CN100369953C - Water-soluble polymer alkanals - Google Patents

Abstract

The present invention is directed to alkanal derivatives of water-soluble polymers such as poly(ethylene glycol), their corresponding hydrates and acetals, and to methods for preparing and using such polymer alkanals. The polymer alkanals of the invention are prepared in high purity and exhibit storage stability.

Description

Water soluble polymer alkanals

Technical Field

The present invention relates to specific aldehyde derivatives of water-soluble polymers, and to methods of making and methods of using the same.

Background

In recent years, human therapeutics have broken through the traditional small molecule drugs into the biopharmaceutical field in the past. The discovery of novel proteins and peptides has led to the development of numerous protein and polypeptide biopharmaceuticals. Unfortunately, proteins and polypeptides, when used as therapeutic agents, often exhibit properties that make them extremely difficult to formulate or administer, such as short circulating half-life, immunogenicity, proteolytic degradation, and low solubility. One way to improve the pharmacokinetic or pharmacokinetic properties of biopharmaceuticals is to conjugate to natural or synthetic polymers such as polyethylene glycol (PEG). Covalent attachment of PEG to therapeutic proteins can result in a number of advantages, such as (i) shielding antigenic epitopes of the protein, thus reducing its reticuloendothelial clearance and recognition by the immune system, (ii) reducing proteolytic enzyme-induced degradation, and (iii) reducing renal filtration.

Much work has been devoted to the development of polymer derivatives coupled to biopharmaceuticals such as peptides, and in particular to the development of polymer derivatives coupled to reactive amino groups of proteins. Such polymer derivatives are referred to as "electrophilically activated" because they carry electrophilic groups suitable for reaction with nucleophiles such as amines. Examples of such PEG derivatives include PEG dichlorotriazine, PEG trifluoroethylsulfonate (tresylate), PEG succinimidyl carbonate, PEG carbonylimidazole, and PEG succinimidyl succinate. Unfortunately, the use of these specific reagents can lead to one or more of the following problems: undesirable side reactions occur under the reaction conditions required to effect coupling, lack of selectivity, and/or formation of weak (i.e., unstable) bonds between the biopharmaceutical and the PEG.

To overcome some of these problems, a number of new or "second generation" electrophilically activated PEGs have been developed, such as PEG propionaldehyde and PEG acetaldehyde (see, e.g., US patents 5,252,714 and 5,990,237, respectively). Aldehyde derivatives are particularly attractive reagents for coupling to proteins and other biomolecules because aldehydes react only with amines (i.e., are selective in their attachment chemistry). The above-described agents offer a number of advantages: they can be prepared without the problems of PEG diol contamination, are not limited to low molecular weight mPEG, form stable amine bonds upon coupling, and are selective. While the above-identified derivatives offer many advantages over the first generation PEG reagents, the applicant has identified some particular drawbacks of these aldehyde reagents, making them less desirable in certain circumstances.

More specifically, the applicant has recognized in a great deal of work directed to these reagents that PEG acetaldehyde is very unstable, particularly in alkaline media, and is difficult to separate due to excessive salt formation caused by neutralization of the reaction mixture. In particular, PEG acetaldehyde is very sensitive to dimerization via aldol condensation. PEG propionaldehyde, while a much better reagent in terms of its stability, has some drawbacks that make it difficult to obtain the PEG propionaldehyde product in high purity due to side reactions that occur during the preparation process.

Morespecifically, applicants have found that when PEG propionaldehyde is prepared in situ from its precursor PEG aldehyde hydrate, the product yield is typically only about 50%, due to the elimination reaction that consumes a larger portion of the acetal reagent. Although it is possible to use an improved synthetic route for the synthesis of PEG propionaldehyde, i.e., utilizing a base catalyzed reaction of 3-hydroxypropanal diethyl acetal with PEG mesylate, applicants have found that this reaction route also results in the production of relatively large amounts of unstable PEG vinyl ethers and the elimination of side reactions that produce the parent dihydroxy PEG (also known as PEG diol) that is difficult to remove. Thus, the yield of this reaction is generally less than about 85 to 90%. In addition, the use of any of the above PEG propionaldehyde syntheses would require hydrolysis of the acetal intermediate at very low pH, e.g., at pH 2 or below 2. Hydrolysis at such low pH is undesirable due to the large amount of base required to neutralize the reaction mixture to a pH suitable for conjugation. In addition, coupling of PEG propionaldehyde to proteins at basic pH is also problematic due to the formation of large amounts of acrolein (resulting from retro-Michael type side reactions) that are quite difficult to remove. The formation of these undesirable by-products requires extensive purification to obtain a pharmaceutically pure grade product.

Thus, there remains a need for improved electrophilically activated polymer derivatives for conjugation to bioactive molecules and surfaces, particularly polymer derivatives meeting the following conditions: (i) are selective in their coupling chemistry, (ii) can be prepared in high yield and with few reaction steps, (iii) are stable over a wide pH range, (iv) can be easily isolated, (iv) can be prepared in high purity (i.e., substantially free of polymer-derived impurities and by-products), and (v) overcome at leastsome of the deficiencies of known polymer derivatives, such as those described above.

Summary of the invention

The present invention provides a unique family of polymer alkanals-i.e., polymers that include at least one aldehyde functional group linked to a polymer segment by one or more intervening carbon atoms.

To a large extent, the polymer alkanals of the invention are less reactive and, therefore, more selective than prior art aldehyde derivatives. In addition, the polymer alkanals of the invention are prepared in high yield, and certain structures can be prepared in a simple single step process. Certain polymeric aldehydes described herein have greater stability at basic pH than the aldehyde derivatives known in the prior art and are formed without significant or even detectable amounts of retro-Michael type reaction byproducts. In addition, the polymer alkanals of the invention are formed from the corresponding acetal precursors by hydrolysis under mildly acidic conditions, i.e., under less severe acidic conditions than are required for PEG acetaldehyde or PEG propionaldehyde. This mild condition allows for direct in situ conjugation of the polymer derivatives of the invention to proteins, peptides, or other molecular targets without the need for intermediate separation steps. The polymer alkanals of the invention are also produced in high purity, such that they are particularly advantageously coupled to drugs and biopharmaceuticals to yield polymer conjugate compositions having a purity sufficient for administration to a mammalian subject.

More specifically, in one aspect, the present invention relates to a water-soluble polymer having the structure:

in the above structure, POLY represents a water-soluble polymer segment; x' is a linker moiety; z' is an integer from 1 to about 21; r¹Independently in each occurrence, H or an organic group selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl; and R²In each case independently H or selected from alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl.

In some cases, a polymer alkanal of the invention will possess certain characteristics. For example, according to one embodiment of the present invention, when POLY is linear: (a) the total number of carbonyl groups present in the polymer (excluding/not counting the aldehyde carbonyl carbons) is 0 or 2 or more, and when X' comprises one or more vicinities (-CH)₂CH₂O-) or (-CH)₂CH₂Except for NH-segments. When X' comprises one or more adjacent (-CH)₂CH₂O-) or (-CH)₂CH₂NH-) segments, then the total number of carbonyl groups present in the polymer is 0, 1, 2, or greater.

In yet another example, according to a further embodiment, when X' is oxygen or comprises at least one (-CH)₂CH₂An O-) segment and z' is 2 to 12, then in at least one case R¹Or R²At least one of which is an organic radical as defined aboveOr the polymer is heterobifunctional, wherein POLY comprises a reactive group at one end that is not a hydroxyl group.

The polymer alkanals provided herein can have any of a number of overall geometries or structures that will be described in greater detail herein. Preferably, when POLY is branched, then (i) R is at least one occurrence¹Or R²Is an organic group as defined above or (ii) for the case where POLY comprises a lysine residue, X' comprises- (CH)₂CH₂O)_b-, wherein b is 1 to about 20. Alternatively, when POLY is branched and has two polymer branches, then for the case where POLY includes "C-H" as the branch point, no polymer branch includes oxygen as the only heteroatom.

In general, the polymer alkanals of the invention have a structure in which z' is within one of the following ranges: z' is from about 2 to about 21, from about 3 to about 12, from about 3 to about 8, or from 3 to about 6.

In a particular embodiment of the invention, the polymer has the following structure:

wherein POLY, X', each R¹Each R²And R³As defined above. In the foregoing structure, C₁Represents the aldehyde carbonyl carbon; c₂Represents a carbon atom with a carbonyl group or C₁Adjacent or at its α th carbon, C₃To representSeparated from the carbonyl carbon by one or the carbon atom at position β, and C₄Represents a carbon atom at the gamma position. A polymer alkanal having the overall structure depicted by I-A is generally referred to herein as a polymer butyraldehyde. In a preferred variant of the above formula I-A, the linkage to C₂R of (A) to¹Is alkyl, and all other R¹And R²The variable is H. Preferably, attached to C₂R of (A) to (B)¹Is a lower alkyl group. Alternatively, the polymer alkanal corresponds to structure I-A above, where it is attached to C₃R of (A) to (B)¹Is alkyl, and all other R¹And R²The variable is H. In yet another preferred embodiment, the polymer alkanal is described by structure I-A, where it is attached to C₄R of (A) to (B)¹Is alkyl, and all other R¹And R²The variable is H.

In yet another particular embodiment, the polymer alkanal of the invention corresponds to formula I, and has an additional carbon atom in the alkylene chain when compared to structure I-A. In this embodiment (see Structure I-B herein), z' is 4, attached to C₂R of (A) to (B)¹Is alkyl, and all other R¹And R²The variable is H. Or, is connected to C₃Or C₄R of (A) to (B)¹Is alkyl, and all other R¹And R²The variable is H.

In yet another particular embodiment belonging to formula I, z' is 5, attached to C₂R of (A) to (B)¹Is alkyl, and all other R¹And R²The variable is H (see structures I-C herein). Or, is connected to C₃Or C₄Or C₅R of (A) to¹One of the variables is alkyl, and all other R¹And R²The variable is H.

In certain embodiments, a polymer alkanal according to formula I has a linker moiety generally described by the following structural formula: - (CH)₂)_c-D_e-(CH₂)_f-or- (CH)₂)_p-M_r-C(O)-K_s-(CH₂)_q-, wherein c is 0 to 8; d is 0, NH, or S; e is 0 or 1; f is 0 to 8; p is 0 to 8; m is-NH or O; k is NH or O; q is 0 to 8, and r and s are each independently 0 or 1. Specific linkers belonging to this formula are described in more detail below.

