JP2008174755A - Hetero-bifunctional polyethylene glycol derivative and method for preparing it - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for preparing a hetero-bifunctional derivative of poly(ethylene glycol) or a related polymer thereof with a high purity and in a high yield. <P>SOLUTION: An intermediate polymer of the formula: W-poly-OH is provided with a removable group W at one terminus without going through a chromatographic purification step. The intermediate polymer W-poly-OH is first modified by converting the OH group into a first functional group X and subsequently removing W to produce a second hydroxyl group. Then, the latter hydroxyl group is converted into a second functional group Y to obtain the desired hetero-bifunctional derivative. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a heterobifunctional poly (ethylene glycol) derivative and a preparation method thereof.

The covalent attachment of hydrophilic polymer poly (ethylene glycol) abbreviation (PEG), also known as poly (ethylene oxide) abbreviation (PEO), to molecules and surfaces has important applications in biotechnology and medicine. In its most common form, PEG is a linear polymer with hydroxyl groups at each end.
HO—CH ₂ —CH ₂ O (CH ₂ CH ₂ O) nCH ₂ CH ₂ —OH This formula can be shown briefly as HO-PEG-OH, where —PEG— is a polymer with no end groups. It is understood to indicate a skeleton.
_{-PEG - = - CH 2 CH 2} O (CH 2 CH 2 O) nCH 2 CH 2 - PEG is methoxy-PEG-OH, i.e. Briefly used commonly by mPEG, where the one end Is a relatively inert methoxy group, while the other end is a hydroxyl group that can undergo chemical modification.
_{CH 3 O- (CH 2 CH 2} O) n-CH 2 CH 2 -OH

  The term poly (ethylene glycol) or PEG is understood by those skilled in the art to indicate or include all of the above forms and also other forms.

Copolymers of ethylene oxide and propylene oxide are closely related to PEG in their chemistry, and they are an alternative to PEG in many of their applications.
_{HO-CH 2 CHRO (CH 2} CHRO) nCH 2 CHR-OH R = H and CH ₃

  PEG is a useful polymer that has water-soluble as well as solubility properties in many organic solvents. PEG is also non-toxic and non-immunogenic. When PEG is chemically attached to a water-insoluble compound, the resulting conjugate is generally water soluble as well as soluble in many organic solvents. If the molecule to which PEG is attached is biologically active, such as a drug, this activity is generally retained after the attachment of PEG, and the conjugate can exhibit altered pharmacokinetics. For example, Bentley et al., Polymer Preprints, 38 (1), 584 (1997) demonstrated that when conjugated to PEG, water-insoluble antemisinin becomes water soluble and exhibits increased antimalarial activity. Davis et al., In US Pat. No. 4,179,337, showed that PEG-conjugated proteins show enhanced blood circulation life due to reduced renal clearance and reduced immunogenicity. The lack of toxicity of PEG and its rapid clearance from the body are advantageous in pharmaceutical applications.

As the application of PEG chemistry has become more sophisticated, the demand for heterobifunctional PEG, ie, PEG with different end groups: X-PEG-Y (where X and Y are different groups) has increased. ing. PEG with backbone ester groups and end groups X and Y: X-PEG-CO ₂ -PEG-Y is heterogeneous even if X and Y are the same because each PEG unit in the backbone is asymmetrically substituted. It can be considered bifunctional.

  Using said heterobifunctional PEG with appropriate functional groups, PEG can be linked to a surface or other polymer, such as a polysaccharide or protein, the other end of which, for example, a drug, liposome, another protein Or attached to a biosensor. If one end is attached to the polymer and the other end is attached to the appropriate functional group, the formation of a useful hydrogel by cross-linking can occur.

However, it is often difficult or impossible to prepare heterobifunctional PEGs with high purity using existing methods. For example, the following reaction can be performed with the goal of preparing the indicated heterobifunctional PEG acetal product using equimolar amounts of each reagent: HO-PEG-OH + ClCH ₂ CH (OC ₂ H ₅ ) ₂ + NaOH →→ HO-PEG-OCH ₂ CH (OC ₂ H ₅ ) ₂ + NaCl + H ₂ O However, in practice, some disubstituted PEG diethyl acetals (C ₂ H ₅ O) ₂ CH ₂ O-PEG _{-OCH 2 CH (OC 2 H 5} ) 2 is also inevitably formed, it will also remain some unreacted PEG. Troublesome chromatography is necessary to separate this mixture.

A chromatographic approach was used by Zalipsky (Bioconjugate Chemistry, 4: 296-299, 1993) and the following heterobifunctional PEG derivative: HO-PEG-CONHCH ₂ CO ₂ H was converted to unreacted PEG and disubstituted carboxylic acid derivatives. And purified from a mixture of reaction products.

In certain applications, it is essential that a minimum amount of HO-PEG-OH is present in the monoalkyl PEG used to prepare the monofunctional activated PEG. This is because the presence of HO-PEG-OH results in a doubly activated PEG derivative resulting in a cross-linked product or has other undesirable effects. In fact, HO-PEG-OH is a common contaminant in monoalkyl PEG. Trityl (Ph ₃ C-derivative) is formed, the derivative is separated by chromatography, and the CH ₃ O-PEG-OH is converted to HO-PEG- by removing the trityl group from CH ₃ O-PEG-OCPh _3. A chromatographic approach, separating from OH, is disclosed in US Pat. No. 5,298,410. Recent patent applications, Suzawa et al (WO No. 96/35451), the benzyl _{PEG (C 6 H 5 -CH 2} -OPEG-OH), having a group having affinity to the target cells at one end, Disclosed as an intermediate in the preparation of heterobifunctional PEGs having a toxin at the other end. However, benzyl PEG was prepared by separating benzyl PEG from dibenzyl PEG and unreacted PEG by benzylation of PEG, followed by exhaustive exhaustive gradient chromatography. The procedure was performed on a small scale and the yield was only 7.8%. Therefore, this method is hardly useful for commercial production.

この方法は、Ｙｏｋｏｙａｍａら（ＢｉｏｃｏｎｊｕｇａｔｅＣｈｅｍｉｓｔｒｙ。３：２７５〜２７６、１９９２）により、一方の末端にヒドロキシル基を有し、他方にアミノ基を有するＰＥＧの調製に使用されている。Ｃａｍｍａｓら（ＢｉｏｃｏｎｊｕｇａｔｅＣｈｅｍｉｓｔｒｙ、６：２２６〜２３０、１９９５）は、この方法を使用して、一方の末端にアミノ基を有し、他方にヒドロキシルまたはメトキシ基を有するＰＥＧを調製した。それは、Ｎａｇａｓａｋｉら（ＢｉｏｃｏｎｊｕｇａｔｅＣｈｅｍｉｓｔｒｙ、６：２３１〜２３３、１９９５）により、一方の末端にホルミル基を有し、他方にヒドロキシル基を有するＰＥＧの調製に使用されている。この方法は、一般に、Ｘが重合を開始するのに適切で望ましい基である場合にのみ有用であり；そうではない場合が多い。また、この方法の成功裡の適用には、ＨＯ−ＰＥＧ−ＯＨの形成を防ぐために厳密な水の除外が必要であり、この問題は、分子量が増加するにつれより深刻となる。また、所望の分子量のＰＥＧ誘導体を得るためには重合度を注意深く制御することが必要である。この方法は、エチレンオキシド重合を直接薬物分子上に実施する場合には、過酷な重合条件下における多くのタイプの薬物分子の分解により制限される。この方法はまた、重合が起こり得る、２つ以上の官能基が存在する場合には、選択性の欠如により制限される。 A second strategy, the polymerization approach, for preparing bifunctional PEGs involves anionic polymerization of ethylene oxide to an anion X-, which ultimately becomes the end group of the polymer.

This method has been used by Yokoyama et al. (Bioconjugate Chemistry. 3: 275-276, 1992) to prepare PEGs having a hydroxyl group at one end and an amino group at the other. Cammas et al. (Bioconjugate Chemistry, 6: 226-230, 1995) used this method to prepare PEG with an amino group at one end and a hydroxyl or methoxy group at the other. It has been used by Nagasaki et al. (Bioconjugate Chemistry, 6: 231-233, 1995) to prepare PEGs having a formyl group at one end and a hydroxyl group at the other. This method is generally useful only when X is a suitable and desirable group for initiating polymerization; often it is not. Also, successful application of this method requires strict exclusion of water to prevent the formation of HO-PEG-OH, and this problem becomes more serious as the molecular weight increases. Further, in order to obtain a PEG derivative having a desired molecular weight, it is necessary to carefully control the degree of polymerization. This method is limited by the degradation of many types of drug molecules under harsh polymerization conditions when ethylene oxide polymerization is carried out directly on drug molecules. This method is also limited by the lack of selectivity when two or more functional groups are present where polymerization can occur.

  It would be desirable to provide additional methods for preparing heterobifunctional PEGs that substantially eliminate at least some of the problems and disadvantages of previous methods.

The present invention provides a method for preparing heterobifunctional poly (ethylene glycol) derivatives via a PEG intermediate having a removable group at one end. A class of PEG derivatives of W-PEG-OH, where W is a group removable by mild chemical methods, is provided, first modifying the OH group to the desired group X and then W Is removed to create a second hydroxyl group. The latter hydroxyl group can then be further converted to a second functional group Y, thus providing the desired bifunctional PEG:
W-PEG-OH → W-PEG-X → HO-PEG-X → Y-PEG-X

Preferred removable groups are benzyloxy groups (C ₆ H ₅ CH ₂ —O—), but 4-methylbenzyl, 3-methylbenzyl, 4-chlorobenzyl, 4-methoxybenzyl, diphenylmethyl, triphenyl Other arylmethyl groups may also be used, including but not limited to methyl, or 1-naphthylmethyl. Diarylmethyl and triarylmethyl groups may also be used. Benzyloxy-PEG-OH (BzO-PEG-PH) can be prepared, for example, in high purity by polymerization of ethylene oxide to the benzyl oxide ion BzO-. By carrying out the reaction under carefully controlled anhydrous conditions, the heterobifunctional derivative product can be prepared with a minimal amount of HO-PEG-OH. The advantage of benzyl and other arylmethyl groups is that they can be removed by catalytic hydrogenolysis under relatively mild conditions or by acid-catalyzed hydrolysis.
In _{BzO-PEG-X + H 2} (cat) → C 6 H 5 -CH 3 + HO-PEG-X BzO-PEG-X + H 2 O (H +) → C 6 H 5 CH 2 OH + HO-PEG-X the above reaction, cat is a catalyst such as carbon palladium.

