L305V / V158T / K337A / M298Q-FVII, L305V / V158T / E296V / K337A-FVII, L305V / V158D / K337A / M298Q-FVII, L305V / V158D / E296V / K337A-FVII L305V / V158D / E296V / M298Q / K337A-FVII (L305V / V158T / E296V / M298Q / K337A-FVII, S314E / K31 6H-FVII, S314E / K316Q-FVII, S314E / L305V-FVII, S314E / K337A-FVII, S314E / V158D-FVII, S314E / E296V-FVII, S314E / M298Q-FVII, S314E / V158T-FVII, K316H / L305V- FVII, K316H / K337A-FVII, K316H / V158D-FVII, K316H / E296V-FVII, K316H / M298Q-FVII, K316H / V158T-FVII, K316Q / L305V-FVII, K316Q / K337A-FVII, K316Q / V158D-FVII, K316Q / E296V-FVII, K316Q / M298Q-FVII, K316Q / V158T-FVII, S314E / L305V / K337A-FVII, S314E / L305V / V158D-FVII, S314E / L305V / E296V-FVII, S314E / L305V / M298Q-FVII, S314E /)
L305V / V158T-FVII, S314E / L305V / K337A / V158T-FVII, S314E / L305V / K337A / M298Q-FVII, S314E / L305V / K337A / E296V-FVII, S314E / L305V / K337A / V158D-FVII, S314E / L305V / V158D / M298Q-FVII, S314E / L305V / V158D / E296V-FVII, S314E / L305V / V158T / M298Q-FVII, S314E / L305V / V158T / E296V-FVII, S314E / L305V / E296V / M298Q-FVII, S314E / L305V / V158D / E296V / M298Q-FVII, S314E / L305V / V158T / E296V / M298Q-FVII, S314E / L305V / V158T / K337A / M298Q-FVII, S314E / L305V / V158T / E296V / K337A-FVII, S314E / L305V / V158D / K337A / M298Q-FVII, S314E / L305V / V158D / E296V / K337A-FVII, S314E / L305V / V158D / E296V / M298Q / K337A-FVII, S314E / L305V / V158T / E296V / M298Q / K337A-FVII, K316H / L305V / K337A-FVII, K316H / L305V / V158D-FVII, K316H / L305V / E296V-FVII, K316H / L305V / M298Q-FVII, K316H / L305V / V158T-FVII, K316H / L305V / K337A / V158T-FVII, K316H / L305V / K337A / M298Q-FVII, K316H / L305V / K337A / E296V-FVII, K316H / L305V / K337A / V158D-FVII, K316H / L305V / V158D / M298Q-FVII, K316H / L305V / V158D / E296V-FVII, K316H / L305V / V158T / M298Q-FVII, K316H / L305V / V158T / E296V-FVII, K316H / L305V / E296V / M29 8Q-FVII, K316H / L305V / V158D / E296V / M298Q-FVII, K316H /
L305V / V158T / E296V / M298Q-FVII, K316H / L305V / V158T / K337A / M298Q-FVII, K316H / L305V / V158T / E296V / K337A-FVII, K316H / L305V / V158D / K337A / M298Q-FVII, K316H / L305V / V158D / E296V / K337A-FVII, K316H / L305V / V158D / E296V / M298Q / K337A-FVII, K316H / L305V / V158T / E296V / M298Q / K337A-FVII, K316Q / L305V / K337A-FVII, K316Q / L305V / V158D- FVII, K316Q / L305V / E296V-FVII, K316Q / L305V / M298Q-FVII, K316Q / L305V / V158T-FVII, K316Q / L305V / K337A / V158T-FVII, K316Q / L305V / K337A / M298Q-FVII, K316Q / L305V / K337A / E296V-FVII, K316Q / L305V / K337A / V158D-FVII, K316Q / L305V / V158D / M298Q-FVII, K316Q / L305V / V158D / E296V-FVII, K316Q / L305V / V158T / M298Q-FVII, K316Q / L305V / V158T / E296V-FVII, K316Q / L305V / E296V / M298Q-FVII, K316Q / L305V / V158D / E296V / M298Q-FVII, K316Q / L305V / V158T / E296V / M298Q-FVII, K316Q / L305V / V158T / K337A / M298Q- FVII, K316Q / L305V / V158T / E296V / K337A-FVII, K316Q / L305V / V158D / K337A / M298Q-FVII, K316Q / L305V / V158D / E296V / K337A-FVII, K316Q / L305V / V158D / E296V / M298Q / K337A- FVII, K316Q / L305V / V158T / E296V / M298Q / K337A-FVII, F374Y / K337A-FVII, F374 Y / V158D-FVII, F374Y / E296V-FVII, F374Y / M298Q-FVII, F374Y / V158T-FVII, F374Y / S314E-FVII, F374Y / L305V-FVII, F374Y / L305V / K337A-FVII, F374Y / L305V / V158D- FVII, F374Y / L305V / E296V-FVII, F374Y / L305V / M298Q-FVII, F374Y / L305V / V158T-FVII, F374Y / L305V / S314E-FVII, F374Y / K337A / S314E-FVII, F374Y / K337A / V158T-FVII, F374Y / K337A / M298Q-FVII, F374Y / K337A / E296V-FVII, F374Y / K337A / V158D-FVII, F374Y / V158D / S314E-FVII, F374Y / V158D / M298Q-FVII, F374Y / V158D / E296V-FVII, F374Y / V158T / S314E-FVII, F374Y / V158T / M298Q-FVII, F374Y / V158T / E296V-FVII, F374Y / E296V / S314E-FVII, F374Y / S314E / M298Q-FVII, F374Y / E296V / M298Q-FVII, F374Y / L305V / K337A / V158D-FVII, F374Y / L305V / K337A / E296V-FVII, F374Y / L305V / K337A / M298Q-FVII, F374Y / L305V / K337A / V158T-FVII, F374Y / L305V / K337A / S314E-FVII, F374Y / L305V / V158D / E296V-FVII, F374Y / L305V / V158D / M298Q-FVII, F374Y / L305V / V158D / S314E-FVII, F374Y / L305V / E296V / M298Q-FVII, F374Y / L305V / E296V / V158T-FVII, F374Y / L305V / E296V / S314E-FVII, F374Y / L305V / M298Q / V158T-FVII, F374Y / L305V / M298Q / S314E-FVII, F374Y / L305V / V158T / S314E-FVII, F374Y / K337A / S314E / V158T-FVII, F374Y / K337A / S314E / M298Q-FVII, F374Y / K337A / S314E / E296V-FVII, F374Y / K337A / S314E / V158D-FVII, F374Y / K337A / V158T / M298Q-FVII, F374Y / K337A / V158T / E296V-FVII, F374Y / K337A / M298Q / E296V-FVII, F374Y / K337A / M298Q / V158D-FVII, F374Y / K337A / E296V / V158D-FVII, F374Y / V158D / S314E / M298Q-FVII, F374Y / V158D / S314E / E296V-FVII, F374Y / V158D / M298Q / E296V-FVII, F374Y / V158T / S314E / E296V-FVII, F374Y / V158T / S314E / M298Q-FVII, F374Y / V158T / M298Q / E296V-FVII, F374Y / E296V / S314E / M298Q-FVII, F374Y / L305V / M298Q / K337A / S314E-FVII, F374Y / L305V / E296V / K337A / S314E-FVII, F374Y / E296V / M298Q / K337A / S314E-FVII, F374Y / L305V / E296V / M298Q / K337A-FVII, F374Y / L305V / E296V / M298Q / S314E-FVII, F374Y / V158D / E296V / M298Q / K337A-FVII, F374Y / V158D / E296V / M298Q / S314E-FVII, F374Y / L305V / V158D / K337A / S314E-FVII, F374Y / V158D / M298Q / K337A / S314E-FVII, F374Y / V158D / E296V / K337A / S314E-FVII, F374Y / L305V / V158D / E296V / M298Q-FVII, F374Y / L305V / V158D / M298Q / K337A-FVII, F374Y / L305V / V158 D / E296V / K337A-FVII, F374Y / L305V / V158D / M298Q / S314E-FVII, F374Y / L305V / V158D / E296V / S314E-FVII, F374Y / V158T / E296V / M298Q / K337A-FVII, F374Y / V158T / E296V / M298Q / S314E-FVII, F374Y / L305V / V158T / K337A / S314E-FVII, F374Y / V158T / M298Q / K337A / S314E-FVII, F374Y / V158T / E296V / K337A / S314E-FVII, F374Y / L305V / V158T / E296V / M298Q-FVII, F374Y / L305V / V158T / M298Q / K337A-FVII, F374Y / L305V / V158T / E296V / K337A-FVII, F374Y / L305V / V158T / M298Q / S314E-FVII, F374Y / L305V / V158T / E296V / S314E- FVII, F374Y / E296V / M298Q / K337A / V158T / S314E-FVII, F374Y / V158D / E296V / M298Q / K337A / S314E-FVII, F374Y / L305V / V158D / E296V / M298Q / S314E-FVII, F374Y / L305V / E296V / M298Q / V158T / S314E-FVII, F374Y / L305V / E296V / M298Q / K337A / V158T-FVII, F374Y / L305V / E296V / K337A / V158T / S314E-FVII, F374Y / L305V / M298Q / K337A / V158T / S314E-FVII F374Y / L305V / V158D / E296V / M298Q / K337A-FVII, F374Y / L305V / V158D / E296V / K337A / S314E-FVII, F374Y / L305V / V158D / M298Q / K337A / S314E-FVII, F374Y / L305V / E296V / M298Q / K337A / V158T / S314E-FVII, F374Y / L305V / V158D / E296V / M298Q / K337A / S314E-FVII, S52A- Factor VII, S60A-Factor VII; and P11Q / K33E-FVII, T106N-FVII, K143N / N145T-FVII, V253N-FVII, R290N / A292T-FVII, G291N-FVII, R315N / V317T-FVII, K143N / N145T / R315N / V317T-FVII; FVII having substitution, addition, or deletion in amino acid sequence 233Thr-240Asn, FVII having substitution, addition, or deletion in amino acid sequence 304Arg-329Cys, and amino acid sequence Ile153-Ar
FVII with a substitution, addition, or deletion at g223.

  In one embodiment, the Factor VII related polypeptide is selected from the group consisting of: R152E-Factor VII, S344A-Factor VII, FFR-Factor VII, and Factor VIIa lacking the Gla domain factor.

  In one embodiment, a Factor VII-related polypeptide is produced in the same cell type when tested in one or both of a hydrolysis assay or a proteolysis assay as described herein. It exhibits at least about 25%, preferably at least 50%, more preferably at least about 75, and most preferably at least about 90% of the specific activity of wild type Factor VIIa.

  In one embodiment, the Factor VII-related polypeptide is the same cell type when tested in one or more of a clotting assay, proteolytic assay, or TF assay as described herein. Less than about 25%, preferably less than about 10%, more preferably less than about 5%, and most preferably less than about 1% of the specific activity of wild-type factor VIIa produced in.

  In different embodiments, the conjugated polypeptide is at least about 110% of the bioavailability of the reference preparation (at least about 120%, or at least about 130% of the bioavailability of the reference preparation, or At least about 140%).

  In one embodiment, the conjugated polypeptide is at least about 125% of the half-life of the reference preparation (at least about 150%, or at least about 200%, or about 250% of the half-life of the reference preparation). Serum half-life is shown.

Functional Properties of Glycoprotein Conjugates Glycoconjugates prepared according to the present invention exhibit improved functional properties compared to a reference preparation. For example, improved functional properties include: a) physical properties such as storage stability; b) pharmacokinetic properties (eg, bioavailability and half-life); and c) immunogenicity in humans. Although it may include, it is not limited to these.

  Reference preparations are those contained in the preparations of the invention to which they are compared (eg, wild-type factor VII or certain mutants or chemically modified forms of non-conjugated forms) A preparation comprising a polypeptide having the same amino acid sequence but not conjugated to any polymer molecule found in the preparation of the present invention. For example, a reference preparation typically includes non-conjugated glycoprotein.

  The storage stability of glycoproteins (eg, Factor VII preparations) is: (a) the time required for 20% of the bioactivity of the preparation to disintegrate when stored as a dry powder at 25 ° C., and / or Or (b) In the preparation, evaluation may be performed by measuring the time required for doubling the ratio of a predetermined decomposition product such as an aggregate.

  In some embodiments, the preparation of the present invention is compared to the time required for the same phenomenon in the reference preparation when both preparations are stored as dry powder at 25 ° C. Exhibit an increase of at least about 30%, preferably at least 60%, and more preferably at least about 100% of the time required for 20% of the bioactivity to decay.

  The bioactivity measurement may be performed according to the type of biological activity associated with the particular protein; for example, in the case of FVII, the bioactivity may be determined by clotting assay, proteolytic assay, TF binding assay, It may be measured using any of the dependent thrombin generation assays.

  In some embodiments, the preparation of the present invention comprises a predetermined degradation product, such as an agglomerate, when compared to a reference sample when both samples are stored as a dry powder at 25 ° C. An increase of at least about 30%, preferably at least 60%, and more preferably at least about 100% of the time required for Aggregate content may be determined, for example, by gel permeation HPLC or another type of well-known chromatographic method. In the case of FVII, aggregates may be determined by gel permeation HPLC on a Protein Pak 300 SW column (7.5 × 300 mm) (Waters, 80013) as follows. The column was equilibrated with eluent A (0.2 M ammonium sulfate, 5% isopropanol, pH adjusted to 2.5 with phosphoric acid, and then pH adjusted to 7.0 with triethylamine), after which 25 g of sample was loaded onto the column. Apply. Elution is with eluent A at a flow rate of 0.5 ml / min for 30 minutes and detection is achieved by measuring absorbance at 215 nm. Aggregate content is calculated as Factor VII Aggregate Peak Area / Factor VII Peak (Monomer and Aggregate) Total Area.

  “Bioavailability” refers to the fraction of the administered dose of conjugate that can be detected in the crystal at a given time after administration. Typically, bioavailability is determined by administering a dose between about 25-250 g / kg of the preparation in a test animal; obtaining a plasma sample at a predetermined time after administration; and an appropriate organism It is measured by determining the content of glycoprotein in a sample or equivalent using an assay or immunoassay. Data are typically presented visually as [glycoprotein] v. Time and bioavailability is expressed as area under the curve (AUC). The relative bioavailability of a test sample refers to the ratio between the AUC of the test sample and that of the reference sample.

  In some embodiments, the preparation of the present invention is at least about 110%, preferably at least 120%, more preferably at least about 130%, and most preferably at least about 140% of the bioavailability of the reference preparation. % Relative bioavailability. Bioavailability may be measured in any mammalian species, preferably dogs, and the predetermined time used to calculate AUC may include different increments of 10 minutes to 8 hours.

Assay materials:
Α2-3,6,8,9-Neuraminidase (EC 3.2.1.18) from Anthrobacter urefaciens was obtained from Calbiochem, CA, USA and is agarose-supported soluble from Vibreo cholerae Neuraminidase and neuraminidase from Clostridium perfringens and bovin catalase (EC 1.11.1.6) were obtained from Sigma-Aldrich. Galactose oxidase (EC 1.1.3.9) was obtained from Worthington Biochemical Corporation, USA. PNGase F was from New England Biolabs Inc. MA (USA). All other chemicals were of standard grade and were obtained from Sigma-Aldrich, Bachem, or Fluka. Galactose oxidase kit A22179 was obtained from Molecular Probes, OR, USA. Proton and carbon nuclear magnetic resonance ( ¹ H and ¹³ C NMR) were recorded with a 300 MHz Bruker or 400 MHz NMR instrument, and the reported chemical shift (δ) is that of a low magnetic field from tetramethylsilane or phosphoric acid. It is. MALDI-TOF spectra were obtained using an Autoflex MALDI-TOF mass spectrophotometer from Spectra Bruker Daltonics Inc. Spectra were recorded in linear mode using α-cyano-4-hydroxycinnamic acid as the matrix.

Pharmacological Methods The following assays were useful for determining the bioactivity, half-life, and bioavailability of Factor VII and Factor VII-related polypeptides.

Assay Method (I)
In Vitro Hydrolysis Assay The following method can be used to assay factor VIIa bioactivity. The assay is performed in microtiter plates (MaxiSorp, Nunc, Denmark). Chromogenic substrate D-Ile-Pro-Arg-p-nitroanilide (S-2288, Chromogenix, Sweden) at 1 mM final concentration, containing 0.1 M NaCl, 5 mM CaCl ₂ , and 1 mg / ml bovine serum albumin Add to factor VIIa (final concentration 100 nM) in 50 mM Hepes, pH 7.4 solution. 405 nm absorbance is measured continuously with a SpectraMax 340 plate reader (Molecular Devices, USA). The absorbance developed during the 20 minute incubation is used to calculate the ratio between the activity of the test and reference factor VIIa after subtracting the absorbance in empty wells without enzyme.

