MXPA06005082A - Method of preparing carboxylic acid functionalized polymers - Google Patents

Abstract

Methods for preparing water soluble, non-peptidic polymers carrying carboxyl functional groups, particularly carboxylic acid functionalized poly(ethylene glycol) (PEG) polymers, are disclosed, as are the products of these methods. In general, an ester reagent R(C=O)OR', where R'is a tertiary group and R comprises a functional group X, is reacted with a water soluble, non-peptidic polymer POLY-Y, where Y is a functional group which reacts with X to form a covalent bond, to form a tertiary ester of the polymer, which is then treated with a strong base in aqueous solution, to form a carboxylate salt of the polymer. Typically, this carboxylate salt is then treated with an inorganic acid in aqueous solution, to convert the carboxylate salt to a carboxylic acid, thereby forming a carboxylic acid functionalized polymer.

Description

METHOD FOR PREPARING POLYMERS WITH FUNCTIONAL CARBOXYLIC ACID GROUP FIELD OF THE INVENTION This invention relates to methods for preparing non-peptidic, water-soluble polymers carrying carboxyl functional groups, in particular poly (ethylene glycol) (PEG) polymers with acid functional group. carboxylic BACKGROUND OF THE INVENTION Poly (ethylene glycol) (PEG) derivatives activated with electrophilic groups are useful for coupling with nucleophilic groups, such as for example amino groups, of biologically active molecules. In particular, active esters and other PEG carboxylic acid derivatives have been used to bind PEG to proteins bearing amino groups. PEG molecules having terminal carboxymethyl groups have been described, for example, by Martinez et al. , U.S. Patent No. 5,681,567, Veronese et al. , Journal of Controlled Relay 10: 145-154 (1989), and Bück ann et al. , Makromol. Chem. 182 (5): 1379-1384 (1981). U.S. Patent No. 5,672,662 (Harris et al.) Discloses PEG derivatives having a terminal entity of pfopionic or butanoic acid. These carboxyl-terminated PEGs are used to prepare active active esters for conjugation with proteins or other molecules bearing amino groups. However, a persistent problem associated with the preparation of carboxyl functional group polymers has been the difficulty in obtaining the desired polymer product at a sufficiently high level of purity. For example, Veronese et al. and Bückmann et al. , cited above, employ a method for synthesizing PEG carboxylic acids which comprises converting PEGm-OH to a carboxylic acid ethyl ester PEGm, by a base-catalyzed reaction of PEGm-OH with an α-haloethyl ester, followed by base-stimulated hydrolysis. ester. However, this process provides PEGm acids of only 85% purity, with the main contaminant being PEGm-OH, which can not be separated from the carboxylic acid PEGm using typical purification methods such as, for example, precipitation, crystallization or extraction. . The elimination of PEGm-OH requires the use of preparative ion exchange column chromatography, which takes time and is expensive. Commercially obtained PEG carboxylic acids frequently contain residual amounts of PEG-OH, which complicates the preparation of derivatives or bioconjugates based on these materials.

U.S. Patent Nos. 5,278,303, 5,605,976 and 5,681,567, report the preparation of PEG carboxylic acids containing little or no starting material (PEG alcohol) by using a tertiary alkyl haloacetate to prepare a PEG with tertiary alkyl ester functional group , which is then hydrolyzed with acid, preferably trifluoroacetic acid (TFA, for its acronym in English). Various treaties on the use of protecting groups observe that tertiary alkyl esters, such as, for example, t-butyl esters, are stable to a soft base hydrolysis typically used to hydrolyze primary alkyl esters, such as, for example, ethylesters. Hydrolysis with strong base, could cause the cleavage of carboxylic acid groups. See, for example, T. W. Greene, Protective Groups in Organic Synthesis, 3rd edition, 1999, p. 406; or P. J. Kociens i, Protecting Groups, 1994, p. 125. Accordingly, these tertiary alkyl esters are conventionally cleaved with acid, typically with TFA. However, the use of trifluoroacetic acid can result in problems of purification and product stability. Trifluoroacetic acid is difficult to remove completely from the final carboxyl functional group polymer, in particular the amount of TFA is suggested in the patents referred to above. The presence of residual trifluoroacetic acid results in poor product stability, due to the degradation of the polymer caused by the auto-oxidation stimulated by the acid. See, for example, M. Donbrow, "Stability of the Polyoxyethylene Chain", in Nonionic Surfactants: Physical Chemistry, M. J. Schick, ed., Marcel Dekker, 1987, pp.1011 ff. This article reports that acids catalyze the formation of hydroperoxides and the rupture of hydroperoxide, leading to the cleavage of polyoxyethylene chains. Although U.S. Patent No. 5,605,976 suggests distillation as a means of separating organic materials from the polymer product, even compounds with very low boiling points are difficult to remove from high molecular weight polymers using a distillation process, and the difficulty increases as the molecular weight of the polymer increases. There is a need in the art for alternative methods to prepare carboxylic acid functional group polymers in high yield and free of significant amounts of polymeric contaminants, particularly significant amounts of the polymeric starting material. There is also a need in the art for alternative methods of synthesis that do not use reagents that are either difficult to remove from the final polymer product or cause product stability problems.

SUMMARY OF THE INVENTION In one aspect, the invention provides a method for preparing a water-soluble, non-peptidic polymer functionalized with a carboxyl group, the method comprising: (i) reacting an ester reagent R (C = 0) OR ', where R' is a tertiary group and R comprises a functional group X, with a polymer POLY-Y non-peptidic, soluble in water, where Y is a functional group that reacts with X to forming a covalent bond, to form a tertiary ester of the polymer; and (ii) treating the tertiary ester of the polymer with a strong base, such as, for example, an alkali metal hydroxide, in aqueous solution, to form a carboxylate salt of the polymer. The method may further comprise the step of (iii) treating the carboxylate salt of the polymer with an inorganic acid in aqueous solution, to convert the carboxylate salt to a carboxylic acid, thereby forming a polymer with a carboxylic acid functional group. The carboxylic acid functional group polymer can then be extracted from the aqueous solution with a suitable solvent, preferably a chlorinated solvent. In one embodiment, X is a leaving group, such as, for example, a halide or a sulfonate ester, and Y is a hydroxyl group. When Y is a hydroxyl group, the reaction (i) is preferably carried out in the presence of a base, for example, a base of the form R'0 ~ M +, where M is a cation. The treatment with a strong base in the reaction (ii) is preferably effective to produce a pH in the reaction of about 11 to 13. The inorganic acid, for example, a mineral acid, in step (iii) is preferably a acid that produces non-nucleophilic anions in aqueous solution. Preferred acids include sulfuric acid, nitric acid, phosphoric acid, and hydrochloric acid. The acid treatment of (iii) is preferably effective to produce a reaction pH of about 1 to 3. The tertiary ester reagent employed in reaction (i) preferably has structure (I): (I) In the structure, X is a leaving group; and each of R1 and R2 is independently selected from hydrogen, alkyl, cycloalkyl, alkoxy, aryl, aralkyl and heterocycle. Preferably, the group (CR1R2) n does not include two heteroatoms attached to the same carbon atom; for example, R1 and R2 in the same carbon atom are preferably not both alkoxy. Each of R3-R5 are independently selected from alkyl, aryl, aralkyl and lower cycloalkyl, wherein any of R3-R5 can be linked to form a ring or ring system, such as, for example, ada antyl. Any of R1 through R5, except hydrogen, can be substituted with a group selected from lower alkyl, lower alkoxy, C3-C6 cycloalkyl, halo, cyano, oxo (keto), nitro and phenyl. The variable n is 1 to approximately 24, preferably 1 to 6, more preferably 1 to 4, and most preferably 1 to 2. In one embodiment, n is 1. In selected modalities of the structure (1), each one of R1 and R2 is independently hydrogen or unsubstituted lower alkyl, preferably hydrogen or methyl, and each of R3 to R5 is independently unsubstituted alkyl or phenyl, preferably methyl, ethyl or phenyl. In one embodiment, each of R1 and R2 is H and n is 1. The leaving group X in structure (I) is preferably a halide or a sulfonate ester. In one embodiment, the reagent of this tertiary ester is a tertiary alkyl haloacetate, such as, for example, a t-butyl haloacetate. The water-soluble non-peptide polymer is preferably selected from the group consisting of poly (alkylene glyols), poly (olefinic) alcohol, poly (vinylpyrrolidone), poly (hydroxyalkyl ethacrylamide), poly (hydroxyalkyl methacrylate), poly (saccharides) ), poly (a-hydroxyacetic acid), poly (acrylic acid), poly (vinyl alcohol), polyphosphazene, polyoxazolines, poly (N-acryloylmorpholine), and copolymers of terpolymers thereof. In a preferred embodiment, the polymer is a poly (ethylene glycol). The poly (ethylene glycol) can be linear and terminated at one end with the functional group Y, and at the other end with another functional group Y 'or a capped group, such as for example, a methoxy group. Alternatively, the poly (ethylene glycol) can be branched, bifurcated or multi-branched. The method may further comprise converting the carboxylic acid of the carboxylic acid functional group polymer to an activated carboxylic acid derivative, for example, an activated ester, such as, for example, an N-succinimidylester, o-, m-, or p Nitrophenylester, 1-benzotriazolyl ester, 1-idazolyl ester, or N-sulfosuccinimidylester. The polymer can then be conjugated to a biologically active molecule, by reacting the carboxylic acid derivative with a functional group, preferably, a nucleophilic group such as, for example, a hydroxyl, thiol, or amino group, on the biologically active molecule . Preferably, the nucleophilic group is an amino group. In a preferred embodiment of the method, as noted above, the polymer is a PEG polymer. In this regard, the invention provides a method for preparing a poly (ethylene glycol) '(PEG) functionalized with a carboxyl group, the method comprising: (i) reacting a tertiary ester reagent R (C = 0) OR', in where R 'is a tertiary alkyl group and R comprises a functional group X, with a PEG-Y polymer, wherein Y is a functional group that reacts with X to form a covalent bond, to form a tertiary PEG ester; and (ii) treating the PEG tertiary ester with a strong base, such as, for example, an alkali metal hydroxide in aqueous solution, to form a PEG carboxylate salt. The method may further comprise (iii) treating the PEG carboxylate salt with an inorganic acid in aqueous solution, to convert the carboxylate salt to a carboxylic acid, thereby forming a PEG carboxylic acid.