In one embodiment of the invention, the method is used to conjugate PEG or related polymers to macromolecules or surfaces such as proteins, lipids, polysaccharides or other polymers. First, the hydroxyl group of immediate BzO-PEG-OH is converted to a first reactive functional group. This reactive functional group allows attachment of BzO-PEG- to the macromolecule. The benzyl group is then removed by hydrogenolysis or hydrolysis without chemically affecting the macromolecule, thus making a new terminal hydroxyl group available on the PEG derivative. This new hydroxyl group can be used directly to attach the end of the PEG derivative to the same or another macromolecule. Alternatively, the hydroxyl group can be further converted to a second reactive functional group, which is then used to link the PEG derivative to the macromolecule. When the second reactive functional group is linked to another polymer, a crosslinked polymer useful as a hydrogel can be created. The reaction scheme can be described in general form as follows:
(1) BzO-PEG-OH → BzO-PEG-X (X = reactive functional group)
(2) BzO-PEG-X → BzO-PEG-M1 (M1 = macromolecule, eg surface, drug, protein or polymer)
(3) BzO-PEG-M1 + H2 (Pd / C) → BzH + HO-PEG-M1 or BzO-PEG-M1 + H2O / H + → BzOH + HO-PEG-M1 (4) HO-PEG-M1 → M2-PEG-M1 (M2 = Macromolecule, eg surface, drug, protein or polymer, or different sites on M1)

If desired, the sequence of reactions can be manipulated to avoid destruction of chemical groups that are sensitive to hydrogenation or hydrolysis.
(1) Bz-PEG-OH-> Bz-PEG-X (2) BzO-PEG-X-> BzO-PEG-M2 (M2 = drug, surface, polymer, or other group not sensitive to hydrogenation or hydrolysis) )
(3) BzO-PEG-M2 + H2 (Pd / C) → BzH (or BzOH) + HO-PEG-M2BzO-PEG-M2 + H2O / H + → BzH (or BzOH) + HO-PEG-M2 (4) HO-PEG-M2 → M1-PEG-M2 (M1 = drug, surface, polymer, or other group sensitive to hydrogenation or hydrolysis)

In another embodiment of the invention, a method for inhibiting the reactivity of HO-PEG-OH in a mixture of W-O-PEG-OH and HO-PEG-OH is disclosed. In this approach, alkylation of W—O—PEG—OH, including HO—PEG—OH, results in a mixture of W—O—PEG—OR and RO—PEG—OR, where R is an alkyl group. .
BzO-PEG-OH + HO-PEG-OH + R-X-> BzO-PEG-OR + RO-PEG-OR + HXX is a leaving group such as mesylate or tosylate.
Catalytic hydrogenation converts BzO-PEG-OR to RO-PEG-OH.
BzO-PEG-OR + RO-PEG-OR + H2 (Pd / C) .fwdarw.RO-PEG-OH + RO-PEG-OR + BzH Thus, a mixture of RO-PEG-OH and RO-PEG-OR results.
Unlike HO-PEG-OH, RO-PEG-OR is inert and non-reactive. This mixture is therefore equivalent to the pure product of RO-PEG-OH in most chemical reactions.

  The foregoing and other objects, advantages and features of the present invention, as well as the manner of doing so, will become more readily apparent in view of the following detailed description of the invention.

The present invention provides high purity and high yield heterobifunctional derivatives of poly (ethylene glycol) or related polymers. This method does not require a chromatographic purification step. According to the method of the present invention, an intermediate polymer having W-poly-OH carrying a removable group W on one side is obtained. The intermediate polymer W-poly-OH is first modified with the first functional group X of the OH group, and then W is removed to obtain a second hydroxyl group. Since the latter hydroxyl group can be further converted to the second functional group Y, the desired heterobifunctional derivative is obtained.
W-Poly-OH → W-Poly-X → HO-Poly-X → Y-Poly-X

  In the discussion below, Poly is often referred to as PEG or poly (ethylene glycol) for convenience. However, it should be understood that other related polymers are also useful in the practice of the present invention and are included in the term PEG or poly (ethylene glycol) but not in this respect.

  Poly (ethylene glycol) or PEG has very desirable properties and is generally useful for biology applications because it is generally approved for biology or biotechnology applications. PEG is typically clear and colorless, water soluble, heat stable, inert to many chemicals, does not hydrolyze or alter, and is nontoxic. Poly (ethylene glycol) is believed to be biocompatible (ie, PEG can coexist without harming living tissues or organs). More specifically, PEG is not immunogenic (ie, PEG does not tend to elicit an immune response in the body). When combined with a moiety that has some desired function in the body, PEG tends to mask that part, reducing or eliminating any immune response so that the organ is resistant to the presence of that part it can. Accordingly, the heterobifunctional derivatives of the present invention should be substantially non-toxic and cause substantially no immune response or clotting or other undesirable effects.

PEG having the formula —CH ₂ CH ₂ — (CH ₂ CH ₂ O) n —CH ₂ CH ₂ —, where n is about 8 to 4000, is one of the useful polymers in the practice of this invention. . Other bifunctional, water-soluble, non-peptidic polymers other than PEG are also useful in the present invention. These other polymers include poly (vinyl alcohol) (“PVA”); other poly (alkylene oxides such as poly (propylene glycol) (“PPG”)); and poly (oxyethylated polyols (poly (oxyethylated)). Glycerol), poly (oxyethylated sorbitol), and poly (oxyethylated glucose), etc. The polymer may be a homopolymer or a random or block copolymer and a linear or branched terpolymer based on monomers of the polymer. It can be.

  Specific examples of suitable additional polymers include poly (oxazoline), difunctional poly (acryloylmorpholine) (“PAcM”), and poly (vinyl pyrrolidone) (“PVP”). PVP and poly (oxazoline) are well known polymers in the art and their preparation should be easy to those skilled in the art. PAcM and its synthesis and use are described in US Pat. Nos. 5,629,384 and 5,631,322, the entire contents of which are hereby incorporated by reference.

  The terms “group”, “functional group”, “moiety”, “active moiety”, and “active site” are all somewhat synonymous in the chemical field and are used in the art and are used herein as molecules. Are units that show distinct and definable parts and some function or activity and are reactive with other molecules or parts of molecules.

  The term “bond” is used to refer to a group that is usually formed as a result of a chemical reaction and is typically a covalent bond.

  “Drug” should be understood as any substance intended to diagnose, cure, alleviate, treat, or prevent the real feeling of humans and other animals or improve physical or mental health.

  The term “macromolecule” is used to mean a macromolecule including but not limited to lipids, polysaccharides, proteins, nucleotide sequences, drugs, polymers, and the like. Often it is desirable to conjugate such polymers with such polymers.

According to the present invention, the removable group W can be removed from the polymer W-Poly-X by a mild chemical reaction. Such chemical reactions can be carried out under conditions that do not cause other modifications of the polymer W-Poly-X, particularly the first functional group X. Preferably, W is of the formula Ar—C (R 1) (R 2) —O—, wherein Ar is phenyl, substituted phenyl, biphenyl, substituted biphenyl, polycyclic aryl, substituted polycyclic aryl, and heterocyclic A moiety selected from the group consisting of aryl, wherein R 1 and R 2 are H, alkyl, or Ar as defined above. Thus, examples of removable groups W include benzyloxy groups (C ₆ H ₅ CH ₂ O) and other arylmethyloxy groups (4-methylbenzyloxy, 3-methylbenzyloxy, 4-chlorobenzyloxy, But includes, but is not limited to, 4-methoxybenzyloxy, diphenylmethyloxy, triphenylmethyloxy, and 1-naphthylmethyloxy). The arylmethyloxy group can be removed from the polymer under relatively mild conditions by catalytic hydrogenolysis or acid-catalyzed hydrolysis.

  In accordance with the present invention, it is preferred to synthesize W-poly-OH, for example, by polymerization of a suitable polymer monomer to aryl methyl oxide. For example, benzyloxy-PEG-OH (BzO-PEG-OH) can be prepared in high purity and yield by polymerization of ethylene oxide to benzyloxy ion BzO-. Preferably, the polymerization reaction is performed under anhydrous conditions. According to this aspect of the invention, the production of HO-PEG-OH is minimal. Extensive gradient chromatographic purification is not required and the yield of BzO-PEG-OH is high. This is in contrast to the prior art, which is a cost-effective and low yield method that is unavoidable for high cost and low yield yielding laborious and extensive gradient chromatography after PEGylation of PEG.

  According to the present invention, the final product of a heterobifunctional derivative of poly (ethylene glycol) or related polymer has the formula Y-Poly-X. Desirably, the first functional group X and the second functional group Y are conjugated with a PEG derivative, for example, other macromolecules (including but not limited to proteins, lipids, other parties, and other polymers). A reactive moiety that can react with other molecules. Examples of the first functional group X include mesylate; tosylate; tresylate, O (CH2) nCO2H (where n = 1-6), O (CH2) nCO2R3 (where n = 1-6, R3 is an alkyl group), NHR4 (wherein R4 is H or alkyl or an amine protecting group such as t-Boc and Fmoc); O (CH2) nCH (ZR5) 2 where n is 1-6 A number, Z is O or S, and R5 is H or an alkyl group; Ar-CH = CH-CH = CH-CO2 wherein Ar is phenyl, substituted phenyl, biphenyl, substituted biphenyl, Represents a moiety selected from the group consisting of polycyclic aryl, substituted polycyclic aryl, and heterocyclic aryl); —O— (CH 2) n —CHO and —O 2 CCH 2 CH 2 CO 2 R 6, wherein R 6 is H or N succinimidyl. Indicate HS is a) include, but are not limited to.

Examples of the second functional group Y include hydroxyl group; mesylate; tosylate; tresylate, O (CH2) nCO2H (where n = 1-6), O (CH2) nCO2R3 (where n = 1-6). There, R3 is an alkyl group), NHR4 (where, R4 is an amine protecting group such as H or alkyl, or t-Boc and _{Fmoc); O (CH 2)} nCH (ZR 5) 2 ( wherein, n 1 to 6; Z is O or S; R ₅ is H or an alkyl group; Ar—CH═CH—CH═CH—CO ₂ (wherein Ar is phenyl, substituted phenyl) shows biphenyl, substituted biphenyl, polycyclic aryls, substituted polycyclic aryl and a moiety selected from the group consisting of heterocyclic _{aryl); - O- (CH 2)} n-CHO and -O ₂ CCH ₂ CH ₂ CO ₂ R ₆ (wherein R ₆ is H or And NH 2 represents N succinimidyl); and CH ₂ ═CH—CO ₂ —. In the poly (ethylene glycol) derivative of the formula Y-Poly-X, it is confirmed that the first functional group X and the second functional group Y are different from each other and thus are heterobifunctional.

X is Ar—CH═CHCH═CH—CO ₂ (Ar is as defined above), and Y is —O— (CH ₂ ) n —CHO or O (CH ₂ ) n CH (ZR ₅ ) ₂ (formula In the formula, n is a number from 1 to 6, Z is O or S, and R ₅ is H or an alkyl group. When X is O (CH ₂ ) nCH (ZR ₅ ) ₂ , where n is a number from 1 to 6, Z is O or S, and R ₅ is H or an alkyl group, Y Is preferably —O ₂ CCH ₂ CH ₂ CO ₂ R ₆ (wherein R ₆ is H or NHS). When X is OCH ₂ CO ₂ CH (CH ₃ ) CH ₂ CONHS, the second functional group Y is preferably CH ₂ ═CHCO ₂ .