Assay method (II)
In Vitro Proteolysis Assay The following method can be used to assay factor VIIa bioactivity. The assay is performed in microtiter plates (MaxiSorp, Nunc, Denmark). 100 μl of Factor VIIa (10 nM) and Factor X (0.8 μM) in a 50 mM Hepes (pH 7.4) solution containing 0.1 M NaCl, 5 mM CaCl 2 and 1 mg / ml bovine serum albumin are incubated for 15 minutes. Factor X cleavage is then stopped by adding 50 μl of 0.1 M NaCl, 20 mM EDTA, and 50 mM Hepes containing 1 mg / ml bovine serum albumin, pH 7.4. The amount of factor Xa produced is determined by adding the chromogenic substrate ZD-Arg-Gly-Arg-p-nitroanilide (S-2765, Chromogenix, Sweden) (final concentration 0.5 mM). 405 nm absorbance is measured continuously with a SpectraMax 340 plate reader (Molecular Devices, USA). The absorbance developed during 10 minutes is used to calculate the ratio between the test and reference factor VIIa proteolytic activity after subtracting the absorbance in empty wells without FVIIa.

Assay method (III)
Measuring in vivo half-life Measuring in vivo biological half-life can be done in a number of ways described in the literature. An example of an assay for measuring the in vivo half-life of rFVIIa and these variants is described in FDA Document No. 96-0597. Briefly, FVIIa clotting activity is measured in withdrawn plasma prior to administration of the conjugate, polypeptide, or composition and during a period of 24 hours after administration. Measure the apparent amount of median distribution at steady state and determine median clearance.

Assay Method (IV)
Bioavailability of Factor VII polypeptide Bioavailability may be measured in a canine model, for example as follows: in 12 beagle dogs divided into 4 groups, performed as a 4-legged cross To study the experimentis. All animals have a glycyl containing sodium chloride (2.92 mg / ml), calcium chloride dihydrate (1.47 mg / ml), mannitol (30 mg / ml), and polysorbate 80 at a dose of approximately 90 μg / kg. Glycine buffer (pH 5.5) solution gives test sample A and reference sample B. Following the first dose, blood samples are withdrawn at 10, 30, and 60 minutes and at 2, 3, 4, 6, and 8 hours. Plasma is obtained from the sample and Factor VII is quantified by ELISA.

Analysis method:
CE-analysis: Capillary electrophoresis is a Hewlett-Packard HP 3DCE system with UV-VIS DAD, fully pneumatically driven, with 200-600 nm range and automatic capillary wash and buffer vial refill I went with the system. The instrument was operated in normal polarity mode at 80-100 μA, + 7.0-7.5 kV / +, and detection was performed on the column at 214 nm with a reference wavelength of 280 nm. CE analysis was performed using a 64,5 cm (55,5 cm effective length) × 75 μm ID fused silica capillary. The capillary was brought to 30 ° C. with a thermostat. The sample was injected at the end of the capillary and further away from the long end injection using +50 mbar for 10 s. HP Chemstation software (Hewlett-Packard) operating on an HP Kayak XA computer (Hewlett-Packard) was used for each measurement control, data collection, and data analysis. Electrophoresis data was collected at a rate of 10 Hz. The electrolyte was prepared by dissolving a known amount of putrescein dichloride in 100 mM phosphate buffer (pH 8.0) and then adjusting to pH 8.0 with 1N sodium hydroxide solution (NK Klausen and T. Kornfelt, J. Chromatography A, 718, 195-202 (1995)).

LCMS analysis:
LC-MS mass spectra were obtained using an instrument consisting of a Hewlett Packard series 1100 G1312A Bin Pump, a Hewlett Packard series 1100 Column compartment, a Hewlett Packard series 1100 G13 15A DAD diode array detector, and a Hewlett Packard series 1100 MSD. The instrument was controlled by HP Chemstation software. The HPLC pump was connected to two eluent reservoirs containing 0.01% aqueous TFA (A) and 0.01% TFA in acetonitrile (B). Analysis was performed at 40 ° C. by injecting the sample onto an appropriate volume (preferably 1 μL) of the column, which was eluted with an acetonitrile gradient. The HPLC conditions, detector settings, and mass spectrometer settings used are as follows:
Column Waters Xterra MS C-18, 50 x 3 mm id
Gradient 10% to 100% acetonitrile linearly at 1.0 ml / min for 7.5 minutes Detection UV: 210 nm (analog output from DAD)
MS ionization mode: API-ES
Scan 100-1000 amu step 0.1 amu.

Analysis of natural and modified N-glycans:
The N-glycan analysis procedure is modified from that of DI Papac et al. Glycobiology 8 (5), 445-454 (1998), and for FVIIa is shown as follows: FVIIa solution (1 mg / ml The pH of 10 mM glycylglycine, 10 mM CaCl ₂ , 50 mM NaCl, pH 6.5) (50 μl) is adjusted to 8.3 by adding 50 mM NaOH. PNGase F (500 U / ml 10 mM Tris acetate buffer, pH 8.3) was added followed by (20 μl, 10 U) followed by Tris acetate pH 8.3 buffer (total volume of reaction mixture: 80 μl). The reaction mixture was incubated overnight at 37 ° C. PVDF membrane (Immobilon-P from Millipore, Millipore Corporation, Bedford, Ma, USA) (1.5 cm ² ) is added and the vial is shaken vigorously for 10 minutes to remove the membrane. Acetic acid 10% is added (10 μl) and the reaction mixture is shaken at RT for 2 h. The reaction mixture is then added to the resin (Dowex 50W-X8, Form H) (0.3 ml). Water (280 μl) is added and the mixture is vortexed. The supernatant is removed, evaporated, redissolved in water (20 μl) and analyzed by MALDI-TOF as described by Papac et al.

Determination of exposed galactose residues:
The amount of free (exposed) galactose residues present on the glycoprotein was determined using the Galactose Assay Kit A22179 and protocol from Molecular Probes. The assay was performed in a microtiter plate format (MaxiSorp, Nunc, Denmark) and data were recorded on a Spectra Plus 384 plate reader (Molecular Device, USA) with an excitation wavelength of 560 nm and an emission wavelength of 590 nm. The corresponding results from a standard galactose oxidase sequence (60, 30, and 15 μM) and a 14.9 μM solution of FVIIa and Asialo FVIIa, respectively, are shown, with a ratio of 3.70-3.90 μmol galactose / μmol asialo FVIIa When assuming a coupled (N145 / 322) dual antenna structure, this corresponds to complete removal of all sialic acid from FVIIa.

  Examples 1-4 show the synthesis of suitable representative O-substituted hydroxylamine nucleophiles for conjugation to galactose oxidized glycans according to the present invention.

Example 1:
O- (N, N-diethylaminoethyl) hydroxylamine

N-hydroxyphthalimide (10.0 g, 2.5 mmol) was dissolved in THF (100 ml) and potassium carbonate (18.63 g; 0.134 mol) was added. Diethylaminoethyl chloride (10.54 g; 61.5 mmol) was added and the mixture was heated to reflux for 4 hours. The mixture was cooled and water (100 ml) was added and stirred until all of the solid was dissolved. The mixture was then extracted twice with DCM. The organic phase was dried over anhydrous sodium sulfate and evaporated to give 8.63 g (53%) of N- (N, N-diethylaminoethoxy) phthalimide as a clear yellow oil. The oil was dissolved in a minimum of acetonitrile and 1.1 N 1N HCl in ethyl acetate was added to precipitate N- (N, N-diethylaminoethoxy) phthalimide hydrochloride. ¹ H-NMR (D ₂ O): δ 1.24 ppm (t, 6H); 3.28 (m, 4H); 3.56 (t, 2H); 4.45 (t, 2H); 7.72 (s, 4H). ¹³ C- NMR (D ₂ O): δ 8.25 ppm; 47.93; 50.46; 72.10; 124.27; 128.34; 135.81;

N- (N, N-diethylaminoethoxy) phthalimide hydrochloride (2,50 g; 8.37 mmol) was dissolved in ethanol (20 ml), and hydrazine monohydrate (1 ml; 20.6 mmol) was added. The mixture was refluxed for 30 minutes. A precipitate formed rapidly. The mixture was cooled on an ice bath. The crystalline material was removed by filtration and the filtrate was dried. A semicrystalline residue was obtained. This was resuspended in cold ethanol (10 ml) and filtered. The filtrate was dried again to give 0.9 g of a clear yellow oil. ¹ H-NMR (CDCl ₃ ): δ 1.05 ppm (t, 6H); 2.58 (m, 4H); 2.65 (t, 2H); 3.75 (t, 2H); 5.12 (bs, 2H).

Example 2:
3-[(2-Aminooxyacetyl)-(2-carboxyethyl) amino] propionic acid

3-Aminopropionic acid tert-butyl ester hydrochloride (7.27 g, 40 mmol) was dissolved in DMF (40 ml). Triethylamine was added (4.05 g, 40 mmol). A solution of tert-butyl acrylate (5.13 g, 40 mmol) in DMF (40 ml) was added to the solution under an inert atmosphere. After stirring at rt for 16 h, the precipitate was filtered and the filtrate was concentrated, dissolved in ethyl acetate (100 ml), washed with sar.NaHCO ₃ (2 × 50 ml), dried over MgSO ₄ and concentrated. The oil was purified by vacuum distillation (0.02 torr, 103-107 ° C.) to give 3- (2-tert-butoxycarbonylethylamino) propionic acid tert-butyl ester as a colorless oil (4.7 g, 43% yield) It was. ¹ H-NMR (CDCl ₃ ): δ 1.45 ppm (s, 18 H), 2.41 (t, J = 6.4 Hz, 4 H), 2.84 (t, J = 6.5 Hz, 4 H). LCMS: m / z = 274 (M + 1), Rt = 2.41 min.

3- (2-tert-butoxycarbonylethylamino) propionic acid tert-butyl ester (0.63 g, 3.07 mmol), N-tert-butoxycarbonylaminooxyacetic acid (1.07 g 5.27 mmol), and 1-hydroxybenzotriazole (0.50 g, 3.68 mmol) was dissolved in DMF (20 ml) and DIEA (0.48 g, 3.68 mmol) and (3-dimethylaminopropyl) -ethylcarbodiimide hydrochloride (0.71 g, 3.68 mmol) were added. The solution was stirred overnight under an inert atmosphere. After concentration of the sample, it was dissolved in ethyl acetate (50 ml) and washed with 5% acetic acid, sat.NaHCO ₃ , and water (2 × 20 ml each). The sample was dried over MgSO ₄ and concentrated to a pale yellow oil 3-[(2-tert-butoxycarbonylaminooxyacetyl)-(2-tert-butoxycarbonylethyl) amino] propionic acid tert-butyl ester (1.05 g, 77% yield). ¹ H-NMR (CDCl ₃ ): δ 1.44 ppm (s, 18 H), 1.47 (s, 9 H), 2.47-2.57 (m, 4 H), 3.48 (t, J = 7.1 Hz, 2 H), 3.56 (t, J = 7.1 Hz, 2 H), 4.60 (s, 2 H), 8.16 (s, 1 H). LCMS: m / z = 469 (M + 23), Rt = 4.30 min.

3-[(2-tert-butoxycarbonylaminooxyacetyl)-(2-tert-butoxycarbonylethyl) amino] propionic acid tert-butyl ester (0.50 g, 1.1 mmol) was triturated at rt for 1 hour under inert atmosphere. Stir with fluoroacetic acid (5 ml). Concentrated hydrochloric acid (2 ml) was added and the sample was concentrated. The sample was dried by adding 10 ml of toluene and concentrating the sample (twice). The residue was dissolved in ethanol (2 ml) and added dropwise to cold diethyl ether. The resulting precipitate can be fixed to the flask and the solvent decanted. The residue was dried under reduced pressure to give a sticky white residue 3-[(2-aminooxyacetyl)-(2-carboxyethyl) amino] propionic acid. ¹ H-NMR (D ₂ O): δ 2.67 ppm (t, J = 6.8 Hz, 2 H), 2.74 (t, J = 6.8 Hz, 2 H), 3.56 (t, J = 6.8 Hz, 2 H) , 3.65 (t, J = 7.2 Hz, 2 H), 4.98 (s, 2 H).
LCMS: m / z = 235 (M + 1), Rt = 0.37 min.

Example 3:
mPEG5000- (CH ₂ ) ₂ -CONH- (CH ₂ ) ₄ -O-NH ₂

  Step 1: O- (4-Aminobutyl) -N- (tert-butoxycarbonyl) hydroxylamine (120 mg, 0.584 mmol) was dissolved in DCM (25 ml) under nitrogen. mPEG (5000) -succinimidylpropionic acid (1 g, 0.195 mmol) was added and the solution was stirred for 16 hours. Diethyl ether (150 ml) was added and the resulting precipitate was filtered and washed with diethyl ether (2 × 60 ml). The precipitate was dried under dry vacuum for 16 hours to give a white solid (911 mg).

mPEG ₍₅₀₀₀₎ -(CH ₂ ) ₂ -CONH- (CH ₂ ) ₄ -O-NH-Boc: ¹ H-NMR (DMSO-d ₆ , 400 MHz): δAmong other signals was observed: 1.40 (s, 9H ), 2.29 (t, 2H), 3.03 (m, 2H), 3.51 (s, 454H), 3.68 (m, 4H), 7.80 (t, 1H), 9.91 (s, 1H).

Step _{2: mPEG5000- (CH 2) 2} -CONH- (CH 2) 4-O-NH-Boc (911mg, 0.174mmol) was dissolved in TFA (8 ml). The solution was stirred at rt for 30 minutes under nitrogen. After adding diethyl ether (150 ml) and stirring for 30 minutes, the resulting precipitate was filtered and washed with diethyl ether (2 × 50 ml). The solid was dried in a vacuum oven at 35 ° C. for 16 hours to give a white solid (800 mg, 90% yield).

_{mPEG 5000 - (CH 2) 2} -CONH- (CH 2) 4 -O-NH 2: ¹ H-NMR (DMSO-d ₆ , 400 MHz): δ Among other signals was observed: 1.43 (m, 2H), 1.55 (m, 2H), 2.29 (t, 2H), 3.05 (m, 2H) , 3.24 (s, 3H), 3.51 (s, 454H), 3.68 (t, 2H), 3.90 (t, 2H), 7.83 (t, 1H), 10.23 (br, 1H).

Example 4:
16-aminooxy-hexadecanoic acid

Step 1: 16-Bromo-hexadecanoic acid methyl ester (1.05 g, 3 mmol) and N-tert-butoxycarbonylhydroxylamine (1 g, 7.5 mmol) were placed in a flask and dissolved in DBU (2.25 ml). The solution was stirred for 3 hours under nitrogen. DCM (100 ml) was added and the solution was washed with 1N HCl (3 × 25 ml) using several brines and methanol to aid phase separation. The solution was dried over MgSO ₄ and concentrated to give an oily crystalline residue (1.09 g, 90% yield).

16- (N-tert-butoxycarbonylaminooxy) hexadecanoic acid methyl ester: ¹ H-NMR (CDCl ₃ , 300 MHz): δ 1.25 (s, 20H, 1.48 (s, 9H), 1.62 (m, 4 H) , 2.30 (t, 2H), 3.67 (s, 3H), 3.84 (t, 2H), 7.16 (s, 1H).
LCMS: m / z = 425 ( M + 23), R t = 6.17.

Step 2: 16- (N-tert-butoxycarbonylaminooxy) hexadecanoic acid methyl ester (1.05 g, 2.62 mmol) was placed in THF (100 ml) and 1N NaOH (2.75 ml) was added. The sample was stirred at rt for 16 hours and then refluxed for 2 hours. After cooling to rt, 4N NaOH (20 ml) and methanol (50 ml) were added and the solution was stirred at rt for 45 min. The solution was neutralized with conc. HCl and cooled in an ice bath. The precipitate is filtered and the filtrate is concentrated under reduced pressure to give a white solid, which is then added to DCM (100 ml) and 0.33N HCl (150 ml) using 10 ml brine to aid in phase separation. divided. The organic phase was dried over MgSO ₄ and concentrated to give an off-white residue (730 mg, 72% yield). The residue was purified via flash chromatography (silicon dioxide, AcOEt / heptane 3: 7 (400 ml) then 1: 1 (200 ml)) to give a white solid (150 mg, 15% yield).

16- (N-tert-butoxycarbonylaminooxy) hexadecanoic acid: ¹ H-NMR (CDCl ₃ , 400 MHz): δ 1.25 (s, 20 H), 1.49 (s, 9H), 1.62 (m, 4H), 2.35 (t, 2H), 3.84 (t, 2H), 7.23 (s, 1H). LCMS: m / z = 410 (M + 23), R _t = 5.53.