The preferred embodiments of the method correspond to those described above. The method may further comprise converting the PEG carboxylic acid to an activated carboxylic acid derivative. Such as, for example, an activated ester, and conjugate the polymer to a biologically active molecule, by reacting the carboxylic acid derivative with a functional group on the molecule, as described above. In one embodiment the poly (ethylene glycol) is linear and terminates at one end with the functional group Y and at the other end with another functional group Y 'or' with a capped group, such as for example, a methoxy group. The molecular weight of the PEG is preferably in the range between about 100 Da to 100 kDa, more preferably in the variation between about 300 Da to 40, 50 or 60 kDa. In other embodiments, the PEG is branched, bifurcated, or has multiple branches, as will be described further below. In a related aspect, the invention provides an isolated polymer product comprising a carboxylic acid functional group polymer prepared by the method set forth herein, wherein the product contains less than 5% by weight of the starting material; that is to say the POLY-Y or PEG-Y polymer, with the remainder consisting essentially of the carboxylic acid functional group polymer. Preferably, the isolated polymer product contains less than 2%, more preferably less than 1%, and most preferably less than 0.5% by weight of the POLY-Y or PEG-Y polymer. In further preferred embodiments, the isolated polymer product contains less than 0.4%, more preferably less than 0.3%, and most preferably less than 0.2% by weight of POLY-Y or PEG-Y polymer. In a further preferred aspect, the isolated polymer product contains practically no amount of low molecular weight organic acid. In one embodiment, the isolated polymer product contains practically no amount of monomeric organic carboxylic acid, such as, for example, trifluoroacetic acid. In one embodiment of the polymer product of the invention, the carboxylic acid functional group polymer is a PEG carboxylic acid. For example, the carboxylic acid functional group polymer can be PEGm-CH2-COOH, and contains less than 5%, preferably less than 2%, more preferably less than 0.5%, and most preferably less than 0.2% in weight of PEGm-OH. Preferably, the product contains practically no amount of trifluoroacetic acid. In another embodiment of the product, the carboxylic acid functional group polymer is HOOC-CH2-PEG-CH2-COOH, and contains less than 5%, preferably less than 2%, more preferably less than 0.5%, and with the maximum preference less than 0.2% by weight of HO-PEG-OH. Preferably, the product contains practically no amount of trifluoroacetic acid. In a further embodiment of the product, the polymer with carboxylic acid functional group is a PEG with functional group of branched multifunctional carboxylic acid, or of multiple branches, represented by PEG- (CH2-COOH) x, where x is 3 to 8 , and contains less than 5%, preferably less than 2%, more preferably less than 0.5%, and most preferably less than 0.2% by weight of PEG- (OH) x. Preferably, the product contains practically no amount of trifluoroacetic acid. The invention further provides an improvement in a method for preparing a poly (ethylene glycol) polymer (PEG) functionalized with a carboxyl group, by the reaction of a tertiary ester reagent R (C = 0) OR ', where R' is a tertiary alkyl group and R comprises a functional group X, with a PEG-Y polymer , wherein Y is a functional group that reacts with X to form a covalent bond, to form a tertiary PEG ester. The improvement comprises treating the PEG tertiary ester with a strong base, preferably an alkali metal hydroxide, in aqueous solution, to form a PEG carboxylate salt. The strong base preferably is one which is a strong base effective to produce a reaction pH between about 11 to 13 in the aqueous solution. The improved method may further comprise treating the PEG carboxylate salt with an inorganic acid in aqueous solution, to convert the carboxylate salt to a carboxylic acid, thereby forming a PEG carboxylic acid. The inorganic acid is preferably a mineral acid selected from the group consisting of sulfuric acid, nitric acid, phosphoric acid, and hydrochloric acid. These and other objects and features of the invention will become more apparent when the following detailed description of the invention is read together with the included drawings.

DETAILED DESCRIPTION OF THE INVENTION The present invention will now be described more fully. This invention may, however, be characterized in many different ways and should not be construed as limiting the embodiments set forth herein; instead, these embodiments are provided in such a way that this disclosure will be thorough and complete, and will fully express the scope of the invention to those skilled in the art. The invention is not limited to the particular polymers, synthetic techniques, active agents, and the like set forth in this disclosure, as such may vary within the scope of the invention as incorporated by the appended claims. The terminology used herein is presented to describe only particular modalities, and is not intended to be limiting.

I. Definitions In order to describe and claim the present invention, the following terminology will be used according to the definitions described below. In the sense in which it is used in this specification, the singular forms "one", "one", and "the" include references to the plural unless the context clearly dictates otherwise. In the sense in which it is used herein, "non-peptide" refers to a polymer virtually free of peptide bonds. However, the polymer may include a smaller number of bonds with separate peptides along the length of the structure, such as, for example, no more than 1 peptide bond per about 50 monomer units. "PEG" or "polyethylene glycol", in the sense in which it is used herein, means that it encompasses any water-soluble poly (ethylene oxide). Typically, the PEGs for use in the present invention will comprise one of the following two structures: -0 (CH2CH20) m- or -CH2CH20 (CH2CH20) m-CH2CH2-, wherein m in general is from 3 to about 3000. In a broader meaning, "PEG" can refer to a polymer that contains a majority, that is, more than 50%, of subunits that are -CH2CH20-. The terminal groups and • the architecture of the total PEG may vary. The PEG may contain an end-capped group on a terminal oxygen which is generally a carbon-containing group typically consisting of 1-20 carbon atoms and is preferably selected from alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl , heterocycle, substituted forms of any of the foregoing. The group topped off at the end can also be a silane. Most preferred are groups capped with alkyl (alkoxy) or aralkyl (aralkoxy), such as, for example, methyl, ethyl or benzyl. The end-capped group may also advantageously comprise a detectable label. These labels include, without limitation, entities for imparting fluorescence, chemiluminescence, used in enzymatic labeling, colorimetric (e.g., dyes), metal ions, radioactive entities, and the like. The other term ("not capped at the end") is typically a hydroxyl, amine or activated group that can be subjected to further chemical modification. Specific PEG forms for use in the invention include PEGs having a variety of molecular weights, structures or geometries (eg, branched, linear, bifurcated, multiple branches). A "multifunctional" polymer has 3 or more functional groups, which may be the same or different. Multifunctional polymers will typically contain approximately 3-100 functional groups, or 3-50 functional groups, or 3-25 functional groups, or 3-15 functional groups, or 3 to 10 functional groups, or they will contain 3, 4, 5, 6, 7, 8, 9 or 10 functional groups. A "difunctional" polymer has two functional groups contained therein, which may be the same (ie, homodifunctional) or different (ie, heterodifunctional). "Molecular mass" or "molecular weight" refers to the average molecular mass of a polymer, typically determined by size exclusion chromatography, light scattering techniques or intrinsic rate determination in 1, 2, 4-trichlorobenzene. Unless otherwise noted, the molecular weight is expressed herein as the number average molecular weight (Mn), which is defined as? NiMi /? Ni, where Ni is the number of polymer molecules (or the number of moles of these molecules) having the molecular weight Mi. The polymers of the invention, or those employed in the invention are typically polydispersed; that is, the numerical average molecular weight and the weighted average molecular weight of the polymers is not the same. The polydispersity values, expressed as a ratio of weight average molecular weight (Mw) to numerical average molecular weight (Mn), (Mw / Mn), are generally low; that is, less than about 1.2, preferably less than about 1.15, more preferably less than about 1.10, even more preferably less than about 1.05, still more preferably less than about 1.03, and most preferably less than about 1.025. . An "activated carboxylic acid" refers to a functional derivative of a carboxylic acid that is more reactive than the original carboxylic acid, in particular with respect to nucleophilic attack. Activated carboxylic acids include, but are not limited to: acid halides (such as, for example, acid chlorides), anhydrides and esters. More generally, the term "activated" or "reactive" when used in conjunction with a particular functional group, refers to a functional group that reacts readily with an electrophile or a nucleophile or another molecule, in contrast to the groups that require strong catalysts or impractical reaction conditions to react (ie, "non-reactive" or "inert" groups). The term "protecting group" or "protecting group" refers to an entity that prevents or blocks the reaction of a particular chemically reactive functional group in a molecule under certain reaction conditions. The protective group will vary depending on the type of chemically reactive group that is being protected, as well as the reaction conditions that will be used and the presence of additional reactive or protective groups in the molecules, if any. Protective groups known in the art can be found in Greene, T. W., et al. , Pro'tective Groups in Organic Synthesis, 3rd ed. , John Wiley & Sons, New York, NY (1999). In the sense in which it is used in the present, the term "functional group" or any synonym thereof means that it encompasses the protected forms thereof. The term "separator" or "separating entity" in the sense in which it is used herein, refers to an atom or a collection of atoms used to link interconnected entities, such as, for example, a term of a soluble polymer moiety in water and an electrophile. A typical spacer includes selected alkylene (carbon-carbon), ether, amino, amide, ester, carbamate, urea and keto bonds, and combinations thereof. A spacer may include alternating short alkylene entities; with, or, flanked by, one or more types of bonds containing heteroatoms listed above. Various examples include: -CH2OCH2CH2CH2-, -CH2C (O) NHCH2-, -C (0) OCH2-, -OC (O) NHCH2CH2-, -CH2CH2NHCH2, -CH2CH2C (0) CH2CH2-, -CH2CH2CH2C (O) NHCH2CH2NH- , and -CH2CH2CH2C (O) NHCH2CH2NHC (O) CH2CH2-. The separating entities of the invention may be hydrolytically stable or may include. a physiologically hydrolysable or enzymatically degradable linkage (e.g., an ester linkage). "Alkyl" refers to a hydrocarbon chain, which typically ranges from 1 to 20 atoms in length. These hydrocarbon chains are preferably, although not necessarily saturated, and may be branched or, preferably, lineable (unbranched). Exemplary alkyl groups include ethyl, propyl, butyl, pentyl, 2-methylbutyl, 2-methylpropyl (isobutyl), 3-methylpentyl, and the like. In the sense in which it is used herein, "alkyl" includes cycloalkyl when referring to three or more carbon atoms. "Lower alkyl" refers to an alkyl group containing 1 to 6 carbon atoms, as exemplified by methyl, ethyl, n-butyl, i-butyl, t-butyl. "Cycloalkyl" refers to a saturated or unsaturated cyclic hydrocarbon chain, which includes fused or spirocyclic source compounds, preferably consisting of from 3 to about 12 carbon atoms, more preferably from 3 to about 8. In the sense in which it is used herein, "alkenyl" refers to a group branched or unbranched hydrocarbon having from 2 to 15 carbon atoms and containing at least one double bond, such as, for example, ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, and similar. The term "alkynyl" in the sense in which it is used herein, refers to an unbranched branched hydrocarbon group having 2 to 15 atoms and containing at least one triple bond, such as, for example, ethinyl, - propinyl, isopentinyl, n-butinyl, octinyl, decinyl, etc. "Alkoxy" refers to a group -OR, wherein R is alkyl or substituted alkyl, preferably C1-C20 alkyl (e.g., methoxy, ethoxy, propyloxy, etc.), more preferably lower alkyl (i.e. -C6). "Aryl" refers to a substituted or unsubstituted monovalent aromatic radical having a single ring (e.g., phenyl) or two fused or fused rings (e.g., naphthyl). The multiple aryl rings may also be un-fused (eg, biphenyl). The term includes heteroaryl groups, which are aromatic ring groups having one or more nitrogen, oxygen or sulfur atoms in the ring, such as for example, furyl, pyrrole, pyridyl, and indole. "Aralkyl" refers to an alkyl, preferably lower alkyl (C? ~ C, more preferably, C? -C2), a substituent that is further substituted with an aryl group; the examples are benzyl and phenethyl. "Aralkoxy" refers to a group of the -O form wherein R is aralkyl; An example is benzyloxy. A "heterocycle" refers to a ring, preferably a 5- to 7-membered ring, whose ring atoms are selected from the group consisting of carbon, nitrogen, oxygen and sulfur. Preferably, the atoms in the ring include from 3 to 6 carbon atoms. Examples of aromatic heterocycles (heteroaryls) were given above; non-aromatic heterocycles include, for example, pyrrolidine, piperidine, piperazine, and morpholine. A "substituted" group or entity is one in which a hydrogen atom has been replaced with a non-hydrogen atom or group, which is preferably a substituent that does not interfere. "Substituents that do not interfere" are those groups that, when present in a molecule, are typically not reactive with other functional groups contained within the molecule. These include, but are not limited to: alkyl, alkenyl, or lower alkyl; lower alkoxy; C3-C6 cycloalkyl; halo, for example, fluorine, chlorine, bromine, or iodine; cyano; oxo (keto); nitro; and phenyl. A "tertiary group" is a group of the form -CR3, where each R is an organic entity linked to C via a carbon atom. Each R can be, for example, alkyl, cycloalkyl, aryl, or aralkyl, substituted or unsubstituted. Examples of tertiary groups include t-butyl, wherein each R is methyl; triphenylmethyl (trityl), wherein each R is phenyl; and dimethoxytrityl (DMT), where two R are p-methoxyphenyl and one is phenyl.