The linker moiety may optionally include a moiety corresponding to the structure- (CH)₂CH₂O)_b-or- (CH)₂CH₂NH)_g-wherein b and g are each independently 1to 20. Preferably, b and g are each independently from about 1 to about 10, and even more preferably from about 1 to about 6. TheseOligomeric linkers provide additional stability to the alkanals of the invention and also provide certain advantages in the synthetic process for making the polymers, as described in detail below.

More specifically, in certain embodiments, X' comprises a moiety corresponding to the structure: - (CH)₂)_c-D_e-(CH₂)_fP-or- (CH)₂)_p-M_r-C(O)-K_g-(CH₂)_q-T-, wherein PAnd T are each independently- (CH)₂CH₂O)_b-or- (CH)₂CH₂NH)_g(ii) a And b and g are each independently 1 to about 20. In particular examples of polymer alkanals according to formula I, X' includes-C (O) NH- (CH)₂)_1-6NH-C (O) -or-NHC (O) NH- (CH)₂)_1-6NH-C(O)-。

Preferably, the water-soluble polymer segment of the polymer alkanal of the invention is a poly (alkylene oxide), poly (vinyl pyrrolidone), poly (vinyl alcohol), polyoxazoline, poly (acryloyl morpholine), or poly (ethoxylated polyol). In a preferred embodiment, the polymer segment is a poly (alkylene oxide), preferably a poly (ethylene glycol).

According to one embodiment, the poly (ethylene glycol) segment comprises the following structure: z- (CH)₂CH₂O)_n-or Z- (CH)₂CH₂O)_n-CH₂CH₂Wherein n is from about 10 to about 4000 and Z is a moiety comprising a functional group selected from the group consisting of hydroxyl, amino, ester, carbonate, aldehyde, alkenyl, acrylate, methacrylate, acrylamide, sulfone, thiol, carboxylic acid, isocyanate, isothiocyanate, hydrazide, maleimide, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, alkoxy, benzyloxy, silane, lipid, phospholipid, biotin, and fluorescein.

Alternatively, POLY may be capped with capping moieties such as alkoxy, substituted alkoxy, alkenyloxy, substituted alkenyloxy, alkynyloxy, substituted alkynyloxy, aryloxy, and substituted aryloxy. Preferred end capping groups include methoxy, ethoxy, and benzyloxy.

Generally, POLY has a nominal average molecular weight that falls within one of the following ranges: from about 100 daltons to about 100,000 daltons, from about 1,000 daltons to about 50,000 daltons, or from about 2,000 daltons to about 30,000 daltons. Preferred molecular weights for POLY include 250 daltons, 500 daltons, 750 daltons, 1kDa, 2kDa, 5kDa, 10kDa, 15kDa, 20kDa, 30kDa, 40kDa, and 50kDa, or even greater.

In yet another specific example, a polymer alkanal of the invention comprises the structure:

wherein POLY, each X ', each (z'), each R¹Each R²And each R³As previously defined. In a particular example, POLY is linear and the polymer is homobifunctional.

As noted above, the polymer segment within the polymer alkanal can have any of a number of geometries, such as linear, branched, forked, multi-branched, or dendritic, as described in more detail below.

Particular embodiments of the invention include polymer alkanals corresponding to the following structure:

in the above structure, PEG is poly (ethylene glycol), and b and g are each independently 0 to 20, and a is 0 to 6. For the generalized structures provided in this section, the variables correspond to the ranges/values provided previously, unless otherwise specified.

In a particular example, a polymer alkanal according to the invention corresponds to the structure:

a particularly preferred polymer alkanal falling within generalized structures III-D has the following structure:

according to another aspect, the present invention is directed to a composition comprising a water-soluble polymer having the structure:

VII

wherein the composition does not contain a detectable amount of iodine-containing material or retro-Michael type reaction products. This is particularly advantageous because iodine-containing species can lead to degradation of the poly (ethylene glycol) chains due to chain scission, resulting in polymer products with high polydispersity values, e.g., greater than about 1.2. Preferably, a polymer alkanal of the invention will have a polydispersity value of less than about 1.2, preferably less than about 1.1, and even more preferably less than about 1.05. Even more preferred are polymer alkanals, such as those described herein, characterized by a polydispersity of 1.04, 1.03, or less.

According to yet another aspect, the present invention relates to a composition comprising a water-soluble polymer having the structure:

wherein POLY is a linear, terminally blocked water-soluble polymer segment and the composition is absent a detectable amount of dialdehyde polymer derivative.

Another feature of the polymer alkanals of the invention is their stability, e.g., storage stability, as compared to other known polymer aldehyde compositions. For example, provided herein are polymer alkanal compositions that exhibit 10% or less than 10% degradation (as measured by NMR) of the polymer aldehyde groups when stored for 15 days at 40 ℃.

In a preferred embodiment, the compositions of the present invention comprise a polymer alkanal corresponding to the structure:

VII-A.

in an even more preferred embodiment, POLY has the structure Z- (CH) according to structure VII-A₂CH₂O)_n-CH₂CH₂Wherein X is O, n is from about 10 to about 4000, and Z comprises a functional group, a target moiety, a reporter group, a capping group, and the like.

Another composition of the invention includes a polymer according to the structure:

in yet another aspect of the invention, provided is a hydrate or acetal form of the above-described polymer alkanal.

Acetals of the present invention include dimethyl acetal, diethyl acetal, diisopropyl acetal, dibenzyl acetal, 2, 2, 2-trichloroethyl acetal, bis (2-nitrobenzyl) acetal, S, S '-dimethyl acetal, S, S' -diethyl acetal, and dioxolane.

More specifically, the acetal or hydrated form of the polymer alkanals of the invention can be generally described by the following structure:

wherein W^aAnd W^bEach of which isIndependently is O or S, and R³And R⁴Each independently is H, or an organic group selected from methyl, ethyl, isopropyl, benzyl, 1, 1, 1-trichloroethyl, and nitrobenzyl, or when joined together is- (CH)₂)₂-or- (CH)₂)₃-, when and W^a、C₁And W^bWhen considered together, form a 5-or 6-membered ring. The polymer acetals are useful precursors for alkanals of the invention and can be hydrolyzed to give polymer alkanals.

In a particular example, provided is a water-soluble polymer having the structure:

in structure IX-A, the alkanal is a methylene group only or- (CH)₂) -an alkanal in which carbon separates the acetal or aldehyde hydrate portion of the molecule from the linker X'.

In addition, the invention relates to conjugates formed by the reaction of a bioactive agent with the polymer alkanals described herein, their hydrates and/or the corresponding alkanals.

Preferably, the conjugate corresponds to the following structure:

wherein "NH-bioactive agent" means a bioactive agent comprising an amino group.

Also forming part of the invention are hydrogels formed from the polymer alkanals described herein or their precursors.

According to yet another aspect, the present invention provides protected aldehyde reagents. These protected aldehyde reagents are particularly useful in forming the polymer alkanals of the invention and generally correspond to the following structures:

wherein G is a functional group, and the remaining variables have the values given above.

In preferred examples of structures XI-A, B, and C, G is a leaving group such as chloro, bromo, p-tolyl sulfonate, methylsulfonyl ester, triflate, and trifluoroethyl sulfonate.

Alternatively, G isselected from-OH, -NH₂-SH, and functional groups in their protected forms.

Another aspect of the invention relates to a method of making a water-soluble polymer alkanal, optionally in protected form. Briefly, the method comprises the steps of: reacting a water-soluble polymer comprising at least one reactive group Y with a protected alkanal reagent comprising from about 2 to 20 carbon atoms and a reactive group K suitable for replacement by Y, or with Y, under conditions effective to form the water-soluble polymer alkanal in protected form. In this process, the activated polymer is coupled to a reagent or precursor thereof containing the alkanal portion of the final product.

Preferably, the reaction is carried out under an inert atmosphere.

In a specific example, POLY-Y is prepared by direct polymerization.

The process may also include the additional step of hydrolyzing the protected water-soluble polymer alkanal, e.g., under acidic conditions, to form the corresponding water-soluble polymer alkanal.

In a preferred embodiment, the hydrolysis step is carried out at a pH of not less than about 3.

Protected forms of alkanal reagents useful in carrying out the process include acetals such as dimethyl acetal, diethyl acetal, diisopropyl acetal, dibenzyl acetal, 2, 2, 2-trichloroethyl acetal, bis (2-nitrobenzyl) acetal, S, S '-dimethyl acetal, and S, S' -diethyl acetal, cyclic acetals, and cyclic thioacetals.

In yet another example, the polymer alkanal produced is recovered by raising the pH of the reaction mixture to about 6.0 to 7.5, extracting the polymer alkanal into an organic solvent, and removing the solvent.

In a preferred embodiment of the method, the water-soluble polymer corresponds to the structure "POLY-Y" and the protected alkanal reagent corresponds to the structure:

preferably, POLY comprises POLY (ethylene glycol) which may or may not be capped.

In a specific embodiment of the method, POLY-Y comprises the structureZ-(CH₂CH₂O)_nH, wherein n is from about 10 to about 4000, and Z is selected from-OCH₃，-OCH₂CH₃and-OCH₂(C₆H₅). In another embodiment, POLY-Y comprises the structure Z- (CH)₂CH₂O)_nCH₂CH₂O^-M⁺Wherein POLY-Y is an alkoxide Z-CH polymerized to the end-capping by anionic ring-opening of ethylene oxide₂CH₂O^-M⁺(use of strong base to make Z-CH₂CH₂terminal-OH group of OH) by metal substitution. M⁺Represents a metal counter ion such as Na⁺，K⁺，Li⁺，Cs⁺，Rb⁺. POLY-Y prepared is suitable for reaction with a protected alkanal reagent as described above.

In another particularly preferred embodiment, the recovered alkanal is absent a detectable amount of unreacted POLY-Y (e.g., Z- (CH)₂CH₂O)_nH) And contrary toMichael type reaction products.