  Step 3: 16- (N-tert-butoxycarbonylaminooxy) hexadecanoic acid (110 mg, .28 mmol) was dissolved in TFA (2 ml) and stirred at rt for 30 min. Diethyl ether (20 ml) was added and the precipitate was filtered and dried under dry vacuum to give a white solid (0.11 g).

16-aminooxyhexadecanoic acid: δ 1.24 (s, 20 H), 1.49 (t, 2H), 1.55 (t, 2H), 2.18 (t, 2H), 3.90 (t, 2H), 10.50 (br-m, 2H), 11.65 (br, 1H). LCMS: m / z = 288 (M + 1), R _t = 3.58.

  Examples 5-15 show enzymatic protocols and chemical conjugation methods to obtain glycoproteins with modified glycan structures according to the present invention.

Desialylation of rFVIIa (Preparation of asialo rFVIIa)
Example 5:
Method of using soluble neuraminidase from Vantellobacter urefaciens
Add 250 μl rFVIIa (1.24 mg / ml, 24 μM) in 10 mM glycylglycine buffer (pH 6.0) containing 10 mM CaCl ₂ and 50 mM NaCl to 25 mM U2-3,6,8,9-neuraminidase The mixture was incubated at 4 ° C. for 32 hours. To monitor the reaction, a small aliquot of the reaction was diluted with the appropriate buffer and IEF gel performed according to the Invitrogen procedure (see FIG. 2). Samples were also analyzed by MALDI-TOF spectroscopy using an α-cyano 4-hydroxycinnamic acid matrix (see FIG. 3). An average decrease in molecular weight of 700 Da was observed.

Example 6:
Method of using soluble neuraminidase from Vibrio cholerae
5 ml of FVIIa solution (1.4 mg / ml 10 mM glycylglycine, 10 mM CaCl ₂ , 50 mM NaCl, pH 6) is added to neuraminidase (20 μl, 300 mM, Vibrio cholerae, type II, Sigma N 6514) and the mixture is Shake for hours at room temperature. The removal of sialic acid was confirmed by IEF-gel electrophoresis using the protocol invented by Invitrogen (Novex mini gel (pI 5.85) → 6.55, FIG. 4, lane 4). The mixture was then cooled on ice and the pH was adjusted to 8.0 by adding 50 mM aqueous NaOH. An aqueous solution of EDTA (450 μl, 100 mM, pH 8.0, equivalent to [Ca ²⁺ ]) is added and the sample (5.1 mS / cm) is loaded onto a 5 ml HiTrap Q HP ion exchange column (Amersham-Biosciences) and 10 mM. Equilibrated with Tris, 50 mM NaCl, pH 8.0. The column was eluted with 10 mM Tris, 50 mM NaCl, pH 8.0 (10 vol, flux: 1 ml / min). The elution buffer was then changed to 10 mM Tris, 50 mM NaCl, 25 mM CaCl ₂ , pH 8.0 (10 vol, flux: 1 ml / min). Pure FVIIa samples were pooled. The buffer was then changed to 10 mM glycylglycine, 10 mM CaCl ₂ , 50 mM NaCl, pH 6 using a NAP-10 column (Amersham), and the sample was stored at −80 ° C. until later use. The amidolytic activity on the peptide substrate S2288 (see in vitro hydrolysis assay section) was determined to be identical to unmodified rFVIIa. A galactose assay kit from Molecular Probes (A22179) used to quantify the amount of galactose residues exposed on a protein (see Analysis Procedures section) 3.70 μmol galactose / μmol FVIIa (FIG. 1) quantification was given, corresponding to complete removal of all sialic acid from FVIIa, assuming the presence of a (N145 / 322) dual antenna structure.

Example 7:
Method of using agarose-supported neuraminidase from Clostridium perfringens Neuraminidase-agarose resin (Bacillus perfringens, Type VI-A, Sigma N 5254) as an ammonium sulfate suspension (2 ml) was washed extensively with MilliQ water and then drained. And added to 5 ml of FVIIa solution (1.4 mg / ml 10 mM glycylglycine, 10 mM CaCl ₂ , 50 mM NaCl, pH 6). The mixture was gently shaken for 48 hours at room temperature and the neuraminidase-agarose resin was filtered. The removal of sialic acid was confirmed by IEF-gel electrophoresis (Novex mini gel (pI 5.85) → 6.55, FIG. 4, lane 8) using the protocol invented by Invitrogen. Amidolytic activity on the peptide substrate S2288 (see in vitro hydrolysis assay section) was determined to be modified and identical to in-court rFVIIa. Samples were stored at −80 ° C. until later use. A galactose assay kit from Molecular Probes (A22179) used to quantify the amount of galactose residues exposed on a protein (see Analysis Procedures section) The ratio of 3.90 μmol galactose / μmol FVIIa (FIG. 1) corresponding to complete removal of all sialic acid from FVIIa was given when assuming the presence of the (N145 / 322) dual antenna structure. Alternatively, removal of sialic acid can be analyzed by CE-protocol as described in the analysis section.

Example 8:
General one-step procedure for the preparation of galactosyl derivatized FVIIa analogs:
Step A (buffer exchange): FVIIa (1ml, 28nmol, 1.4mg / ml) 10mM glycylglycine buffer (pH 6.0) containing 10 mM CaCl ₂ and 50 mM NaCl solution of 15ml containing 10 mM CaCl ₂ and 50 mM NaCl Add to a 1 ml NAP-10 column (Amersham Bioscience) pre-calibrated with 10 mM MES buffer (pH 6.0). The solution is passed through the column bed and then 1.5 ml of 10 mM MES buffer (pH 6.0) containing 10 mM CaCl ₂ and 50 mM NaCl is added, while 1.5 ml of eluate is collected.

  Step B (neuraminidase treatment): The eluate is added to 75 mU neuraminidase (EC 3.2.1.18 from Vibrio cholerae) and the mixture is incubated for 24 hours at room temperature.

Step C (reaction with galactose oxidase + nucleophile): 12U galactose oxidase (EC. 1.1.3.9) and 240U catalase (EC.1.11.1.6) and O-substituted hydroxylamine derivative (500 nmol or oxidized) Add about 200 μl freshly prepared MES buffer (10 mM MES, 10 mM CaCl ₂ , and 50 mM NaCl, pH 6.0) solution of about 5 equivalents per galactose residue. The degree of modification is done by analysis techniques appropriate for the particular modification in question (eg, IEF gel for charged moieties, or reduced / non-reduced SDS-PAGE gel for larger size hydroxylamines). To obtain the conversion, it is typically between 8 and 24 hours at room temperature. N-glycans from small aliquots are subsequently cleaved using the PNGase F protocol, analyzed using the procedure described in the analysis section, and then the FVIIa modified end product is performed Purified as described in Example 6 and stored at −80 ° C. until further use.

Example 9:
General procedure for one-pot galactose oxidation of asialo rFVIIa and reaction with hydroxylamine (RO-NH ₂ ):
250 μl of desialylated rFVIIa (24 μM) in 10 mM MES buffer (pH 6.0) containing 10 mM CaCl ₂ and 50 mM NaCl (prepared as described above) was added 930 mM galactose oxidase and catalase (145 U) 1. React with 10 mM MES buffer (pH 6.0) solution containing 20 mM (50 ×) O-derivatized hydroxylamine. The reaction is placed at 4 ° C. for 24-72 hours. The progress of the reaction is performed by capillary electrophoresis using the method specified in the material section, for small aliquots according to Invitrogen procedures, or by IEF gel (or SDS-PAGE gel depending on the type of hydroxylamine) Monitor by either. When the reaction is complete, the glycan derivatized FVIIa analog is obtained by ion exchange chromatography according to L. Thim et al. Biochemistry, 1988, 27, 7785-7793 or as described in Example 6. Purify. The product is finally characterized by capillary electrophoresis, MALDI-TOF, or other suitable method as described in the analysis section for protein analysis. The modified N-glycans are cleaved using the PNGase F protocol and analyzed by the MALDI-time-of-flight method described in the analysis section.

Example 10:
General procedure for one-pot galactose oxidation of asialoglycoprotein and reaction with nucleophiles (eg R-CO-NHNH ₂ , R-NHNH ₂ or RO-NH ₂ ):
250 μl of asialoglycoprotein (24 μM) in 10 mM MES buffer (pH 6.0) and 1.20 mM (50 ×) nucleophile (eg R-CO-NHNH ₂ , R-NHNH ₂ or RO-NH ₂ ) React with galactose oxidase and catalase in 10 mM MES buffer (pH 6.0). The reaction is placed at 4 ° C. for 24-72 hours. The progress of the reaction is performed by capillary electrophoresis using the method specified in the material section, for small aliquots according to Invitrogen procedures, or by IEF gel (or SDS-PAGE gel depending on the type of hydroxylamine) Monitor by either. When the reaction is complete, the glycan derivatized glycoprotein is purified by appropriate chromatographic techniques. The product is finally characterized by capillary electrophoresis, MALDI-TOF, or other suitable method as described in the analysis section for protein analysis. The modified N-glycans are cleaved using the PNGase F protocol and analyzed by the MALDI-time-of-flight method described in the analysis section.

Example 11:
General procedure for one-pot galactose oxidation of asialoglycoproteins and reactions with nucleophiles (eg R-CO-NHNH2, R-NHNH2, or RO-NH2) using bead-supported enzymes:
250 μl of asialoglycoprotein (24 μM) in 10 mM MES buffer (pH 6.0) was added to agarose-immobilized galactose oxidase (100 mg, typically with 70 U / g support activity) and agarose-immobilized catalase ( MES buffer containing 100 mg (typically 3000 U / g support activity) and 1.20 mM (50 ×) nucleophile (eg R-CO-NHNH2, R-NHNH2 or RO-NH2) React in (pH 6.0). The reaction is placed at 4 ° C. for 24-72 hours. The progress of the reaction is performed by capillary electrophoresis using the method specified in the material section, for small aliquots according to Invitrogen procedures, or by IEF gel (or SDS-PAGE gel depending on the type of hydroxylamine) Monitor by either. When the reaction is complete, the glycan derivatized glycoprotein is purified by appropriate chromatographic techniques. The product is finally characterized by capillary electrophoresis, MALDI-TOF, or other suitable method as described in the analysis section for protein analysis. The modified N-glycans are cleaved using the PNGase F protocol and analyzed by the MALDI-time-of-flight method described in the analysis section.

Example 12:
MPEG ₅₀₀₀ with respect to _{FVIIa - (CH 2) 2 -CONH-} (CH 2) a ₄ -O-NH ₂ ligation step 1 (Preparation of asialo FVIIa): neuraminidase (Agarose on Clostridium perfringens, Sigma N5254) a milli-Q water ( 3 × 15 ml) and added to a solution of FVIIa (11 ml, 1.4 mg / ml in Gly-gly buffer). Samples were gently shaken for 16 hours. Neuraminidase is filtered and the buffer is exchanged for MES buffer (10 mM MES, 10 mM CaCl ₂ , 50 mM NaCl, pH 6) using NAP-25 and NAP-10 columns (Amersham biosciences) and Asialo FVIIa Of MES buffer was obtained. Analysis using IEF-gel shows changes in protein pI.

Step 2 (one-spot galactose oxidation and oxime formation with mPEG ₍₅₀₀₀₎ -(CH ₂ ) ₂ -CONH- (CH ₂ ) ₄ -O-NH ₂ ): galactose some asialo FVIIa (5 ml) from above Oxidase (1.28 mg 51 U / mg), catalase (1183 U / mg 9.24 mg), and mPEG ₍₅₀₀₀₎ -(CH ₂ ) ₂ -CONH- (CH ₂ ) ₄ -O-NH ₂ were added. The sample was allowed to stand for 20 hours at rt. The pH was adjusted to 8 with 50 mM NaOH, 100 mM EDTA (500 μl) was added, and then the pH was adjusted again to 8. Samples using 5 ml Hi-Trap Q column (Amersham Biosciences), start buffer (10 mM TRIS, 50 mM NaCl, pH 8.0), and elution buffer (10 mM TRIS, 50 mM NaCl, 25 mM CaCl ₂ , pH 8.0) Purified. Fractions containing FVIIa were collected and analyzed by SDS-PAGE. An increase in product mass was visible by SDS-PAGE (Figure 5).

Example 13:
Large scale method for preparing aminoxyacetic acid derivatized FVIIa Asialo FVIIa (10.5 mg, Gly-Gly buffer pH 6.0 (7.5 ml) solution) prepared in Example 2 or 3 was added to 10 mM MES, 10 mM CaCl ₂ Three NAP-25 (Amersham) columns pre-equilibrated with 50 mM NaCl, pH 6.0 were used for buffer exchange to 10 mM MES, 10 mM CaCl ₂ , 50 mM NaCl, pH 6.0 buffer. Then 2.25 ml of a 10 mM MES, 10 mM CaCl ₂ , 50 mM NaCl, pH 6.0 solution of 10 mM aminoxyacetic acid was added followed by 135 U galactose oxidase (1.93 mg, 78 U / mg) and 6039 U catalase (2.57 mg, 2350 U / mg). mg) 2.25 ml of 10 mM MES, 10 mM CaCl ₂ , 50 mM NaCl, pH 6.0 solution was added. The mixture was incubated for 48 hours at 4 ° C. with occasional shaking. The pH was raised to 8.0 using 50 mM NaOH. Calcium ions were removed from the solution by adding 100 mM EDTA solution at pH 8.0 (1.7 ml). The conductivity of the solution was measured at 8.3 mS / cm. The solution was then loaded onto a 5 ml HiTrap Q HP ion exchange column (Amersham-Biosciences) equilibrated with 10 mM Tris, 50 mM NaCl, pH 8.0. The column was eluted with 10 mM Tris (50 mM NaCl), pH 8.0 (10 vol, flux: 1 ml / min). The elution buffer was then changed to 10 mM Tris, 50 mM NaCl, 25 mM CaCl ₂ , pH 8.0 (10 vol flux: 1 ml / min). Fractions were analyzed by IEF gel (FIG. 6) and pure aminoxyacetic acid modified FVIIa samples (having about 5.8 pI on the gel) were pooled. The buffer was then changed to 10 mM Gly-Gly, 10 mM CaCl ₂ , 50 mM NaCl, pH 7 using a NAP-10 column (Amersham), and samples were stored at −80 ° C. until further use. Peptidolytical activity was measured as 54% of the starting material using the S2288 peptide substrate (see in vitro assay section).

Example 14:
One-pot method for derivatization with p-nitrobenzyloxyamine Step A (buffer exchange): 10 mM glycylglycine buffer containing 10 mM CaCl ₂ and 50 mM NaCl in FVIIa (1 ml, 28 nmol, 1.4 mg / ml) The pH 6.0) solution is added to a 1 ml NAP-10 column (Amersham Bioscience) that has been callibrated with 15 ml of 10 mM MES buffer (pH 6.0) containing 10 mM CaCl ₂ and 50 mM NaCl. The solution is passed through the column bed and then 1.5 ml of 10 mM MES buffer (pH 6.0) containing 10 mM CaCl ₂ and 50 mM NaCl is added, while 1.5 ml of eluate is collected.

  Step B (neuraminidase treatment): The eluate is added to 75 mU neuraminidase (EC 3.2.1.18 from Vibrio cholerae) and the mixture is incubated for 24 hours at room temperature.

Step C (reaction with galactose oxidase + nucleophile): 12U galactose oxidase (EC.1.1.3.9) and 240U catalase (EC.1.11.1.6) and O-substituted hydroxylamine derivative (500 nmol or oxidized galactose residue) Add approximately 5 equivalents of freshly prepared 200 μl MES buffer (10 mM MES, 10 mM CaCl ₂ , and 50 mM NaCl, pH 6.0) solution. The mixture was reacted at rt for 24 hours.

  Step D (Analysis of derivatized N-glycans): All samples were used for analysis. N-glycans were cleaved using PNGase F and the N-glycans released by MALDI-TOF spectroscopy were identified using the method described in the analysis section. Two derivatized glycan structures were identified that correspond to dual antennas modified with Gal and GalNAc modifications, respectively (FIG. 7).

Example 15:
One-pot derivatization with aminoxyacetic acid Step A (buffer exchange): FVIIa (1 ml, 28 nmol, 1.4 mg / ml) in 10 mM glycylglycine buffer (pH 6.0) containing 10 mM CaCl ₂ and 50 mM NaCl in 10 mM CaCl ₂ And add to a 1 ml NAP-10 column (Amersham Bioscience) pre-calibrated with 15 ml 10 mM MES buffer (pH 6.0) containing 50 mM NaCl. The solution is passed through the column bed and then 1.5 ml of 10 mM MES buffer (pH 6.0) containing 10 mM CaCl ₂ and 50 mM NaCl is added, while 1.5 ml of eluate is collected.