Also included are groups wherein one or more R forms a ring or ring system, such as, for example, adamantyl.

A "tertiary ester" is an ester having a tertiary group in its alcoholic moiety, ie R '- (C = 0) -OCR3, where CR3 is a "tertiary group" as defined above, and R' is the moiety acid of the ester. A "carboxyl group", in the sense in which it is used herein, refers to the group -C (= 0) OH (carboxylic acid) or -C (= 0) 0-M '-, where M' is a positively charged ion, such as, for example, an alkali metal ion (carboxylate group). An "low molecular weight" organic acid refers to an acidic organic compound having a molecular weight of less than about 400, preferably, less than about 300, and more preferably less than about 200. The term typically refers to a non-polymeric or non-oligomeric acid, and generally refers to an acid used as a reagent. Examples include formic acid, acetic acid, trifluoroacetic acid (TFA), and p-toluenesulfonic acid. An "electrophile" is an atom or a collection of atoms that has an electrophilic center, that is, a center that is electron-seeking or capable of reacting with a nucleophile. A "nucleophile" refers to an atom or a collection of atoms that has a nucleophilic center, that is, a center that is a seeker of an electrophilic center or capable of reacting with an electrophile. A "physiologically cleavable" or "hydrolyzable" bond is a relatively weak bond that reacts with water (i.e., hydrolyzes) under physiological conditions. The tendency of a bond to hydrolyze in water will depend not only on the general type of bond that connects two central atoms but will also depend on the substituents attached to these central atoms. Suitable hydrolytically unstable or weak linkages include, but are not limited to, carboxylates, phosphatesters, anhydrides, acetals, ketals, acyloxyalkylether, acids, and orthoesters. An "enzymatic degradable link between" is a bond that is subject to degradation by one or more enzymes. A "hydrolytically stable" bond or linkage refers to a chemical bond, typically a covalent bond that is substantially stable in water, ie, does not undergo hydrolysis under physiological conditions to any appreciable extent over an extended period of time. In general, a hydrolytically stable linkage is one that exhibits a hydrolysis rate of less than about 1-2% per day under physiological conditions. Examples of hydrolytically stable bonds include carbon-carbon bonds, ethers, amines, and amides. Hydrolysis rates of representative chemical bonds can be found in most standard chemistry textbooks. A product that contains "virtually no amount" of a specific component either does not contain any specific component amount, or does not contain any amount that can be detected by conventional methods of analysis of the product, and / or has no detectable effect on the product. the properties or stability of the product. For example, a product that has never been known or deliberately exposed or is in contact with a particular substance could be considered as containing practically no amount of the substance. Each of the terms "drug", "biologically active molecule", "biologically active entity", and "biologically active agent", when used in the present, means any substance that can affect any physical or biochemical property of a biological organism, where the organism can be selected from viruses, bacteria, fungi, plants, animals, and humans. In particular, in the sense in which it is used herein, biologically active molecules include any substance intended for diagnosis, mitigation for cure, treatment, or prevention of a disease in humans or other animals, or to otherwise improve the physical or mental well-being of humans or animals. Examples of biologically active molecules include, but are not limited to: peptides, proteins, enzymes, small molecule drugs, dyes, lipids, nucleosides, oligonucleotides, polynucleotides, nucleic acids, cells, viruses, liposomes, microparticles and micelles. The classes of biologically active agents that are suitable for use with the invention include, but are not limited to: antibiotics, fungicides, antiviral agents, anti-inflammatory agents, anti-tumor agents, cardiovascular agents, anti-anxiety agents, hormones, growth factors, steroidal agents and the similar. Also included are food, food supplements, nutrients, nutritional products, drugs, vaccines, antibodies, vitamins, and other beneficial agents. The term "conjugate" refers to an entity formed as a result of the covalent attachment of a molecule, for example, a biologically active molecule, to a reactive polymer molecule, preferably a poly (ethylene glycol). "Pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to an excipient that can be included in the compositions of the invention and that does not cause any significant adverse toxicological effects to a patient. "Pharmacologically effective amount", "physiologically effective amount" and "therapeutically effective amount" in the sense in which they are used herein, refer to the amount of a conjugate of polymeric active agent present in a pharmaceutical preparation that is necessary to provide a desired level of the active agent and / or conjugate in the blood stream or in the intended tissue. The precise amount will depend on many factors, for example, the particular active agent, the components and physical characteristics of the pharmaceutical preparation, the patient population to which it is intended, the patient's considerations, and the like, and can be easily determined. by someone with experience in the technique, based on the information provided in the present and available in the relevant literature. The term "patient" refers to a living organism that suffers from or is prone to a condition that can be prevented or treated by administration of a biologically active agent or a conjugate thereof, and includes both humans and animals.

II. METHOD FOR PREPARING POLYPERS WITH CARBONYLIC ACID FUNCTIONALITY GROUP. SUMMARY The present invention provides, in one aspect, a method for preparing a water-soluble non-peptide polymer functionalized with a carboxyl group, ie a carboxylate or carboxylic acid salt. The method involves reacting a tertiary ester reagent R (C = 0) OR ', where R' is a tertiary group, as defined above, and R includes a functional group X, with a non-peptidic POLY-Y polymer, soluble in water, wherein Y is a functional group that reacts with X to form a covalent bond, to form a tertiary ester of the polymer, which can be represented as POLY-R- (C = 0) OR '. The nature of the bond between POLY and R depends on the functional groups Y and X. The starting material of the reaction, represented by POLY-Y, or by PEG-Y when the polymer is a polyethylene glycol, can include more than one functional group And, in various configurations. Examples include linear, branched, and multi-branched PEGs containing multiple hydroxyl groups, as will be discussed further below. The product of the reaction, ie the carboxyl functional group polymer, contains several carboxyl groups which are equal to the number of functional groups Y in the starting material (or greater than Y, if the starting material has existing carboxyl groups). Preferably, the functional group Y of the polymer is a hydroxyl group, or other nucleophilic groups, and the functional group X of the tertiary ester reagent is a leaving group capable of being displaced by Y. Other possible combinations of functional groups will be described below. Once the tertiary ester group is bound to the polymer, it is converted to a carboxylate by hydrolysis by base in aqueous solution, which is preferably followed by acidification to produce the carboxylic acid. Surprisingly, it has been found that the tertiary ester, while stable in the presence of the base used in the initial nucleophilic substitution reaction, can be removed by base-stimulated hydrolysis. As noted above, tertiary alkyl esters, such as, for example, t-butyl esters, are conventionally believed to be resistant to base hydrolysis. The following general reaction scheme represents a preferred embodiment of the method of the invention, wherein Y is hydroxyl and X is a leaving group, and the ester reagent has the structure shown as (I). m (i) Reaction Scheme I B. Reaction Components The preferred ester reagent (I), each of R1 and R2 is independently selected from H, alkyl, cycloalkyl, alkoxy, aryl, aralkyl, and lower heterocycle; and each of R3-R5 is independently selected from alkyl, aryl, and lower aralkyl, each as defined above. Preferably, the group (CR1R2) n does not include two heteroatoms attached to the same carbon atom for example, R1 and R2 at the same carbon atom are preferably not both alkoxy. None of R1 through R5, except hydrogen, can be substituted with a non-interfering substituent, as defined above. Preferably, each of R1 and R2 is independently hydrogen or unsubstituted lower alkyl, and each of R3 to R5 is independently alkyl or unsubstituted lower phenyl. In selected embodiments, each of R1 and R2 is independently hydrogen or methyl, more preferably hydrogen, and each of R3 to R5 is independently methyl, ethyl, or phenyl. The variable n is 1 to approximately 24, preferably 1 to approximately 12. In the selected modalities, n is 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8 , 1 to 9, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 up to 22, 1 to 23, or 1 to 24. In additional selected modes, n is 1 to 6; preferably, n is 1 to 4; and more preferably, n is 1 to 2. When n is greater than 1, the entity - (CRxR2) n- preferably includes at most two, and most preferably at most one, hydrogen-free mode of R1 or R2. In further embodiments, n is 1, and R1 and R2 are independently hydrogen or methyl. In one of these embodiments, when both of R1 and R2 are hydrogens, the product (IV) contains a carboxymethyl group. Preferably, the functional group X in the ester reagent (II) is a leaving group, such as, for example, halo, chloro or bromo, or sulfonate ester, such as, for example, p-toluenesulfonyl (tosyl), methanesulfonyl (mesyl) , trifluorosulfonyl, or trifluoroethylsulfonyl (tresyl).