  Although the reaction scheme of this method is shown above: W-Poly-OH → W-Poly-X → HO-Poly-X → Y-Poly-X, any two formations in the formula It should be understood that there can be more than one chemical reaction step between objects. For example, several successive reactions can occur to convert the terminal hydroxyl group of W-Poly-OH to the first functional group X. Similarly, the second functional group Y can be obtained by performing several reaction steps to modify the novel hydroxyl group of HO-Poly-X.

Furthermore, in one embodiment of the invention, prior to the removal step of the removable group W, the polymer Y-Poly-X is placed between the first reactive functional group X and a suitable moiety on the polymer surface. It is possible to bind to the polymer or the surface by the bond formed (hence the conjugation of the polymer W-Poly- moiety and the polymer: W-Poly-M ₁ (M ₁ is a protein, peptide, Lipids, drugs, polysaccharides, or other polymers, or macromolecules such as the surface of substances (eg, microorganisms)). The removable group W in the conjugate W-Poly-M ₁ is then removed by a mild chemical reaction, such as catalytic hydrogenolysis or acid catalyzed hydrolysis. The resulting —OH reacts directly with, for example, another macromolecule M ₂ (such as the surface of a protein, peptide, lipid, drug, polysaccharide, or other polymer, or substance (eg, microorganism)). Thus, M ₂ -Poly-M ₁ can be formed. If conjugation to another polymer is preferred, the —OH group can optionally be converted to an inert, non-reactive group capped, for example by alkylation. Alternatively, the resulting OH group can be converted to the reactive functional group Y: Y-Poly-M ₁ . The functional group Y can then be reacted with M ₂ to form M ₂ -Poly-M ₁ .
(1) WPolyOH ---> WPolyX (X = reactive functional group)
(2) WPolyX ---> WPolyM ₁ (M ₁ = macromolecule such as surface, drug protein, or polymer)
(3) WPolyM ₁ + H ₂ (Pd / C) → WH + HOPolyM ₁ or WPPolyM ₁ + H ₂ O / H + → WOH + HOPolyM ₁
(4) HOPolyM ₁ → Y ₂ PolyM ₁
(5) YPolyM ₁ + M ₂ → M ₂ PolyM ₁ (M ₂ = macromolecule such as surface, drug, protein or polymer, or different sites on M ₁ )

  In this method, a hydrogel can be made by cross-linking a plurality of different polymers with a PEG related polymer Poly. However, according to the present invention, two functional groups X and Y can be attached to the same polymer, thereby forming a polymer shell on the polymer by conjugation of the PEG-related polymer on the polymer. It should be understood that it can be done.

  In another embodiment of the present invention, the hetero-path bifunctional derivative Y-Poly-X of PEG or related polymers made as described above is a functional group X and Y and reaction on macromolecules or other materials. It can react with the macromolecule or other molecules through the moiety. For example, X and Y can be selected such that different types of macromolecules or other materials bind to X and Y, respectively. It is also possible to select X and Y so as to react with the same type of polymer.

According to another aspect of the present invention, heterobifunctional derivatives of poly (ethylene glycol) or related polymers are obtained. Such a polymer is represented by the formula Y-Poly-X, where Poly represents poly (ethylene glycol) or related compounds as defined above. X and Y are mesylate; tosylate; tresylate, O (CH ₂ ) nCO ₂ H (where n = 1 to 6), O (CH ₂ ) nCO ₂ R ₃ (where n = 1 to 6) , R ₃ is an alkyl group), NHR ₄ (wherein R ₄ is H or alkyl or an amine protecting group such as t-Boc and Fmoc); O (CH ₂ ) nCH (ZR ₅ ) ₂ (wherein n is a number from 1 to 6, Z is O or S, R ₅ is H or an alkyl group; Ar—CH═CH—CH═CH—CO ₂ (wherein Ar is phenyl, Represents a moiety selected from the group consisting of substituted phenyl, biphenyl, substituted biphenyl, polycyclic aryl, substituted polycyclic aryl, and heterocyclic aryl); —O— (CH ₂ ) n —CHO and —O _{2 CCH 2 CH 2 CO 2 R 6} ( wherein, R ₆ is H or NHS Ri, n represents a reactive functional group selected from the group consisting of 1 to 6). Preferably X and Y are different.

In some embodiments, when X is Ar—CH═CHCH═CH—CO ₂ (Ar is defined as above), Y is —O— (CH ₂ ) n —CHO or O (CH ₂ ). nCH (ZR ₅ ) ₂ (wherein n is a number from 1 to 6, Z is O or S, R ₅ is H or an alkyl group), and X is O (CH ₂ ) NCH (ZR ₅ ) ₂ , wherein n is a number from 1 to 6, Z is O or S, and R ₅ is H or an alkyl group, Y is —O ₂ CCH ₂ CH ₂ CO ₂ R ₆ (wherein R ₆ is H or NHS), and when X is OCH ₂ CO ₂ CH (CH ₃ ) CH ₂ CONHS, the second functional group Y is It is preferred that CH ₂ = CHCO ₂ .

In yet another embodiment, in formula _{XPolyaOCHR 5 (CH 2) nCO 2} PolybX ( wherein, Polya and Polyb show the same type of polymers represented by the above Poly, n is 0-6, R ₅ is Provided is a method for preparing poly (ethylene glycol) or related polymers having H or alkyl and X being a reactive functional group. Substantially pure forms of heterobifunctional derivatives can be made in high purity and yield without relying on chromatographic purification steps.

In this method, a first polymer of the formula ArC (R ₁ ) (R ₂ ) OPolibU and a second polymer of the formula ArC (R ₁ ) (R ₂ ) OPolia-CHR ₅ (CH ₂ ) nCO-V (wherein , R ₁ and R ₂ are H, alkyl, or Ar (Ar is phenyl, substituted phenyl, biphenyl, substituted biphenyl, polycyclic aryl, substituted polycyclic aryl, and heterocyclic aryl), U And V so that the first polymer reacts with the _second polymer to form a polymer of ArC (R ₁ ) (R ₂ ) OPoliaOCHR ₅ (CH ₂ ) nCO ₂ PolybOC (R ₁ ) (R ₂ ) -Ar. Is the selected part). As described above, the first polymer and the second polymer are converted from the allylmethyl oxide ion ArC (R ₁ ) (R ₂ ) O— to the product ArC (R ₁ ) (R ₂ ) O-Polya-OH or ArC. (R ₁ ) (R ₂ ) Separate reaction of O-Polyb-OH with Polyb and Polya, and optionally subsequent terminal hydroxyl moieties U and V such that the two polymers are linked by, for example, an ester bond Can be made by individual modifications. The bound polymer can then be modified by removal of the ArC (R ₁ ) (R ₂ ) O- moiety by catalytic hydrogenolysis or acid-catalyzed hydrolysis. The resulting OH group can optionally be converted to other reactive functional groups. Examples of reactive functional groups X include —OH; CH ₂ ═CR ₅ CO ₂ (wherein R ₅ is H or alkyl); O (CH ₂ ) n CH (ZR) ₂ (wherein R ₅ Is H or alkyl, Z is O or S, and n is 1 to 6); NHS—O ₂ CO— (where NHS represents N-succinimidyl) Not. In a preferred embodiment, U is —OH and V is a halogen group such as —Cl.

In yet another aspect of the present invention, the formula R ₈ OPolyOCHR ₅ (CH ₂ ) nCO ₂ PolybY (wherein Polya, Polyb, n, R ₅ are as defined above, R ₈ is H or an alkyl group) A process for the preparation of poly (ethylene glycol) or related polymers having Y is a reactive functional group. This method does not require a chromatography step. In the present method, a first polymer of ArC (R ₁ ) (R ₂ ) OPolybU is obtained as described above. (Or R ₈ is H or an alkyl group, Ar (wherein, Ar is as above _{defined)) R 8 OPolya-CHR 5} (CH 2) nCO-V also obtained a second polymer. The moieties U and V react with each other so that the first polymer combines with the second polymer to form a polymer of R ₈ OPolyOCHR ₅ (CH ₂ ) nCO ₂ PolybOC (R ₁ ) (R ₂ ) -Ar. Thus, for example, an ester bond can be formed. The ArC (R ₁ ) (R ₂ ) O— moiety can then be converted to the desired functional group. Preferably U is -OH and V is a halogen group such as -Cl. Y is —OH; CH ₂ ═CR ₅ CO ₂ (wherein R ₅ is H or alkyl); O (CH ₂ ) n CH (ZR) ₂ (wherein R is H or alkyl, Z is O or S, n represents 1 to 6); and -O- (CH ₂₎ in n-CO ₂ H (wherein, n can be a functional group such as 1 to 6).

In accordance with yet another aspect of the present invention, there is provided a method for making a polymer of R ₉ O-Poly-OH having HO-Poly-OH reactivity without contamination of the polymer. Poly is as defined above and is an R ₉ alkyl group or an aryl group. As disclosed in the art, removal of HO-Poly-OH, such as HO-PEG-OH, from the preparation of PEG derivatives usually requires extensive and laborious purification steps using, for example, chromatography. is there. The method of the present invention does not require that. In this method, ArCR ₁ R ₂ OPolyOH is first synthesized by formation of the polymer Poly on the arylmethyloxy ion ArCR ₁ R ₂ O—. The ArCR ₁ R ₂ OPolyOH is then alkylated and converted to the polymer Ar—CR ₁ R ₂ —OPEGOR ₉ . Any impurities in HO-Poly-OH are converted to R ₉ OPEGOR ₉ during alkylation. The next step converts the ArCR ₁ R ₂ O moiety to —OH by acid-catalyzed hydrolysis or hydrogenolysis to form a new mixture of R ₉ O-PEG-OH and R ₉ O-PEG-OR _9. It is to be. Since R ₉ O-PEG-OR ₉ is a most chemical reaction-inert mixture is pure R ₉ O-PEG-OH and chemically equivalent. Optionally, it is possible further to convert the R ₉ O-PEG-OH in R ₉ O-PEG-CHO.