  Step B (neuraminidase treatment): The eluate is added to 150 mU neuraminidase (EC 3.2.1.18 from Vibrio cholerae) and the mixture is incubated for 24 hours at room temperature.

Step C (reaction with galactose oxidase + nucleophile): 12U galactose oxidase (EC.1.1.3.9) and 240U catalase (EC.1.11.1.6) and O-substituted hydroxylamine derivative (500 nmol or oxidized galactose residue) Add approximately 5 equivalents of freshly prepared 200 μl MES buffer (10 mM MES, 10 mM CaCl ₂ , and 50 mM NaCl, pH 6.0) solution. The mixture was reacted at rt for 24 hours. An aliquot collected from each step was analyzed by IEF-gel analysis (Figure 8). Also, Step C was performed using 30 mU galactose oxidase.

Example 16:
Desialylation of a sialylated glycoprotein (preparation of asialoglycoprotein) using neuraminidase supported on a solid:
Add 250 μl rFVIIa (1.24 mg / ml, 24 μM) in 10 mM glycylglycine buffer (pH 6.0) containing 10 mM CaCl ₂ and 50 mM NaCl to 25 mM U2-3,6,8,9-neuraminidase The mixture was incubated at 4 ° C. for 32 hours. To monitor the reaction, a small aliquot of the reaction was diluted with the appropriate buffer and IEF gel performed according to the Invitrogen procedure. Samples were also analyzed by capillary electrophoresis according to the methods described in the materials section.

Example 17:
The following examples relate to the synthesis of dendrimer compounds.

Examples and general procedures refer to intermediate compounds and end products identified in structural details and in synthetic schemes. The preparation of the dendrimer compounds of the present invention is described in detail using the following examples, but the chemical reactions described are generally described below for the preparation of selected branched polymers of the present invention. It discloses about what can be applied in general. From time to time, the reactions described for each compound included within the disclosed scope of the invention may not be applicable. The compounds for which this occurs are readily recognized by those skilled in the art. In these cases, the reaction is successfully performed by conventional modifications known to those skilled in the art, i.e. by appropriate protection of interfering groups, by changing to other conventional reagents, or by routine modification of reaction conditions. be able to. Alternatively, other reactions disclosed herein or otherwise conventional would be applicable to the preparation of the corresponding compounds of the invention. In all preparative methods, all starting materials are known or may be readily prepared from known starting materials. All temperatures are given in degrees Celsius, and unless otherwise stated, all parts and percentages are by weight when stated in terms of yield, and all parts and percentages when referring to solvents and eluents. Part is by volume. All reagents were of standard grade supplied by Aldrich, Sigma, etc. Proton, carbon, and phosphorus nuclear magnetic resonances ( ¹ H-, ¹³ C-, and ³¹ P NMR) were recorded on a Bruker NMR instrument, and the reported chemical shift (δ) is tetramethylsilane or phosphate. From low magnetic fields. LC-MS mass spectra were obtained using the following equipment and preparation conditions:
・ Hewlett Packard Series 1100 G1312A Bin Pump
・ Hewlett Packard Series 1100 Column compartment ・ Hewlett Packard Series 1100 G13 15A DAD diode array detector ・ Hewlett Packard Series 1100 MSD
The instrument was controlled by HP Chemstation software.

The HPLC pump was connected to two eluent reservoirs including:
A: 0.01% TFA aqueous solution
B: A solution of 0.01% TFA in acetonitrile.

Analysis was performed at 40 ° C. by injecting the sample onto an appropriate volume (preferably 1 μL) of the column, which was eluted with an acetonitrile gradient. The HPLC conditions, detector settings, and mass spectrometer settings used are as follows:
Column Waters Xterra MS C-18, 50 x 3 mm id
Gradient 10% to 100% acetonitrile linearly at 1.0 ml / min for 7.5 minutes Detection UV: 210 nm (analog output from DAD)
MS ionization mode: API-ES
Scan 100-1000 amu step 0.1 amu.

Some of the NMR data shown in the following examples are only selected. In the examples, the following terms are intended to have the following general meaning:
Abbreviation
Boc: tert-butoxycarbonyl
CDI: Carbonyldiimidazole
DCM: dichloromethane, methylene chloride
DIC: Diisopropylcarbodiimide
DIPEA: N, N-diisopropylethylamine
DhbtOH: 3-hydroxy-1,2,3-benzotriazine-4 (3H) -one
DMAP: 4-dimethylaminopyridine
DMF: N, N-dimethylformamide
DMSO: Dimethyl sulfoxide
DTT: Dithiothreitol
EtOH: ethanol
Fmoc: 9-fluorenylmethyloxycarbonyl
HOBt: 1-hydroxybenzotriazole
MeOH: methanol
NMP: N-methyl-2-pyrrolidone
NEt ₃ : Triethylamine
THF: tetrahydrofuran
TFA: trifluoroacetic acid
TSTU: 2-succinimide-1,1,3,3-tetramethyluronium tetrafluoroborate.

  The following non-limiting examples illustrate monomer synthesis and polymerization techniques using solid phase synthesis or solution phase synthesis.

Synthesis of monomer building blocks and linkers Example 17
2- [2- (2-Chloroethoxy) ethoxymethyl] oxirane

  2 (2-Chloroethoxy) ethanol (100.00 g; 0.802 mol) was dissolved in a catalytic amount (2.28 g; 16 mmol) of dichloromethane (100 ml) and borane trifluride etherate. The clear solution was cooled to 0 ° C. and epibromhydrin (104.46 g; 0.762 mol) was added dropwise to maintain the temperature at 0 ° C. The clear solution was stirred at 0 ° C. for a further 3 hours and the solvent was removed by rotary evaporation. The residual oil was once evaporated from acetonitrile to give crude 1-bromo-3- [2- (2-chloroethoxy) ethoxy] propan-2-ol, which was redissolved in THF (500 ml). Then powdered potassium tert-butoxide (85.0 g; 0.765 mmol) was added and the mixture was heated to reflux for 30 minutes. Insoluble salts were removed by filtration and the filtrate was concentrated in vacuo to give a clear yellow oil. The oil was further purified by vacuum distillation to give 56.13 g (41%) of pure title material.

bp = 65-75 ° C (0.65 mbar). ¹ H-NMR (CDCl ₃ ): δ 2.61 ppm (m, 1H); 2.70 (m, 1H); 3.17 (m, 1H); 3.43 (dd, 1H) 3.60-3.85 (m, 9H). ¹³ C-NMR (CDCl ₃ ): δ 42.73 ppm; 44.18; 50.80; 70.64 & 70,69 (may collaps); 71.37;

Example 18
1,3-bis [2- (2-chloroethoxy) ethoxy] propan-2-ol

2- [2- (2-Chloroethoxy) ethoxymethyl] oxirane (2.20 g; 12.2 mmol) was dissolved in DCM (20 ml) and 2- (2-chloroethoxy) ethanol (1.52 g; 12.2 mol) was added. . The mixture was cooled to 0 ° C., and a catalytic amount of borane trifluride etherate (0.2 ml; 1.5 mmol) was added. The mixture was stirred for 2 hours at 0 ° C. and the solvent was removed by rotary evaporation. The remaining borane trifluoride etherate was removed by coevaporating twice from acetonitrile. The oil thus obtained was purified by Kuglerohr destilation. The title material was obtained in 2.10 g (45%) yield as a clear sticky oil. bp. = 270 ° C., 0.25 mbar. _{^{1 H-NMR (CDCl 3)}} :. Δ3.31 (bs, 1H); 3.55 ppm (ddd, 4H); 3.65-3.72 (m, 12H); 3.75 (t, 4H); 3.90 (m, 1H) 13 C-NMR (CDCl ₃ ): δ 43.12 ppm; 69.92; 70.95; 71.11; 71.69;

Example 19
1,3-bis [2- (2-azidoethoxy) ethoxy] propan-2-ol

1,3-bis [2- (2-chloroethoxy) ethoxy] propan-2-ol (250 mg; 0.81 mmol) dissolved in DMF (2.5 ml), sodium azide (200 mg; 3.10 mmol) and sodium iodide (100 mg; 0.66 mmol) was added. The suspension was heated to 100 ° C. (internal temperature) overnight. The mixture was then cooled and filtered. The filtrate was dried and the semicrystalline oil was resuspended in DCM (5 ml). Insoluble salts were removed by filtration; the filtrate was evaporated to dryness to give the pure title material as a colorless oil. Yield: 210 mg (84%). ¹ H-NMR (CDCl ₃ ): δ 3.48 ppm (t, 4H); 3.60-3.75 (m, 16H); 4.08 (m, 1H). ¹³ C-NMR (CDCl ₃ ): δ 51.05 ppm; 69.10 70.24; 70.53; 70.78; 71.37. LC-MS (any-one): m / e = 319 (M + 1) ⁺ ; 341 (M + Na) ⁺ ; 291 (MN ₂ ) ⁺ . R _t = 2.78 min .

Example 20
1,3-bis [2- (2-azidoethoxy) ethoxy] propan-2-yl-p-nitrophenyl carbonate

1,3-bis [2- (2-azidoethoxy) ethoxy] propan-2-ol (2.00 g; 6.6 mmol) was dissolved in THF (50 ml) and added to diisopropylethylamine (10 ml). The clear yellow solution was then added to 4-dimethylaminopyridine (1.60 g; 13.1 mmol) and p-nitorphenylchloroformiate (2.64 g; 13.1 mmol) and stirred at ambient temperature. A precipitate formed rapidly. The suspension was stirred at room temperature for 5 hours, then filtered and concentrated in vacuo. The residue was further purified by chromatography using ethyl acetate-heptane-triethylamine (40/60/2) as eluent. The product was obtained as a clear yellow oil in 500 mg (16%) yield. ¹ H-NMR (CDCl ₃ ): δ 3.38 ppm (t, 4H); 3.60-3.72 (m, 12H); 3.76 (m, 4H); 5.12 (q, 1H); 7.41 (d, 2H); 8.28 ( d, 2H). LC-MS (any-one): m / e = 506 (M + Na) ⁺ ; 456 (MN ₂ ) ⁺ .R _t = 4.41 min.

Example 21
1,3-bis [2- (2-azidoethoxy) ethoxy] propan-2-ylchloroformate

  Trichloroacetyl chloride (1,42 g, 7.85 mmol) was dissolved in THF (10 ml) and the solution was cooled to 0 ° C. A solution of 1,3-bis [2- (2-azidoethoxy) ethoxy] propan-2-ol (1.00 g; 3.3 mmol) and triethylamine (0,32 g, 3.3 mmol) in THF (5 ml) is slowly added over 10 minutes. And added dropwise. Cooling was removed and the resulting suspension was stirred at ambient temperature for 6 hours. The mixture was filtered and the filtrate was evaporated to give a light brown oil. After evaporation, the oil was treated twice with acetonitrile and the product was used without further purification.

¹ H-NMR (CDCl ₃ ): δ 3.40 (t, 4H); 3,55-3,71 (m, 12H); 3,75 (d, 4H); 5.28 (m, 1H).

Example 22
2- (1,3-Bis [2- (2-azidoethoxy) ethoxy] propan-2-yloxy) acetic acid

Sodium hydride (7.50 g; 80% oil suspension) was washed twice with heptane and resuspended in dry THF (100 ml). Then a solution of 1,3-bis [2- (2-azidoethoxy) ethoxy] propan-2-ol (10.00 g; 33.0 mmol) in dry THF (100 ml) was added slowly dropwise over 30 minutes at room temperature. . Then a solution of bromoacetic acid (6.50 mg; 47 mmol) in THF (100 ml) was added dropwise over 20 minutes → slightly exothermic. A cream colored suspension was formed. The mixture was stirred overnight at ambient temperature. As the mixture was cooled, excess sodium hydride was carefully destroyed with water (20 ml). The suspension was dried by rotary evaporation and the residue was partitioned between DCM and water. The aqueous phase was then extracted twice with DCM and acidified by adding acetic acid (25 ml). The aqueous phase was then momentarily extracted with DCM and the combined organic phases were dried over sodium sulfate and evaporated to dryness. The residual oil at this point contained the title material as well as bromoacetic acid. The latter was removed by redissolving the oil in DCM (50 ml) solution containing piperidine (5 ml); stirred for 30 minutes, then the organic solution was washed (1 ×) with 1N aqueous HCl. The pure title material was then obtained after drying of the solvent (Na ₂ SO ₄ ) and evaporation. Yield: 7.54 g (63%). ¹ H-NMR (D ₂ O): δ 3.11 ppm (t, 4H); 3.53-3.68 (m, 16H); 3.80 (m, 1H); 4.25 (s, 2H). ¹³ C-NMR (D ₂ O ): δ 38.18 ppm; 65.43; 66.09; 68.55: 69.13; 69.23; 77.18; 173.42.

Example 23
Imidazole-1-carboxylic acid 1,3-bis (2- (2-azidoethoxy) ethoxy) propan-2-yl ester

1,3-bis [2- (2-azidoethoxy) ethoxy] propan-2-ol (1.00 g; 3.3 mmol) was dissolved in DCM (5 ml) and carbonyldiimidazole (1.18 g, 6.3 mmol) was added. . The mixture was stirred at room temperature for 2 hours. The solvent was removed and the residue was dissolved in methanol (20 ml) and stirred for 20 minutes. The solvent was removed and the clear oil thus obtained was further purified by column chromatography on silica using 2% MeOH in DCM as eluent. Yield: 372.4 mg (35%). ¹ H-NMR (CDCl ₃ ): δ 3.33 (t, 4H); 3,60-3,75 (m, 12H); 3,80 (d, 4H); 5.35 (m, 1H); 7.06 (s, 1H); 7.43 (s, 1H); 8.16 (s, 1H).
LC-MS (any-one): m / e = 413 (M + 1) ⁺ ; R _t = 2.35 min.

Example 24
t-Butyl 2- (1,3-bis [2- (2-azidoethoxy) ethoxy] propan-2-yloxy) acetate

2- (1,3-bis [2- (2-azidoethoxy) ethoxy] propan-2-yloxy) acetic acid (5.0 g; 13.28 mmol) is dissolved in toluene (20 ml) and the reaction mixture is stirred under an inert atmosphere. Heated to reflux. N, N-dimethylformamide-di-tert-butylacetal (13 ml; 54.21 mmol) was then added dropwise over 30 minutes. Reflux was continued for 24 hours. The dark brown solution was then filtered through celite. The solvent was removed under reduced pressure and the oily residue was purified by flash chromatography on silica using 3% methanol dichloromethane as eluent. Pure fractions were pooled and evaporated to dryness. The title material was obtained as a yellow clear oil. Yield: 5.07 g (88%). ¹ H-NMR (CDCl ₃ ): δ 1.42 ppm (s, 9H); 3.35 (t, 4H); 3.54-3.69 (m, 16H); 3.75-3.85 (m, 1H); 4.16 (s, 2H). ¹³ C-NMR (CDCl ₃ , selected peaks): δ 30.35 ppm; 52.93; 70.65; 72.25; 73.12; 73.90; 80.44; 83.55; 172.28. R _f = 0.33 in ethyl acetate-heptane (1: 1).

Example 25
t-Butyl 2- (1,3-bis [2- (2-aminoethoxy) ethoxy] propan-2-yloxy) acetate

Dissolve t-butyl 2- (1,3-bis [2- (2-azidoethoxy) ethoxy] propan-2-yloxy) acetate (5.97 g, 11.7 mmol) in ethanol-water (25 ml; 2: 1). Acetic acid (5 ml) was added followed by an aqueous suspension of Raney nickel (5 ml). The mixture was then hydrogenated using a Parr apparatus for 16 hours at 3 atmospheres. The catalyst was then removed by filtration and the reaction mixture was dried by rotary evaporation. The oily residue was dissolved in water and lyophilized to give a quantitative yield of the title material. ¹ H-NMR (CDCl ₃ ): δ 1.45 ppm (s, 9H); 3.15 (bs, 4H); 3.48-3.89 (broad m, 17H); 4.15 (s, 2H). ¹³ C-NMR (CDCl ₃ , selected peaks): δ 28.44 ppm; 39.81; 68.17; 70.58; 70.79; 70.99; 78.81; 82.31;

Example 26
2- (1,3-Bis [2- (2-aminoethoxy) ethoxy] propan-2-yloxy) acetic acid

2- (1,3-Bis [2- (2-azidoethoxy) ethoxy] propan-2-yloxy) acetic acid (1.00 g; 2.65 mmol) was dissolved in 1N aqueous hydrochloric acid (10 ml) and dissolved on carbon (1 ml). A 50% aqueous suspension of 5% palladium was added. The mixture was hydrogenated using a Parr apparatus at 3.5 atmospheres. After 1 hour, the reaction was stopped and the catalyst was removed by filtration. The solvent was removed by rotary evaporation and the residue was evaporated twice from acetonitrile. Yield: 930 mg (88%). ¹ H-NMR (D ₂ O): δ 3.11 ppm (t, 4H); 3.53-3.68 (m, 16H); 3.80 (m, 1H); 4.25 (s, 2H). ¹³ C-NMR (D ₂ O ): Δ 38.18 ppm; 65.43; 66.09; 68.55: 69.13; 69.23; 77.18; 173.42.