However, other functional groups capable of reacting with a functional group on the polymer could also be used to form a covalent bond. Preferably, the functional group on the polymer is a nucleophilic group, such as for example, amine, hydrazide (-C (= 0) NHNH2), or thiol, and the functional group X on the ester reagent is an electrophilic group. . In addition to the leaving groups such as for example those described above, the electrophilic groups include carboxylic ester, including imidaester, orthoester, carbonate, isocyanate, isothiocyanate, aldehyde, ketone, thione, alkenyl, acrylate, methacrylate, acrylamide, sulfone, maleimide, disulfide, iodine, epoxy, thiosulfonate, silane, alkoxysilane, halosilane, and phosphoramidate. More specific examples of these groups include succinimidylester or carbonate, imidazolyster or carbonate, benzotriazolester or carbonate, p-nitrophenylcarbonate, vinylsulfone, chloroethylsulphone, vinylpyridine, pyridyl disulfide, iodo, acetamide, glyoxal and dione. Also included are other activated carboxylic acid derivatives, as well as hydrates or protected derivatives of any of the foregoing entities (eg, hydrated aldehyde, hemiacetal, acetal, hydrated ketone, hemiketal, ketal, thioketal, thioacetal). Preferred electrophilic groups include succinimidylcarbonate, succinimidylester, maleimide, benzotriazolecarbonates, glycidyl ethers, i-isololyester, p-nitrophenylcarbonate, acrylate, aldehyde and orthopyridyl disulfide. In general, the functional group X on the reagent is selected such that it reacts with the functional group Y on the polymer much more easily than the functional group Y could react with the t-butylester portion of the reagent. When the polymeric functional group Y is a nucleophile, such as, for example, hydroxyl, X is more suitably a good leaving group such as, for example, halo or sulfonate. Particularly preferred ester reagents include t-butyl haloacetates, such as, for example, t-butyl bromoacetate, t-butyl chloroacetate, and t-butyl iodoacetate. These t-butyl haloacetates are available, for example, from Sigma Chemical Co., St. Louis, Mo. In scheme I, POLY-OH is a non-peptidic polymer, soluble in water, such as, for example, PEGm-OH. In general, the polymer can be any non-peptidic polymer, soluble in water, having any available geometrical configuration (eg, linear, branched, bifurcated, etc.), as will be discussed further below. For simplicity, the reaction scheme provided above utilizes a polymer with a single hydroxyl group. However, as would be appreciated by one of ordinary skill in the art, the polymer may comprise more than one hydroxyl group, such as, for example, 1 to about 25 hydroxyl groups (eg, 1, 2, 3, 4, 5). , 6, 7, 8, 9, 10 or more hydroxyl groups). Also, the hydroxyl group could be replaced with any nucleophilic functional group reactive with the functional group X on the tertiary ester reagent. These nucleophilic functional groups include thiols, amines, and stabilized carbanions.

C. Reaction Process For the first stage of the process, shown in the top line of Scheme I of the previous example, the components are preferably dissolved in a suitable organic solvent, such as, for example, t-butanol, benzene, toluene, xylenes , tetrahydrofuran (THF), dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and the like. As shown in the embodiment of the invention represented by Scheme I, the reaction of a polymeric hydroxyl group with a tertiary ester reagent is typically carried out in the presence of a base. Example bases include potassium t-butoxide, butyl lithium, sodium amide, and sodium hydride. Other strong bases could also be used. The reaction is typically carried out at a temperature of about 0-120 ° C, preferably about 20-80 ° C, more preferably about 25-50 ° C, although the reaction conditions will vary based on the polymer and functional groups in reaction. As shown in the Examples provided below, the reactions of the PEG containing hydroxy with t-butyl bromoacetate were carried out effectively at temperatures between room temperature and about 45 ° C. The reaction time is typically from about 0.5 hours to about 24 hours; for example, approximately 1 to 20, 3 to 18, 4 to 12 or 6 to 8 hours. Typical reaction times for the reaction of a hydroxylated polymer with t-butyl bromoacetate, as shown in the following examples, range from 12 to 20 hours. The reaction can be monitored to be completed according to standard methods. Preferably, the reaction is carried out under an inert atmosphere such as, for example, nitrogen or argon. The preferred reaction employs a molar excess of the ester reagent (eg, a molar excess of 2 times, 3 times, 6 times, 10 times or 20 times, up to about 30 times) to ensure complete conversion of the material is achieved polymeric starting After the reaction step, the organic solvent is removed, typically by evaporation or distillation. The ester-containing product (III) is dissolved in water, preferably distilled or deionized water, during the second stage of the process, in which the ester-containing polymer is subjected to hydrolysis stimulated by base by treatment with a strong base , such as, for example, hydroxide, in aqueous solution. The hydrolysis with base is typically carried out at a pH between about 9 or higher, preferably between about 10 or higher, and most preferably between about 11 or higher (eg, between about 11 to 13). Therefore, the base is one that is strong enough to produce a pH in this variation in aqueous solution. In one embodiment, the pH is adjusted to be in the range between about 12 to 12.5. Preferably, the base is added as necessary throughout the reaction to maintain the pH in this variation. The base is also effective to hydrolyze any remaining ester reagent. The base must produce a fairly soluble salt in water when neutralized with acid in the step following hydrolysis. Preferred are alkali metal hydroxides, such as, for example, sodium hydroxide (NaOH) or potassium hydroxide (KOH). Also preferred is the use of distilled or deionized water, or water that does not have detectable levels of divalent cations such as, for example, calcium ions, magnesium. The hydrolysis step with base is typically conducted at a temperature of about 0-50 ° C, preferably about 10-30 ° C. The reaction time is typically between about 12 to 36 hours; for example between approximately 18 to 24 hours. The polymeric carboxylate salt produced by the base hydrolysis can be isolated and stored as the salt, or, preferably, directly converted to the carboxylic acid by acid treatment, as will be described below. In general, the carboxylic acid is more suitable for an additional derivation than the carboxylate salt. The carboxylate-containing polymer is treated with aqueous acid to convert the salt to the free acid form. The acid is preferably one that produces a non-nucleophilic anion in aqueous solution. Mineral acids (ie, inorganic acids) are preferred such as, for example, sulfuric acid, nitric acid, phosphoric acid, hydrochloric acid, and the like. Typically, sufficient acid is added to adjust the pH of the solution to about 1-3, more preferably about 2-3, which is effective to convert the polymeric carboxylate salt to a free acid form, as well as to neutralize (and convert to a water soluble salt freely) any base that remains in solution. The acidification step is typically conducted at a temperature between about 0o to 50 ° C, preferably about 10 ° to 30 ° C. The carboxylic acid-containing polymer is then separated using a conventional organic extraction step, preferably employing a halogenated solvent such as, for example, dichloromethane or chloroform. The polymer product is extracted in the organic phase, while any hydrolyzed reagent and excess mineral acid or its salt remains in the aqueous phase. In this way, the separation of the mineral acid from the polymer product is relatively simple. The organic extract is dried and concentrated, and the polymer product is then purified using standard methods, for example, the polymer can be isolated by precipitation, followed by filtration and drying. The choice of precipitating solvent will depend on the nature of the polymer; for the PEG polymers, as described in the following examples, the ethyl ether, a suitable precipitating solvent. Recrystallization from solvents such as, for example, ethyl acetate or ethanol can also be used for purification.

D. Reaction Products Using the method of the invention, carboxyl functional group polymers with high purity are produced, typically with a purity of at least about 95%, preferably at least about 96%, 97% or 98%, of higher preferably at least about 99%, and most preferably at least about 99.5% by weight. In selected embodiments, the polymer product contains at least about 99.6%, 99.7%, 99.8% or 99.9% by weight of the desired carboxyl functional group polymer. Accordingly, the product of the synthetic method set forth herein contains less than 5%, preferably less than 4%, 3%, or 2%, more preferably less than 1%, and most preferably less than 0.5% in weight of the starting polymer (e.g., PEGm-OH, PEG diol, or multifunctional PEG polyol) or other polymer impurities. In selected embodiments, the product contains less than 0.4%, 0.3%, 0.2% or 0.1% by weight of the polymeric starting material (e.g., PEGm-OH) or other polymer impurities. By "product" or "polymer product" is meant the material obtained by carrying out the synthetic process set forth above, including routine preparation procedures such as, for example, extraction, precipitation and solvent removal. As shown in the following examples, reaction mixtures containing the products of the methods set forth herein were prepared by extraction with a chlorinated solvent, followed by precipitation of the product from ethyl ether. The ion exchange chromatographic analysis of these products showed practically 100% of the desired PEG-carboxylic acid product, without any detectable amount of starting material or other polymeric impurity present. Accordingly, polymeric products having the purities discussed above are obtained without the need to remove the polymeric impurities, such as, for example, the starting material. These products are often used directly for an additional derivation and / or conjugation, as will be described later. An additional advantage of this process is that it provides high purity polymeric carboxylic acids, such as, for example, PEGm carboxylic acids, from inexpensive starting materials such as for example PEGm-OH, in contrast to the use of available polymeric carboxylic acids. They tend to be expensive and often also contain residual amounts of the polymeric hydroxyl compound. As described above, the reagents employed in the synthetic process set forth herein are easily removed from the polymer product. In particular, none of the low molecular weight organic acids, such as, for example, TFA, are used in the process. Accordingly, the carboxyl-containing polymeric products of this invention do not contain trace amounts of low molecular weight organic acids, such as, for example, TFA, as would generally be present in the polymeric carboxylic acids prepared using a hydrolysis process which Use this reagent. The products herein therefore do not suffer from the disadvantage of reduced stability associated with the presence of residual acids, as described above. For example, the polymer described in Example 4 below showed no sign of degradation (by GPC analysis) after 8 months of storage at -20 ° C.