The following examples are set forth to illustrate the present invention but should not be construed as limiting the invention.
Example 1. Synthesis of HOPEGNH ³⁺ Cl- Example 2 Synthesis of HOPEG OCH ₂ CO ₂ H Example 3 _{Cl-H 3 N + PEGOCH 2} CO 2 H Synthesis Example 4. _{Cl-H 3 N + PEGOCH 2} CH 2 CO 2 H Synthesis Example 5. _{C 6 H 5 CH = CHCH =} CHCO 2 PEGOCH 2 CH (OC 2 H 5) 2 of Example 6. Synthesis of NHSO ₂ COPEGOCH ₂ CO ₂ PEGOCO ₂ NHS (NHS = Nsuccinimidyl) Example 7 _{CH 2 = CHCO 2 PEGOCH 2 CO} 2 PEGO 2 CCH = Synthesis Example of CH ₂ 8. Synthesis of CH ₃ OPEGOCH ₂ CH ₂ CO ₂ PEGOH Example 9 Synthesis of NHSO ₂ CCH ₂ CH ₂ COOPEGOCH ₂ CH ₂ CH (OC ₂ H ₅ ) ₂ Example 10 _{CH 2 = CHCO 2 PEGOCH 2 CO} 2 PEGOCH (CH 3) CH 2 CO 2 Synthesis Example 11 of NHS. Application of alkylation to prepare CH ₃ OPEGOH without HOPEGOH from a mixture of BzOPEGOH and HOPEGOH

Example 1 Preparation Reaction of HOPEGNH ³⁺ Cl-

a) Preparation of BzOPEGOM: A 150 ml toluene solution of BzOPEGOH (MW = 3400, 34 g, 10 mmole) was azeotroped under nitrogen for 2 hours and the solution was cooled to room temperature. To this solution was added 40 ml dry methylene chloride and 2.1 ml dry triethylamine (15 mmole). The solution was cooled in an ice bath and 1.2 ml of dry mesyl chloride (15 mmole) was added dropwise. The solution was stirred overnight at room temperature under nitrogen and quenched by the addition of 2 ml absolute ethanol. The mixture was evaporated under reduced pressure to remove especially solvents other than toluene, filtered, concentrated again under reduced pressure and precipitated in 100 ml of ethyl ether. The product was collected by filtration and dried in vacuo. Yield was 34 g (100%). 1H nmr (DMSOd6) :( 3.5 ( br m, PEG), 4.31 (t, OCH 2 CH 2 OMs) ,? 4.49 (s, C 6 H 5 CH 2 OPEG), 7.33 ( s + complex multi., C ₆ H ₅ CH ₂ OPEG).

b) Preparation of BzOPEGNH ₂ : BzOPEGOMs (25 g, 7.35 mmole) were dissolved in 500 ml aqueous ammonia containing 5 g ammonium chloride and the solution was stirred at room temperature for 72 hours. The solution was then extracted 3 times with methylene chloride. The organic layer was dried over sodium sulfate, filtered, concentrated under reduced pressure and the product was precipitated with 100 ml of ethyl ether. The product was collected by filtration and dried in vacuo. Yield: 23 g (92%). 1 H nmr (DMSOd 6): (3.5 (br m, PEG), 2.9 (t, CH ₂ NH ₂ ), 4.49 (s, C ₆ H ₅ CH ₂ OPEG), 7.33 (s + complex multit , C ₆ H ₅ CH ₂ OPEG).

c) Preparation of HOPEGNH ³⁺ Cl-: A solution of BzOPEGNH ₂ (46 g, 14 mmoles) in 200 ml concentrated HCl (12 M) was stirred at room temperature for 44 hours. This was then diluted to 1200 ml with water and NaCl was added to make a 15% solution. The aqueous solution was extracted three times with methylene chloride and the combined extracts were dried over sodium sulfate. The methylene chloride was concentrated under reduced pressure and the product was precipitated by the addition of ether. The product was collected by filtration and dried in vacuo at room temperature. Yield: 42 g (95%). 1H nmr (DMSOd6) :( 2.96 ( t, CH 2 N), 3.5 (br m, PEG), 4.6 (br, OH), 7.9 (br, NH 3+).

Example 2 Preparation Reaction of HOPEGOCH ₂ CO ₂ H

a) Preparation of BzOPEGOCH ₂ CO ₂ C (CH ₃ ) ₃ : BzOPEGOH (MW = 3400, 40 g, 11.7 mmole) was azeotroped with 250 ml of toluene under N ₂ . After 2 hours, the solution was cooled to room temperature. To the PEG solution was added potassium tert-butoxide (2.8 g, 23.5 mmole) dissolved in 90 ml tert-butanol and 90 ml toluene. The mixture was stirred at room temperature for 2 hours. tert- butyl bromoacetate (4ml, 26.3mmole) was added and the solution was stirred overnight at room temperature under N _2. The solution was filtered, concentrated under reduced pressure and precipitated in 300 ml of ether. The product was collected by filtration and dried in vacuo. 1H nmr (DMSOd6) :( 1.5 ( s, tBu), 3.51 (m, PEG), 3.98 (s, OCH 2 CO 2), 4.49 (s, C 6 H 5 CH 2 O ), 7.33 (s + comp.mult. , C 6 H 5 CH 2 O-).

b) Preparation of HOPEG OCH ₂ CO ₂ H: BzOPEGOCH ₂ CO ₂ C (CH ₃ ) ₃ (10 g) was dissolved in 100 ml hydrochloric acid (37%) and the solution was stirred at room temperature for 48 hours. The solution was diluted with 1 liter of distilled water and the pH was adjusted to 2 with 1N sodium hydroxide. The solution was then extracted 3 times with methylene chloride. The organic layer was dried over anhydrous sodium sulfate, filtered to remove salts, concentrated under reduced pressure, and precipitated with ether. The product was collected by filtration and dried in vacuo. Yield: 8.5 g (85%). 1H nmr (DMSOd6) :( 3.51 ( br m, PEG), 4.01 (s, -PEGOCH 2 COOH).

Example 3 Preparation of ClH ₃ N + PEGOCH ₂ CO ₂ H

a) Preparation of HOPEG ₃₄₀₀ OCH ₂ CO ₂ CH ₃ : HOPEGOCH ₂ CO ₂ H (15 g) prepared in Example 2 was dissolved in 75 ml of methanol, and 3 ml of concentrated H ₂ SO ₄ was added to the resulting solution. did. The solution was stirred at room temperature for 1.5 hours and 180 ml of 5% aqueous NaHCO ₃ was carefully added. Sodium chloride (25 g) was then added and the pH of the resulting solution was adjusted to 7 with 5% Na ₂ HPO ₄ . The solution was extracted with methylene chloride and the combined organic layers were dried over Na ₂ SO ₄ . The methylene chloride solution was then evaporated to 20 ml and the product was precipitated with 300 ml of cold ethyl ether. The product was collected by filtration and dried in vacuo at room temperature to obtain 13.5 g of product, which was shown by GPC to be 100% pure.

b) Preparation of MsOPEGOCH ₂ CO ₂ CH ₃ : HOPEG ₃₄₀₀ OCH ₂ CO ₂ CH ₃ (13.5 g) was dissolved in 400 ml of CHCl ₃ and about 200 ml of solvent was distilled. The remaining solution was cooled to room temperature and triethylamine (0.72 ml) was added followed by 0.38 ml of MsCl. The reaction mixture was stirred overnight at room temperature under N ₂ , 2 ml of ethanol was added and the resulting mixture was stirred for 15 minutes. After evaporation of the solvent under reduced pressure (55 ° C. bath), the resulting precipitate was collected by filtration and dried in vacuo at room temperature. Yield was 14 g and 1H nmr spectrum showed 100% mesylation.

_{c) H 2 NPEG 3400 CO 2} - NH 4+ _{Preparation: MsO-PEG-OCH 2 CO} 2 CH 3 and (13 g), was dissolved in H ₂ O in 70 ml, the pH was adjusted to 12. After stirring for 1.5 hours while maintaining pH 12, 250 ml of NH ₄ OH solution containing 5% NH ₄ Cl was added. The reaction mixture was then stirred for about 40 hours and NaCl was added to a concentration of about 8%. The resulting solution was extracted with CH ₂ Cl ₂ and dried over Na ₂ SO ₄ . The CH ₂ Cl ₂ layer was evaporated to about 20 ml and precipitated with about 300 ml of cold ethyl ether. The precipitated product was collected by filtration and dried in vacuo at room temperature. The yield was 12.5g. The purity was 97% for 1H nmr and 95% for GPC.

d) Preparation of Cl—H ₃ N ⁺ -PEG ₃₄₀₀ OCH ₂ CO ₂ H: H ₂ NPEGCO ₂ —NH ₄ ⁺ (12 g) was dissolved in H ₂ O (50 ml) containing 15% NaCl. The pH was adjusted to 3.0 with 1N HCl and the resulting solution was extracted with CH ₂ Cl ₂ . The CH ₂ Cl ₂ extract was dried over Na ₂ SO ₄ , evaporated to about 20 ml, the product precipitated with about 300 ml of ethyl ether and dried in vacuo at room temperature. The purity at 1 H nmr was 95%.

Preparation Example _{4 Cl-H 3 N + PEGOCH} 2 CH 2 CO 2 H

a) Preparation of HOPEG ₃₄₀₀ OCH ₂ CH ₂ CO ₂ H: BzOPEGOH (100 g) was dissolved in 100 ml of H ₂ O, 5 ml of 40% aqueous KOH solution was added to the resulting solution and the mixture was stirred for 1 hour. Stir. The solution was then cooled to 0 ° C. and 50 ml of acrylonitrile was added under argon. After stirring for 3 hours, 20% NaCl aqueous solution was added, and the pH was adjusted to 7.0 with 10% NaH ₂ PO ₄ . The solution was then extracted with CH ₂ Cl ₂ and the extract was dried over Na ₂ SO ₄ . After evaporation of the solvent under reduced pressure, the residue was dissolved in 500 ml concentrated HCl and stirred at room temperature for 60 hours. The solution was then added to a 1.2 liter aqueous solution of 240 g NaOH and NaCl was added to make an 8% solution and the pH was adjusted to 7.0. The solution was extracted with CH ₂ Cl ₂ and the extract was dried over Na ₂ SO ₄ and dried by evaporation under reduced pressure. The residue was stirred in 1.5 l 8% KOH for 20 hours and 180 ml concentrated HCl and NaCl (8%) were added. The pH was adjusted to 3.0 and the product was extracted with methylene chloride. The extract was dried over Na ₂ SO ₄ , evaporated and the product was precipitated with ethyl ether. The product was collected by filtration and dried in vacuo to give 90 g of product containing 91% yield of HOPEGOCH ₂ CH ₂ CO ₂ H by GPC.

b) Preparation of Cl − + H ₃ NPEGOCH ₂ CH ₂ CO ₂ H: HOPEGOCH by the same procedure as the conversion of HOPEGOCH ₂ CO ₂ H in Example 3 to Cl—H ₃ N + PEG ₃₄₀₀ OCH ₂ CO ₂ H ₂ CH ₂ CO ₂ H was converted to Cl − + H ₃ NPEGOCH ₂ CH ₂ CO ₂ H.