In yet another embodiment of the method, POLY-Y corresponds to PEG-diol, i.e., POLY-Y has the structure HO- (CH)₂CH₂O)_nH, wherein n is from about 10 to about 4000, K is selected from:

Cl，Br，

and the process results in the formation of a protected polymer alkanal having the structure:

in yet another embodiment of the method, POLY-Y comprises the structure Z- (CH)₂CH₂O)_nH, wherein n is from about 10 to about 4000, and Z is a protected hydroxyl group. In this case, a preferred embodiment of the process comprises deprotecting the protected hydroxyl group after the reaction step, optionally followed by conversion of the terminal hydroxyl group of the poly (ethylene glycol) to a functional group other than a hydroxyl group.

Exemplary functional groups include amino, ester, carbonate, aldehyde, alkenyl, acrylate, methacrylate, acrylamide, sulfone, thiol, carboxylic acid, isocyanate, isothiocyanate, maleimide, vinyl sulfone, dithiopyridine, vinyl pyridine, iodoacetamide, and silane. Preferably, the functional group is selected from the group consisting of N-hydroxysuccinimide ester, benzotriazolyl carbonate, amine, protected amine, vinyl sulfone, and maleimide.

According to yet another embodiment of the invention, "Y" in POLY-Y is an ionizable group or is a derivative of an ionizable group such as a carboxylic acid, an active ester, or an amine. Preferably, POLY-Y has been chromatographically purified prior to use in the reacting step. In a particular embodiment, POLY-Y is purified by ion exchange chromatography prior to use. Desirably, such chromatographically purified POLY-Y used in the reacting step is substantially free of detectable amounts of polymeric impurities. In one such embodiment of this method, POLY-Y is end-capped and is substantially absent detectable amounts of PEG-diol or bifunctional PEG impurities.

Alternatively, in practicing the methods of the invention, the alkanal reagent comprises the structure:

or

Wherein g and b are each independently from about 1 to about 20. For example, preferred alkanal reagents correspond to the following structure:

and the product of this reaction step has the following generalized structure:

in yet another approach to preparing a polymer alkanal of the invention, the polymer alkanal described herein is prepared by building the polymer segment directly onto an acetal precursor, e.g., by direct polymerization. More specifically, this method comprises the following steps:

(i) providing an acetal precursor comprising at least one active anionic site suitable for initiating a polymerization reaction,

(ii) contacting the acetal precursor with a reactive monomer capable of polymerization, thereby initiating polymerization of the reactive monomer onto the acetal precursor,

(iii) as a result of the contacting step, additional reactive monomers are added to the acetal precursor to form polymer chains,

(iv) allowing the contacting to continue until the desired length of polymer chain is reached, and

(v) the reaction is terminated to obtain the polymeric aldehyde precursor of the present invention.

If desired, the polymer aldehyde precursor formed can be further hydrolyzed to the corresponding alkanal as described above.

In a particular embodiment of the above process, the reactive monomer is ethylene oxide and the reactive anionic site contained within the acetal precursor is an alkoxide anion (O-), preferably accompanied by an alkali metal or other suitable counterion. The alkoxide end groups present in the acetal precursor are active for anionic ring-opening polymerization of ethylene oxide to form the polymer alkanals of the invention.

The acetal precursor generally has a structure corresponding to the formula:

wherein the variables have the values as described above, with the exception that X 'is oxygen anion or O-terminated (e.g., in its neutral form, X' is typically hydroxyl or-OH terminated, and is converted to the corresponding alkoxide in the presence of a strong base). Suitable counterions include Na⁺，K⁺，Li⁺And Cs⁺. The terminating step generally includes neutralizing the reaction, for example, by adding an acid. Optionally, the polymer segment may be capped by the addition of an alkylating agent or other agent suitable for providing a non-reactive terminus.

These and other objects and features of the present invention will become more apparent when read in conjunction with the following detailed description.

Brief description of the drawings

FIG. 1 is a general reaction scheme for preparing a polymer alkanal of the invention by anionic ring-opening polymerization of Ethylene Oxide (EO) onto an acetal precursor having anionic sites.

Detailed description of the invention

Before describing the present invention in detail, it is to be understood that this invention is not limited to particular polymers, synthetic techniques, activators, and the like, as such may vary. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

It must be noted that, as used in this specification, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polymer" includes a single polymer and two or more of the same or different polymers, reference to "a conjugate" includes a single conjugate and two or more of the same or different conjugates, reference to "an excipient" includes a single excipient and two or more of the same or different excipients, and the like.

In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

Definition of

The following terms used herein have the meanings indicated.

As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, "PEG" or "poly (ethylene glycol)" is meant to include any water-soluble poly (ethylene oxide). Typically, the PEG used in the present invention comprises one of the following structures: "- (CH)₂CH₂O)_n- "or" (CH)₂CH₂O)_n-1CH₂CH₂- ", depending on whether the terminal oxygen is replaced, for example during synthetic conversion. The variable (n) is 3-3000, and the end groups and structure of the overall PEG can vary. When the PEG further comprises a linker moiety (described in more detail below), the atoms comprising the linker (X') when covalently attached to the PEG segment do not result in the formation of (i) an oxygen-oxygen linkage (-O-O-, peroxy linkage), or (ii) a nitrogen-oxygen linkage (N-O, O-N). "PEG" means a polymer containing a majority, i.e., greater than 50%, of the subunit-CH₂CH₂A polymer of O-. PEGs for use in the present invention include PEGs having various molecular weights, structures, or geometries (e.g., branched, linear, branched PEGs, dendrimers, etc.), as described in more detail below.

"Water-soluble" or "water-soluble polymer segment" in the context of the polymers of the present invention is any segment or polymer that is soluble in water at room temperature. Typically, the water-soluble polymer or segment will transmit at least about 75%, more preferably at least about 95%, of the light transmitted by the same solution after filtering. The water-soluble polymer or segment thereof is preferably at least about 35% (by weight) soluble in water, more preferably at least about 50% (by weight) soluble in water, even more preferably about 70% (by weight) soluble in water, and even more preferably about 85% (by weight) soluble in water. Most preferably, however, the water-soluble polymer or segment is about 95% (by weight) soluble or completely soluble in water.

An "end-capping" group or "end-capped" group is an inert group present on the end of a polymer such as PEG. End capping groups are groups that do not readily undergo chemical transformation under typical synthetic reaction conditions. The end capping group is generally alkoxy, -OR, where R is an organic group of 1-20 carbons and is preferably lower alkyl (e.g., methyl, ethyl) OR benzyl. "R" may be saturated or unsaturated, and includes aryl, heteroaryl, cyclic, heterocyclic, and substituted versions of any of the foregoing. For example, the capped PEG will typically comprise the structure "RO- (CH)₂CH₂O)_n- ", whichWherein R is as defined above. Additionally, the end capping group may also advantageously comprise a detectable label_(label). When the polymer has an end-capping group comprising a detectable label, the amount or position of the polymer and/or the moiety (e.g., activator) to which the polymer is coupled can be determined by using a suitable detector. Such labels include, but are not limited to, optical brighteners, chemiluminescent agents, moieties for use in enzyme labeling, colorimetry (e.g., dyes), metal ions, radioactive moieties, and the like.

By "non-natural" with respect to the polymers of the present invention is meant that the polymer is not found in nature in its entity. The non-natural polymers of the present invention may, however, contain one or more subunits or one or more segments of subunits that are natural, so long as the entire polymer structure is not found in nature.

"molecular weight" in relation to a water-soluble polymer of the invention, such as PEG, refers to the nominal average molecular weight of the polymer, typically determined by size exclusion chromatography, light scattering techniques, or characteristic velocity determination in 1, 2, 4-trichlorobenzene. The polymers of the present invention are typically polydisperse, having low polydispersity values of less than about 1.20.

The term "reactive" or "activated" when used in conjunction with a particular functional group refers to a functional group that readily reacts with, undergoes conversion by, and is typically reactive with, an electrophilic or nucleophilic group present on another molecule. This is in contrast to those groups that require strong catalysts or harsh reaction conditions for the reaction (i.e., "non-reactive" or "inert" groups).

The term "protected" or "protecting group" refers to the presence of a moiety (i.e., a protecting group) that prevents or blocks the reaction of a particular chemically reactive functional group under certain reaction conditions. The protecting group will vary depending on the chemically reactive group being protected as well as the reaction conditions used and the presence of additional reactive or protecting groups, if any, in the molecule. Protecting GROUPS known IN the prior art can be found IN Greene, T.W. et al, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, third edition, John Wiley&Sons, Inc., New York, NY (1999).

The term "functional group" or any synonym thereof as used herein is meant to include protected forms thereof.

The term "linker moiety" is used herein to refer to an atom or atoms that are optionally used to link interconnecting moieties such as polymer segments and alkanals. The linker moieties of the invention are hydrolytically stable or may include physiologically hydrolyzable or enzymatically degradable linkages.

A "physiologically cleavable" or "hydrolyzable" or "degradable" bond is a weaker bond that reacts with water (i.e., hydrolyzes) under physiological conditions. The tendency of a bond to hydrolyze in water depends not only on the general type of linkage connecting the two central atoms but also on the substituents attached to the two central atoms. Suitable hydrolytically unstable or weak linkages include, but are not limited to, carboxylate esters, phosphate esters, anhydrides, acetals, ketals, acyloxyalkyl ethers, imines, orthoesters, peptides and oligonucleotides.

"enzymatically degradable linkage" refers to a linkage that is degraded by one or more enzymes.

By "hydrolytically stable" linkage or bond is meant a chemical bond, typically a covalent bond, which is sufficiently stable in water, i.e., does not undergo hydrolysis to any significant extent under physiological conditions over an extended period of time. Examples of hydrolytically stable linkages include, but are not limited to, the following: carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides, urethanes, and the like. Generally, a hydrolytically stable linkage is one that exhibits a rate of hydrolysis of less than about 1-2% per day under physiological conditions. The hydrolysis rates of representative chemical bonds can be found in most standard textbooks.

"alkanal" refers to the aldehyde portion (CHO) of the water-soluble polymer of the invention, including the carbonyl carbon and any additional methylene or substituted methylene (-C (R)¹)(R²) -) up to linking of alkanal moieties of the polymerThe linker moiety attached to the polymer segment isAnd (4) stopping. In naming the alkanal segment, C₁Corresponding to the carbonyl carbon. The term "alkanal" as used herein is intended to include hydrated and protected forms of the aldehyde group, as well as chalcogen (chalcogen) analogs. A particularly preferred protected form of the alkanals of the invention is an acetal.