III. Non-Peptide Polymers, Water-soluble, Suitable Any of a variety of water-soluble, non-peptide polymers can be used in the present invention. The polymer could be non-toxic and biocompatible, which means that it is capable of coexisting with living tissues or organisms without causing damage. Examples of suitable polymers include, but are not limited to: poly (alkylene glycols), copolymers of ethylene glycol and propylene glycol, poly (olefinic alcohol), 'poly (vinylpyrrolidone), poly (hydroxyalkyl methacrylamide), poly (hydroxyalkyl methacrylate), poly (saccharides), poly (a-hydroxyacetic acid), poly (acrylic acid), poly ( vinyl alcohol), polyphosphazene, polyoxazolines, poly (N-acryloylmorpholine), such as, for example, those described in U.S. Patent No. 5,629,384 which is incorporated herein by reference in its entirety, and copolymers, terpolymers, and mixtures thereof. The molecular weight of the polymer will vary, depending on the desired application, the configuration of the polymer structure, the degree of branching, and the like. In general, polymers having a molecular weight between about 100 Da to 100,000 Da are useful in the present invention, preferably between about 200 Da to 60,000 Da, and most preferably between about 300 Da to 40,000 Da. Exemplary polymer modalities have a molecular weight of approximately 200 Da, 350 Da, 550 Da, 750 Da, 1,000 Da, 2,000 Da, 3,000 Da, 4,000 Da, 5,000 Da, 7,500 Da, 10,000 Da, 15,000 Da, 20,000 Da , 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 50,000 Da, 55,000 Da, and 60,000 Da. The polymer preferably comprises at least one hydroxyl group, capable of reacting with a tertiary ester reagent carrying a leaving group, as described herein, in a nucleophilic substitution reaction. However, other functional groups capable of reacting with a functional group of the tertiary ester reagent could also be used. These include other nucleophilic groups, such as, for example, amine, hydrazide (-C (= 0) NHNH2), and thiol; and electrophilic groups, such as for example, carboxylic ester, including imidaester, orthoester, carbonate, isocyanate, isothiocyanate, aldehyde, ketone, thione, alkenyl, acrylate, methacrylate, acrylamide, sulfone, maleimide, disulfide, iodine, epoxy, sulfonate, thiosulfonate , silane, alkoxysilane, halosilane and phosphoramidate. More specific examples of these groups include succinimidylester or carbonate, imidazolyster or carbonate, benzotriazolester or carbonate, p-nitrophenylcarbonate, vinylsulfone, chloroethylsulfone, vinylpyridine, pyridyl disulfide, iodoacetamide, glyoxal, dione, mesylate, tosylate and tresylate. Also included are other activated carboxylic acid derivatives, as well as hydrates or protected derivatives of any of the foregoing entities (eg, hydrated aldehyde, hemiacetal, acetal, hydrated ketone, hemiketal, ketal, thioketal, thioacetal). Preferred electrophilic groups include, succinimidylcarbonate, succinimidylester, maleimide, benzotriazolecarbonate, glycidyl ether, imidazoyl ether, p-nitrophenylcarbonate, acrylate, tresylate, aldehyde, and orthopyridyl disulfide. The functional groups are selected such that a nucleophilic group on the polymer reacts with an electrophilic group on the tertiary ester reagent, or vice versa. The reaction between the two functional groups is preferably a displacement reaction of a leaving group by a nucleophile, although it could also be, for example, a condensation or addition reaction. The polymer preferably comprises at least one nucleophilic group, such as, for example, a hydroxyl group. For ease of reference, the hydroxyl groups will be analyzed later, although other functional groups could be used. A polymer can also include different functional groups within the same molecule. Preferably, these have similar functional groups, for example, both nucleophilic, such as, for example, the hydroxyl group or an amino group. Preferably, the polymer is a poly (ethylene glycol) polymer (ie, PEG). As noted above, the term PEG includes poly (ethylene glycol) in any of a number of geometries or shapes, including linear, branched or multi-branched forms (e.g., bifurcated PEG or PEG attached to a polyol core), pendant PEG , or PEG with degradable links in it. The number and position of the hydroxyl groups (and / or other functional groups) carried by the polymer may vary. Typically, the polymer comprises 1 to about 25 hydroxyl groups, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 hydroxyl groups. Linear polymers, such as, for example, linear PEG polymers, typically comprise one or two hydroxyl groups, each placed at one end of the polymer chain. If the PEG polymer is monofunctional (ie, PEGm), the polymer includes a single hydroxyl group. If the PEG polymer is difunctional, the polymer contains two hydroxyl groups, one at each terminus of the polymer chain, or contains a single hydroxyl group and a different functional group at the opposite term. Polymers with multiple or branched branches may comprise a greater number of hydroxyl groups. Multi-branched or branched PEG molecules are described, for example, in U.S. Patent No. 5,932,462 which is incorporated herein by reference in its entirety. Generally speaking, a multi-branched or branched polymer possesses two or more polymeric "branches" that extend from a central branching point, which preferably comprises a hydrolytically stable bond structure. An example branched PEG polymer is lysine disubstituted with methoxy poly (ethylene glycol). In another multi-branched embodiment, the polymer comprises a central core molecule derived from a polyol or polyamine, the core molecule derived from a polyol or polyamine, the central core molecule provides a plurality of suitable binding sites to covalently link the polymeric branches to the core molecule in order to form a multi-branched polymer structure. Depending on the desired number of polymeric branches, the polyol or polyamine typically will comprise 3 to about 25 hydroxyl or amino groups, preferably 3 to about 10, most preferably 3 to about 8 (eg, 3, 4, 5, 6, 7 or 8). Multi-branched polymers are further described, for example, in the United States patents Nos. 2002/0156047 and 2002/0156047, which are incorporated herein by reference. The PEG polymer may alternatively comprise a bifurcated PEG. Generally speaking, a polymer having a bifurcated structure is characterized by having a polymer chain attached to two or more functional groups via covalent bonds that extend from a stable hydrolytically stable branch point in the polymer. U.S. Patent No. 6,362,254, the content of which is incorporated by reference herein, discloses various bifurcated PEG structures capable of being used in the present invention. The PEG polymer can also be a pendant PEG molecule, which has reactive groups (eg, hydroxyl groups) covalently linked along the length of the PEG structure instead of the PEG chain end. The pendant reactive groups can be attached to the PEG structure directly or through a linking entity, such as, for example, an alkylene group. Different polymers can be incorporated in the same polymer structure. For example, one or more of the PEG molecules in the branched structures described above can be replaced with a different type of polymer. The polymer can also be prepared with one or more hydrolytically stable or degradable bonds in the polymer structure. For example, PEG can be prepared with ester linkages in the polymer structure that are subject to hydrolysis. Other hydrolytically degradable linkages that may be incorporated include carbonate, imine, phosphate ester, hydrazone, acetal, orthoester, and phosphorus-idate linkages. The term poly (ethylene glycol) or PEG includes any or all of the variations described above. Generally preferred PEG structures include linear monofunctional, branched monofunctional, and difunctional or trifunctional linear branched or bifurcated PEGs. Because end-capped polyethylene glycol starting materials, such as, for example, PEGm (methoxy-PEG) or PEGb (benzyloxy-PEG), may contain detectable amounts of PEG diol impurity, which leads to side products that Frequently they are difficult to analyze or separate, the PEG starting material is, in a preferred embodiment, a diol-free benzyloxy-PEG as described in co-pending United States Patent No. 6,448,369.

IV. Derivation and additional conjugation of the polymers with carboxylic acid functional group. A. Summary If desired, a carboxylic acid functional group polymer prepared by the method of the invention can be further modified to form useful reactive derivatives of carboxylic acids using methodology known in the art. The preparation of these derivatives is facilitated by the high purity of the carboxylic acid functional group polymers of the invention, as compared to the prior art products containing, for example, polymer with residual starting material and / or reagents residuals such as for example TFA. This is a significant benefit, particularly for a pharmaceutical product, because the presence and quantities of these contaminants can be quite variable, thus leading to the product not being able to be reproduced. Accordingly, the method of the invention, wherein a polymer with carboxylic acid functional group is prepared, can further comprise the steps of (i) modifying the carboxylic acid to form a reactive derivative and (ii) conjugating the reactive derivative to a pharmacologically relevant molecule having a corresponding reactive functional group. Steps (i) and (ii) can be performed in situ wherein the carboxylic acid is converted to an activated derivative using one or many activating reagents known in the art, then immediately reacting with the molecule to be conjugated. The carboxylic acid can be derivatized to form, for example, acyl halides, acyl pseudohalides, such as for example, acyl cyanide, acyl isocyanate, and acyl azide, neutral salts, such as, for example, alkali metal salts or alkaline earth metal (eg, calcium, sodium, or barium salts), esters, anhydrides, amides, imides, hydrazides and the like. In a preferred embodiment, the acid is esterified to form an active ester, such as, for example, an n-succinimidylester, o-, m-, or p-nitrophenyl ester, 1-benzotriazolyl ester, imidazolyl ester, or N-sulfosuccinimidylester. In one embodiment, the additional polymer derivative is a PEG polymer having the structure: PEGm-0- (CR ^ 2) n-C (= 0) -Z (V) where Ri, R2 and n are as described above. The Z-entity is preferably selected from the group consisting of halo, amino, substituted amino, -NCO, -NCS, N3, -CN, and -OR ', wherein R' is selected from N-succinimidyl, nitrophenyl, benzotriazolyl , imidazolyl, N-sulfosuccinimidyl, N-phthalimidyl, N-glutarimidyl, N-tetrahydrophthalimidyl, N-norbornene-2,3-dicarboximidyl, and hydroxy-7-azabenzotriazolyl. The carboxyl-containing polymer produced by the method of the invention, or a reactive derivative thereof, can be used to form conjugates with biologically active molecules, in particular biologically active molecules carrying nucleophilic functional groups, such as, for example, amino groups , hydroxyl, or mercapto (thiol). Often, the molecule that will be conjugated is a protein. The proteins are conjugated via reactive amino acids, such as for example, lysine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, cysteine, the N-terminal amino group, and the C-terminal carboxylic acid. Carbohydrate entities on glycosylated proteins can also be used as conjugation sites. For the reaction with an activated carboxylic acid, the most suitable groups are the N-terminal amino group, the side chains containing amine on lysine, histidine or arginine, the side chains containing hydroxyl on serine, threonine and tyrosine, and the chains lateral thiol on cysteine. Although the preferred conjugation methods of the carboxyl-containing polymers of the invention employ derivatives of activated carboxylic acid, which reacts with nucleophilic groups on the molecule to be conjugated, it is also possible to derive the terminal carboxyl group to contain any variety of functional groups. For example, in one embodiment, entity Z in structure (V) above has the structure -NHR6, wherein R6 is an organic group that contains a reactive functional group (eg, aldehyde, maleimide, mercapto, hydroxyl, amino, etc.), the functional groups that will be separated from the nitrogen atom by an alkylene chain (for example, Cl-6) and, optionally, an additional binder, such as, for example, a short PEG chain and another alkylene chain ( for example, alkylene-PEG-alkylene).

B. Exemplary conjugation methods These polymer conjugates can be formed using known techniques for the covalent attachment of an activated polymer, such as, for example, an activated PEG, to a biologically active agent. See, for example, Poly (ethy lene glycol): Chemisty and Biological Applications, J.M. Harris and S. Zalipsky, editors, American Chemical Society, Washington, DC (1997) or Bioconjugate Techniques, G.T. Hermanson, Academic Press (1996). In general, conjugation reactions are typically carried out in a buffer, such as, for example, a phosphate or acetate buffer, at or near room temperature, although the conditions will depend on the particular reaction to be carried out. An excess of the polymeric reagent is typically combined with the active agent. In some cases, however, it is preferred to have stoichiometric amounts of the reactive groups on the polymeric reagent and on the active agent. The progress of a conjugation reaction can be monitored by mass spectrometry SDS-PAGE, MALDI-TOF, or any other suitable analytical method. Once a scenario is reached with respect to the amount of the conjugate formed or the amount of the remaining unconjugated polymer, it is assumed that the reaction will be complete. The product mixture is purified, if necessary, to remove excess reagents, unconjugated reagents (e.g., active agent), unwanted multi-jumbled species, and / or unreacted polymer, using known methods. For example, conjugates having different molecular weights can be separated using gel filtration chromatography. Fractions can be analyzed by several different methods, for example (i) OD at 280 nm for protein content, (ii) protein BSA analysis, (iii) iodine test for PEG content (Sims et al., Anal Biochem. 107: 60-63, 1980), or (iv) SDS-PAGE, followed by staining with barium iodide. The separation of positional isomers (i.e., conjugates of the same or substantially the same molecular weight having a polymer attached to different positions on a molecule) can be carried out by reverse phase HPLC or ion exchange chromatography. The conjugate can be lyophilized for storage, with or without residual buffer. In some cases, it is preferable to exchange a buffer used for conjugation, such as, for example, sodium acetate, with a volatile buffer, such as, for example, ammonium carbonate or ammonium acetate, which can be easily removed during lyophilization. Alternatively, a buffer exchange step can be used using a formulation buffer, such that the lyophilized conjugate is in a form suitable for reconstitution in a formulation buffer and ultimately for administration to a mammal.