Example 27:
2- (1,3-Bis [2- (2- {9-fluorenylmethyloxycarbonylamino} ethoxy) ethoxy] propan-2-yloxy) acetic acid

  2- (1,3-bis [2- (2-aminoethoxy) ethoxy] propan-2-yloxy) acetic acid (9.35 g; 28.8 mmol) was added to DIPEA (10 ml; 57 mmol). The reaction mixture was cooled on an ice bath and chlorotrimethylsilane (15 ml; 118 mmol) dissolved in DCM (50 ml) was added dropwise via DIPEA (11 ml; 62.7 mmol). To the almost clear solution, a solution of Fmoc-Cl (15.0 g; 57 mmol) in DCM (50 ml) was added dropwise. The reaction mixture was stirred overnight, then diluted with DCM (500 ml) and added to 0.01 N aqueous solution (500 ml). The organic layer was separated; washed with water (3 × 200 ml) and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation.

The crude product was purified by flash chromatography on silica using ethyl acetate-heptane (1: 1) as eluent. The pure fractions were collected and dried to give 9.20 g (42%) of the title material.

¹ H-NMR (D ₂ O): δ 3.34 ppm (t, 4H); 3.45-3.65 (m, 16H); 3.69 (bs, 1H); 4.20 (t, 2H); 4.26 (s, 2H); 4.38 (D, 4H); 5.60 (t, 2H); 7.30 (t, 4H); 3.35 (t, 4H); 7.58 (d, 4H); 7.72 (d, 4H). ¹³ C-NMR (D ₂ O; selected peaks): δ 21.20 ppm; 30.75; 34.64; 67.66; 68.90; 70.38; 70.51; 80.02; 120.37; 125.54; 127.48; 128.09; 128.67; 136.27; 141.69;

Example 28
2- [2- (2-Azidoethoxy) ethoxy] ethanol

A slurry of 2- (2-(-2-chloroethoxy) ethoxy) ethanol (25.0 g, 148 mmol) and sodium azide (14.5 g, 222 mmol) in dimethylformamide (250 ml) was left at 100 ° C. overnight. The reaction mixture was cooled in an ice bath, filtered and the organic solvent was evaporated in vacuo. The residue is dissolved in dichloromethane (200 ml), washed with water (75 ml), the aqueous phase is further extracted with dichloromethane (75 ml), and the combined organic phases are dried over magnesium sulfate (MgSO ₄ ), filtered. Evaporated in vacuo to give an oil that was used without further purification. Yield: 30.0 g (100%).

¹³ C-NMR (CDCl ₃ ): δ 72.53; 70.66-70.05; 61.74; 50.65.

Example 29
(2- [2- (2-Azidoethoxy) ethoxy] ethoxy) acetic acid

2- [2- (2-Azidoethoxy) ethoxy] ethanol (26 g, 148 mmol) is dissolved in tetrahydrofuran (100 ml) and slowly added to ice-cold sodium hydride (24 g, 593 mmol, 60% oil) under a nitrogen atmosphere. (This was prewashed with a solution of heptane (2 × 100 ml) in tetrahydrofuran (250 ml)). The reaction mixture was allowed to stand for 40 minutes, then subcooled in an ice bath, followed by the slow addition of bromoacetic acid (31 g, 223 mmol) dissolved in tetrahydrofuran (150 ml) and left at RT for about 3 hours. The organic solvent was evaporated in vacuo. The residue was suspended in dichloromethane (400 ml). Water (100 ml) was added slowly, after which the mixture was left under mechanical stirring for 30 minutes. The aqueous phase was separated, acidified with hydrochloride (4N) and extracted with dichloromethane (2 × 75 ml). All combined organic phases were evaporated in vacuo to give a yellow oil. Piperidine (37 ml, 371 mmol) in dichloromethane (250 ml) was slowly added to the oil and the mixture was left under mechanical stirring for 1 hour. The clear solution was diluted with dichloromethane (100 ml) and washed with hydrochloride (4N, 2 × 100 ml). The aqueous phase was further extracted with dichloromethane (2 × 75 ml) and the combined organic phases were evaporated in vacuo to give a yellow oil that was used without further purification. Yield: 27.0 g (66%). ¹³ C-NMR (CDCl ₃ ): δ 173.30; 71.36; 70.66-70.05; 68.65; 50.65.

Example 30
(S) -2,6-bis- (2- {2- [2- (2-azidoethoxy) ethoxy] ethoxy} acetylamino) hexanoic acid methyl ester

The above (2- [2- (2-azidoethoxy) ethoxy] ethoxy) acetic acid (13 g, 46.9 mol) was dissolved in dichloromethane (100 ml). N-hydroxysuccinimide (6.5 g, 56.3 mmol) and 1-ethyl-3- (3-dimethylaminopropylcarbodiimide hydrochloride (10.8 g, 56.3 mmol) were added and the reaction mixture was allowed to stand for 1 hour. 39 ml, 234 mmol) and L-lysine methyl ester dihydrochloride (6.0 g, 25.8 mmol) were added and the reaction mixture was allowed to stand for 16 h The reaction mixture was diluted with dichloromethane (300 ml), water (100 ml), Extracted with hydrochloride (2N, 2 × 100 ml), water (100 ml), 50% saturated sodium hydrogen carbonate (100 ml), and water (2 × 100 ml) The organic phase was dried over magnesium sulfate, filtered Evaporated in vacuo to give an oil that was used without further purification Yield: 11 g (73%) LCMS: m / z = 591 ¹³ C-NMR (CDCl ₃ ): (selected ) Δ 172.48; 169.87; 169 .84; 71.093-70.02; 53.51; 52.34; 51.35; 50.64; 38.48; 36.48; 31.99; 31.40; 29.13;

Example 31
((S) -2,6-Bis- (2- {2- [2- (2-t-butyloxycarbonylaminoethoxy) ethoxy] ethoxy} acetylamino) hexanoic acid methyl ester

  (S) -2,6-bis- (2- {2- [2- (2-azidoethoxy) ethoxy] ethoxy} acetylamino) hexanoic acid methyl ester (1.0 g, 1.7 mmol) ethyl acetate (15 ml) ) The solution was added to di-tert-butyl dicarbon at (0.9 g, 4.24 mmol) and 10% Pd / C (0.35 g). Hydrogen was then constantly bubbled through the solution for 3 hours. The reaction mixture was filtered and the organic solvent was removed in vacuo. The residue was purified by flash chromatography using ethyl acetate / methanol 9: 1 as eluent. Fractions containing product were pooled and the organic solvent was removed in vacuo to give an oil. Yield: 0.60 g (50%). LC-MS: m / z = 739 (M + 1).

Example 32
(S) -2,6-Bis- (2- {2- [2- (2-aminoethoxy) ethoxy] ethoxy} acetylamino) hexanoic acid methyl ester

  (S) -2,6-bis- (2- {2- [2- (2-t-butyloxycarbonylaminoethoxy) ethoxy] ethoxy} acetylamino) hexanoic acid methyl ester (0.6 g, 0.81 mmol) Dissolved in dichloromethane (5 ml). Trifluoroacetic acid (5 ml) was added and the reaction mixture was allowed to stand for about 1 hour. The reaction mixture was evaporated in vacuo to give an oil which was used without further purification: 0.437 g (100%). LC-MS m / z = 539 (M + 1).

Example 33
(S) -2,6-bis- (2- {2- [2- (2-azidoethoxy) ethoxy] ethoxy} acetylamino) hexanoic acid

  (S) -2,6-bis- (2- {2- [2- (2-azidoethoxy) ethoxy] ethoxy} acetylamino) hexanoic acid methyl ester (2.0 g, 3.47 mmol) in methanol (10 ml) Sodium hydroxide (hydroxide) (4N, 1.8 ml, 6.94 mmol) was added and the reaction mixture was allowed to stand for 2 hours. The organic solvent was evaporated in vacuo and the residue was dissolved in water (45 ml) and acidified with hydrogen chloride (4N). The mixture was extracted with dichloromethane (150 ml), which was washed with a saturated aqueous sodium chloride solution (2 × 25 ml). The organic phase was dried over magnesium sulfate, filtered and evaporated in vacuo to give an oil. LC-MS m / z = 577 (M + 1).

Example 34
N- (tert-Butyloxycarbonylaminoxybutyl) phthalimide

To a stirred mixture of N- (4-bromobutyl) phthalimide (18.9 g, 67.0 mmol), MeCN (14 ml), and N-Boc-hydroxylamine (12.7 g, 95.4 mmol), partially DBU (15.0 ml, 101 mmol) Was added. The resulting mixture was stirred at 50 ° C. for 24 hours. Water (300 ml) and 12M HCl (10 ml) were added and the product was extracted 3 times with AcOEt. The combined extracts were washed with brine, dried (MgSO ₄ ) and concentrated under reduced pressure. The resulting oil (28 g) was purified by chromatography (140 g SiO ₂ with heptane / AcOEt, gradient elution). 17.9 g (80%) of the title compound was obtained as an oil. ¹ H NMR (DMSO-d ₆ ) δ 1.36 (s, 9H), 1.50 (m, 2H), 1.67 (m, 2H), 3.58 (t, J = 7 Hz, 2H), 3.68 (t, J = 7 Hz, 2H), 7.85 (m, 4H), 9.90 (s, 1H).

Example 35
4- (tert-Butyloxycarbonylaminoxy) butylamine

To a solution of N- (tert-butyloxycarbonylaminoxybutyl) phthalimide (8.35 g, 25.0 mmol) in EtOH (10 ml) was added hydrazine hydrate (20 ml) and the mixture was stirred at 80 ° C. for 38 hours. The mixture was concentrated and the residue was coevaporated with EtOH and PhMe. EtOH (50 ml) was added to the residue and the precipitated phthalhydrazide was filtered and washed with EtOH (50 ml). Concentration of the combined filtrate gave 5.08 g of oil. This oil was mixed with aqueous K 2 CO 3 (10 g) (20 ml) and the product was extracted with H ₂ Cl ₂ . Drying (MgSO4) and concentration afforded 2.28 g (45%) of the title compound as an oil that was used without further purification. ¹ H NMR (DMSO-d ₆ ) δ 1.38 (m, 2H), 1.39 (s, 9H), 1.51 (m, 2H), 2.51 (t, J = 7 Hz, 2H), 3.66 (t, J = 7 Hz, 2H).

Example 36
2- (2-Trityloxyethoxy) ethanol

Trityl chloride (10 g, 35.8 mmol) was dissolved in dry pyridine, diethylene glycol (3.43 mL, 35.8 mmol) was added and the mixture was stirred overnight under nitrogen. The solvent was removed in vacuo. The residue was dissolved in dichloromethane (100 mL) and washed with water. The organic phase was dried over Na ₂ SO ₄ and the solvent was removed in vacuo. The crude product was purified by recrystallization from heptane / toluene (3: 2) to give the title compound. ¹ H NMR (CDCl ₃ ): δ 7.46 (m, 6H), 7.28, (m, 9H), 3.75 (t, 2H), 3.68 (t, 2H), 3.62 (t, 2H), 3.28 (t, 2H LC-MS: m / z = 371 (M + Na); R _t = 2.13 min.

Example 37
2- [2- (2-Trityloxyethoxy) ethoxymethyl] oxirane

2- (2-Trityloxyethoxy) ethanol (6.65 g, 19 mmol) was dissolved in dry THF (100 mL). A 60% NaH-oil suspension (0.764 mg, 19 mmol) was added slowly. The suspension was stirred for 15 minutes. Epibromohydrin (1.58 mL, 19 mmol) was added and the mixture was stirred overnight at room temperature under nitrogen. The reaction was quenched with ice and partitioned between diethyl ether (300 mL) and water (300 mL). The aqueous phase was extracted with dichloromethane. The organic phase was collected, dried (Na ₂ SO ₄ ), the solvent was removed in advance to give an oil, purified on a silica gel column eluting with DCM / MeOH / Et 3 N (98: 1: 1), The title compound was obtained. ¹ H NMR (CDCl ₃ ): δ 7.45 (m, 6H), 7.25, (m, 9H), 3.82 (dd, 1H), 3.68 (m, 6H), 3.45 (dd, 1H), 3.25 (t, 2H ), 3.15 (m, 1H) , 2.78 (t, 1H), 2.59 (m, 1H) LC-MS:. m / z = 427 (M + Na); R t = 2.44 min.

Example 38
1,3-bis [2- (2-trityloxyethoxy) ethoxy] propan-2-ol

2- (2-Trityloxyethoxy) ethanol (1.14 g, 3.28 mmol) was dissolved in dry DMF (5 mL). A 60% NaH-oil suspension (144 mg, 3.61 mmol) was added slowly and the mixture was stirred for 30 minutes at room temperature under nitrogen. The mixture was heated to 40 ° C. 2- [2- (2-Trityloxyethoxy) ethoxymethyl] oxirane (1.4 g, 3.28 mmol) was dissolved in dry DMF (5 mL) while stirring and added dropwise to the solution under nitrogen. After the addition was complete, the mixture was stirred at 40 ° C. overnight under nitrogen. After removing heat and cooling to room temperature, the reaction was quenched with ice and poured into saturated aqueous NaHCO ₃ (100 mL). The mixture was extracted with diethyl ether (3 × 75 mL). The organic phase was collected and dried (Na ₂ SO ₄ ) and the solvent removed in vacuo to give an oil that was purified on a silica gel column eluting with EtOAc / Heptane / Et3N (49: 50: 1). The title compound was obtained. ¹ H NMR (CDCl ₃ ): δ 7.45 (m, 12H), 7.25, (m, 18H), 3.95 (m, 1H), 3.78-3.45 (m, 16H), 3.22 (t, 4H), LC-MS : M / z = 775 (M + Na); R _t = 2.94 min.

Example 39
1,3-bis [2- (2-trityloxyethoxy) ethoxy] propan-2-yloxy β-cyanoethyl N, N-diisopropyl phosphoramidite

1,3-bis [2- (2-trityloxyethoxy) ethoxy] propan-2-ol (0.95 g, 1.26 mmol) was evaporated twice from dry pyridine and once from dry acetonitrile. The residue was dissolved in dry THF (15 mL) while being stirred under nitrogen. Diisopropylethylamine (1.2 mL, 6.95 mmol) was added. The mixture was cooled to 0 ° C. with an ice bath and 2-cyanoethyldiisopropylchloro-phosphoramidite (0.39 mL, 1.77 mmol) was added under nitrogen. The mixture was stirred at 0 ° C. for 10 minutes followed by 30 minutes at room temperature. Aqueous NaHCO ₃ solution (50 mL) was added and the mixture was extracted with DCM / Et 3 N (98: 2) (3 × 30 mL). The organic phase is collected, dried (Na ₂ SO ₄ ) and the solvent removed in vacuo to give an oil that is purified on a silica gel column eluting with EtOAc / Heptane / Et3N (35: 60: 5). Gave 703 mg of the title compound. ³¹ P-NMR (CDCl ₃ ): δ 149.6 ppm.

Example 40
2- (1,3-Bis [2- (2-hydroxyethoxy) ethoxy] propan-2-yloxy) acetic acid tert-butyl ester

1,3-bis [2- (2-trityloxyethoxy) ethoxy] propan-2-ol (0.3 g, 0.40 mmol) was evaporated once from dry pyridine and once from dry acetonitrile. Under nitrogen (60% NaH), the residue was dissolved in dry DMF (2 mL) -oil suspension (24 mg, 0.6 mmol) was added. The mixture was stirred for 15 minutes at room temperature. tert-Butyl bromoacetate (0.07 mL, 0.48 mmol) was added and the mixture was stirred for an additional 60 minutes. The reaction was quenched with ice and partitioned between diethyl ether (100 mL) and water (100 mL). The organic phase was collected and dried (Na ₂ SO ₄ ) and the solvent removed in vacuo to give an oil that was eluted on a silica gel column with EtOAc / Heptane / Et 3 N (49: 50: 1). . Fractions containing the main product were collected. The solvent was removed in vacuo and the residue was dissolved in 80% aqueous acetic acid (5 mL) and stirred at room temperature overnight. The solvent was removed in vacuo and the crude material was dissolved in diethyl ether (25 mL) and washed with water (2 × 5 mL). The aqueous phase was collected and the water was removed by rotary evaporation to give 63 mg of the title compound. ¹ H NMR (CDCl ₃ ): δ 4.19 (s, 2H), 3.78-3.55 (m, 21H), 1.49 (s, 9H).