Preparation reaction of Example _{5 C 6 H 5 CH = CHCH} = CHCO 2 PEGOCH 2 CH (OC 2 H 5) 2:

a) Preparation of BzOPEGOCH ₂ CH (OC ₂ H ₅ ) ₂ : A 500 ml round bottom three-neck flask was charged with 300 ml dioxane and 14 g BzOPEGOH (MW = 3400, 0.0040 moles). Then azeotroped resultant solution with a solvent and under N ₂ for 130 ml. After cooling the solution, finely powdered NaOH (0.8 g, 0.02 moles) and ClCH ₂ CH (OC ₂ H ₅ ) ₂ (3 ml, 0.02 moles) were added under N ₂ and the resulting suspension was obtained. The suspension was stirred rapidly while refluxing for 24 hours. 30 ml of dioxane was then removed by distillation and the rapidly stirred solution was refluxed under N ₂ for an additional 24 hours. The suspension was then cooled and filtered by addition of Celite (trade name). The filtrate was evaporated under reduced pressure and 200 ml of ethyl ether was added to the residual oil. The resulting precipitate was collected by filtration and dried under reduced pressure at room temperature to obtain a tan powder (13.6 g). The powder was dissolved in CH ₂ Cl ₂ (35 ml) and reprecipitated by the addition of 500 ml cold ethyl ether. The precipitate was collected by filtration and dried in vacuo at room temperature to give 13.0 g of BzOPEGOCH ₂ CH (OC ₂ H ₅ ) ₂ as a white powder (purity 9498% according to 1H nmr). 1H nmr (DMSOd6) :( 1.11 ( t, OCH 2 CH 3); 3.51 (br m, OCH 2 CH 2 O), 4.48 (s, C 6 H 5 CH 2 O); _{4.55 (t, CH (OC 2} H 5) 2), 7.32 (s, C 6 H 5).

_{b) HOPEGO CH 2 CH (OC} 2 H 5) 2 _{Preparation: BzOPEGOCH 2 CH (OC 2 H} 5) 2 (13 g) was dissolved in 95% ethanol 150 ml, 10% of 6.5g under N ₂ Carbon supported Pd catalyst was added. The suspension was shaken under H ₂ (40 psi) for 70 hours and the suspension was filtered. The remaining catalyst was washed with 2 × 25 ml of boiled chloroform, and the washed product was combined with ethanol, filtered, and concentrated under reduced pressure to obtain a colorless and transparent oil. 400 ml of cold ethyl ether was added to the oil and the resulting precipitate was collected by filtration to give 11.3 g of HOPEGOCH ₂ CH (OC ₂ H ₅ ) ₂ as a white powder after drying under reduced pressure at room temperature (1H Purity 92% according to nmr). 1H nmr (DMSOd6) :( 1.10, (t, OCH 2 CH 3), 3.51 (br m, OCH 2 CH 2 O), 4.55, (m, HO + CH (OCH 2 CH 3) ₂ ).

_{c) C 6 H 5 CH =} CHCH = CHCO 2 PEGOCH 2 CH (OC 2 H 5) 2 Preparation: cinnamylidene acetic acid (1.7g, 0.01 moles) and thionyl chloride (3 ml, 0.04 moles) of The 50 ml hexane solution was refluxed under N ₂ for 4 hours, filtered to remove a small amount of dark solid and the filtrate was evaporated under reduced pressure. The residue was dried under reduced pressure at room temperature overnight to obtain 1.5 g of cinnamylidene acetyl chloride (melting point: 51-52 ° C.) as a yellow solid.

A toluene solution (50 ml) of HOPEG-OCH ₂ CH (OC ₂ H ₅ ) ₂ (3.4 g, 1.0 mmole) was azeotroped for 2 hours under nitrogen to remove traces of water and cooled to room temperature. Triethylamine was distilled from KOH under nitrogen and 0.28 ml (2 mmole) of fresh distillate was poured into a toluene solution of HOPEGO CH ₂ CH (OC ₂ H ₅ ) ₂ under nitrogen. Cinnamylidene acetyl chloride (C ₆ H ₅ CH═CHCH═CHCOCl) was added dropwise with rapid stirring of the resulting solution at room temperature and under nitrogen. Stirring was continued for 3 days under the same conditions, and the white precipitate was removed by filtration. The filtrate was evaporated to 20 ml under reduced pressure and 300 ml of cold ether was added. The pale yellow precipitate was collected by filtration and dried under reduced pressure to obtain 3.4 g of a pale yellow powder. The powder was dissolved in methylene chloride and extracted once with 50 ml of saturated aqueous sodium chloride solution and once with water. The organic layer was dried over sodium sulfate, evaporated to 25 ml and 300 ml of cold ether was added with stirring. The resulting precipitate was collected by _{filtration, C 6 H 5 CH = CHCH} = CHCO 2 PEG-OCH 2 CH of 3.05 g (86%) as a pale yellow powder was dried under reduced pressure at room temperature (OC ₂ H _{5) 2} got. 1 H nmr (DMSOd 6): δ 1.11 ppm (t, C H ₃ CH ₂ O—, 3.51 ppm (m, PEGOC H ₂ C H ₂ O + CH ₃ C H ₂ O); 4.20 ppm (t, − C H ₂ O ₂ C—), 4.52 ppm (t, —CH (OC ₂ H ₅ ) ₂ ), 6.11 (d, = C H ), 7.57-7.12 (comp. Purity according to C ₆ H ₅ + = C H ) nmr: 89-96%.

Example 6 Preparation Reaction of NHSO ₂ COPEGOCH ₂ CO ₂ PEGOCO ₂ NHS (NHS = N Succinimidyl):

a) Preparation of BzOPEGOCH ₂ CO ₂ H: BzOPEGOCH ₂ CO ₂ C (CH ₃ ) ₃ (20 g) was dissolved in distilled water, and the pH was adjusted to 12.0 with 1N NaOH solution. The solution was maintained at pH 12.0 for 2 hours by continuous addition of NaOH and the solution was stirred overnight. The pH of the solution was adjusted to 2.5 by addition of 1N NaOH solution and the solution was extracted 3 times with methylene chloride. The combined organic methylene chloride layers were dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the product was precipitated with ethyl ether. The product was collected by filtration and dried in vacuo at room temperature. The yield was 18 g (90%). 1H nmr (DMSOd6) :( 3.5 ( br m, PEG), 4.01 (s, PEGOCH 2 COOH), 4.49 (s, C 6 H 5 CH 2 OPEG), 7.33 (s + com, C ₆ H ₅ CH ₂ OPEG).

b) Preparation of BzOPEGOCH ₂ CO ₂ PEGOBz: A toluene solution of BzOPEGOCH ₂ CO ₂ H (MW = 3400, 3.4 g, 1 mmol) was dried azeotropically in a 100 ml round bottom flask. Thionyl chloride (2M, 4 ml, 8mmole) methylene chloride solution was injected and the mixture was stirred overnight under N _2. The solvent was concentrated on a rotary evaporator and the syrup was dried in vacuo with P ₂ O ₅ powder for about 4 hours. To the residue was added 5 ml of anhydrous methylene chloride and BzOPEGOH (MW = 3400, 2.55 g, 0.75 mmol) was dried azeotropically in toluene (20 ml). After dissolution of BzOPEGOCH ₂ COCl, freshly distilled triethylamine (0.6 ml) was added and the mixture was stirred overnight. The triethylamine salt was removed by filtration and the product was recovered by precipitation with ethyl ether. This was further purified by dissolution in water and extraction with methylene chloride. Gel permeation chromatography of the product showed that 100% BzOPEGOH was converted to the ester. The mixture was then chromatographed on an ion exchange column (DEAE Sepharose fastflow, Pharmacia) to remove BzOPEGOCH ₂ CO ₂ H to obtain pure BzOPEGOCH ₂ CO ₂ PEGOBz. Yield: 4.1 grams (80%). 1H nmr (DMSO-d6) :( 3.5 (br m, PEG), 4.14 (s, PEGOCH 2 COOPEG), 4.18 (t, PEGOCH 2 COOCH 2 CH 2 OPEG),), 4 .48 (s, ArCH ₂ O) 7.32 (s, C ₆ H ₅ ).

c) HOPEGOCH ₂ CO ₂ PEGOH Preparation _{of: BzOPEGOCH 2 CO 2 PEGOBz (MW} = 6800,2g, 0.59mmole) in 1,4-dioxane (20ml) and H _{2 (2} atm) and 1 gram Pd / C Hydrogenolysis overnight (10%). The catalyst was removed by filtration and the product was precipitated in ethyl ether after most of the solvent was precipitated on a rotary evaporator. Pure HOPEGOCH ₂ CO ₂ PEG-OH was collected by filtration and dried in vacuo at room temperature to give 1.5 g (75%) of HOPEGOCH ₂ CO ₂ PEGOH. 1H nmr (DMSOd6) :( 3.5 ( br m, PEG), 4.14 (s, PEGOCH 2 COOPEG), 4.18 (t, PEGOCH 2 COOCH 2 CH 2 OPEG).

d) Preparation of NHSO ₂ COPEGOCH ₂ CO ₂ PEGOCO ₂ NHS: HOPEGOCH ₂ CO ₂ PEGOH (2 g, 0.29 mmole) was azeotroped with 100 ml of acetonitrile and slowly cooled to room temperature. Disuccinimidyl carbonate (621 mg, 1.17 mmole) and pyridine (0.3 ml) were added to the resulting solution and the solution was stirred at room temperature overnight. The solvent was removed under reduced pressure and 35 ml of dry methylene chloride was added to the residue. The insoluble solid was removed by filtration and the filtrate was washed with sodium chloride saturated acetate buffer at pH 4.5. The organic layer was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. Ethyl ether was added and the precipitate was collected by filtration and dried in vacuo. Yield: 1.8 g (90%). 1H nmr (DMSOd6) :( 3.5 ( br m, PEG), 4.14 (s, PEGOCH 2 COOPEG), 4.18 (t, PEGOCH 2 COOCH 2 CH 2 OPEG), 4.45 (t, PEGOCH ₂ CH ₂ OCONHS), 2.81 (s, NHS).

Preparation reaction of Example _{7 CH 2 = CHCO 2 PEGOCH 2} CO 2 PEGO 2 CCH = CH 2:

_{CH 2 = CHCO 2 PEGOCH 2 CO} 2 PEGO 2 CCH = a CH _{2 Preparation: HOPEGOCH 2 CO 2 PEGOH (M.W.} = 6800,1.5g, 0.44mmole end groups) for 2 hours co of toluene 100ml Boiled dry. The solution was cooled to room temperature under N ₂ and 25 ml of methylene chloride and triethylamine (TEA, 0.56 mmole) were added. The solution was cooled in an ice bath, and a solution of acryloyl chloride (1.5 mmole) in CH ₂ Cl ₂ was added dropwise to the solution. After the addition of acryloyl chloride, the ice bath was removed and the solution was stirred at room temperature overnight. 1 ml of ethanol was added to consume excess acryloyl chloride, and methylene chloride was concentrated under reduced pressure. The salt was removed by filtration and the remaining solution was precipitated with 100 ml of ether. The product was collected by filtration and dried in vacuo. The product was dissolved in 50 ml of chloroform and sodium carbonate (1.3 g) was added. The mixture was stirred vigorously at room temperature overnight. The salt was removed by filtration and the solvent was removed under reduced pressure. The residue was dissolved in 5 ml methylene chloride and the solution was added to 100 ml ethyl ether. The resulting precipitate was collected by filtration, washed with 20 ml 2-propanol and washed with 40 ml ether. Finally, the product was vacuum dried. Yield 1.35 g (90%). ¹ H nmr (DMSO-d ₆ ): δ 3.5 (br m, PEG), 4.14 (t, PEGOC H ₂ COOPEG), 4.18 (t, PEGOCH ₂ COOC H ₂ CH ₂ OPEG), 4. _{21 (t, CH 2 = CHCOOC} H 2 CH 2 OPEG, 4H), 5.85-6.45 (m, C H 2 = C H COOPEG).