"Total number of carbonyl groups" in reference to certain polymer alkanals of the invention is the total number of carbonyl groups contained in the polymer alkanal, not counting aldehyde carbons.

"branched" when referring to the geometry or overall structure of a polymer refers to a polymer having two or more polymer "branches". The branched polymer may have 2 polymer branches, 3 polymer branches, 4 polymer branches, 6 polymer branches, 8 polymer branches, or more. One particular type of highly branched polymer, which is a dendritic polymer or dendrimer for the purposes of the present invention, is believed to have a structure that is different from that of the branched polymer.

A "branch point" refers to a bifurcation point comprising one or more atoms where the polymer splits or branches from a linear structure into one or more additional polymer branches.

A "dendrimer" is a spherical, size monodisperse polymer having a regular branching pattern and having a number of repeating units each constituting a branch point, wherein all of the bonds emanate radially from a central focal point or core. Dendrimers exhibit certain dendritic properties such as the surrounding of the core, making them behave differently than other types of polymers.

"predominantly" or "substantially" refers to almost the total or all, e.g., 95% or more than 95%, of a given amount.

"retro-Michael type product" refers to a product obtained from the reverse of a Michael type addition reaction. The Michael addition reaction (forward direction) refers to the addition of a nucleophilic carbon species to an electron-withdrawing double bond. Typically, but not necessarily, the nucleophile is an enolate or an enamine, although the nucleophile can also be an alkoxide or amine or otherwiseFrom the reverse (or backward direction) of the Michael type addition reaction described above, i.e., an elimination reaction that results in the loss of a nucleophilic carbon species (which may but need not be an enolate or enamine) and the formation of an electron-withdrawing double bond such as α unsaturated ketone, etc., the resulting product is considered to be a retro-Michael type product₂＝CH-CHO)。

"alkyl" refers to a hydrocarbon chain, typically having a length of about 1 to 20 atoms. Such hydrocarbon chains are preferably, but not necessarily, saturated and may be branched or straight chain, although typically straight chain is preferred. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl, 3-methylpentyl, and the like. As used herein, "alkyl" when referring to three or more carbon atoms includes cycloalkyl.

"lower alkyl" means an alkyl group containing from 1 to 6 carbon atoms and may be straight or branched, for example, methyl, ethyl, n-butyl, isobutyl, tert-butyl.

"cycloalkyl" refers to a saturated or unsaturated cyclic hydrocarbon chain, including bridged, fused, spiro cyclic compounds, preferably consisting of 3 to about 12 carbon atoms, more preferably 3 to about 8 carbon atoms.

"non-interfering substituents" are groups that, when present in a molecule, are typically unreactive with other functional groups contained within the molecule.

The term "substituted" as in, for example, "substituted alkyl" refers to a moiety (e.g., alkyl) substituted with one or more non-interfering substituents such as, but not limited to: c₃-C₈Cycloalkyl groups, such as cyclopropyl, cyclobutyl, and the like; halogen, for example, fluorine, chlorine, bromine, and iodine; a cyano group; alkoxy, lower phenyl; substituted phenyl; and so on. To pairIn the substitution on the phenyl ring, the substituents may be in any orientation (i.e., ortho, meta, or para).

"alkoxy" refers to the group-O-R, where R is alkyl or substituted alkyl, preferably C₁-C₂₀Alkyl (e.g., methoxy, ethoxy, propoxy, benzyl, etc.), preferably C₁-C₇。

As used herein, "alkenyl" refers to a branched or unbranched hydrocarbon group having a length of 1 to 15 carbon atoms containing at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, and the like.

The term "alkynyl" as used herein refers to a branched or unbranched hydrocarbon group having a length of 2 to 15 carbon atoms containing at least one triple bond, such as ethynyl, n-propynyl, isopropynyl, n-butynyl, isobutynyl, octynyl, decynyl and the like.

"aryl" means one or more aromatic rings, each ring having 5 or 6 carbon atoms in the core. Aryl includes multiple aryl rings, which may be fused, as in naphthyl, or unfused, as in biphenyl. The aryl ring may also be fused or unfused with one or more cyclic hydrocarbons, heteroaryl groups, or heterocyclic rings. As used herein, "aryl" includes heteroaryl.

"heteroaryl" is an aryl group containing one to four heteroatoms (preferably N, O or S, or a combination thereof). Heteroaryl rings may also be fused with one or more cyclic hydrocarbon, heterocyclic, aryl or heteroaryl rings.

"heterocycle" or "heterocyclic" means one or more rings having 5 to 12 atoms, preferably 5 to 7 atoms, with or without unsaturation or aromatic character and having at least one ring atom other than carbon. Preferred heteroatoms includesulfur, oxygen, and nitrogen.

"substituted heteroaryl" is heteroaryl having one or more non-interfering groups as substituents.

A "substituted heterocycle" is a heterocycle having one or more side chains formed from non-interfering substituents.

"electrophile" refers to an ion, atom, or aggregate of atoms, which can be ionic, having an electrophilic center, i.e., a center that seeks electrons, capable of reacting with a nucleophile.

"nucleophile" refers to an ion, atom, or aggregate of atoms, which may be ionic, have a nucleophilic center, i.e., a center that seeks an electrophilic center, and are capable of reacting with an electrophile.

As used herein, "active agent" includes any agent, drug, compound, composition of matter or mixture that provides some pharmacological (often beneficial) effect, as indicated in vivo or in vitro. This includes foods, food supplements, nutrients, nutraceuticals, drugs, vaccines, antibodies, vitamins, and other beneficial agents. As used herein, these terms further include any physiologically or pharmacologically active substance that produces a local or systemic effect in a patient.

By "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" is meant an excipient that can be included in the compositions of the present invention without causing a significant adverse toxicological effect to the patient.

"pharmacologically effective amount," "physiologically effective amount," and "therapeutically effective amount" are used interchangeably herein to refer to the amount of PEG-activator conjugate present in a pharmaceutical formulation that is required to provide a desired level of activator and/or conjugate in the blood or in a target tissue. The precise amount depends on many factors, e.g., the particular active agent, the composition and physical characteristics of the pharmaceutical formulation, the intended patient population, patient considerations, etc., and can be readily determined by one skilled in the art based on the information provided herein and available in the relevant literature.

By "multifunctional" in reference to the polymers of the present invention is meant a polymer backbone having 3 or more than 3 functional groups contained therein, wherein the functional groups may be the same or different, and are typically present on the polymer termini. The polyfunctional polymers of the present invention typically contain about 3 to 100 functional groups, or 3 to 50 functional groups, or 3 to 25 functional groups, or 3 to 15 functional groups, or 3 to 10 functional groups, or contain 3, 4, 5, 6, 7, 8, 9, or 10 functional groups in the polymer backbone.

"bifunctional" polymer refers to a polymer having two functional groups contained therein, typically on the polymer termini. When the functional groups are the same, the polymers are said to be homobifunctional. When the functional groups are different, the polymer is said to be non-homobifunctional.

The basic or acidic reactants described herein include neutral, charged, and any corresponding salt forms thereof.

"polyolefinic alcohol" refers to a polymer comprising an olefinic polymer backbone, such as polyethylene, having a plurality of pendant hydroxyl groups attached to the polymer backbone. An exemplary polyolefinic alcohol is polyvinyl alcohol.

As used herein, "non-peptidic" means that the polymer backbone is substantially free of peptide bonds. However, the polymer may include a small number of peptide bonds spaced along the repeating monomer subunits, for example, no more than 1 peptide bond per about 50 monomer units.

As used herein, "hydrate" refers to a hydrated aldehyde formed by the addition of a water molecule to an aldehyde group, which replaces the carbonyl functional group with two hydroxyl groups. The aldehydes are in equilibrium with the corresponding hydrates n water.

The term "chalcogen analog" refers to an aldehyde analog in which an oxygen atom is replaced with another heteroatom (typically sulfur, selenium, or tellurium).

The term "patient" refers to a living organism suffering from or susceptible to a condition which can be prevented or treated by administration of a polymer of the invention (typically but not necessarily in the form of a polymer-activator conjugate) and includes humans and animals.

"optional" or "optionally" means that the subsequently described circumstance may or may not occur, such that the description includes instances where the circumstance occurs and instances where it does not.

Polymer and method of making same

In a first aspect, the present invention provides a water-soluble polymer having reactive aldehyde groups. The polymers of the present invention are unique in many respects. They are not only prepared in high yield, but are also storage stable because there are no deleterious reaction by-products that can lead to polymer chain degradation and poor polymer polydispersity. The polymers, particularly end-capped polymers, are additionally prepared in high purity, e.g., in the absence of detectable amounts of PEG-diol derived and other polymer impurity species. This feature is particularly advantageous for preparing high molecular weight end-capped PEG polymers, for example, having a molecular weight of about 30kDa or greater, where the amount of PEG diol impurity in a starting material, such as mPEG, can be about 2 wt% to 30 wt% or greater, dependingon the supplier. Moreover, in certain embodiments, the polymers of the present invention are less reactive than other known polymer aldehydes, making them more distinct in conjugation reactions and more stable in conversion, handling, and post-reaction treatments.

General structural features and alkanal moieties

In general, the polymers of the present invention have a polymer segment linked via an intermediate linker moiety to from about 1 to about 21 contiguous methylene groups or substituted methylene groups terminating at an aldehyde functionality (i.e., alkanal moiety). The generalized structure corresponding to the polymers of the present invention is provided below as structure I.

Structure I

With reference to the description above in connection with Structure I, the polymer segment is represented by POLY, the linker moiety is represented by X', and the contiguous methylene group (forming the alkylene chain) or substituted methylene group (forming the substituted alkylene chain) is represented by-C (R)¹)(R²) -represents. More specifically, in structure I, POLY is a water-soluble polymer segment; x' is a linker moiety; and z' is an integer from 1 to about 21. R¹In each case independently H or an organic radical, e.g. alkyl, substituted alkyl, alkenyl, substitutedAlkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl. R²And in each case is also independently H or an organic radical such as alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl. While many of the structures specifically provided herein are aldehydes, it is to be understood that these same structures and indeed theinvention extends generally to the corresponding aldehyde hydrates, protected forms of aldehydes, and chalcogen analogues, wherein the carbonyl oxygen in structure I is replaced by sulfur, selenium,or tellurium substitution.