Example 41:
N, N-bis (2- (2-phthalimidoethoxy) ethyl) -O-tert-butylcarbamic acid

  N, N-bis (2-hydroxyethyl) -O-tert-butylcarbamic acid is dissolved in a polar aprotic solvent such as THF or DMF. Sodium hydride (60% mineral oil suspension) is slowly added to the solution. The mixture is stirred for 3 hours. Add N- (2-bromoethyl) phthalimide. The mixture is stirred until the reaction is complete. The reaction is quenched by the slow addition of methanol. Add ethyl acetate. The solution is washed with aqueous sodium bicarbonate. The organic phase is filtered, dried and then concentrated under reduced pressure whenever possible. The crude compound is purified by standard column chromatography.

Example 42:
N, N-bis (2- (2-aminoethoxy) ethyl) -O-tert-butylcarbamic acid

  N, N-bis (2- (2-phthalimidoethoxy) ethyl) -O-tert-butylcarbamic acid is dissolved in a polar solvent such as ethanol. Add hydrazine (or another known agent to remove the phthaloyl protecting group). The mixture is stirred at room temperature (or elevated temperature if necessary) until the reaction is complete. The mixture is concentrated as much as possible under reduced pressure. The crude compound is purified by standard column chromatography or, if possible, by vacuum distillation.

Example 43:
N, N-bis (2- (2-benzyloxycarbonylaminoethoxy) ethyl) -O-tert-butylcarbamic acid

  N, N-bis (2- (2-aminoethoxy) ethyl) -O-tert-butylcarbamic acid is dissolved in a mixture of aqueous sodium hydroxide and THF or in a mixture of aqueous sodium hydroxide and acetonitrile. Add chlorocarbonate containing benzyl group. The mixture is stirred at room temperature until the reaction is complete. If necessary, reduce the volume in vacuum. Add ethyl acetate. The organic phase is washed with brine. The organic phase is dried, filtered and then concentrated in vacuo as much as possible. The crude compound is purified by standard column chromatography.

Example 44:
Bis (2- (2-phthalimidoethoxy) ethyl) amine

  Bis (2- (2-phthalimidoethoxy) ethyl) -tert-butylcarbamic acid is dissolved in trifluoroacetic acid. The mixture is stirred at room temperature until the reaction is complete. Concentrate the mixture in vacuo as much as possible. The crude compound is purified by standard column chromatography.

Example 45:
11-oxo-17-phthalimido-12- (2- (2-phthalimidoethoxy) ethyl) -3,6,9,15-tetraoxa-12-azaheptadecanoic acid

  3,6,9-Trioxaundecanoic acid is dissolved in dichloromethane. Add carbodiimide (eg, N, N-dicyclohexylcarbodiimide or N, N-diisopropylcarbodiimide). The solution is stirred overnight at room temperature. Filter the mixture. The filtrate can be concentrated in vacuo as needed. Acylation of amines with intramolecular anhydrides formed is known from the literature (eg Cook, RM; Adams, JH; Hudson, D. Tetrahedron Lett., 1994, 35, 6777-6780 or Stora, T Dienes, Z .; Vogel, H .; Duschl, C. Langmuir 2000, 16, 5471-5478). The anhydride is mixed with an aprotic solvent solution of bis (2- (2-phthalimidoethoxy) ethyl) amine, such as dichloromethane or N, N-dimethylformamide. The mixture is stirred until the reaction is complete. The crude compound is purified by extraction and subsequent standard column chromatography.

Example 46:
5-Oxo-11-phthalimido-6- (2- (2-phthalimidoethoxy) ethyl) -3,9-dioxa-6-azaundecanoic acid

  An aprotic solvent solution of diglycolic anhydride, such as dichloromethane or N, N-dimethylformamide, is added to a non-protic solvent solution of bis (2- (2-phthalimidoethoxy) ethyl) amine, such as dichloromethane or N, N-dimethylformamide. Add dropwise to the proton solvent solution. The mixture is stirred until the reaction is complete. The crude compound is purified by extraction and subsequent standard column chromatography.

Example 47
1,2,3-Benzotriazine-4 (3H) -on-3-yl 2- [2- (2-methoxyethoxy) ethoxy] acetate

3-hydroxy-1,2,3-benzotriazin-4 (3H) -one (10.0 g; 61.3 mmol) and 2- [2- (2-methoxyethoxy) ethoxy] acetic acid (10.9 g; 61.3 mmol) in DCM (125 ml) and DIC (7,7 g; 61.3 mmol) was added. The mixture was stirred overnight at ambient temperature in a dry atmosphere. A diisopropylurea precipitate was formed and filtered. The organic solution was washed extensively with saturated aqueous sodium bicarbonate, then dried (Na ₂ SO ₄ ) and evaporated in vacuo to give the title product as a clear yellow oil. The yield was 16.15 g (81%). ¹ H-NMR (CDCl ₃ ): δ 3.39 ppm (s, 3H); 3.58 (t, 2H); 3.68 (t, 2H); 3.76 (t, 2H); 3.89 (t, 2H); 4.70 (s, . 2H); 7.87 (t, 1H); 8.03 (t, 1H); 8.23 (d, 1H); 8.37 (d, 1H) 13 C-NMR (CDCl 3, selected peaks): δ 57.16 ppm; 64.96; 68.71 68.79; 69.59; 69.99; 120.32; 123.87; 127.17; 130.96; 133.63; 142.40; 148.22; 164.97.

Oligomer Product Solid Phase Oligomer Formation: All of the reactions described below are performed on polystyrene functionalized with Wang linkers. The reaction also generally works on other types of solid supports, as well as linkers with other types of functionality.

Reduction of solid phase azide:
The reaction is known (Schneider, SE et al. Tetrahedron, 1998, 54 (50) 15063-15086) and is a mixture solution of excess triphenylphosphine in THF and water at room temperature for 12-24 hours. This can be done by processing the bound azide. Alternatively, an aqueous THF solution of trimethylphosphine can be used as described by Chan, TY et al Tetrahedron Lett. 1997, 38 (16), 2821-2824. In addition, reduction of azide can be achieved by using sulfides such as dithiothreitol (Meldal, M. et al. Tetrahedron Lett. 1997, 38 (14), 2531-2534), 1,2-dimercaptoethane and 1,3- Dimercaptopropane (Meinjohanns, E. et al. J. Chem. Soc, Perkin Trans 1, 1997,6, 871-884), or tin (II) salts such as tin (II) chloride (Kim, JM et al. Tetrahedron Lett, 1996, 37 (30), 5305-5308).

Solid phase carbamate formation:
The reaction is known and is usually carried out by reacting an activated carbonate or haloformate derivative with an amine, preferably in the presence of a base.

Example 48
3- (1,3-bis {2- [2- (1,3-bis [2- (2- {2- [2- (2-methoxyethoxy) ethoxy] acetamino} ethoxy) ethoxy] propane-2- Yloxy) acetylamino] ethoxy]) ethoxy} propan-2-yloxy) acetylamino) propanoic acid

  This example is the synthesis of a second generation amide based on a dendrimer capped with 2- [2- (2-methoxyethoxy) ethoxy] acetic acid. [Azidoethoxyethyl] propan-2-yloxy) acetic acid monomer building blocks are used. Coupling chemistry is based on standard solid phase peptide chemistry and protection methods are based on solid phase azide reduction steps as described above.

  Step 1: Fmoc-βala-Wang resin (100 mg; filling 0.31 mmol / g BACHEM) was suspended in dichloromethane for 30 minutes and washed twice with DMF. 20% piperidine in DMF was added and the mixture was shaken for 15 minutes at ambient temperature. This process was repeated and the resin was washed with DMF (3 ×) and DCM (3 ×).

  Step 2: Coupling of monomer building blocks: 2- (1,3-bis [azidoethoxyethyl] propan-2-yloxy) acetic acid (527 mg; 1,4 mmol, 4 ×) and DhbtOH (225 mg; A solution of 1,4 mmol, 4 ×) was dissolved in DMF (5 ml) and DhbtOH (225 mg; 1,4 mmol, 4 ×) was added. The mixture was left for 10 minutes (pre-activation) and then added to the resin with DIPEA (240 μl; 1,4 mmol, 4 ×). The resin was shaken for 90 minutes, then drained and washed with DMF (3 ×) and DCM (3 ×).

  Step 3: Capping with acetic anhydride: The resin was then treated with a solution of acetic anhydride, DIPEA, DMF (12: 4: 48) for 10 minutes at ambient temperature. The solvent was removed and the resin was washed with DMF (3x) and DCM (3x).

Step 4: Deprotection (Reduction of azide group): The resin was treated with a solution of DTT (2M) and DMF in DIPEA (1M) at 50 ° C. for 1 hour. The resin was then washed with DMF (3x) and DCM (3x). A small amount of resin was again aspirated and treated with a solution of benzyl chloride (0.5M) and DMF in DIPEA (1M) for 1 hour. The resin was cleaved with 50% TFA / DCM and the dibenzylated product was analyzed by NMR and LC-MS. ¹ H-NMR (CDCl ₃ ): 3.50-3.75 (m, 20H); 3.85 (s, 1H); 4.25 (d, 2H); 6.95 (t, 1H); 7.40-7.50 (m, 6H); 7.75 ( m, 4H). LC-MS (any-one): m / e = 576 (M + 1) ⁺ ; R _t = 2.63 min.

  Steps 5-7 were performed as in steps 2-4 using twice the amount of reagent but using the same amount of solvent.

  Step 8: Capping with 2- [2- (2-methoxyethoxy) ethoxy] acetic acid: 2- [2- (2-methoxyethoxy) ethoxy] acetic acid (997 mg; 5.6 mmol, 16 × for resin loading) and DhbtOH (900 mg; 5.6 mmol, 16 ×) is dissolved in DMF (5 ml) and DIC (864 μl, 5.6 mmol, 16 ×) is added. The mixture is left for 10 minutes (pre-activation) and then added to the resin with DIPEA (960 μl; 5.6 mmol, 16 ×). The resin is shaken for 90 minutes, then drained and washed with DMF (3 ×) and DCM (3 ×).

  Step 9: Cleavage from resin: Treat resin with 50% TFA-DCM solution at ambient temperature for 30 minutes. The solvent is collected and the resin is washed an additional number of times with 50% TFA-DCM. The combined filtrate is evaporated to dryness and the residue is purified by chromatography.

Example 49
3- (1,3-bis {2- [2- (1,3-bis [2- (2- {2- [2- (2-methoxyethoxy) ethoxy] acetamino} ethoxy) ethoxy] propane-2- Yloxycarbonyl) amino] ethoxy]) ethoxy} propan-2-yloxycarbonyl) amino) propanoic acid

  This example is for the synthesis of a second generation carbamate capped with 2- [2- (2-methoxyethoxy) ethoxy] acetic acid and the 1,3-bis [2- (2- Azidoethoxy) ethoxy] propan-2-yl-p-nitrophenyl carbonate monomer building block is used. Coupling chemistry is based on standard solid phase carbamate chemistry and protection methods are based on solid phase azide reduction steps as described above.

  Step 1: Fmoc-βala-Wang resin (100 mg; filling 0.31 mmol / g BACHEM) was suspended in dichloromethane for 30 minutes and washed twice with DMF. 20% piperidine in DMF was added and the mixture was shaken for 15 minutes at ambient temperature. This process was repeated and the resin was washed with DMF (3 ×) and DCM (3 ×).

  Step 2: Coupling of monomer building blocks: DIPEA with a solution of 1,3-bis [azidoethoxyethyl] propan-2-yl-p-nitrophenylcarbamic acid (527 mg; 1,4 mmol, 4 ×) (240 μl; 1,4 mmol, 4 ×) was added to the resin. The resin was shaken for 90 minutes, then drained and washed with DMF (3 ×) and DCM (3 ×).

  Step 3: Capping with acetic anhydride: The resin was then treated with a solution of acetic anhydride, DIPEA, DMF (12: 4: 48) for 10 minutes at ambient temperature. The solvent was removed and the resin was washed with DMF (3x) and DCM (3x).

Step 4: Deprotection (Reduction of azide group): The resin was treated with a solution of DTT (2M) and DMF in DIPEA (1M) at 50 ° C. for 1 hour. The resin was then washed with DMF (3x) and DCM (3x). A small amount of resin was again aspirated and treated with a solution of benzyl chloride (0.5M) and DMF in DIPEA (1M) for 1 hour. The resin was cleaved with 50% TFA / DCM and the dibenzylated product was analyzed by NMR and LC-MS. ¹ H-NMR (CDCl ₃ ): 3.50-3.75 (m, 20H); 3.85 (s, 1H); 4.25 (d, 2H); 6.95 (t, 1H); 7.40-7.50 (m, 6H); 7.75 ( m, 4H). LC-MS (any-one): m / e = 576 (M + 1) ⁺ ; R _t = 2.63 min.

  Steps 5-7 were performed as in steps 2-4 using twice the amount of reagent but using the same amount of solvent.

  Step 8: Capping with 2- [2- (2-methoxyethoxy) ethoxy] acetic acid: 2- [2- (2-methoxyethoxy) ethoxy] acetic acid (997 mg; 5.6 mmol, 16 × for resin loading) and DhbtOH (900 mg; 5.6 mmol, 16 ×) is dissolved in DMF (5 ml) and DIC (864 μl, 5.6 mmol, 16 ×) is added. The mixture is left for 10 minutes (pre-activation) and then added to the resin with DIPEA (960 μl; 5.6 mmol, 16 ×). The resin is shaken for 90 minutes, then drained and washed with DMF (3 ×) and DCM (3 ×).

  Step 9: Cleavage from resin: Treat resin with 50% TFA-DCM solution at ambient temperature for 30 minutes. The solvent is collected and the resin is washed an additional number of times with 50% TFA-DCM. The combined filtrate is evaporated to dryness and the residue is purified by chromatography.

Example 50
3- [2- (1,3-Bis [2- (2- {2- [2- (2-methoxyethoxy) ethoxy] acetylamino} ethoxy) ethoxy] propan-2-yloxy) acetylamino] propanoic acid

  Step 1: Fmoc-β-alanine linked Wang resin (A22608, A22608, NovaBiochem, 3.00 g; loading, 0.83 mmol / g) was dissolved in DCM for 20 minutes, then DCM (2 × 20 ml) and NMP (2 × 20 ml) ). The resin was then treated twice with 20% piperidine in NMP (2 × 15 min). The resin was washed with NMP (3 × 20 ml) and DCM (3 × 20 ml).

  Step 2: 2- (1,3-bis [2- (2-azidoethoxy) ethoxy] propan-2-yloxy) acetic acid (3.70 g; 10 mmol) to NMP (30 ml) and DhbtOH (1.60 g; 10 mmol) Dissolved and DIC (1.55 ml; 10 mmol) was added. The mixture is stirred at ambient temperature for 30 minutes and then added to the resin obtained in step 1 with DIPEA (1.71 ml; 10 mmol). The reaction mixture was shaken for 1.5 hours, then drained and washed with NMP (5 × 20 ml) and DCM (3 × 20 ml).

Step 3: A solution of SnCl ₂ .2H ₂ O (11.2 g; 49.8 mmol) in NMP (15 ml) and DCM (15 ml) was then added. The reaction mixture was shaken for 1 hour. The resin was drained and washed with NMP: MeOH (5 × 20 ml; 1: 1). The resin was then dried in vacuo.

  Step 4: A solution of 2- [2- (2-methoxyethyl) ethoxy] acetic acid (1.20 g; 6.64 mmol), DhbtOH (1.06 g; 6.60 mmol) and DIC (1.05 ml; 6.60 mmol) in NMP (10 ml) Mix for 10 minutes at room temperature and then connect to the 3- [2- (1,3-bis [2- (2-aminoethoxy) ethoxy] propan-2-yloxy) acetylamino] propanoic acid obtained in step 3 Wang resin (1.0 g; 0.83 mmol / g) was added. DIPEA (1.15 ml, 6.60 mmol) was added and the reaction mixture was shaken for 2.5 hours. The solvent was removed and the resin was washed with NMP (5 × 20 ml) and DCM (10 × 20 ml).