C. Exemplary conjugation agents A biologically active agent for use in coupling to the polymer formed by the method of the invention can be any one or more of the following. Suitable agents can be selected from, for example, hypnotic and sedatives, psychic energizers, tranquilizers, drugs, respiratory anticonvulsants, muscle relaxants, antiparkinson agents (dopamine antagonists), analgesics, anti-inflammatories, antianxiety drugs (anxiolytics), suppressant appetite, anti-migraine agents, muscle contractors, 'anti-infectious' agents (antibiotics, antivirals, antifungals, vaccines) antiarthritics, antimalarials, antimicrobials, antiepileptics, bronchodilators, cytokines, growth factors, anti-cancer, antithrombotic agents, anti-hypotensive drugs , cardiovascular, - antiarrhythmic agents, antioxidants, antiasthma, hormonal agents including contraceptives, sympathomimetics, diuretics, lipid regulating agents, antiandrogenic, antiparasitic, anticoagulant, neoplastic, antineoplastic, hypoglycemic, nutritional and supplement agents, growth supplements, antienteritis agents, vaccines, antibodies, diagnostic agents, and agents for contrast. More particularly, the active agent can be in various structural classes, including but not limited to: small molecules, peptides, polypeptides, proteins, antibodies, polysaccharides, spheroids, nucleotides, oligonucleotides, polynucleotides, fats, electrolytes, and the like. Preferably, an active agent for coupling to a carboxyl-containing polymer of the invention possesses a natural amino, hydroxyl, or thiol group, or is modified to contain at least one of these groups. Specific examples of active agents include, inter alia: aspariginase, doxovir (DAPD), antide, becaplermin, calcitonins, cyanovirin, denileukin diftitox, erythropoietin (EPO), EPO agonists (eg, peptides of about 10-40 amino acids of length and comprising a particular core sequence as described in WO 96/40749), dornase alfa, erythropoiesis-stimulating protein (NESP), coagulation factors such as, for example, Factor V, Factor VII, Factor Vlla, Factor VIII, Factor IX, Factor 'X, Factor XII, Factor XIII, von Willebrand factor; ceredasa, cerezima, alpha-glucosidase, collagen, cyclosporine, alpha-defensins, beta-defensins, exedin-4, granulocyte colony-stimulating factor (GCSF), thrombopoietin (TPO), alpha-1 inhibitor proteinase, elcatonin, granulocyte-macrophage colony-stimulating factor (GMCSF), fibrinogen, filgrastim, growth hormone, human growth hormone (hGH), growth hormone-releasing hormone (GHRH, by its acronym in English), GRO-beta, antibody GRO-beta, oseomorphogenic proteins such as for example, protein-2 oseomorphogenic, protein-6 oseomorphogenic, OP-1, growth factor of acid fibroblasts, growth factor of basic fibroblasts, CD-40 ligand, heparin, human serum albumin, low molecular weight heparin (LMWH), interferons such as, for example, interferon alpha, beta interferon, interferon gamma, interferon omega rón, interferon tau, consensual interferon; interleukins and interleukin receptors such as, for example, interleukin-1 receptor, interleukin-2, interleukin-2 fusion proteins, interleukin-1 receptor antagonist, interleukin-3, interleukin-4, interleukin-4 receptor, interleukin-6, interleukin-8, interleukin-12, interleukin-13 receptor, interleukin-17 receptor, lactoferrin and fragments • of lactoferrin, hormone-releasing hormone (LHRH), insulin, pro insulin, insulin analog (e.g., mono-acylated insulin as described in U.S. Patent No. 5,922,675), ilin, C-peptide, somatostatin, somatostatin analogues including octreotide, vasopressin, hormone-stimulating hormone follicles (FSH), influenza vaccine, insulin-like growth factor (IGF), insulintropin, macrophage colony stimulating factor (M-CSF, for short) in English), plasminogen activators, such as, for example, alteplase, urokinase, reteplase, streptokinase, pamiteplase, lanoteplase and teneteplase, nerve growth factor (NGF), osteoprotegerin, platelet-derived growth factor , tissue growth factors, transforming growth factor-1, vascular endothelial growth factor, leukemia inhibitory factor, keratinocyte growth factor (KGF), viral growth factor (GGF), acronyms in English), T lymphocyte receptors, molecules / CD antigens, tumor necrosis factor (TNF), monocyte chemoattractant protein-1, endothelial growth factors, parathyroid hormone (PTH), English), glucagon-like peptide, somatotropin, thymosin alfa • 1, inhibitor Ilb / IIIa of thymosin alfa-1 ,. thymosin beta-10, thymosin beta-9, thymosin beta-4, alpha-1 antitrypsin, phosphodiesterase compounds (PDE), VLA-4 (very late antigen-4), inhibitors of VLA-4, bisphosphonates, antibody, virus respiratory syncytial, cystic fibrosis transmembrane regulator gene (CFTC), deoxyribonuclease (Dnasa), bactericidal / permeability enhancement protein (BPI), and anti-CMV antibody. Exemplary monoclonal antibodies include etanercept (a dimeric fusion protein consisting of an extracellular ligand binding portion of human 75 kD TNF receptor linked to the Fe portion of IgGl), abciximab, afeliomomab, basiliximab, daclizumab, infliximab, ibritumomab tiuexetan, mitumomab, muromonab-CD3, iodine 131 conjugated tositumomab, olizumab, rituximab, and trast.uzumab (herceptin). Additional agents suitable for covalent attachment to a polymer include amifostine, amiodarone, aminocaproic acid, aminohipuratosodium, aminoglutethimide, aminolevulinic acid, aminosalicylic acid, amsacrine, anagrelide, anastrozole, asparaginase, anthracyclines, bexarotene, bicalutamide, bleomycin, buserelin, busulfan, cabergoline , capecitabine, carboplatin, carmustine, chlorambucin, cilastatinasodium, cisplatin, cladribine, clodronate, cyclophosphamide, cyproterone, cytarabine, camptothecins, 13-cis retinoic acid, all trans-retinoic acids; dacarbazine, dactinomycin, daunorubicin, deferoxamine, dexamethasone, diclofenac, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estramustine, etoposide, exemestane, fexofenadine, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, epinephrine, L-Dopa, hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan, itraconazole, goserelin, letrozole, leucovorin, levamisole, lisinopril, lovothyroxinasodium, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, metaraminol bitartrate, methotrexate, metoclopramide, mexiletine, mitomycin, mitotane, mitoxantrone, naloxone, nicotine, nilutamide, octreotide, oxaliplatin, pamidronate, pentostatin, pilcamycin, porfimer, prednisone, procarbazine, prochlorperazine, ondansetron, raltitrexed, sirolimus, streptozocin, tacrolimus, tamoxifen, temozolomide, teniposide, testosterone, tetrahydrocannabinol, thalidomide, thioguanine, thiotepa, topotecan, tretinoin, valrubicin , vinblastine, vincristine, vindesine, vinorelbine, dolasetron, granisetron; formoterol, fluticasone, leuprolide, midazolam, alprazolam, amphotericin B, podophyllotoxins, nucleoside antivirals, aroyl hydrazones, sumatriptan; macrolides such as, for example, erythromycin, oleandomycin, troleandomycin, roxithromycin, clarithromycin, davercin, azithromycin, fluritromycin, dirithromycin, josamycin, spiromycin, midecamycin, leucomycin, mycocaine, rochytamycin, andazithromycin, and swinolide A; fluoroquinolones such as, for example, ciprofloxacin, ofloxacin, levofloxacin, trovafloxacin, alatrofloxacin, moxifloxacin, norfloxacin, enoxacin, grepafloxacin, gatifloxacin, lomefloxacin, sparfloxacin, temafloxacin, pefloxacin, amifloxacin, fleroxacin, tosufloxacin, prulifloxacin, irloxacin, pazufloxacin, clinafloxacin, and sitafloxacin; aminoglycosides such as, for example, gentamicin, netilmicin, paramecin, tobramycin, amikacin, kanamycin, neomycin, and streptomycin, vancomycin, teicoplanin, rampolanin, mideplanin, colistin, daptomycin, gramicidin, colistimethate; polymyxins such as, for example, polymyxin B, capromycin, bacitracin, penems; penicillins including penicillinase-sensitive agents similar to penicillin G, penicillin V; penicillinase-resistant agents similar to miticillin, oxacillin, cloxacillin, dicloxacillin, floxacillin, nafcillin; active agents of gram-negative microorganisms similar to ampicillin, amoxicillin, and hetacillin, cillin, and galampicillin; antipseudomonal penicillins similar to carbenicillin, ticarcillin, azlocillin, mezlocillin, and piperacillin; Cephalosporin similar to cefpodoxime, cefprozil, ceftbuten, ceftizoxime, ceftriaxone, cephalothin, cephapirin, cephalexin, cephrhydrin, cefoxitin,. cefamandole, cefazolin, cephaloridine, cefaclor, cefadroxil, cephaglycine, cefuroxime, ceforanide, cefotaxime, cefatrizine, cefacetril, cefepime, cefixime, cephonic acid, cefoperazone, cefotetan, cefmetazole, ceftazidime, loracarbef, and moxalactam, monolactams similar to aztreonam; and carbapenems such as, for example, imipenem, meropenem, pentamidine isethiouate, albuterol sulfate, lidocaine, metaproterenol sulfate, beclomethasone diprepionate, triamcinolone acetamide, budesonide ketonide, fluticasone, ipratropium bromide, flunisolide, cromolynodium, and ergotamine tartrate; taxanes such as, for example, paclitaxel; SN-38, and tyrphostins. The above biologically active agents mean that they encompass, where applicable, analogs, agonists, antagonists, inhibitors, isomers, and pharmaceutically acceptable salt forms thereof. With reference to peptides and proteins, the invention is intended to encompass recombinant, natural, glycosylated and non-glycosylated synthetic forms, as well as biologically active fragments thereof. The above biologically active proteins further means that they encompass variables having one or more substituted amino acids (eg, cysteine), deleted or the like, so long as the resulting variable protein possesses at least some degree of activity of the parent protein (natural ).