The present invention provides considerable flexibility with respect to the size of the alkylene chain attached to the aldehyde group. The carbon chain length is understood to be the carbonyl carbon (C)₁) Plus the number of intermediate carbon atoms linking the carbonyl carbon to the linking group, (e.g., [ -C (R) constituting the polymer¹)(R²)]Total number of C of z' portion). The carbon chain length typically has from 3 to about 22 carbon atoms, or more typically from about 4 to about 13 carbon atoms. With reference to structure I above, this means that the value of z' is typically from 2 to about 21, or more typically from about 3 to 12. More specifically, the value of z' is most typically one of the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or greater. Most preferred are z' values in the range of from 2 to about 8. A particularly preferred polymer alkanal of the invention is one in which z' is 3.

With reference to structure I above, certain types of alkanals are particularly preferred. Such compounds include alkanals as described above having at least one organic group located on at least one "C" in the carbon chain. The organic group can be any of the organic groups described above, such as alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl, with alkyl being preferred. Typically, the alkyl group is a linear lower alkyl group or a branched lower alkyl group, such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, pentyl, and the like, with linear being generally preferred. One particularly preferred alkyl substituent is methyl.

While the alkanal portion of the polymer may have more than one organic group located on one or more "C" s in the carbon chain, one type of preferred alkanal is where only one "C" in the carbon chain is substituted with an organic group and all other R's are¹And R²An alkanal that is H. For example, regardless of the length of the alkylene chain, it is preferred that all R's therein¹And R²Alkanals in which the variable is H, with the exception of: (i) r on C-2¹Or R²Is alkyl, or (ii) R is located on C-3¹Or R²One of which is alkyl; or (iii) R is at C-4¹Or R²One of which is alkyl; or (iv) R on C-5¹Or R²One is an alkyl group, and the like. One particularly preferred type of substituent in this regard is lower alkyl such as methyl, ethyl, or propyl. The synthesis of an exemplary 2-methyl substituted alkanal of the invention, mPEG-2-methylbutyraldehyde, is described in example 17.

Focusing now on the alkanal portion of the polymer, certain preferred alkanals are shown below.

Structure I-a.

Structure I-A is an alkanal in which z' in structure I has a value of 3. This structure, regardless of C₂，C₃Or C₄Whether or not any one or more of them is substituted with an alkyl or other organic group as described above, is referred to herein as "butyraldehyde (butyraldehyde)" or as "butyraldehyde (butanal)". Exemplary polymer butyraldehydes of the invention include those in which the alkanal portion of the polymer is 2-methylbutyraldehyde, isovaleraldehyde, or 4-methylbutyraldehyde, 2-ethylbutyraldehyde, 3-ethylbutyraldehyde, or 4-ethylbutyraldehyde.

Structure I-B

Structure I-B is one where z' in structure I has a value of 4. This structure, regardless of C₂，C₃，C₄Or C₅Whether or not any one or more of (a) is substituted with an alkyl or other organic group as described above, is referred to herein as "pentanal" or as "pentanal". Exemplary polymer valeraldehydes of the invention include those where the alkanal portion of the polymer is 2-methyl valeraldehyde, 3-methyl valeraldehyde, 4-methyl valeraldehyde, or 5-methyl valeraldehyde. Additional polymer pentanals include those where the alkanal portion of the polymer is 2-ethyl pentanal, 3-ethyl pentanal, 4-ethyl pentanal, or 5-ethyl pentanal.

Structure I-C is one where z' in structure I has a value of 5. This structure, regardless of C₂，C₃，C₄，C₅Or C₆Whether or not any one or more of them is substituted with an alkyl group or other organic group as described above, is referred to herein as "hexanal". Exemplary polymer hexanals of the invention include those in which the alkanal portion of the polymer is 2-methylhexanal, 3-methylhexanal, 4-methylhexanal, 5-methylhexanal, 6-methylhexanal, 2-ethylpentanal, 3-ethylpentanal,4-ethyl pentanal, or 5-ethyl pentanal.

Additional alkanal components of the polymers of the invention include heptanal, octanal, nonanal, and the like.

Linker moieties

Referring now to the linker moiety, the linker moiety or simply "linker" of the present invention is generally represented by the variable X'. The linker moiety is that portion of the overall polymer that links the alkanal portion of the polymer to the polymer segment (described in more detail below). The linking group of the present invention may be a single atom, such as oxygen or sulfur, two atoms, or a plurality of atoms. The linking group is typicallyBut not necessarily linear. The total length of the linker is typically 1 to about 40 atoms, where length refers to the number of atoms in a single chain, not counting substituents. For example, -CH₂-considered as one atom for the entire linker length, -CH₂CH₂O-is considered to be 3 atoms long. Preferably, the linker will have a length of about 1 to about 20 atoms, or about 2 to about 15 atoms.

The linking group of the present invention can be a single functional group such as an amide, ester, urethane, or urea, or can contain methylene or other alkylene groups attached flanking either side of the single functional group. Alternatively, the linking groups may contain a combination of the same or different functional groups. In addition, the linking group of the present invention can be an alkylene chain, optionally containing one or more oxygen or sulfur atoms (i.e., an ether or thioether). Preferred linkers are those that are hydrolytically stable. When viewed within the context of structure I, a linker is one that, when considered as part of the overall polymer, does not result in an overall structure containing a peroxide linkage (-O-) or-N-O-or-O-N-linkage.

Exemplary linking groups X' are those corresponding to any one of the following structures:

-(CH₂)_c-D_e-(CH₂)_f-or- (CH)₂)_p-M_r-C(O)-K_g-(CH₂)_q-。

When referring to the linker structure above, the variable "c" is 0 to 8; "D" is O, NH, or S; the variable "e" is 0 or 1; the variable "f" is 0 to 8; the variable "p" is 0 to 8; "M" is-NH or O; "K" is NH or O; the variable "q" is 0 to 8, and the variables "r" and "s" are each independently 0 or 1.

Within the scope of structure I, the linker X' of the invention may be any of the following: -O-, -NH-, -S-, -C (O) -, C (O) -NH, NH-C (O) -NH, O-C (O) -NH, -C (S) -, -CH₂-，-CH₂-CH₂-，-CH₂-CH₂-CH₂-，-CH₂-CH₂-CH₂-CH₂-，-O-CH₂-，-CH₂-O-，-O-CH₂-CH₂-，-CH₂-O-CH₂-，-CH₂-CH₂-O-，-O-CH₂-CH₂-CH₂-，-CH₂-O-CH₂-CH₂-，-CH₂-CH₂-O-CH₂-，-CH₂-CH₂-CH₂-O-，-O-CH₂-CH₂-CH₂-CH₂-，-CH₂-O-CH₂-CH₂-CH₂-，-CH₂-CH₂-O-CH₂-CH₂-，-CH₂-CH₂-CH₂-O-CH₂-，-CH₂-CH₂-CH₂-CH₂-O-，-C(O)-NH-CH₂-，-C(O)-NH-CH₂-CH₂-，-CH₂-C(O)-NH-CH₂-，-CH₂-CH₂-C(O)-NH-，-C(O)-NH-CH₂-CH₂-CH₂-，-CH₂-C(O)-NH-CH₂-CH₂-，-CH₂-CH₂-C(O)-NH-CH₂-，-CH₂-CH₂-CH₂-C(O)-NH-，-C(O)-NH-CH₂-CH₂-CH₂-CH₂-，-CH₂-C(O)-NH-CH₂-CH₂-CH₂-，-CH₂-CH₂-C(O)-NH-CH₂-CH₂-，-CH₂-CH₂-CH₂-C(O)-NH-CH₂-，-CH₂-CH₂-CH₂-C(O)-NH-CH₂-CH₂-，-CH₂-CH₂-CH₂-CH₂-C(O)-NH-，-C(O)-O-CH₂-，-CH₂-C(O)-O-CH₂-，-CH₂-CH₂-C(O)-O-CH₂-，-C(O)-O-CH₂-CH₂-，-NH-C(O)-CH₂-，-CH₂-NH-C(O)-CH₂-，-CH₂-CH₂-NH-C(O)-CH₂-，-NH-C(O)-CH₂-CH₂-，-CH₂-NH-C(O)-CH₂-CH₂，-CH₂-CH₂-NH-C(O)-CH₂-CH₂，-C(O)-NH-CH₂-，-C(O)-NH-CH₂-CH₂-，-O-C(O)-NH-CH₂-，-O-C(O)-NH-CH₂-CH₂-，-NH-CH₂-，-NH-CH₂-CH₂-，-CH₂-NH-CH₂-，-CH₂-CH₂-NH-CH₂-，-C(O)-CH₂-，-C(O)-CH₂-CH₂-，-CH₂-C(O)-CH₂-，-CH₂-CH₂-C(O)-CH₂-，-CH₂-CH₂-C(O)-CH₂-CH₂-，-CH₂-CH₂-C(O)-，-CH₂-CH₂-CH₂-C(O)-NH-CH₂-CH₂-NH-，-CH₂-CH₂-CH₂-C(O)-NH-CH₂-CH₂-NH-C(O)-，-CH₂-CH₂-CH₂-C(O)-NH-CH₂-CH₂-NH-C(O)-CH₂-, divalent cycloalkyl radicals, -N (R)⁶)-，-CH₂-CH₂-CH₂-C(O)-NH-CH₂-CH₂-NH-C(O)-CH₂-CH₂-，-O-C(O)-NH-[CH₂]_h-(OCH₂CH₂)_j-, and combinations of any two or more of the foregoing, wherein (h) is 0 to 6, (j) is 0 to 20, R⁶Is H or an organic group selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl. Other specific linkers have the following structure: -C (O) NH- (CH)₂)_1-6NH-C (O) -, or-NHC (O) NH- (CH)₂)_1-6NH-C (O) -or-OC (O) NH- (CH)₂)_1-6NH-C (O) -, where the subscript value after each methylene group indicates the possible number of methylene groups contained in the linker structure, e.g., (CH)₂)_1-6Meaning that the linker may contain 1, 2, 3, 4, 5 or 6 methylene groups.