Step 5: The resin product of Step 4 was treated with TFA: DCM (10 ml, 1: 1) for 1 hour. The resin was filtered and washed once with TFA: DCM (10 ml, 1: 1). The combined filtrate and washed, then dried to give a yellow oil (711 mg). The oil was dissolved in 10% acetonitrile-water (20 ml) and purified over two runs on a preparative HPLC apparatus using a C18 column and a 15-40% acetonitrile-water gradient. The fractions were then analyzed by LC-MS. Fractions containing product were pooled and dried. Yield: 222 mg (37%). LC-MS: m / z = 716 (m + 1), Rt = 1.97 min. ¹ H-NMR (CDCl ₃ ): δ 2.56 ppm (t, 2H); 3.36 (s, 6H); 3.46-3.66 (m, 39H); 4.03 (s, 4H); 4.16 (s, 2H); 7.55 ( 8.05 (t, 1H). ¹³ C-NMR (CDCl ₃ , selected peaks): δ 33.71 ppm; 34.90; 58.89; 68.94; 69.40; 69.98; 70.09; 70.33; 70.74; 70.91; 71.07; 79.07; 171.62; 171.97; 173.63.

Example 51
3- (1,3-bis {2- (2- [2- (1,3-bis [2- (2- {2- [2- (2-methoxyethoxy) ethoxy] acetamino} ethoxy) -ethoxy] Propan-2-yloxy) acetylamino] ethoxy) ethoxy} propan-2-yloxy) acetylamino) propanoic acid

This material is obtained by repeating steps 2-5 with twice the amount of reagent used, and 3- [2- (1,3-bis [2- (2-amino) obtained in step 3 of Example 50. Prepared from wang resin (1.0 g; 0.83 mmol / g) coupled to ethoxy) ethoxy] propan-2-yloxy) acetylamino] propanoic acid. Yield: 460 mg (33%). MALDI-MS (-cyanohydroxysinapic acid matrix): m / z = 1670 (M + Na ⁺ ). ¹ H-NMR (CDCl ₃ ): δ 2.57 ppm (t, 2H); 3.38 (s, 12H); 3.50-3.73 (m, 85 H); 4.05 (s, 8H); 4.17 (s, 2H); 4.19 (S, 4H); 7.48 (m, 4H); 7.97 (m, 3H). ¹³ C-NMR (CDCl ₃ , selected peaks): δ 38.81 ppm; 58.92; 69.46; 69.92; 70.05; 70.05; 70.13; 70.73; 70.97; 71.11; 71.88; 76.74; 77.06; 77.38; 171.33; 172.02.

Example 52
3 (1,3-Bis {2 (2 [2 (1,3-bis {2 (2 [2 (1,3-bis [2 (2 {ethoxy2 [2 (2-methoxyethoxy)]-acetamino} Ethoxy) ethoxy] propan-2-yloxy) acetylamino] ethoxy) ethoxy} propan-2-yloxy) acetylamino) ethoxy) ethoxy} propan-2-yloxy) acetylamino) propanoic acid

This material is obtained in Step 3 of Example 50 by repeating steps 2-3 with twice the amount of reagent used and then repeating steps 2-5 with twice the amount of reagent used. Prepared from wang resin (1.0 g; 0.83 mmol / g) linked to 3- [2- (1,3-bis [2- (2-aminoethoxy) ethoxy] propan-2-yloxy) acetylamino] propanoic acid . Yield: 84 mg (4%). LC-MS: (m / 2) + 1 = 1758; (m / 3) + 1 = 1172; (m / 4) + 1 = 879; (m / 5) + 1 = 704. Rt = 2.72 min. ¹ H-NMR (CDCl ₃ ): δ 2.51 ppm (t, 2H); 3.33 (s, 24H); 3.44-3.70 (m, 213H); 3.93 (s, 16H); 4.08 (s, 14H); 7.25 ( 7.69 (m, 7H). ¹³ C-NMR (CDCl ₃ , selected peaks): δ 38.94 ppm; 59.33; 69.78; 70.08; 70.37; 70.44; 70.56; 70.82; 71.10; 71.26; 71.51; 79.24; 170.60; 171.22.

Example 53
N-hydroxysuccinimidyl 3- [2- (1,3-bis [2- (2- {2- [2- (2-methoxyethoxy) ethoxy] acetylamino} ethoxy) ethoxy] propan-2-yloxy ) Acetylamino] propanoate

  3- [2- (1,3-Bis [2- (2- {2- [2- (2-methoxyethoxy) ethoxy] acetylamino} ethoxy) ethoxy] propan-2-yloxy) acetylamino] propanoic acid ( 67 mg; 82 μmol) was dissolved in THF (5 ml). The reaction mixture was cooled on an ice bath. DIPEA (20 μl; 120 μmol) and TSTU (34 mg; 120 μmol) were added. The mixture was stirred overnight at ambient temperature, at which time the reaction was complete according to LC-MS. LC-MS: m / z = 813 (M + H) +; Rt = 2.22 min.

Example 54
N-hydroxysuccinimidyl 3- (1,3-bis {2- (2- [2- (1,3-bis [2- (2- {2- [2- (2-methoxyethoxy) ethoxy] Acetamino} ethoxy) ethoxy] propan-2-yloxy) acetylamino] ethoxy) ethoxy} propan-2-yloxy) acetylamino) propanoate

As described in Example 53, 3- (1,3-bis {2- (2- [2- (1,3-bis [2- (2- {2- [2- (2-methoxy Prepared from ethoxy) ethoxy] -acetamino} ethoxy) ethoxy] propan-2-yloxy) acetylamino] ethoxy) ethoxy} propan-2-yloxy) acetylamino) propanoic acid and TSTU. LC-MS: (m / 2 ) +1 = 873, R t = 2.55 min.

Example 55
N-hydroxysuccimidyl 3- (1,3-bis {2- (2- [2- (1,3-bis {2- (2- [2- (1,3-bis [2- (2- {2- [2- (2-methoxyethoxy) ethoxy] -acetamino} ethoxy) ethoxy] propan-2-yloxy) acetylamino] ethoxy) ethoxy} propan-2-yloxy) acetylamino) ethoxy) ethoxy} propane-2 -Iloxy) acetylamino) propanoate

As described in Example 53, N-hydroxysuccinimidyl 3- (1,3-bis {2- (2- [2- (1,3-bis {2- (2- [2- (1,3-bis [2- (2- {2- [2- (2-methoxyethoxy) ethoxy] -acetamino} ethoxy) ethoxy] propan-2-yloxy) acetylamino] ethoxy) ethoxy} propane-2- Prepared from yloxy) acetylamino) ethoxy) ethoxy} propan-2-yloxy) acetylamino) propanoic acid and TSTU. LC-MS: (m / 4 ) +1 = 903, R t = 2.69 min.

Example 56
N- (4-tert-Butoxycarbonylaminoxybutyl) 3- (1,3-bis {2- (2- [2- (1,3-bis [2- (2- {2- [2- (2 -Methoxyethoxy) ethoxy] acetamino} ethoxy) ethoxy] propan-2-yloxy) acetylamino] ethoxy) ethoxy} propan-2-yloxy) acetylamino) propanamide

N-hydroxysuccinimidyl 3- (1,3-bis {2- (2- [2- (1,3-bis [2- (2- {2- [2- (2-methoxyethoxy) ethoxy] -Acetamino} ethoxy) ethoxy] propan-2-yloxy) acetylamino] ethoxy) ethoxy} propan-2-yloxy) acetylamino) propanoate (105 mg; 0.06 mmol) was dissolved in DCM (2 ml). A solution of 4- (tert-butyloxycarbonylaminoxy) butylamine (49 mg; 0.24 mmol) (49 mg; 0.24 mmol) was then added followed by DIPEA (13 μl; 0.07 mmol). The mixture was stirred at ambient temperature for 1 hour and then concentrated under reduced pressure. Dissolve the residue in 20% acetonitrile-water (4 ml) and prepare using a C18 column and a stepwise gradient of 0, 10, 20, 30, and 40% (10 ml elution each) of acetonitrile-water It refine | purified with the HPLC apparatus. Fractions containing pure product were concentrated and dried in a vacuum oven for 16 hours to give a yellow oil. Yield: 57 mg (51%). LC-MS: (m / 2) + 1 = 918, Rt = 2.75 min. ¹ H-NMR (CDCl ₃ ): δ 1.42 ppm (s, 9H); 2.40 (t, 2H); 3.21 (dd, 2H); 3.33 (s, 12H); 3.38-3.72 (m, 99H); 3.80 ( 3.95 (s, 8H); 4.08 (s, 6H); 6.99 (m, 1H); 7.23 (m, 4H); 7.69 (m, 2H); 7.85 (m, 1H); 8.00 (m , 1H). ¹³ C-NMR (CDCl ₃ , selected peaks): δ 28.27 ppm; 38.58; 58.97; 69.42; 69.72; 70.01; 70.08; 70.20; 70.41; 70.46; 70.73; 70.91; 71.16; 170.89.

Example 57
N- (4-aminoxybutyl) 3- (1,3-bis {2- (2- [2- (1,3-bis [2- (2- {2- [2- (2-methoxyethoxy) Ethoxy] acetamino} ethoxy) ethoxy] propan-2-yloxy) acetylamino] ethoxy) ethoxy} propan-2-yloxy) acetylamino) propanamide

  N- (4-tert-Butoxycarbonylaminoxybutyl) 3- (1,3-bis {2- (2- [2- (1,3-bis [2- (2- {2- [2- (2 -Methoxyethoxy) ethoxy] acetamino} ethoxy) ethoxy] propan-2-yloxy) acetylamino] ethoxy) ethoxy} propan-2-yloxy) acetylamino) propanamide (19 mg; 10 μmol) in 50% TFA / DCM (10 ml) and the clear solution was stirred for 30 minutes at ambient temperature. The solvent was removed by rotary evaporation and the residue was removed twice from DCM to give a quantitative yield (19 mg) of the title product. LC-MS: (m / 2) + 1 = 868, (m / 3) + 1 = 579, Rt = 2,35 min.

  The following examples show solution phase conjugation to peptides or proteins.

Example 58
t-Butyl 2- (1,3-bis [2- (2- {2- [2- (2-methoxyethoxy) ethoxy] acetamino} ethoxy) -ethoxy] propan-2-yloxy) acetate

t-Butyl 2- (1,3-bis [2- (2-aminoethoxy) ethoxy] propan-2-yloxy) acetate (1.74 g; 4.5 mmol) and 1,2,3-benzotriazine-4 (3H) -On-3-yl 2- [2- (2-methoxyethoxy) ethoxy] acetate (2.94 g; 9 mmol) was dissolved in DCM (100 ml). DIPEA (3.85 ml; 22.3 mmol) was added and the celar mixture was stirred at room temperature for 90 minutes. The solvent was removed in vacuo and the residue was purified by chromatography on silica using MeOH-DCM (1:16) as eluent. Pure fractions were pooled and dried to give the title material as a clear oil. The yield was 1.13 g (36%). ¹ H-NMR (CDCl ₃ ): δ 1.46 ppm (s, 9H); 3.38 (s, 6H); 3.49-3.69 (m, 37H); 4.01 (s, 4H); 4.18 (s, 2H); 7.20 ( bs, 2H).

Example 59
2- (1,3-bis [2- (2- {2- [2- (2-methoxyethoxy) ethoxy] acetamino} ethoxy) ethoxy] propan-2-yloxy) acetic acid:

Dissolve t-butyl 2- (1,3-bis [2- (2-aminoethoxy) ethoxy] propan-2-yloxy) acetate (470 mg; 0.73 mmol) in DCM-TFA (25 ml, 1: 1). The mixture was stirred at ambient temperature for 30 minutes. The solvent was removed in vacuo and the residue was removed from DCM twice. LC-MS: (m + 1) = 645, Rt = 2,26 min. ¹ H-NMR (CDCl ₃ ): δ 3.45 ppm (s, 6H); 3.54-3.72 (m, 37H); 4.15 (s, 4H); 4.36 (s, 2H).

Example 60
N-hydroxysuccimidyl 2- (1,3-bis [2- (2- {2- [2- (2-methoxyethoxy) ethoxy] acetamino} ethoxy) ethoxy] propan-2-yloxy) acetate

  2- (1,3-bis [2- (2- {2- [2- (2-methoxyethoxy) ethoxy] acetamino} ethoxy) ethoxy] propan-2-yloxy) acetic acid (115 mg; 0.18 mmol) in THF (5 ml). The reaction mixture was placed on an ice bath. TSTU (65 mg, 0.21 mmol) and DIPEA (37 μl; 0.21 mmol) were added and the reaction mixture was stirred at 0 ° C. for 30 minutes and then at room temperature overnight. The reaction was then dried to give 130 mg of the title material as a clear oil. LC-MS: (m + 1) = 743, (m / 2) + 1 = 372, Rt = 2,27 min.

Example 61
tButyl 3- (1,3-bis {2- (2- [2- (1,3-bis {2- (2- [2- (1,3-bis [2- (2- {2- [ 2- (2-methoxyethoxy) ethoxy] -acetamino} ethoxy) ethoxy] propan-2-yloxy) acetylamino] ethoxy) ethoxy} propan-2-yloxy) acetylamino) ethoxy) ethoxy} propan-2-yloxy) acetate

  The material is 2 equivalents of N-hydroxysuccimidyl 2- (1,3-bis [2- (2- {2- [2- (2-methoxyethoxy) ethoxy] acetamino} ethoxy) ethoxy] propane-2- (Iloxy) acetate and 1 equivalent of t-butyl 2- (1,3-bis [2- (2-aminoethoxy) ethoxy] propan-2-yloxy) acetate followed by the protocol and purification method described in Example 58. Use and adjust. Subsequent removal of the t-butyl group is performed as described in Example 59, and N-hydroxysuccimidyl ester formation is performed as described in Example 60.

Example 62
(S) -2,6-bis- (2- [2- (2- [2- (2,6-bis- [2- (2- [2- (2-azidoethoxy) ethoxy] ethoxy) acetylamino ] Hexanoylamino) ethoxy] ethoxy) ethoxy] acetylamino) hexanoic acid methyl ester

  (S) -2,6-bis- (2- {2- [2- (2-azidoethoxy) ethoxy] ethoxy} acetylamino) hexanoic acid (1.8 g, 3.10 mmol) 1: 3 (10 ml) dimethylformamide In dichloromethane / dichloromethane mixture, adjust the pH to basic reaction using diisopropylethylamine, add N-hydroxybenzotriazole and 1-ethyl-3- (3-dimethylaminopropylcarbodiimide hydrochloride) to react The mixture was then allowed to stand for 30 minutes and then the reaction mixture was converted to (S) -2,6-bis- (2- {2- [2- (2-aminoethoxy) ethoxy] ethoxy} acetylamino) hexanoic acid methyl ester (0.37 g, 0.70 mmol) in dichloromethane solution and the reaction mixture was allowed to stand overnight The reaction mixture was diluted with dichloromethane (150 ml), water (2 × 40 ml), 50% saturated sodium bicarbonate (2 × 30ml), and water (3 × 40ml) The organic phase was dried over magnesium sulfate, filtered and evaporated in vacuo to give an oil, yield: 1.6 g (89%), LC-MS: m / z = 1656 (M + 1) ) And m / z = 828.8 (M / 2) +1 and m / z = 553 (M / 3) +1.

Example 63
((S) -2,6-bis- (2- [2- (2- [2-((S) -2,6-bis- [2- (2- [2- (2-tert-butoxycarbonyl Aminoethoxy) ethoxy] ethoxy) acetylamino] hexanoylamino) ethoxy] ethoxy) ethoxy] acetylamino) hexanoic acid methyl ester

(S) -2,6-bis- (2- [2- (2- [2-((S) -2,6-bis- [2- (2- [2- (2-azidoethoxy) ethoxy) ] Ethoxy) acetylamino] hexanoylamino) ethoxy] ethoxy) ethoxy]
Acetylamino) hexanoic acid methyl ester (1.6 g, 0.97 mmol) in ethyl acetate (60 ml) contains di-tert-butyl dicarbonate (1.0 g, 4.8 mmol) and Pd / C (10%, 1.1 g) Was added. Hydrogen was constantly bubbled through the reaction mixture for 2 hours. The reaction mixture was filtered and the organic solvent was removed in vacuo to give an oil that was used without further purification. Yield: 1.8 g (98%). LC-MS: m / z = 1953 (M + 1) and m / z = 977 (M / 2) +1.

Example 64
(S) -2,6-bis- (2- [2- (2- [2-((S) -2,6-bis- [2- (2- [2 (2aminoethoxy) ethoxy] ethoxy) Acetylamino] hexanoylamino) ethoxy] ethoxy) ethoxy] acetylamino) hexanoic acid methyl ester

  (S) -2,6-bis- (2- [2- (2- [2-((S) -2,6-bis- [2- (2- [2- (2-tert-butoxycarbonyl) Amino-ethoxy) ethoxy] ethoxy) acetylamino] hexanoylamino) ethoxy] ethoxy) ethoxy] -acetylamino) hexanoic acid methyl ester was dissolved in dichloromethane (20 ml) and trifluoroacetic acid (20 ml) was added. The reaction mixture was left for 2 hours. The organic solvent was evaporated in vacuo to give an oil. Yield: 1.4 g (100%). LC-MS: m / z = 1552 (M + 1); 777.3 (M / 2) +1; 518.5 (M / 3) +1 and 389.1 (M / 4) +1.