Example 8 Preparation Reaction of CH ₃ OPEGOCH ₂ CH ₂ CO ₂ PEGOH:

a) Preparation of CH ₃ OPEGOCH ₂ CH ₂ CO ₂ PEGOBz: In a 100 ml round bottom flask, a solution of CH ₃ OPEGOCH ₂ CH ₂ CO ₂ H (MW = 2000, ₂ g, 1 mmole) was dissolved in toluene and mixed for 2 hours. Boiled dry. After slowly cooling to room temperature, this solution was added thionyl chloride (3 ml, 6 mmole) in methylene chloride was stirred overnight under N _2. The solvent was then removed by rotary evaporator and the remaining syrup was vacuum dried with P ₂ O ₅ powder for about 4 hours. To the solid was added 5 ml of anhydrous methylene chloride and a toluene solution (20 ml) of azeotropically dried BzOPEGOH (MW = 3400, 2.04 g, 0.60 mmol). To the resulting solution 0.6 ml freshly distilled triethylamine was added and the solution was stirred overnight. The triethylamine salt was removed by filtration and the crude product was precipitated with ethyl ether and collected by filtration. The mixture was then purified by ion exchange chromatography (DEAE Sepharose fast flow column, Pharmacia). Pure CH ₃ OPEGOCH ₂ CH ₂ CO ₂ PEGOBz was obtained. Yield: 2.6 g (80%). lH nmr (DMSOd6) :( 3.5 ( br.mult., PEG), 3.24 (s, CH 3 OPEG), 4.48 (s, -PEGOCH 2 C 6 H 5), 7.33 (s _{+ comp.mult., PEGOCH 2 C 6} H 5), 2.55 (t, OCH 2 CH 2 CO 2 PEG), 4.13 (t, PEGCO 2 CH 2 CH 2 OPEG).

_{b) CH 3 OPEGOCH 2 CH 2} CO 2 PEGOH Preparation of: 2 g of _{CH 3 OPEGOCH 2 CH 2 CO 2} Pd / C (10% 1,4-dioxane 1 gram PEGOBz) on at H ₂ (2 atm) For overnight hydrogenolysis. The catalyst was removed by filtration, the solvent was concentrated under reduced pressure, and the solution was added to ethyl ether. The product was collected by filtration and dried in vacuo at room temperature to give 1.5 g (75%) of CH ₃ OPEGOCH ₂ CH ₂ CO ₂ PEGOH. 1H nmr (DMSOd6) :( 3.5 ( br.mult.PEG), 3.24 (s, CH 3 OPEG), 2.55 (t, OCH 2 CH 2 CO 2 PEG), 4.13 (t, PEGCO ₂ CH ₂ CH ₂ OPEG).

Example 9 Preparation Reaction of NHSO ₂ CCH ₂ CH ₂ COOPEGOCH ₂ CH ₂ CH (OC ₂ H ₅ ) ₂

a) Preparation of BzOPEGOMs: A 150 ml toluene solution of BzOPEGOH (MW = 3400, 25 g, 7.35 mmol) was azeotroped for 1 hour under nitrogen and the solution was cooled to room temperature. To the solution was added dropwise 20 ml dry methylene chloride, 1.14 ml dry triethylamine (8.16 mmol), and 0.61 ml dry mesyl chloride (7.86 mmol). The solution was stirred overnight at room temperature under nitrogen and quenched by addition of 5 ml absolute ethanol. The mixture was concentrated under reduced pressure, filtered, concentrated again under reduced pressure and precipitated in ethyl ether. The product was collected by filtration and dried in vacuo. Yield: 23 g (100%). 1H nmr (DMSOd6) :( 3.5 ( br m, PEG), 4.31 (t, OCH 2 CH 2 OMs), 4.49 (s, C 6 H 5 CH 2 OPEG), 7.33 (s _{+ comp m., C 6 H} 5 CH 2 OPEG).

_{b) BzOPEGOCH 2 CH 2 CH (} OC 2 H 5) 2 Preparation: 3,3-diethoxy-propanol (9.806g, 66.2mmol) was azeotroped for 1 hour in toluene 90ml under nitrogen. After cooling to room temperature, the solution was added to a 50 ml anhydrous toluene dispersion of sodium hydride (60% mineral oil solution, 2.75 g, 68.7 mmol). The solution was mixed for 2 hours with gentle heating to 35 ° C. and filtered. The filtrate was added to a 150 ml toluene azeotrope solution of BzOPEGOMs (23 g, 6.76 mmol). The mixture was concentrated under a nitrogen atmosphere at 125 ° C. for 20 hours, and the residue was dissolved in 80 ml of methylene chloride. The solution was filtered and the product was precipitated with 1 liter of cold isopropyl alcohol. The product was collected by filtration and dried in vacuo. The powder was dissolved in 100 ml deionized water and extracted three times with 200 ml methylene chloride. The mixture was concentrated under reduced pressure, filtered and precipitated with ethyl ether. The product was collected by filtration and dried in vacuo. Yield 19 g (100%). 1H nmr (DMSOd6) :( 1.10 ( t, -CH (OCH 2 CH 3) 2, 1.73 (q, OCH 2 CH 2 CH), 3.5 (br m, PEG), 4.49 ( _{s, C 6 H 5 CH 2} OPEG), 4.56 (m, CH (OCH 2 CH 3) 2), 7.33 (s + comp m, C 6 H 5 CH 2 OPEG).

_{c) HOPEGOCH 2 CH 2 CH (} OC 2 H 5) 2 _{Preparation: BzOPEGOCH 2 CH 2 CH (OC} 2 H 5) 2 (10g, a 2.94 mmol) was dissolved in 96% ethanol 100 ml, under a nitrogen 5.0 g of 10% carbon supported Pd catalyst was added. The suspension was shaken under H ₂ (40 psi) for 48 hours and the suspension was filtered. The residue was washed with methylene chloride. The product in the combined filtrate of methylene chloride and ethanol was concentrated under reduced pressure and filtered. The viscous solution was precipitated with cold ethyl ether and the product was collected by filtration and dried in vacuo. Yield: 15g. 1H nmr (DMSOd6) :( 1.10 ( t, CH (OCH 2 CH 3) 2, 1.72 (q, -OCH 2 CH 2 CH) .3.5 (br m, PEG), 4.55 ( _{m, CH (OCH 2 CH 3} ) 2).

_{d) HO 2 CCH 2 CH 2} CO 2 PEGOCH 2 CH 2 CH (OCH 2 CH 3) 2 _{Preparation: HOPEGOCH 2 CH 2 CH (OC} 2 H 5) 2 (3g, 0.88mmol) and BHT to (5 mg) And dissolved in 20 ml of anhydrous toluene and azeotroped at 120 ° C. for 1 hour under nitrogen. After cooling the solution to 75 ° C., pyridine (0.36 ml) and succinic anhydride (0.353 g) were added and stirred at 75 ° C. for 24 hours. The solution was concentrated under reduced pressure, filtered and precipitated into cold ethyl ether. The precipitate was collected by filtration and dried in vacuo. The powder was reconstituted in 50 ml of deionized water and 1 M sodium hydroxide was added dropwise to maintain the pH at 7.2 for 1 hour. 1N HCl was immediately added dropwise to pH 3.0 and then immediately extracted three times with 100 ml of methylene chloride. The product in the organic layer was dried over sodium sulfate, concentrated in vacuo, precipitated with cold ethyl ether, collected by filtration and dried in vacuo. Yield: 2.0 g (88%). 1H nmr (DMSOd6) :( 1.10 ( t, CH (OCH 2 CH 3) 2, 1.72 (q, OCH 2 CH 2 CH), 3.5 (br m, PEG), 4.12 (t , CO ₂ CH ₂ ), 4.55 (t, CH (OCH ₂ CH ₃ ) ₂ ).

_{e) NHSO 2 CCH 2 CH 2} CO 2 PEGOCH 2 CH 2 CH (OCH 2 CH 3) 2 _{Preparation: HO 2 CCH 2 CH 2 CO} 2 PEGOCH 2 CH 2 CH (OCH 2 CH 3) 2 (2.0g, 0.56 mmol) was dissolved in 20 ml of anhydrous methylene chloride under a nitrogen atmosphere. N-hydroxysuccinimide (105 mg, 0.91 mmol) was first added to the solution, followed by dicyclohexylcarbodiimide (174 mg, 0.84 mmol). The solution was stirred overnight at room temperature under a nitrogen atmosphere. The product was concentrated in vacuo, filtered, precipitated into cold ethyl ether, collected by filtration and dried in vacuo. Yield: 1.5 g: (99%). 1H nmr (DMSOd6) :( 1.10 ( t, CH (OCH 2 CH 3) 2, 1.72 (q, OCH 2 CH 2 CH), 2.80 (s, NHS), 3.5 (br m , PEG), 4.12 (t, CO ₂ CH ₂ ), 4.55 (t, CH (OCH ₂ CH ₃ ) ₂ ).

Example _{10 CH 2 = CHCO 2 PEGOCH 2} CO 2 CH (CH 3) CH 2 CO 2 NHS Preparation Reaction:

a) Preparation of BzOPEGOCH ₂ CO ₂ CH (CH ₃ ) CH ₂ CO ₂ H: BzOPEGOCH ₂ CO ₂ H (MW = 3400, 15 g, 4.4 mmole) was azeotroped with 60 ml of toluene under N ₂ . After 2 hours, the solution was slowly cooled to room temperature. To this solution was added thionyl chloride (18 ml, 36 mmole). The resulting solution was stirred overnight, the solvent was concentrated on a rotary evaporator, and the syrup was dried in vacuo with P ₂ O ₅ powder for about 4 hours. 3-Hydroxybutyric acid (1.45 g, 13.5 mmole) was azeotropically dried with 70 ml of 1,4-dioxane and added to dry BzOPEGOCH ₂ COCl. After dissolving the PEG acrylic chloride, 4.5 ml of dry triethylamine was injected into the system and the solution was stirred overnight. The salt was removed by filtration and the filtrate was concentrated on a rotary evaporator at 55 ° C. and dried in vacuo. The crude product was then dissolved in 100 ml distilled water and the pH of the solution was adjusted to 3.0. The aqueous layer was extracted 3 times with a total of 80 ml of methylene chloride. The organic layer was dried over sodium sulfate, filtered, concentrated on a rotary evaporator and precipitated with 100 ml of ethyl ether. The product was collected by filtration and dried in vacuo at room temperature. Yield: 14 g (93%). 1H nmr (DMSOd6) :( 3.5 ( br m, PEG), 2.58 (d, PEGCOOCH (CH 3) CH 2 COOH), 5.14 (h, PEGCOOCH (CH 3) CH 2 COOH), 1 .21 (d, PEGCOOCH (CH ₃ ) CH ₂ COOH), 4.055 (s, PEGOCH ₂ COO), 4.49 (s, C ₆ H ₅ CH ₂ OPEG), 7.33 (s + comp.multi., _{C 6 H 5 CH 2 OPEG)} .

b) Preparation of HOPEGOCH ₂ CO ₂ CH (CH ₃ ) CH ₂ CO ₂ H: 4 g of a solution of BzOPEGOCH ₂ CO ₂ OCH (CH ₃ ) CH ₂ CO ₂ H (8 g) in benzene (50 ml) at room temperature for 48 hours. Hydrogenolysis with H ₂ ( ₂ atm) over Pd / C (10%). The catalyst was removed by filtration, the solvent was concentrated and the solution was precipitated with ethyl ether. The product was collected by filtration and dried in vacuo at room temperature. Yield: 6.6 grams (83%). 1H nmr (DMSOd6) :( 3.5 ( br m, PEG), 2.51 (d, PEGCO 2 CH (CH 3) CH 2 CO 2 H), 5.16 (h, PEGCO 2 CH (CH 3) _{CH 2 CO 2 H), 1.22} (ds PEGCO 2 CH (CH 3) CH 2 CO 2 H), 4.06 (s, PEGOCHCO 2 CH (CH 3) -.