However, for the present disclosure, when a series of atoms is immediately adjacent to the polymer segment POLYAnd the series of atoms is another monomer such that the proposed linker moiety represents only an extension of the polymer chain, the series of atoms should not be considered as a linker moiety. For example, assume a partial structure "POLY-X' -", where in this casePOLY at (A) is defined as "CH₃O(CH₂CH₂O)_n- ", the linker moiety will not be" -CH₂CH₂O- ", since this definition merely represents an extension of the polymer. However, this is not to say that the linker of the invention cannot have one or more adjacent-CH₂CH₂An O-moiety. For example, the linker may contain one or more (-CH)₂CH₂O-) subunits flanked on one or both sides by one or more combinations of the exemplary linkers exemplified above.

That is, the linking group as described above can also include oligomers such as- (CH)₂CH₂O)_b-or- (CH)₂CH₂NH)_gWherein b and g are each independently 1 to about 20. Applicants have discovered that inclusion of such oligomers within the linker provides stability to the final polymer alkanal product by extending the distance between the aldehyde functionality and any reactive groups contained within the linker. In this way, intramolecular interactions are impaired, resulting in increased yields and improved stability of the polymer alkanal product during manufacture. Preferably, the variables b and g are from about 1 to about 10, or in some cases from about 1 to about 6. Having four vicinities- (CH) in the linking group₂CH₂The synthesis of an exemplary polymer alkanal of O) -unit is described in example 5.

Containing- (CH) is given below₂CH₂O)_b-or- (CH)₂CH₂NH)_g-further examples of specific linkers of the oligomer segment, wherein X' comprises or is defined by the formula:

-(CH₂)_c-D_e-(CH₂)_f-P-or- (CH)₂)_p-M_r-C(O)-K_s-(CH₂)_q-T-。

In the above exemplary structures, P and T are each independently- (CH)₂CH₂O)_b-or- (CH)₂CH₂NH)_g-, b and g are each independently 1 to 20, and the remaining variables are as defined above. An example of a preferred linker of this type is-O-C(O)-NH-(CH₂CH₂O)_b-，-C(O)-NH-(CH₂CH₂O)_b-，-NH-C(O)-NH-(CH₂CH₂O)_b-，-O-C(O)-NH-(CH₂CH₂NH)_g-，-C(O)-NH-(CH₂CH₂NH)_g-, and-NH-C (O) -NH- (CH)₂CH₂NH)_g-。

In some cases, for example, when POLY represents a linear polymer segment, then it is preferred that the total number of carbonyl groups present in the polymer alkanal be 0 or 2 or greater, wherein the total number of carbonyl groups excludes aldehyde carbonyl groups. However, when the linking group X' includes one or more vicinities (-CH)₂CH₂In the case of O-) segments, it is preferred that the total number of carbonyl groups present in the polymer alkanal is 0, or 1,or 2,or 3, or greater.

Referring back to structure I, in another preferred embodiment of the present invention, when X' is oxygen or comprises at least one (-CH)₂CH₂In the O-) segment, and z' is 2 to 12, then in at least one case R¹Or R²Is an organic group as defined above, or the polymer is heterobifunctional. In the case where the polymer is heterobifunctional, the polymer segment POLY preferably has a reactive group other than a hydroxyl group at one end.

Preferably, the linker is hydrolytically stable and may contain one or more of the following functional groups: an amide, a urethane, an ether, a thioether, or a urea. However, hydrolytically degradable linkages, such as carboxylate esters, phosphate esters, orthoesters, anhydrides, imines, acetals, ketals, oligonucleotides, or peptides, may also be present in the linkers of the invention. Heteroatom linkers such as O or S are particularly preferred for the oligomers containing- (CH) S as described above₂CH₂O)_b-or- (CH)₂CH₂NH)_gThis is the case for the linker of the segment.

Polymer segment/polymer for preparing polymer alkanals

As shown in the above exemplary structures, representative POLYs include POLY (alkylene glycols) such as POLY (ethylene glycol), POLY (propylene glycol) ("PPG"), copolymers of ethylene glycol and propylene glycol, POLY (alkylene alcohols), POLY (vinyl pyrrolidone), POLY (hydroxyalkyl methacrylamide), POLY (hydroxyalkyl methacrylates), POLY (sugars), POLY (α -hydroxy acids), POLY (vinyl alcohol), polyphosphazenes, polyoxazolines, POLY (N-acryloylmorpholine). POLY can be a homopolymer, an alternating copolymer, a random copolymer, a block copolymer, an alternating terpolymer, a random terpolymer, or a block terpolymer of any of the above.

The polymer segment can have any of a number of different geometries, for example, POLY can be linear, branched, or forked. Most typically, POLY is linear or branched, e.g., having 2 polymer branches. While much of the discussion herein is directed to PEG as an exemplary POLY, the discussion and structure given herein can be readily extended to include any of the water-soluble polymer segments described above.

Any water-soluble polymer having at least one reactive terminus can be used to prepare a polymer alkanal according to the invention and the invention is not limited in this respect. Although water-soluble polymers bearing only a single reactive terminus can be used, polymers bearing two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more reactive termini suitable for conversion to a polymer alkanal as described herein can be used. Advantageously, as the number of hydroxyl groups or other reactive moieties on the water-soluble polymer segment increases, the number of available sites for introducing the alkanal groups increases. Non-limiting examples of upper limits on the number of hydroxyl and/or reactive moieties associated with the water-soluble polymer segment include 500, 100, 80, 40, 20, and 10.

Referring now to the preferred POLY, PEG, "PEG" includes POLY (ethylene glycol) s in any of linear, branched, or multi-branched forms, including capped PEG, forked PEG, branched PEG, pendant PEG, and PEG's containing one or more degradable linkages separating the monomeric subunits, as described more fully below.

To prepare the polymer alkanals of the invention, one commonly used PEG starting material is free PEG, a linear polymer terminated with a hydroxyl group at each end:

HO-CH₂CH₂O-(CH₂CH₂O)_m-CH₂CH₂-OH。

the above polymer α -, omega-dihydroxypoly (ethylene glycol) can be represented in simple form as HO-PEG-OH, also referred to herein as PEG-diol, where "-PEG-" in "HO-PEG-OH" corresponds to:

-CH₂CH₂O-(CH₂CH₂O)_n-1-CH₂CH₂-

and (n) is typically from about 3 to about 4,000, or from about 3 to about 3,000, or more preferably from about 20 to about 1,000. POLY can be, for example, a hydroxyl terminated PEG such as HO-CH, see Structure I₂CH₂O-(CH₂CH₂O)_n-1-CH₂CH₂-。

Another type of PEG useful in preparing the polymer alkanals of the invention is end-capped PEG, where the PEG is end-capped with an inert end-capping group. Preferred capped PEGs are those having as a capping moiety a group such as alkoxy, substituted alkoxy, alkenyloxy, substituted alkenyloxy, alkynyloxy, substituted alkynyloxy, aryloxy, substituted aryloxy. Preferred are end-capping groups such as methoxy, ethoxy, and benzyloxy.

Referring now to structures I and I-a through I-C, POLY is or includes, in certain embodiments, a POLY (ethylene glycol) corresponding to the structure:

“Z-(CH₂CH₂O)_n"or" Z- (CH)₂CH₂O)_nCH₂CH₂-”，

Wherein n is from about 3 to about 4000, or from about 10 to about 4000, and Z is or includes a functional group such as hydroxyl, amino, ester, carbonate, aldehyde, alkenyl, acrylate, methacrylate, acrylamide, sulfone, thiol, carboxylic acid, isocyanate, isothiocyanate, hydrazide, maleimide, vinyl sulfone, dithiopyridine, vinyl pyridine, iodoacetamide, alkoxy, benzyloxy, silane, lipid, phospholipid, biotin, and fluorescein. Again, the POLY structure shownimmediately above may represent a linear polymer segment or may form part of a branched or bifurcated polymer segment. For the case where the polymer segment is branched, the POLY structure immediately above may be, for example, corresponding to a polymer branch that forms part of the overall POLY structure. Alternatively, for the case where POLY has a branched structure, the above POLY structure may correspond to, for example, a linear portion of the polymer segment before the branch point.

POLY can also correspond to a branched PEG molecule having 2 branches, 3 branches, 4 branches, 5 branches, 6 branches, 7 branches, 8 branches, or more. The branched polymers used to prepare the polymer alkanals of the invention can have from about 2 to about 300 reactive end groups. Preferred are branched polymer segments having 2 or 3 polymer branches. Exemplary branched POLY as described in US patent No.5,932,462 corresponds to the following structure:

in these expressions, R "is a non-reactive moiety, such as H, methyl or PEG, and P and Q are non-reactive linkages. In a preferred embodiment, the branched PEG polymer segment is methoxy poly (ethylene glycol) disubstituted lysine.

In the branched configurations specified above, the branched polymer segment has a single reactive site extending from the "C" branch point, the latter serving to position the reactive alkanal group via a linker. Branched PEGs such as those used in the present invention typically have less than 4 PEG branches, and more preferably, will have 2 or 3 PEG branches. Such branched PEGs offer the advantage of having a single reactive site to which is attached a larger, denser polymer cloud than their linear PEG counterparts.

One particular type of branched PEG alkanal corresponds to the structure: (MeO-PEG-)₁G-X' -alkanal in which i is equal to 2 or 3, and G is lysine or another suitable amino acid residue.

Exemplary branched polymer alkanals of the invention have the structure shown below:

in this case, the linker corresponds to C (O) -NH, optionally containing oligo- (CH) s between the amide nitrogen and the alkanal portion of the polymer₂CH₂O)_b-or- (CH)₂CH₂NH)_g-a segment as shown in the following structure V-B. Exemplary oligomer segments will have b or g values in the range of about 1 to about 40, or about 1 to about 30. Preferably b or g have a value below 20 or 20. Preferably, b or g will have one of the following values: 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In a particularly preferred embodiment, b or g is 2 to 6, and mPEG_aAnd mPEG_bAre the same or different.

Certain embodiments are preferred for polymer alkanals having a branched structure. For example, in a particular embodiment, such as when POLY in structure I is branched, then R is at least one occurrence¹Or R²Is an organic group as defined above. In another preferred embodiment, for example, where POLY comprises a lysine residue, when POLY in structure I is branched, then X' comprises- (CH)₂CH₂O)_b-, wherein b is 1 to about 20. Incidentally, when POLY has 2 polymer branches, it is preferable that the polymer branch includes oxygen as the only one for the case where POLY includes "C-H" as the branch pointA heteroatom.