Example 65
(S) -2,6-bis- (2- [2- (2- [2-((S) -2,6-bis- [2- (2- [2- (2- (2- (2 -(2-Methoxyethoxy) ethoxy) acetylamino) ethoxy) ethoxy] ethoxy) acetylamino] hexanoylamino) ethoxy] ethoxy) ethoxy] acetylamino) hexanoic acid methyl ester

  To a solution of 2- (2- (methoxyethoxy) ethoxy) acetic acid (1.3 g, 7.32 mmol) in dichloromethane and dimethylformamide 3: 1 (20 ml) was added N-hydroxysuccinimide (0.8 g, 7.32 mmol) and 1- Ethyl-3- (3-dimethylaminopropylcarbodiimide hydrochloride (1.4 g, 7.32 mmol) was added.The reaction mixture was allowed to stand for 1 hour, after which the mixture was (S) -2,6-bis- (2- [2- (2- [2-((S) -2,6-bis- [2- (2- [2 (2aminoethoxy) ethoxy] ethoxy) acetylamino] hexanoylamino) ethoxy] ethoxy) ethoxy] Acetylamino) hexanoic acid methyl ester (1.42 g, 0.92 mmol) and diisopropylethylamine (2.4 ml, 14.64 mmol) were added to a solution of dichloromethane (10 ml) The reaction mixture was left overnight. Dilute with water (3 x 25 ml) The combined aqueous phases were further extracted with dichloromethane (2 × 75 ml) The residue was flashed using 500 ml of ethyl acetate as eluent followed by 500 ml of ethyl acetate / methanol 9: 1 and finally methanol. Product containing fractions were evaporated in vacuo to give an oil, yield: 0.75 g (38%), LC-MS: m / z = 1097 (M / 2) + 1; 732 (M / 3) +1 and 549 (M / 4) +1.

  (S) -2,6-bis- (2- [2- (2- [2-((S) -2,6-bis- [2- (2- [2- (2- (2- (2 -(2-Methoxyethoxy) ethoxy) acetylamino) ethoxy) ethoxy] ethoxy) acetylamino] hexanoylamino) ethoxy] ethoxy) ethoxy] acetylamino) hexanoic acid methyl ester can be saponified to free acid and active It can be used via a conjugated ester to attach to the amino group of a peptide or protein. The activated ester is made by standard coupling methods known in the art, such as diisopropylethylamine and N-hydroxybenzotriazole or other activation conditions, and attached to the amino group of the peptide or protein. Also good.

  Thus, further transformations according to FIG. 16, or the analogs of Example 53, and the analogs of Examples 56 and 57 result in dendritic materials that can be used to prepare conjugates according to the present invention. .

Example 66

2- (1,3-bis [2- (2-hydroxyethoxy) ethoxy] propane-2-oxy) acetic acid tert-butyl ester (63 mg, 0.16 mmol) was evaporated twice from dry acetonitrile. 1,3-bis [2- (2-trityloxyethoxy) ethoxy] propane-2-oxy β-cyanoethyl N, N-diisopropyl phosphoramidite (353 mg, 0.37 mmol) was evaporated twice from dry acetonitrile and dried Dissolved in acetonitrile (2 mL) and added. A solution of tetrazole in dry acetonitrile (0.25M, 2.64 mL) was added under nitrogen and the mixture was stirred for 1 hour at room temperature. 5.5 mL of I ₂ solution (0.1 M 7: 2: 1 THF / lutidine / water) was added and the mixture was stirred for an additional hour. The reaction mixture was diluted with ethyl acetate (20 mL) and washed with 2% aqueous sodium sulfite until the iodine color disappeared. The organic phase was dried (Na ₂ SO ₄ ) and the solvent removed in vacuo. The residue was dissolved in 80% aqueous acetic acid (5 mL) and stirred at room temperature overnight. The solvent was removed in vacuo and to the crude material was added diethyl ether (25 mL) and water (10 mL). The aqueous phase was collected and the water was removed in vacuo. The product was purified on a reverse phase preparative HPLC C-18 column, 0-40% acetonitrile gradient containing 0.1% TFA to give a tert-butyl protected second generation dendrimer product. HPLC-MS: m / z = 1171 (M + Na); 1149 (M +), 1093 (lost of tert-butyl in the MS) R t = 2.76 min.

  The β-cyanoethyl group was then deprotected and the tert-butyl ester group removed using conventional base and acid treatments known to those skilled in the art.

  Thus, further transformations according to FIG. 16, or the analogs of Example 53, and the analogs of Examples 56 and 57 result in dendritic materials that can be used to prepare conjugates according to the present invention. .

Example 67
A solution of asialo rFVIIa (10.2 mg, 0.2 μmol) in 13.5 ml TRIS buffer (10 mM Cacl ₂ , 10 mM TRIS, 50 mM NaCl, 0.5% Tween 80, pH 7.4) was cooled in an ice bath. N- (4-aminoxybutyl) 3- (1,3-bis {2- (2- [2- (1,3-bis [2- (2- {2- [2- (2-methoxyethoxy) A lith buffer solution of ethoxy] acetamino} ethoxy) ethoxy] -propan-2-yloxy) acetylamino] ethoxy) ethoxy} propan-2-yloxy) acetylamino) propanamide (17 mg; 10.0 μmol, 50 ×) Subsequently, 2.5 ml of a Tris buffer solution of galactose oxidase (135 U) and catalase (7500 U) was added. The reaction mixture was gently shaken at 4 ° C. for 48 hours. The slightly opaque solution was then filtered through a 0.45 μm filter (Sartorius Minisart®). The buffer was then exchanged for MES (10 mM CaCl ₂ , 10 mM MES, 50 mM NaCl, pH 6.0) using a NAP-10 column (Amersham). The mixture was then cooled on ice and an aqueous solution of EDTA (3.5 ml, 100 mM, pH 8.0, corresponding to [Ca ²⁺ ]) was added. The pH is adjusted to 7.6 by adding 1 M NaOH aqueous solution and the sample (6.8 mS / cm) is equilibrated with 5 ml HiTrap Q HP ion exchange column (Amersham-Biosciences) (10 mM Tris, 50 mM NaCl, pH 7.4). Filled). The column was eluted with 10 mM Tris, 50 mM NaCl, pH 7.4 (10 vol, flux: 1 ml / min). The elution buffer was then changed to 10 mM Tris, 50 mM NaCl, 25 mM CaCl ₂ , pH 7.4 (10 vol, flux: 1 ml / min). The eluate was monitored by UV and each fraction containing protein was analyzed by SDS-PAGE gel electrophoresis. Pure samples of N-glycan modified rFVII were pooled and stored at -80 ° C.

Claims (16)

A sugar comprising a glycoprotein having an increased in vivo plasma half-life compared to a non-conjugated glycoprotein and having at least one terminal galactose or derivative thereof and a retarding group covalently bound thereto A method for producing a protein conjugate comprising:
(A) contacting a glycoprotein having at least one terminal galactose or a derivative thereof with galactose oxidase to form an oxidized terminal galactose or a derivative thereof having a reactive aldehyde functional group;
(B) contacting the glycoprotein product produced in step (a) with a reactant X capable of reacting with an aldehyde group and containing a retarding group, represented by the formula (glycoprotein)-(retarding group) Forming a joined body;
Comprising a method. A sugar comprising a glycoprotein having an increased in vivo plasma half-life compared to a non-conjugated glycoprotein and having at least one terminal galactose or derivative thereof and a retarding group covalently bound thereto A method for producing a protein conjugate comprising:
(A) contacting a glycoprotein having at least one terminal galactose or a derivative thereof with galactose oxidase to form an oxidized terminal galactose or a derivative thereof having a reactive aldehyde functional group;
(B) Reactant X comprising a linking moiety that is capable of reacting the glycoprotein product produced in step (a) with an aldehyde group and further comprising a protected second reactive group. Contacting with a glycoprotein to form a conjugate of the linking moiety;
(C) contacting the product of step (b) with a retarding group capable of reacting with the second reactive group of the linking moiety, represented by the formula (glycoprotein)-(linking moiety)-(retarding group) Forming a joined body;
Comprising a method. 3. The method according to claim 1 or 2, further comprising the following steps (a1) and (a2) performed before the step (a) or the step (c), respectively. :
(A1) Contacting a glycoprotein with one or more of sialidase, galacosidases, N-acetylhexosaminidase, fucosidases, mannosidase, endo-H, and endo-F3 to remove some of the glycan structures Forming a glycoprotein that is treated;
(A2) contacting the product of step (a1) with a galactosyltransferase and a galactose substrate to form a glycoprotein comprising at least one terminal galactose or derivative thereof. The method according to claim 1 or 2, further comprising the following first step (a3) performed before the step (a):
(A3) contacting a glycoprotein having at least one terminal sialic acid residue with a sialidase or another reagent capable of removing sialic acid from glycans to form an asialoglycoprotein comprising at least one terminal galactose or derivative thereof Process.   5. The method of any one of claims 1, 3, or 4, wherein the reactant X has the formula nuc-R, where nuc can react with an aldehyde to form a covalent bond. A method wherein the functional group and R is a retarding group.   5. The method according to any one of claims 2, 3 or 4, wherein the reactant X has the formula nuc-L1-R, wherein nuc reacts with an aldehyde to form a covalent bond. A method in which R is a retarding group and L1 is a linking moiety. The method according to claim 5: wherein the reactant X _{is, H 2 NR, HR1N-R} , H 2 NOR, HR1N-OR, H 2 N-NH-COR, H 2 N-CHR1-CHR-SH Selected from the group consisting of H ₂ N—CHR—CHR 1 —SH, H ₂ N—NH—SO ₂ R, and Z′—CH ₂ —Z ″ —R; wherein R is a retarding group; R1 is H or a second retarding group; Z ′ and Z ″ represent an electron withdrawing group such as, for example, COOEt, CN, NO ₂ ; and one or both of the Z groups are bonded to the R group How you can. The method of claim 6 wherein the reactant X _{is, H 2 N-L1-R} , HR1N-L1-R, H 2 NO-L1-R, HR1N-O-L1-R, H 2 N -NH-CO-L1-R, H ₂ N-CHR1-CH (L1-R) -SH, H ₂ N-CH (L1-R) -CHR1-SH, H ₂ N-NH-SO ₂ -L1- R, selected from the group consisting of Z′-CH ₂ —Z ″ -L1-R; wherein L1 is a linking moiety, R is a delay group, and R1 is H or a second delay group Z ′ and Z ″ represent electron withdrawing groups such as COOEt, CN, NO _2, etc., and one or both of the Z groups can be connected to the R group; Claim 2, or a method according to any one of 4: Shared the reactant X has the formula nuc-L1-R _n, where the nuc is reacted with an aldehyde A functional group capable of forming a bond, R is a retarding group, L1 is a multifunctional linking moiety that joins one or more retarding groups (R) to a reactive functional group (nuc), and L1 may or may not contribute to protraction; n represents an integer from 1 to 25, such as n = 1-10.   12. The method according to any one of claims 1 to 11, wherein the retarding group is selected from the group consisting of: dendrimer, polyalkylene oxide (PAO), polyalkylene glycol (PAG), polyethylene glycol. (PEG), polypropylene glycol (PPG), branched PEG, polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolidone, polyethylene-co-maleic anhydride, polystyrene-co-maleic anhydride, dextran, carboxymethyl Dextran; serum protein binding ligands such as fatty acids, C5-C24 fatty acids, aliphatic diacids (eg C5-C24), compounds that bind albumin, glycans are receptors (eg asialoglycoprotein receptors and mannose receptors) ) Structures that inhibit binding to (eg sialic acid derivatives or mimetics), Small organics with neutral substituents that prevent glycan-specific recognition, such as moieties that alter charge properties under physiological conditions such as rubonic acid or amines, or small alkyl substituents (eg C1-C5 alkyl) Molecules, small organic charged radicals (eg C1-C25) that may contain one or more carboxylic acids, amines of sulfo groups, phosphonic acids, or combinations thereof; low molecular neutral hydrophilic molecules such as cyclodextrins (eg C1 -C25), or optionally branched polyethylene chain; polyethylene glycol with an average molecular weight of 2-40 KDa; exact molecular weight in the range between 700-20.000 Da, or more preferably 700-100.000 Da A well-defined and precise polymer such as a dendrimer with a protein; and substantially non-immunogenic, such as albumin or a portion of an antibody or optionally an antibody containing an Fc-domain Polypeptide.   11. The method according to any one of claims 1 to 10, wherein the glycoprotein is selected from the group consisting of: FVII, FVIII, FIX, FX, FII, FV, protein C, protein S. , TPA, PAI-1, tissue factor, FXI, FXII, FXIII, and sequence variants thereof; colony stimulating factors including immunoglobulins, interleukins, α-, β-, and γ-interferons, granulocyte colony stimulating factor , Cytokines such as platelet-derived growth factor, and phospholipase activating protein (PUP), plant proteins such as insulin, lectin and ricin, tumor necrosis factor and related alleles, soluble forms of tumor necrosis factor receptor, interleukin receptor Hormones of soluble forms of the body and interleukin receptors, growth factors such as TGFa's or TGFps and tissue growth factors such as epidermal growth factor General biological and therapeutic including matomedin, erythropoietin, pigment hormone, hypothalamic release factor, antidiuretic hormone, prolactin, chorionic gonadotropin, follicle stimulating hormone, thyroid stimulating hormone, tissue plasminogen activator, etc. An immunoglobulin of interest including other proteins and peptides of interest, IgG, IgE, IgM, IgA, IgD, and fragments thereof.   Conjugates of glycoproteins having an increased in vivo plasma half-life compared to non-conjugated glycoproteins, to which at least one terminal galactose or derivative thereof, optionally via a linking moiety A conjugate comprising a glycoprotein having a covalently bonded retarding group.   13. The glycoprotein conjugate according to claim 12, wherein the retarding group is a conjugate selected from the group consisting of: dendrimer, polyalkylene oxide (PAO), polyalkylene glycol (PAG), polyethylene glycol (PEG ), Polypropylene glycol (PPG), branched PEG, polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolidone, polyethylene-co-maleic anhydride, polystyrene-co-maleic anhydride, dextran, carboxymethyl dextran; Serum protein binding ligands such as compounds that bind albumin, such as fatty acids, C5-C24 fatty acids, aliphatic diacids (eg C5-C24), glycans to receptors (eg asialoglycoprotein receptors and mannose receptors) Structures that inhibit binding (eg sialic acid derivatives or pseudo ), Moieties that alter charge properties under physiological conditions such as carboxylic acids or amines, or neutral substituents that prevent glycan-specific recognition such as small alkyl substituents (eg, C1-C5 alkyl) Small organic molecules, small organic charged groups (eg C1-C25) that may contain one or more carboxylic acids, sulfo amines, phosphonic acids, or combinations thereof; low molecular neutral hydrophilic molecules such as cyclodextrins ( E.g. C1-C25), or optionally branched polyethylene chain; polyethylene glycol with an average molecular weight of 2-40 KDa; an accuracy in the range between 700-20.000 Da, or more preferably 700-10.000 Da Well-defined precise polymers such as dendrimers with variable molecular weights; and substantially non-exempts, such as albumin or antibody or optionally part of an antibody containing an Fc-domain Immunogenic polypeptide.   14. Glycoprotein conjugate according to any one of claims 12 or 13, wherein the glycoprotein is a conjugate selected from the following group: FVII, FVIII, FIX, FX, FII, FV, protein C, Protein S, tPA, PAI-1, tissue factor, FXI, FXII, FXIII, and their sequence variants; colonies containing immunoglobulins, interleukins, α-, β-, and γ-interferons, granulocyte colony-stimulating factor Cytokines such as stimulatory factors, platelet-derived growth factor, and phospholipase activating protein (PUP), plant proteins such as insulin, lectin and ricin, tumor necrosis factor and related alleles, soluble forms of tumor necrosis factor receptor, interleukins Growth factors such as soluble forms of receptors and interleukin receptors, TGFa's or TGFps and tissue growth factors such as epidermal growth factor General biological and hormones, including somatomedin, erythropoietin, pigment hormones, hypothalamic release factor, antidiuretic hormone, prolactin, chorionic gonadotropin, follicle stimulating hormone, thyroid stimulating hormone, tissue plasminogen activator, etc. Immunoglobulins of interest including other proteins and peptides of therapeutic interest, IgG, IgE, IgM, IgA, IgD, and fragments thereof.   A glycoprotein conjugate obtainable by the method according to any one of claims 1 to 11.   A pharmaceutical composition comprising the glycoprotein conjugate according to any one of claims 12 to 15.

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