_{c) CH 2 = CHCO 2 PEGOCH} 2 CO 2 CH (CH 3) CH 2 CO 2 H Preparation _{of: HOPEGOCH 2 CO 2 CH (CH} 3) CH 2 CO 2 H (3g, the 0.88mmole), about 15ml Azeotroped with 40 ml of toluene under N ₂ until in solution. The solution was then cooled to room temperature under N ₂ and 25 ml of methylene chloride and triethylamine (1.5 mmole) were added. The solution was cooled in an ice bath and acryloyl chloride (2 mmole) was added dropwise. After the addition of acryloyl chloride, the ice bath was removed and the solution was stirred at room temperature overnight. The methylene chloride was then partially removed under reduced pressure, the salt was removed by filtration, and the filtrate was added to 100 ml of ether. The precipitated product was collected by filtration and dried in vacuo. The product was then dissolved in sodium acetate buffer (0.1M, pH 5.5), stirred for 30 minutes and extracted three times with methylene chloride. The organic layer was dried over sodium sulfate, filtered, concentrated and precipitated in 100 ml ethyl ether. The precipitate was collected by filtration and dried in vacuo at room temperature. Yield: 2.4 g (80%). 1H nmr (DMSOd6) :( 3.5 ( br m, PEG), 2.51 (d, CH 2 CO 2 H), 5.16 (h, CH (CH 3), 1.22 (d, CH ( _{CH 3)), 4.06 (s} , PEGOCH 2 CO 2 PEG), 4.21 (t, CO 2 CH 2 CH 2 O), 5.856.45 (m, CH 2 = CH).

_{d) CH 2 = CHCO 2 PEGOCH} 2 CO 2 CH (CH 3) CH 2 CO 2 NHS _{Preparation: CH 2 = CHCO 2 PEGOCH 2} CO 2 CH (CH 3) CH 2 CO 2 H (1.4g, approximately 0 .4 mmole) and N-hydroxysuccinimide (51 mg, 0.43 mmole) were dissolved in 30 ml of dry methylene chloride. To this solution was added 5 ml of a dry methylene chloride solution of dicyclohexylcarbodiimide (95 mg, 0.45 mmole). The solution was stirred overnight under nitrogen and the solvent was removed by rotary evaporator. The resulting syrup was dissolved in 10 ml of dry toluene and the insoluble solid was removed by filtration. The filtrate was added to 100 ml of dry ethyl ether and the precipitated product was collected by filtration and dried in vacuo at room temperature. Yield 0.94 g (94%). 1H nmr (DMSOd6): (3.5 (br m, PEG), 3.03.2 (m, PEGCOOCH (CH ₃ ) CH ₂ COONHS), 5.26 (h, PEGCOOCH (CH ₃ ) CH ₂ COONHS) , 1.3 (d, PEGCOOCH (CH ₃ ) CH ₂ COONHS), 4.10 (s, PEGOCH ₂ COO (CM)), 2.81 (s, NHS), 4.21 (t, CH ₂ = CHCOOCH _{2 CH 2 OPEG, 4H),} 5.856.45 (m, CH 2 = CHCOOPEG).

ＢｚＯＰＥＧＯＣＨ₃＋ＣＨ₃ＯＰＥＧＯＣＨ₃の別の合成

いずれかの経路由来の生成物が水素化分解される。

Example 11 Preparation of CH ₃ OPEGOH without HOPEGOH from a mixture of BzOPEGOH and HOPEGOH Reaction:

Alternative synthesis of BzOPEGOCH ₃ + CH ₃ OPEGOCH ₃

The product from either pathway is hydrocracked.

a) Preparation of BzOPEGOMs: BzOPEGOH (MW = 5000, 50 g, 10 mmoles) containing 6 wt% HOPEGOH was dissolved in toluene and the solution was azeotropically dried under nitrogen for 2 hours and cooled to room temperature. To this solution was added 50 ml of anhydrous methylene chloride and 2.1 ml of anhydrous triethylamine (15 mmoles). The resulting solution was cooled in an ice bath and 1.2 ml mesyl chloride (15 mmoles) was added dropwise. The solution was then stirred overnight at room temperature and quenched by the addition of 2 ml absolute ethanol. The mixture was concentrated under reduced pressure to remove 100 ml of solvent, filtered and added to 800 ml of cold ether. The precipitated product was collected by filtration and dried under reduced pressure. Yield 48.3 g (96.6%). 1H nmr (DMSOd6) :( 3.5 ( br m, PEG), 4.31 (t, OCH 2 CH 2 OMs), 4.49 (s, C 6 H 5 CH 2 OPEG), 7.33 (s + complex mul., C ₆ H ₅ CH ₂ OPEG).

b) MsOPEGOCH ₃ Preparation: 6 wt% of MsOPEGOMs (MW = 5078,45g, 8.86mmoles) was 2 hours azeotropically dried toluene solution of 250ml of BzOPEGOMs including. To the resulting solution was added a methanol solution of 25 wt% sodium methoxide (11.5 g, 53.2 mmoles, 6-fold excess) and the resulting solution was heated at 120-122 ° C. under nitrogen for 20 hours. The resulting solution was then cooled to room temperature, 2 ml of water was added and the mixture was stirred for 15 minutes. The mixture was then concentrated in vacuo to remove 100 ml of solvent, filtered and the filtrate added to 700 ml of cold ether. The precipitated product was collected by filtration and dried under reduced pressure. Yield: 42.8g. 1H nmr (DMSOd6) :( 3.24 ( s, PEG), 3.51 (br.mult., PEG), 4.49 (s, C 6 H 5 CH 2 OPEG), 7.33 (s + comp.mult , C ₆ H ₅ CH ₂ OPEG).

c) Preparation of BzOPEGOCH ₃ from BzOPEGOH: A 250 ml toluene solution of BzOPEGOH containing 6 wt% HOPEGOH (MW = 10,000,50 g, 5.0 moles) was azeotroped under nitrogen for 2 hours and the solution was brought to room temperature Cooled down. Potassium tert-butoxide (1.0 M tert-butanol solution, 25 ml, 25 mmole) was added and the mixture was stirred for 15 minutes. Methyl iodide (7.1 g, 50 mmole) was then added and the mixture was stirred in the dark at room temperature under nitrogen for 20 hours. The solvent was removed under reduced pressure and the residue was dissolved in 100 ml methylene chloride and added to 800 ml cold ether. The precipitated product was collected by filtration and dried under reduced pressure. Yield: 46.8g. 1H nmr (DMSOd6) :( 3.24 ( s, CH 3 OPEG), 3.51 (br.mult., PEG), 4.49 (s, C 6 H 5 CH 2 OPEG), 7.33 (s + comp .Mul., C ₆ H ₅ CH ₂ OPEG).

d) HOPEGOH the contained no CH ₃ OPEGOH Preparation of 6 wt% of _{CH 3 OPEGOCH 3 (40g, MW} = 10,000, was dissolved BzOPEGOCH ₃ comprising mmoles) in ethanol 400 ml, 4g of activated carbon supported Pd catalyst ( 10% Pd) was added. The mixture was hydrocracked at room temperature (800 psi). The mixture was then filtered and the solvent removed under reduced pressure. Yield: 37.1 g. 1H nmr (DMSOd6):. ( (S, CH 3 OPEG), 3.51 (br.mult, PEG), 4.58 (t, OH).

  The invention has been described in particular embodiments. However, the above is not intended to limit the invention to the illustrated embodiments, and those skilled in the art will recognize that variations can be made within the scope and purpose of the invention described in the foregoing specification. Should be done. On the contrary, the invention includes all modifications, modifications, and equivalents, which can be included within the scope and spirit of the invention as defined by the claims.

Claims (37)