Branched PEGs for use in preparing polymer alkanals of the invention additionally include those more generally represented by the general formula G (PEG)_nThose represented, wherein G is a central or core molecule from which 2 or more PEG branches extend. The variable n represents the number of PEG branches, where the polymer branches can independently be end-capped or otherwise have a reactive functional group at its end, such as an alkanal or other reactive functional group. Branched PEGs as generally represented by the above formula G (PEG)_nThose represented have from 2 polymer arms to about 300 polymer arms (i.e., n is from 2 to about 300). Branched PEGs such as these PEGs preferably have from 2 to about 25 polymer branches, more preferably from 2 to about 20 polymer branches, and more preferably from 2 to about 15 polymer branches or less. Most preferred are multi-branched polymers having 3, 4, 5, 6, 7 or 8 branches.

The preferred core molecule in the branched PEG described above is a polyol. Such polyols include aliphatic polyols having 1 to 10 carbon atoms and 1 to 10 hydroxyl groups including ethylene glycol, alkane diols, alkyl diols, alkylidene alkyl diols, alkyl cycloalkane diols, 1, 5-decahydronaphthalene diols, 4, 8-bis (hydroxymethyl) tricyclodecane, cycloalkylidene diols, dihydroxyalkanes, trihydroxyalkanes, and the like. Cycloaliphatic polyols may also be used, including linear or closed ring sugars and sugar alcohols such as mannitol, sorbitol, inositol, xylitol, quebrachitol, threitol, arabitol, erythritol, adonitol, dulcitol, facose, ribose, arabinose, xylose, lyxose, rhamnose, galactose, glucose, fructose, sorbose, mannose, pyranose, altrose, talose, tagatose (tagitose), pyranoside, sucrose, lactose, maltose, and the like. Additional aliphatic polyols include derivatives of glyceraldehyde, glucose, ribose, mannose, galactose, and related stereoisomers. Other core polyols that may be used include crown ethers, cyclodextrins, dextrins and other carbohydrates such as starch and amylose. Preferred polyols include glycerol, pentaerythritol, sorbitol, and trimethylolpropane.

Multi-branched PEGs useful in preparing polymer alkanals of the invention include multi-branched PEGs available from Nektar, Huntsville, Alabama. In a preferred embodiment, the multi-branched polymer alkanals of the invention correspond to the formula wherein the alkanal portion of the molecule is detailed

n＝0to 4

Structure XIII-a.

Alternatively, the polymer alkanal can have an overall branched structure. Examples of bifurcated PEGs correspond to the structure: PEG-Y-CH- (X' - [ C (R))¹)(R²)]_z-CHO)₂Wherein PEG is any form of PEG described herein, Y is a linking group, preferably a hydrolytically stable linkage, and another variable corresponds to the linking group and the alkanal moiety being as defined above.

Further exemplary branched PEG alkanal derivatives correspond to the formula:

PEG-Q-CH-[(CH₂)_m-X_0.1-C(O)-Y-V-alkanal]₂

structure XIII-B.

Wherein the PEG is any of the forms of PEG described herein. Q is a hydrolytically stable linkage, such as oxygen, sulfur, or-C (O) -NH-; m is 1-10, (i.e., m can be equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), but preferably, m is 1, 2, 3, or 4; x is an optional atom, and when present, is O or N; y is NH or O; and V isan optional oligomeric segment such as- (CH) as previously described₂CH₂O)_b-or (CH)₂CH₂NH)_g-. An exemplary branched PEG corresponding to "PEG" in the above structural formula is mPEG disubstituted lysine, wherein "PEG" corresponds to：

Alternatively, the PEG polymer segment used to prepare a polymer alkanal of the invention can be a PEG molecule having pendant reactive groups along the length of the PEG chain rather than at the ends, resulting in a polymer alkanal having one or more pendant alkanal groups attached to the PEG chain by a linker X'.

In addition, the polymer segments may have one or more weak or degradable linkages, such as ester linkages that are susceptible to hydrolysis. Other hydrolytically degradable linkages that may be included in POLY include carbonates, imines, phosphates, and hydrazones.

In general, the nominal average molecular weight of the water-soluble polymer segment, POLY, will vary. The nominal average molecular weight of POLY typically falls within one or more of the following ranges: from about 100 daltons to about 100,000 daltons, from about 500 daltons to about 80,000 daltons, from about 1,000 daltons to about 50,000 daltons, from about 2,000 daltons to about 25,000 daltons, from about 5,000 daltons to about 20,000 daltons. Exemplary nominal average molecular weights for the water-soluble polymer segment, POLY, include about 1,000 daltons, about 5,000 daltons, about 10,000 daltons, about 15,000 daltons, about 20,000 daltons, about 25,000 daltons, about 30,000 daltons, and about 40,000 daltons. The low molecular weight has a molecular weight of about 250, 500, 750, 1000, 2000, or 5000 daltons.

Representative Polymer alkanals

In light of the foregoing general description, the following are illustrative of some exemplary structures of preferred polymer alkanals according to the invention.

For example, a polymer alkanal of the invention, when linear, can have a homobifunctional or heterobifunctional structure according to structure II below. The homobifunctional structure according to the following structure is one in which both ends are the same.

X' and-CR¹R²Preferred values of-are as defined above. Particularly preferred structures according to structure II are those wherein POLY is POLY (ethylene glycol), and X' is-O-C (O) -NH-, -NH-C (O) -NH-, -O-C (O) -NH- (CH)₂CH₂O)_b-，-C(O)-NH-(CH₂CH₂O)_b-, or-NH-C (O) -NH- (CH)₂CH₂O)_b-, and z' is 2 to about 12, and more preferably 2, 3, 4, 5 or 6. More specifically, representative polymer alkanals of the invention include the following:

structure III-A

Structure IV-A

The structure of the compound has a structure IV-C,

wherein PEG is poly (ethylene glycol), b and g are each independently 0 to 20, a is 0-6, and the remaining variables are as previously defined. Preferred are structures wherein b and g are in the range of 1 to 8 or alternatively in the range of 1 to about 6. Although z 'is from 1 to about 21, those structures are preferred wherein z' is from 2 to 6, e.g., 3 or 4.

The following provides a schematic representation of the structure of two polymer alkanals in which the variable "a" (as shown in the structure immediately above) is zero.

Structure III-B.

Structure IV-B.

An exemplary polymer butyraldehyde has the following structure:

structure III-D.

Particularly preferred PEGs corresponding to structures III-D above include Z- (CH)₂CH₂O)_n-or

Further branching structures according to the invention are those wherein b is typically from 0 to 20, a is typically from 0 to 6, and d is 1, 2 or 3,

structure VI-A

Or

Structure VI-B

In structures VI-A and B above, the PEG can be shaped or branched. Preferably, R¹And R²In each case H, and z' is from 3 to 12, and more preferably 3, 4, 5, or 6. As an example, a polymer according to structure VI-B satisfies one of the following conditions:

PEG corresponds to the following structure:

wherein Z is a capping group or functional group as defined previously.

Another exemplary branched polymer alkanal of the invention has the structure shown below:

structure XIV

In the above embodiments, the polymer alkanal also has a bifurcated structure and is adapted to be covalently linked to two biologically active agents. The above structure contains, in the part extending from the-CH-branch point, oligo- (CH)₂CH₂O) -linking groups of segments, wherein the number of such segments in each portion is 3. The number of such oligomeric segments in the above structure can vary according to the generalized description provided above.

Other exemplary polymer alkanal structures include the following, where the variables are as previously defined:

structure VII

Structure VII-A

Structure VIII.

Preferably, any of the above structures are provided as a composition having one or more unique compositional features, as described in more detail below.

Features of the Polymer alkanal compositions formed and methods of preparation

The polymer alkanals of the invention have several advantages over previously prepared polymer aldehydes. First, polymer alkanals are produced in very high yields, due in part to the simplicity of the synthetic process used, particularly for alkanals having oxygen as part of the linker moiety. In addition, in examining the stability of butyraldehyde of the present invention, it was found that these types of alkanals are more stable under basic conditions than previously known polymeric aldehyde derivatives (e.g., propionaldehyde, acetaldehyde), and are formed without significant or even detectable amounts of retro-Michael type reaction by-products. For example, as illustrated in example 3, under basic conditions, mPEG propionaldehyde undergoes a retro-Michael type reaction, producing larger amounts of mPEG-OH and elimination product (acrolein) (after 24 hours at room temperature and pH8, almost 40% of the PEG-propionaldehyde has decomposed). In contrast, mPEG butyraldehyde is significantly more stable under basic conditions, indicating that there is essentially no decomposition of this type under the conditions used.

In addition, the butyraldehyde polymer derivative of the invention reaches equilibrium in water at about 50% hydrate, with its corresponding hydrate, much lower than the 70% hydrate equilibrium exhibited by propionaldehyde and the 100% hydrate exhibited by acetaldehyde. The lower reactivity of the polymer derivatives of the invention can also be demonstrated by the significantly greater stability of the derivatives of the invention under alkaline conditions (see example 3 below). No acrolein by-product is observed in the conjugation reaction between the aldehyde derivative of the invention and the protein or other molecule at alkaline pH. The lower reactivity of the aldehyde derivatives of the invention indicates that the derivatives of the invention are more selective, meaning that the derivatives of the invention are able to react with a particular amino group, especially the N-terminal amino group, on a protein or peptide with greater selectivity or specificity, as opposed to non-selective or random reactions between a number of amino groups on a protein or peptide molecule. In many applications, selective N-terminal attachment of the polymer backbone may better preserve protein conformation and biological activity.

In addition, the polymer alkanals of the invention are formed from the corresponding acetal precursors by hydrolysis under mildly acidic conditions, i.e., under less severe acidic conditions than are required for PEG acetaldehyde or PEG propionaldehyde. This mild condition allows direct in situ conjugation of the polymer derivatives of the invention to proteins, peptides, or other molecular targets, if desired, without theneed for an intermediate separation step. In addition, due to the synthetic method used, the polymer alkanals of the invention can also be provided in high purity, often in the absence of iodine-containing species or species capable of promoting decomposition of the polymer segment.