V. Pharmaceutical compositions and methods of administration The present invention also includes pharmaceutical preparations comprising a conjugate as provided herein in combination with a pharmaceutical excipient. Exemplary excipients include, without limitation, those selected from the group consisting of carbohydrates, antimicrobial agents, antioxidants, surfactants, buffers and combinations thereof. A carbohydrate such as for example a sugar, a sugar derivative such as for example, alditol, aldonic acid, an esterified sugar and / or a sugar polymer may be present as an excipient. Specific carbohydrate excipients include, for example: monosaccharides, such as, for example, fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as, for example, lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as, for example, raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as, for example, mannitol, xylitol, maltitol, lactitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like. The excipient may also include an inorganic salt or buffer such as, for example, citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, monobasic sodium phosphate, dibasic sodium phosphate and combinations thereof. The preparation may also include an antimicrobial agent to prevent or deter microbial growth. Non-limiting examples of antimicrobial agents suitable for the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, timersol, and combinations thereof. An antioxidant may also be present in the preparation. Antioxidants are used to prevent oxidation, thus preventing deterioration of the conjugate or other components of the preparation. Suitable antioxidants for use in the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hydrophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium sulfoxylate formaldehyde, sodium metabisulfite, and combinations thereof. themselves. ' A surfactant may be present as an excipient. Exemplary surfactants include: polysorbates, such as, for example, "Tween 20" and "Tween 80", and pluronics such as, for example, F68 and F88 (both available from BASF, Mount Olive, New Jersey); esters of sorbitan; lipids, such as for example, phospholipids such as for example, lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably not in liposomal form), fatty acids and fatty esters; spheroids, such as, for example, cholesterol; and chelating agents, such as, for example, EDTA, zinc and others of these suitable cations. The acids or bases may be present as an excipient in the preparation. Non-limiting examples of acids that can be used include those acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, petroric acid , phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof. Examples of suitable bases include without limitation: bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, citrate of sodium, sodium formate, sodium sulfate, potassium sulfate, potassium fumarate, and combinations thereof. Pharmaceutical preparations encompass all types of formulations and in particular those that are suitable for injection, for example, powders that can be reconstituted as well as suspensions and solution. The amount of the conjugate (ie, the conjugate formed between the active agent and the polymer described herein) in the composition will vary, depending on several factors, although optimally it will be a therapeutically effective dose when the composition is stored in a dosing container. unit (for example, a vial). In addition, the pharmaceutical preparation can be housed in a syringe. A therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of the conjugate in order to determine which amount produces a clinically desired endpoint. The amount of any individual excipient in the composition will vary, depending on the activity of the excipient and the particular needs of the composition. Typically, the optimum amount of any individual excipient is determined through routine experimentation, that is, by preparing compositions contai varying amounts of the excipient (ranging from low to high), exami stability and other parameters, and then determine the variation in which optimal performance is achieved without significant adverse effects. In general, however, the excipient will be present in the composition in an amount between about 1% and 99% by weight, preferably 5% -98% by weight, more preferably about 15-95% by weight, with concentrations more preferred less than 30% by weight. The above pharmaceutical excipients and others are described in Remington: The Science &; Practice of Pharmacy, 19th ed., Williams & Williams, (1995), the Physics Desk Reference, 52nd ed. , Medical Economics, Montvale, NJ (1998), and Kibbe, A.H., Handbook of Pharmaceutical Excipients, 3rd Edition, American Pharmaceutical Association, Washington, D.C., 2000. The pharmaceutical preparations of the present invention typically, but not necessarily, are administered via injection and therefore are generally liquid solutions or suspensions immediately prior to administration. The pharmaceutical preparation can also take other forms such as, for example, syrups, creams, ointments, tablets, powders, and the like. Other forms of administration are also included, such as, for example, pulmonary, rectal, transdermal, transmucosal, oral, intrathecal, subcutaneous, intra-arterial, etc. Formulation types suitable for parenteral administration include: solutions ready for injection, dehydrated powders for combination with a solvent before use, suspensions ready for injection, dry insoluble compositions for combination with a vehicle before use, and emulsions and liquid concentrates for dilution before the administration, among others. The invention also provides a method for administering a conjugate as provided herein to a patient suffering from a condition responding to treatment with a conjugate, as determined by those skilled in the art. The method comprises administering, generally via injection, a therapeutically effective amount of the conjugate, preferably provided as part of a pharmaceutical preparation. The actual dose that will be administered will vary depending on the age, weight, and general condition of the subject, as well as the severity of the condition that will be treated, the judgment of the health care professional, and the conjugate that will be administered. The therapeutically effective amounts of particular drugs are known to those skilled in the art and / or are described in the relevant reference texts and literature. In general, a therapeutically effective amount of the conjugate will vary between about 0.001 mg to 100 mg, preferably at doses of 0.01 mg / day to 75 mg / day, and most preferably at doses of 0.10 mg / day to 50 mg / day. The unit dose of any given conjugate (again, preferably provided as part of a pharmaceutical preparation) can be administered in a variety of dosing schedules depending on the judgment of the physician, the needs of the patient, etc.

EXAMPLES The following examples are provided to illustrate the invention although they should not be considered as limiting the invention. For example, although it is used PEG In the examples, the use of other non-peptidic, water-soluble polymers is encompassed by the invention, as described above. All PEG reagents referenced in these examples are available from Nektar AL, Huntsville, AL. All NMR data were generated by a 300 or 400 MHz NMR spectrometer manufactured by Bruker. Example 1 illustrates the reaction of PEGm-OH with tert-butyl bromoacetate in the presence of a base to form a finished ter-butylester polymer. After , the polymer is subjected to base-stimulated hydrolysis using NaOH as the base, followed by acidification using phosphoric acid, to form the finished carboxylic acid polymer. Examples 2 and 3 exemplify a similar reaction of a difunctional PEG starting material (PEG diol, HO-PEG-OH). Example 4 illustrates the reaction of a multifunctional 4-branched PEG starting material based on a pentaerythritol core and having four reactive hydroxyls, one at the end of each PEG "branch".

Example 1: PEGm (30,000) -carboxylic acid A solution of PEGm-30,000 (50 g, 0.00167 moles) (NOF Corporation) in toluene (600 ml) was azeotropically dried by distilling off 300 ml of toluene. T-butanol (70 ml), potassium tert-butoxide (95%, 1.75 g, 0.0148 moles, 8.9 times excess) and tert-butyl bromoacetate (3.3 g) were added., 0.0169 moles, excess 10.1 times), and the mixture was stirred overnight at 45 ° C under the argon atmosphere. The solvent was removed by distillation under reduced pressure, and the residue was dissolved in distilled water (1000 'ml). The pH of the aqueous solution was adjusted to 12 with 1M sodium hydroxide, and the solution was stirred for 18 h, maintaining the pH at 12 by the periodic addition of 1M sodium hydroxide. The pH was adjusted to 3 with 5% phosphoric acid, and the product was extracted with dichloromethane. The extract was dried with anhydrous magnesium sulfate and added to ethyl ether. The precipitated product was removed by filtration and dried under reduced pressure, yielding 46.6 g. NMR (d6-DMSO): 3.24 ppm (s, -OCH3), 3.51 ppm (s, PEG structure), 4.01 ppm (s, -CH2-COO-). The chromatographic analysis of anion exchange: PEGm (30, 000) - 100% carboxylic acid. This analysis showed that there was practically no starting material or other polymer impurity present in the product precipitated with ether.

Example 2: PEG (10,000) -dicarboxylic acid PEG-10,000 (35.25 g, 0.00705 eq) (NOF Corporation) (terminated at both ends with hydroxyl) was dissolved in toluene (600 ml) and dried azeotropically by distillation extraction of toluene. The residue was redissolved in anhydrous toluene (500 ml). Tert-butanol (40 ml), potassium tert-butoxide (4 g, 0.0356 moles, excess of 5.1 times) and anhydrous toluene (40 ml) were combined and added to the above mixture, followed by stirring for approximately 3.5. hours. T-Butyl bromoacetate (7 mL, 0.0474 moles, 6.7 fold excess) was added, and the mixture was stirred overnight at 40 ° C under an argon atmosphere. The solvent was removed by distillation under reduced pressure, and the residue was dissolved in distilled water (1000 ml). The pH of the aqueous solution was adjusted to 12.1 with 1M sodium hydroxide, and the solution was stirred overnight, maintaining the pH at 12.1 by the periodic addition of 1M sodium hydroxide. The pH was adjusted to 1.0 with 1M hydrochloric acid, and the product was extracted with dichloromethane. The extract was dried with anhydrous sodium sulfate, concentrated, and added to ethyl ether. The precipitated product was removed by filtration and dried under reduced pressure to provide 33 g.

NMR (d6-DMSO): 3.51 ppm (s, PEG structure), 4.01 ppm (s, -CH2-COO-). Anion exchange chromatographic analysis: PEG (10, 000) -dicarboxylic acid 100%.

Example 3: PEG (5,000) -dicarboxylic acid A solution of PEG-5,000 (35 g, 0.01400 equivalent) (NOF Corporation) in acetonitrile (800 ml) was azeotropically dried by distilling off acetonitrile, and the residue was returned to dissolve in anhydrous toluene (300 ml). T-butanol (50 ml), potassium tert-butoxide (4.7 g, 0.0419 moles, 2.99 fold excess), and anhydrous toluene (50 ml) were combined and added to the above mixture, followed by approximately 3.5 hours of stirring . T-butyl bromoacetate (7.2 ml, 0.0488 moles, 3.48 fold excess) was added, and the mixture was stirred for 20 hours at room temperature under an argon atmosphere. The solvent was removed by distillation under reduced pressure, and the residue was dissolved in distilled water (1000 ml). The pH of the aqueous solution was adjusted to 12.0 with 1M sodium hydroxide, and the solution was stirred overnight, maintaining the pH at 12.0 by the periodic addition of 1M sodium hydroxide. The pH was adjusted to 2.0 with 1M hydrochloric acid and the product was extracted with dichloromethane. The extract was dried with anhydrous sodium sulfate, concentrated and added to ethyl ether. The precipitated product was removed by filtration and dried under reduced pressure to provide 32 g. NMR (d6-DMSO): 3.51 ppm (s, PEG structure), 4.01 ppm (s, -CH2-COO-). Anion exchange chromatographic analysis: PEG (5, 000) -dicarboxylic acid 100%.

Example 4: 4-branching-PEG (10,000) -tetracarboxylic acid A solution of multiple branched (4-branched) PEG, 10 kDa MW (Nektar, Huntsville AL) (160 g, 0.064 equivalent) in toluene (2,300) ml) was dried azeotropically by extraction by distillation of 1,000 ml of toluene at 80 ° C under reduced pressure. In another vessel, tert-butanol (17.3 ml) and potassium tert-butoxide (7.18 g, 0.128 mole, excess of 2.00 times) were mixed and then added to the dry toluene solution of the above. The resulting solution was stirred for about 3.5 hours at 45 ° C. T-Butyl bromoacetate (20.8 mL, 0.141 moles, 2.20 fold excess) was added, and the mixture was stirred 12 hours at 45 ° C under argon atmosphere. The solvent was removed by distillation under reduced pressure, and the residue was dissolved in distilled water (1,600 ml). The pH of the aqueous solution was adjusted to 12.0 with 1M sodium hydroxide, and the solution was stirred for 17 hours while maintaining the pH at 12.0 by the periodic addition of 1M sodium hydroxide. The pH was then adjusted to 1.5 with 1M phosphoric acid, and the product was extracted with dichloromethane. The extract was dried with anhydrous sodium sulfate, concentrated and added to ethyl ether. The precipitated product was removed by filtration and dried under reduced pressure to provide 15.5 g. NMR (d6-DMSO): 3.51 ppm (s, PEG structure), 4.01 ppm (s, -CH2-COO-), 100% substitution. After 8 months of storage at -20 ° C, the GPC analysis was identical to the original product. Therefore, no detectable degradation occurred during storage. Many modifications and other embodiments of the invention will come to the mind of one skilled in the art to which this invention pertains having the benefit of the teachings presented in the foregoing description. Therefore, the invention will not be limited to the specific embodiments set forth, and it is intended that modifications and other modalities be included within the scope of the appended claims.