Formula X- poly _{a-O-CHR 5 (CH} 2) n '' CO 2 - a process for preparing a bifunctional polymer derivative having poly b-X, free of chromatography step in the preparation,
In the above formula, poly a and poly b are water-soluble non-peptide polymers, which may be the same or different, n ″ is 0-6, R ₅ is H or alkyl. Yes, X is a functional group,
The first polymer is Ar—C (R ₁ ) (R ₂ ) O-poly b-U and the second polymer is Ar—C (R ₁ ) (R ₂ ) O-poly a-CHR ₅ (CH ₂ ) _n CO -V, R ₁ and R ₂ are H, alkyl or Ar, where Ar is phenyl, substituted phenyl, biphenyl, substituted biphenyl, polycyclic aryl, substituted polycyclic aryl, and heterocyclic aryl U and V are selected from the group consisting of Ar—C (R ₁ ) (R ₂ ) O-poly aO—CHR ₅ (CH ₂ ), wherein the first polymer reacts with the second polymer. providing a polymer that is a moiety selected to form a polymer of _n CO ₂ -poly b-OC (R ₁ ) (R ₂ ) -Ar;
It said first polymer is reacted with the second polymer, wherein the _{Ar-C (R 1) (} R 2) O- poly _{a-O-CHR 5 (CH} 2) n CO 2 - poly b-OC (R ₁₎ (forming a R ₂₎ -Ar,
And a step of converting the Ar—C (R ₁ ) (R ₂ ) O— moiety into a functional group. U is -OH, V is halogen, the method of claim 1. X is OH, The method of claim 1. X _{is, CH 2 = CR 5 CO 2} - a, where R ₅ in is H or alkyl, The method of claim 1. X is _{-O- (CH 2) n -CH (} ZR) 2, wherein the R is H or alkyl, Z is O or S, and n is 1 to 6, claim 1 The method described in 1. X is NHS-O ₂ CO-, NHS is N- succinimidyl group, The method of claim 1. Wherein R ₈ O-Poly _{a-O-CHR 5 (CH} 2) n CO 2 - a process for preparing a bifunctional polymer derivative having poly b-Y, excluding the chromatography step in the preparation,
In the above formula, poly a and poly b are water-soluble non-peptide polymers, which may be the same or different, n is 0-6, R ₅ and R ₈ are H or alkyl Y is a functional group, the first polymer is Ar—C (R ₁ ) (R 2) O-poly b-U, and the second polymer is R ₈ O-poly a-CHR ₅ (CH ₂ ) _n CO -V, R ₈ is H or alkyl, R ₁ and R ₂ are H, alkyl or Ar, Ar is phenyl, substituted phenyl, biphenyl, substituted biphenyl, polycyclic aryl, substituted polycyclic Selected from the group consisting of formula aryl and heterocyclic aryl, U and V are selected from the group consisting of R ₈ O-poly aO—CHR ₅ (CH ₂ ) _n CO, wherein the first polymer reacts with the second polymer; ₂ -poly-b-OC (R1) (R2) -Ar Providing a polymer that is a moiety selected to form a polymer;
The first polymer is reacted with the second polymer to form the R ₈ O-poly a-O—CHR ₅ (CH ₂ ) _n CO ₂ -poly b-OC (R ₁ ) (R ₂ ) —Ar A method for preparing a bifunctional polymer derivative, comprising a step of forming a polymer and a step of converting an Ar—C (R ₁ ) (R ₂ ) O— moiety into a functional group. 8. The method of claim 7 , wherein U is -OH and V is -Cl. The method of claim 7 , wherein Y is OH. Y _{is, CH 2 = CR 5 CO 2} - a, where R ₅ in is H or alkyl, The method of claim 7. Y is —O— (CH ₂ ) _n —CH (ZR) ₂ , wherein R is H or alkyl, Z is O or S, and n is 1-6. 8. The method according to 7 . A _{Ar-C (R 1) (} R 2) O-PEG-OH and HO-PEG-OH method of inhibiting the reactivity of HO-PEG-OH in a mixture of, where PEG is, -CH ₂ CH ₂ O (CH ₂ CH ₂ O) _{n ′} —CH ₂ CH ₂ —, where n ′ is 8 to 4000, Ar is phenyl, substituted phenyl, biphenyl, substituted biphenyl, polycyclic aryl And R ₁ and R ₂ are H, alkyl, or Ar as defined above, wherein R ₁ and R ₂ are selected from the group consisting of: substituted polycyclic aryl, and heterocyclic aryl;
Ar—C (R ₁ ) (R ₂ ) O-PEG-OH and HO-PEG-OH in the mixture are alkylated to give Ar—C (R ₁ ) (R ₂ ) O-PEG-OR ₉ and R Forming each of ₉ O-PEG-OR ₉ ;
Converting the Ar—C (R ₁ ) (R ₂ ) O— moiety to —OH by acid-catalyzed hydrolysis or hydrogenolysis;
A new mixture of R ₉ O-PEG-OH and inert R ₉ O-PEG-OR _{9 (} R ₉ represents an alkyl group or Ar, respectively, Ar represents phenyl, substituted phenyl, biphenyl, substituted biphenyl, Forming a method selected from the group consisting of cyclic aryl, substituted polycyclic aryl, and heterocyclic aryl. Further comprising the method of claim 12 to convert the R ₉ O-PEG-OH of the new mixture to R ₉ O-PEG-CHO. A method of conjugating a polymer to a macromolecule,
Ar—C (R ₁ ) (R ₂ ) O-poly-OH, where Ar consists of phenyl, substituted phenyl, biphenyl, substituted biphenyl, polycyclic aryl, substituted polycyclic aryl and heterocyclic aryl Selected from the group, R ₁ and R ₂ are H, alkyl or Ar, Ar is as defined above, and poly is poly (ethylene glycol), poly (alkylene oxide), poly (oxyethylated polyol) ), Poly (olefin alcohol), and poly (acryloylmorpholine), providing a polymer; and chemically converting the —OH group to produce a first functional group; Linking the first macromolecule to the first functional group; removing the Ar—CR ₁ R ₂ O— group to generate a new hydroxyl group; A method comprising the steps of converting the new hydroxyl group into a second functional group and linking a second macromolecule to the second functional group.   15. The method of claim 14, wherein the first and second macromolecules are selected from the group consisting of proteins, lipids, polysaccharides, oligonucleotides, and drugs. The method of claim 14, wherein the poly is represented by —CH ₂ CH ₂ O (CH ₂ CH ₂ O) _{n ′} —CH ₂ CH ₂ —, wherein n ′ is 8-4000.   15. The method of claim 14, wherein the poly is a copolymer of ethylene oxide and propylene oxide. The first functional group is
Mesylate, tosylate, tresylate,
_{-O- (CH 2) n -CO 2} H, where n is 1-6,
_{-O- (CH 2) n -CO 2} R 3, where n is 1 to 6, R ₃ is an alkyl group, -NHR _4, where R ₄ is H or alkyl or an amine protecting group,
_{-O- (CH 2) n -CH (} ZR 5) 2, where n is an integer from 1 to 6, Z is O or S, R ₅ is H or an alkyl group,
Ar—CH═CH—CH═CH—CO ₂ —, wherein Ar is selected from the group consisting of phenyl, substituted phenyl, biphenyl, substituted biphenyl, polycyclic aryl, substituted polycyclic aryl, and heterocyclic aryl Shows the part
-O- (CH _{2) n} -CHO, wherein n is 1-6, and,
_{-O 2 CCH 2 CH 2 CO 2} R 6, wherein R ₆ is H or N- succinimidyl group is selected from the group consisting of The method of claim 14. The second functional group is
Mesylate, tosylate, tresylate,
_{-O- (CH 2) n -CO 2} H, where n is 1-6,
_{-O- (CH 2) n -CO 2} R 3, where n is 1 to 6, R ₃ is an alkyl group, -NHR _4, where R ₄ is H or alkyl or an amine protecting group,
_{-O- (CH 2) n -CH (} ZR 5) 2, where n is an integer from 1 to 6, Z is O or S, R ₅ is H or an alkyl group,
Ar—CH═CH—CH═CH—CO ₂ —, wherein Ar is selected from the group consisting of phenyl, substituted phenyl, biphenyl, substituted biphenyl, polycyclic aryl, substituted polycyclic aryl, and heterocyclic aryl Shows the part
_{CH 2 = CH-CO 2 -} ,
-O- (CH _{2) n} -CHO, wherein n is 1-6, and,
_{-O 2 CCH 2 CH 2 CO 2} R 6, wherein R ₆ is H or N- succinimidyl group is selected from the group consisting of The method of claim 14. The first functional group is Ar—CH═CH—CH═CH—CO ₂ —, and the second functional group is —O— (CH ₂ ) _n —CH (ZR ₅ ) ₂ , where The method according to claim 14, wherein n is a number from 1 to 6, Z is O or S, and R ₅ is H or an alkyl group. The first functional group is Ar—CH═CH—CH═CH—CO ₂ —, and the second functional group is —O— (CH ₂ ) _n —CHO, where n is 1-6. 15. A method according to claim 14, wherein: The first functional group is —O— (CH ₂ ) _n —CH (ZR ₅ ) ₂ , where n is a number from 1 to 6, Z is O or S, and R ₅ is H or alkyl. a group, the second functional group is a _{-O 2 CCH 2 CH 2 CO 2} R 6, wherein R ₆ is H or N- succinimidyl group, the method of claim 14. The first functional group is CH ₂ ═CH—CO ₂ —, the second functional group is —O—CH ₂ —CO ₂ CH (CH ₃ ) CH ₂ CO—NHS, where NHS is an N-succinimidyl group. The method of claim 14, wherein A polymer derivative having an R—O—poly-R ′ structure, wherein poly represents a water-soluble non-peptide polymer, R represents an alkyl or aryl group, and R ′ represents a functional group, and R—O—poly- A polymer derivative having an OR structure, wherein the poly represents a water-soluble non-peptide polymer, and R represents an alkyl or aryl group;
A polymer composition comprising a mixture of:   The polymer composition according to claim 24, wherein the poly is polyethylene glycol.   26. The polymer composition according to claim 24 or 25, wherein R is a methyl group. R ′ is a hydroxyl group, mesylate, tosylate, tresylate,
-O-CO ₂ R ₃ , wherein R ₃ is an H alkyl group or an N-succinimidyl group,
_{-O- (CH 2) n -CO 2} R 3 -, where n is 1 to 6, R ₃ is H, alkyl or N- succinimidyl group,
-NHR _4, where R ₄ is H, an alkyl group or an amine protecting group,
_{-O- (CH 2) n -CH (} ZR 5) 2, where n is 1 to 6, Z is O or S, R ₅ is H or an alkyl group,
Ar—CH═CH—CH═CH—CO ₂ —, wherein Ar is selected from the group consisting of phenyl, substituted phenyl, biphenyl, substituted biphenyl, polycyclic aryl, substituted polycyclic aryl, and heteropolycyclic aryl Shows the part to be
-O- (CH _{2) n} -CHO, wherein n is 1-6,
_{-O 2 CCH 2 CH 2 CO 2} R 6, wherein R ₆ is H or N- succinimidyl group,
_{CH 2 = CH-CO 2 -} , and,
_{-O-CH 2 -CO 2 CH (} CH 3) CH 2 CO 2 -NHS, wherein the NHS of claim 24, 25 or 26, characterized in that it is selected from the group consisting of N- succinimidyl group Polymer composition. 28. The polymer composition according to claim 24, wherein R ′ is —O—CO ₂ R ₃ or —O— (CH ₂ ) _n —CO ₂ R ₃ . 28. The polymer composition according to claim 24, wherein R ′ is —O— (CH ₂ ) _n —CHO. The poly is polyethylene glycol, the R is a methyl group, and the R ′ is —O— (CH ₂ ) _n —CHO, where n is 1 to 6. 25. The polymer composition according to 24. R-O-poly-M1 structure, poly represents a water-soluble non-peptide polymer, R represents an alkyl or aryl group, and M ₁ is selected from the group consisting of proteins, peptides, lipids, drugs and polysaccharides A conjugated polymer;
A polymer derivative having an R-O-poly-O-R structure, wherein poly represents a water-soluble non-peptide polymer, and R represents an alkyl or aryl group;
A conjugated polymer composition comprising a mixture of: Wherein M ₁ is, conjugated polymer composition according to claim 31, characterized in that a macromolecule.   The conjugated polymer composition according to claim 31 or 32, wherein the poly is polyethylene glycol.   34. The conjugated polymer composition according to claim 31, 32 or 33, wherein R is a methyl group.   A heterobifunctional polymer derivative prepared by the method of any of claims 1-11.   24. A conjugated polymer prepared by the method of any of claims 14-23. The method according to claim 12, wherein each R ₉ is a methyl group.