Claims (60)

1. NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and, therefore, the content of the following CLAIMS is claimed as property: 1. A method for preparing a non-peptidic polymer, soluble in water, functionalized with a carboxyl group, the method characterized in that it comprises: (i) reacting an ester reagent R (C = 0) OR ', wherein R' is a tertiary group and R comprises a functional group X, with a polymer POLY-Y not peptidic, soluble in water, where Y is a functional group that reacts with X to form a covalent bond, to form a tertiary ester of the polymer; and (ii) treating the tertiary ester of the polymer with a strong base, in aqueous solution, to form a carboxylate salt of the polymer.
2. 2. The method according to claim 1, further characterized in that it comprises: iii) treating the carboxylate salt of the polymer with an inorganic acid in aqueous solution, to convert the carboxylate salt to a carboxylic acid, thereby forming a polymer with group Functional carboxylic acid.
3. The method according to claim 1 or 2, characterized in that X is a leaving group and Y is a hydroxyl group.
4. 4. The method according to claim 1 or 2, characterized in that the strong base is an alkali metal hydroxide.
5. The method according to claim 1 or 2, characterized in that the treatment with a strong base is effective to produce a reaction pH of about 11 to 13.
6. The method according to claim 2, characterized in that the inorganic acid is an acid that produces non-nucleophilic anions in aqueous solution.
7. The method according to claim 6, characterized in that the acid is selected from the group consisting of sulfuric acid, nitric acid, phosphoric acid, and hydrochloric acid.
8. The method according to claim 1, characterized in that the tertiary ester reagent has the structure: where: X is a leaving group; each of R1 and R2 is independently selected from hydrogen, alkyl, cycloalkyl, -r-alkoxy, aryl, aralkyl and heterocycle; each of R3-R5 are independently selected from alkyl, aryl, aralkyl, and lower cycloalkyl, wherein any of R3-R5 can be linked to form a ring or ring system; wherein any of R1 through R5, except hydrogen, can be substituted with a group selected from lower alkyl, lower alkoxy, C3-C6 cycloalkyl, halo, cyano, oxo (keto), nitro and phenyl; and n is 1 to approximately 24.
9. The method according to claim 8, characterized in that n is 1 to 6.
10. The method according to claim 9, characterized in that n is 1 or 2.
11. 11. The method according to any of the claims 8-10, characterized in that each of R1 and R2 is independently hydrogen or unsubstituted lower alkyl, and each of R3 and R5 is independently alkyl or unsubstituted lower phenyl.
12. The method according to any of claims 8-10, characterized in that each of R1 and R2 is independently hydrogen or methyl, and each of R3 to R5 is independently methyl, ethyl or phenyl.
13. 13. The method according to claim 12, characterized in that each of R1 and R2 is H and n is 1.
14. The method according to claim 13, characterized in that the tertiary ester reagent is a t-butyl haloacetate.
15. The method according to claim 1 or 2, characterized in that the polymer is selected from the group consisting of poly (alkylene glycols), poly (olefinic alcohol), 'poly (vinylpyrrolidone), poly (hydroxyalkyl methacrylamide), poly (hydroxyalkyl methacrylate), poly (saccharides), poly (hydroxy-acetic acid), poly (acrylic acid), poly (vinyl alcohol), polyphosphazene, polyoxazolines, poly (N-acryloylmorpholine), and terpolymer copolymers thereof.
16. 16. The method according to claim 15, characterized in that the polymer is a poly (ethylene glycol).
17. The method according to claim 16, characterized in that the poly (ethylene glycol) is linear and terminates at one end with the functional group Y, and at the other end with another functional group Y 'or a capped group.
18. 18. The method according to claim 2, further characterized in that it comprises converting the carboxylic acid to an activated carboxylic acid derivative.
19. 19. The method according to claim 18, characterized in that the derivative is an activated ester.
20. The method according to claim 18, further characterized in that it comprises conjugating the polymer with a biologically active molecule, by reacting the carboxylic acid derivative with a functional group on the molecule.
21. The method according to claim 20, characterized in that the carboxylic acid derivative is an activated ester, and the functional group on the molecule is a nucleophilic group.
22. 22. The method according to claim 21, characterized in that the nucleophilic group is an amino group, a hydroxyl group, or a thiol.
23. 23. A method for preparing a poly (ethylene glycol) (PEG) functionalized with a carboxyl group, the method characterized in that it comprises: i) reacting a tertiary ester reagent R (C = 0) OR ', wherein R' is a tertiary a group and R comprises a functional group X, with a PEG-Y polymer, wherein Y is a functional group that reacts with X to form a covalent bond, to form a tertiary ester PEG; and ii) treating the PEG tertiary ester with a strong base in aqueous solution, to form a PEG carboxylate salt.
24. The method according to claim 23 or 24, further characterized in that it comprises: iii) treating the PEG carboxylate salt with an inorganic acid in aqueous solution, to convert the carboxylate salt to a carboxylic acid, thereby forming a PEG carboxylic acid.
25. 25. The method according to claim 23 or 24, characterized in that X is a leaving group and Y is a hydroxyl group.
26. 26. The method according to claim 23, characterized in that the strong base is an alkali metal hydroxide.
27. The method according to claim 24, characterized in that the acid is selected from the group consisting of sulfuric acid, nitric acid, phosphoric acid, and hydrochloric acid.
28. The method according to claim 23, characterized in that the tertiary ester reagent has the structure: where: X is a leaving group; each of R1 and R2 is independently selected from hydrogen, a, cycloa, alkoxy, aryl, ara and heterocycle; each of R3-R5 are independently selected from a, aryl, ara, and lower cycloa, wherein any of R3-R5 can be linked to form a ring or ring system; wherein any of R1 through R5, except a hydrogen, can be substituted with a group selected from lower a, lower alkoxy, C3-C6 cycloa, halo, cyano, oxo (keto), nitro and phenyl; and n is 1 to approximately 24.
29. 29. The method according to claim 28, characterized in that n 1 to 6.
30. 30. The method according to claim 29, characterized in that n 1 or 2.
31. 31. The method according to any of claims 28- 30, characterized in that each of R1 and R2 is independently hydrogen or unsubstituted lower a, and each of R3 to R5 is independently a or unsubstituted lower phenyl.
32. 32. The method according to any of claims 28-30, characterized in that each of R1 and R2 is H and n is 1.
33. 33. The method according to claim 32, characterized in that the tertiary ester reagent is a t-butyl haloacetate.
34. The method according to any of claims 23, 24 or 33, characterized in that the poly (ethylene glycol) is linear and ends at one end with the functional group Y, and at -the other end with another functional group Y 'or a group topped.
35. 35. The method according to any of claims 23, 24 or 33, characterized in that PEG has a molecular weight between about 100 to 100,000 Da.
36. 36. The method according to claim 35, characterized in that PEG has a molecular weight between about 300 to 60,000 Da.
37. 37. The method according to claim 24, further characterized in that it comprises converting the PEG carboxylic acid to an activated carboxylic acid derivative.
38. 38. The method according to claim 37, characterized in that the derivative is an activated ester.
39. 39. The method according to claim 37, further characterized in that it comprises conjugating the PEG with a biologically active molecule, by reacting the carboxylic acid derivative with a functional group on the molecule.
40. 40. An isolated polymeric product comprising a carboxylic acid functional group polymer, prepared by the method according to claim 2, characterized in that the product contains less than 5% by weight of the polymer POLY-Y, with the remainder consisting essentially of the polymer with carboxylic acid functional group.
41. 41. The polymer product according to claim 40, characterized in that it contains less than 2% by weight of POLY-Y polymer.
42. 42. The polymeric product according to claim 40, characterized in that it contains less than 0.5% by weight of the POLY-Y polymer.
43. 43. The polymeric product according to any of claims 40-42, characterized in that they practically do not contain any amount of organic acid of low molecular weight.
44. 44. The polymer product according to any of claims 40-42, characterized in that they practically do not contain any amount of monomeric organic carboxylic acid.
45. 45. The polymer product according to any of claims 40-42, characterized in that it practically does not contain any amount of trifluoroacetic acid.
46. 46. The polymer product according to claim 40, characterized in that the polymer with carboxylic acid functional group is a PEG carboxylic acid.
47. 47. The polymer product according to claim 46, characterized in that the polymer with carboxylic acid functional group is PEGm-CH2-COOH, and the polymeric product contains less than 5% by weight of PEG -OH.
48. 48. The polymer product according to claim 47, characterized in that it contains less than 2% by weight of PEGm-OH.
49. 49. The polymer product according to claim 48, characterized in that it contains less than

    0. 5% by weight of PEGm-OH.
50. 50. The polymer product according to any of claims 47-49, characterized in that it practically does not contain any amount of trifluoroacetic acid.
51. 51. The polymer product according to claim 46, characterized in that the polymer with carboxylic acid functional group is HOOC-CH2-PEG-CH2-COOH, and the product contains less than 5% by weight of HO-PEG-OH.
52. 52. The polymer product according to claim 51, characterized in that it contains less than 0.5% by weight of HO-PEG-OH.
53. 53. The polymer product according to claim 51 or 52, characterized in that it practically does not contain any amount of trifluoroacetic acid.
54. 54. The polymeric "product" according to claim 46, characterized in that the polymer with carboxylic acid functional group is a PEG with functional group of branched or multi-branched multifunctional carboxylic acid represented by PEG- (CH2-COOH) x wherein x is 3 to 8, and the product contains less than 5% by weight of PEG- (OH) x.
55. 55. The polymer product according to claim 54, characterized in that it practically does not contain any amount of trifluoroacetic acid.
56. 56. In a method for preparing a poly (ethylene glycol) (PEG) polymer functionalized with a carboxyl group, by the reaction of a tertiary ester reagent R (C = 0) OR ', where R' is a tertiary alkyl group and R comprises a functional group X, with a PEG-Y polymer, wherein Y is a functional group that reacts with X to form a covalent bond, to form a PEG tertiary ester, an improvement characterized in that it comprises: treating the PEG tertiary ester with a strong base in aqueous solution, to form a PEG carboxylate salt.
57. 57. The improvement according to claim 56, characterized in that the method further comprises: treating the PEG carboxylate salt with an inorganic acid in aqueous solution, to convert the carboxylate salt to a carboxylic acid, thereby forming a PEG carboxylic acid.
58. 58. The improvement according to claim 56, characterized in that the strong base is an alkali metal hydroxide.
59. 59. The improvement according to claim 56, characterized in that the treatment with a strong base is effective to produce a reaction pH of about 11 to 13. The improvement according to claim 57, characterized in that the acid is selected from the group consisting of , sulfuric acid, nitric acid, phosphoric acid, and hydrochloric acid.