Due to the mildness of the synthetic methods employed, and further due to the stable nature of the structures provided herein, the polymer alkanals of the invention are additionally obtained as compositions in the substantial absence of retro-Michael type reaction products. Thus, the polymer alkanal compositions provided herein are particularly storage-stable, exhibiting a very limited amount, if any, of polymer decomposition. As an example, based on stability data collected over time, the polymer alkanals of the invention were found to exhibit less than about 10% degradation of the polymer aldehyde groups when stored for 15 days at 40 ℃. This percentage degradation rate was determined by NMR analysis. Additionally, provided are linear mPEG polymer alkanals of the invention that are substantially free of the corresponding PEG-dialkanal (i.e., the same type of bifunctional PEG impurity due to the presence of a certain amount of PEG-diol in the mPEG-OH starting material).

More specifically, the process for making the polymer alkanals of the invention will now be described, the polymer alkanals generally being prepared by reacting a water-soluble polymer having at least one reactive group Y with a protected alkanal reagent containing a reactive group K suitable for replacement by or reaction with Y under conditions effective to form the water-soluble polymer alkanal in protected form. In general, the protected alkanal reagent will have from about 2 to about 20 carbon atoms. The water-soluble polymer alkanal in protected form so formed is then typically hydrolyzed, e.g., under basic conditions, to form the desired water-soluble polymer alkanal.

Typically, the coupling reaction (i.e., the coupling of the reactive polymer and the protected alkanal reagent) is carried out in an organic solvent such as toluene, chloroform, dichloromethane, acetonitrile, acetone, dioxane, methanol, and ethanol. The reaction is preferably carried out at a temperature of from about 20 ℃ to about 150 ℃ under an inert atmosphere. The hydrolysis to form the desired alkanal is typically facilitated by an acid and is carried out at a pH of less than 7.0, with a preferred pH being from about 3 to about 6.5. The hydrolysis can be carried out at a pH of about 3, 4, 5 or 6, with lower pH around 3 being preferred.

Detailed examples of the synthetic methods given above are provided in examples 1, 2, 5, and 17.

Most typically, coupling of the polymer segment to the protected alkanal reagent is via Williamson ether synthesis. More specifically, the reactive group Y of the polymer is a hydroxyl group (which is converted to its corresponding anionic or alkoxy form in the presence of a strong base) and the reactive group K on the protected alkanal reagent acetal is a good leaving group such as a halogen (preferably Cl-or Br-) or methylsulfate (sulfonate) which can be readily replaced by an oxyanion located at the end of the polymer. The preferred linkage formed is an ether linkage (O-) linking POLY to the alkanal.

After attachment to POLY, the protected alkanal is hydrolyzed at acidic pH to form the corresponding aldehyde or alkanal functionality. As noted above, alkanal acetals such as butyraldehyde acetals hydrolyze under milder conditions than the corresponding propionaldehyde or acetaldehyde acetals. For example, when z' is 3 or greater, the alkanal acetals of the invention are capable of hydrolysis at a pH of about 3 or 4 or greater, particularly when R¹And R²In all cases H. As demonstratedin examples 2 and 4, the butyraldehyde acetal group described therein is hydrolyzed at pH3 over a period of about 3 hours. The ability to form this alkanal functionality under mildly acidic conditions is advantageous because it enables the aldehyde-functionalized polymer to be used in situ for conjugation to a protein or a protein thereof after neutralization of the polymer alkanal-containing solution to a pH suitable for conjugation (typically a pH of about 5 to about 10)It is a biologically active molecule. In contrast, the linear polymer propionaldehyde requires separation prior to joining due to the amount of base required to neutralize the low pH solution and the corresponding large amount of salt produced in this neutralization step.

In the method employed, the protected alkanal reagent is typically an acetal such as dimethyl acetal, diethyl acetal, diisopropyl acetal, dibenzyl acetal, 2, 2, 2-trichloroethyl acetal, bis (2-nitrobenzyl) acetal, S, S '-dimethyl acetal, and S, S' -diethyl acetal. Alternatively, the acetal may be a cyclic acetal or cyclic thioacetal.

More specifically, the alkanals to be protected generally have the following structure:

structures XI-D.

In this structure, z' is an integer from 1 to about 21. For the polymeric aldehydes provided above, R¹Independently each occurrence is H or an organic group selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl; and R²And, in each case, independently is H or an organic group selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl. Wherein W^aAnd W^bEach independently is O or S, and R³And R⁴Each independently is H, or an organic group selected from methyl, ethyl, isopropyl, benzyl, 1, 1, 1-trichloroethyl, and nitrobenzyl, or when joined together is- (CH)₂)₂-or- (CH)₂)₃-, when and W^a、C₁And W^bWhen considered together, form a 5-or 6-membered ring.

Preferably, K is one of the following reactive groups:

Cl，Br，

in this process, the polymer alkanal in protected form is typically formed in greater than about 85% yield, and even more preferably in greater than about 90-95% yield.

After hydrolysis to obtain the desired aldehyde-functionalized polymer, the product may be isolated, if desired, by neutralizing the reaction mixture, e.g., raising the pH to about 6.0 to about 7.5, followed by extraction of the polymer alkanal into an organic solvent, and removal of the solvent, e.g., by rotary evaporation, freeze-drying, or distillation.

Due to the simplicity of this process, which uses neither a direct oxidation process nor an iodine-containing material to obtain the desired aldehyde functionality, the resulting product is of high purity, exhibits improved storage stability compared to other known polymeric aldehydes, and has low polydispersity (polydispersity of less than about 1.5, preferably less than about 1.2, and typically less than about 1.1, 1.08, 1.05, 1.04, and 1.3). Thus polymers with polydispersities as low as 1.03, 1.02 and 1.01 were prepared.

The isolated polymer alkanal of the invention preferably has a purity of at least about 95% based on polymer contaminants.

Examples 1 and 2 illustrate the formation of mPEG polymer alkanals. In the case where the polymeric starting material is a PEG-diol, one of the PEG hydroxyl groups is typically protected prior to reaction with the protected alkanal reagent, and subsequently deprotected after coupling. All exemplary polymer protection alkanals formed are represented by structures XI-E below.

If desired, the hydroxyl end of the PEG can be branched to a functional group to give the protected alkanal either homobifunctional or heterobifunctional. Suitable functional groups include amino, ester, carbonate, aldehyde, alkenyl, acrylate, methacrylate, acrylamide, sulfone, thiol, carboxylic acid, isocyanate, hydrazide, isothiocyanate, maleimide, vinyl sulfone, dithiopyridine, vinyl pyridine, iodoacetamide, and silane. Preferred are functional groups such as N-hydroxysuccinimidyl ester, benzotriazolyl carbonate, amine, vinyl sulfone, and maleimide, N-succinimidyl carbonate, hydrazide, succinimidyl propionate, succinimidyl butyrate, succinimidyl succinate, succinimidyl ester, glycidyl ether, oxycarbonylimidazole, p-nitrophenyl carbonate, aldehyde, o-pyridyl (ortho-pyridyl) -disulfide, and acryloyl.

Other representative alkanal reagents are described by the following structures:

structures XI-F

Structures XI-G.

In yet another approach, the polymer alkanals of the invention are advantageously prepared from POLY-Y purified by chromatographic separation. In this way, polymer impurities, if present, and especially bifunctional impurities derived from PEG-diol, are removed, resulting in the formation of a very pure polymer alkanal product as previously described. This process is illustrated in example 5. An overview of the overall synthetic method used, its advantages, its applicability to the general method described herein, and the details of the reactions carried out are provided in example 5.

Although any chromatographic method can be used, ion exchange chromatography is particularly preferred, wherein Y in POLY-Y is an ionizable group or is a derivative of an ionizable group such as carboxylic acids, active esters, amines, and the like.

Exemplary polymer alkanal acetals of the invention can have any of the following structures, where the variables have been previously described:

or

Structure IX-A

In yet another approach to preparing a polymer alkanal of the invention, the polymer alkanal can be prepared by directly building the polymer segment, POLY, onto an acetal precursor, e.g., by direct polymerization. More specifically, in this process, an acetal precursor having at least one active anionic site suitable for initiating polymerization is first provided. The anionic sites of the acetal precursor are then contacted with a reactive monomer capable of polymerization, thereby initiating polymerization of the reactive monomer onto the acetal precursor, as a result of the contacting step, additional reactive monomer is added to the acetal precursor to form a polymer chain, the contacting is continued until the desired polymer chain length is reached, and the reaction is subsequently terminated to provide the polymeric aldehyde precursor of the invention.

If desired, the polymer aldehyde precursor formed can be further hydrolyzed to the corresponding alkanal as described above. Most preferably, the reactive monomer is ethylene oxide and the reactive anionic site contained within the acetal precursor is an alkoxide anion (O-), preferably accompanied by an alkali metal or other suitable counterion. The alkoxide end groups present in the acetal precursor are active for anionic ring-opening polymerization of ethylene oxide to form the polymer alkanals of the invention.

More specifically, the acetal precursor generally has a structure corresponding to the formula:

wherein the variables have the values stated above, with the exception that X' is an oxyanion or O^-(e.g., in its neutral form X' typically terminates in a hydroxyl or-OH group, and in the presence of a strong baseTo the corresponding alkoxide). Suitable counterions include Na⁺，K⁺，Li⁺And Cs⁺. The terminating step generally includes neutralizing the reaction, for example, by adding an acid. Optionally, the polymer segment may be capped by the addition of an alkylating agent or other agent suitable for providing a non-reactive terminus.

In a specific embodiment of the above method, POLY-Y corresponds to the structure Z- (CH)₂CH₂O)_nH, wherein n is from about 10 to about 4000, and Z is selected from-OCH₃，-OCH₂CH₃and-OCH₂(C₆H₅). In another embodiment, POLY-Y corresponds to the structure Z- (CH)₂CH₂O)nCH₂CH₂O^-M⁺Wherein POLY-Y is polymerized to a capped alkoxide such as Z-CH by anionic ring opening polymerization of ethylene oxide₂CH₂O^-M⁺(reaction of Z-CH with a strong base₂CH₂terminal-OH group of OH) by metal substitution. M⁺Represents a metal counter ion such as Na⁺，K⁺，Li⁺，Cs⁺，Rb⁺. POLY-Y prepared is suitable for reaction with a protected alkanal reagent as described above.

A generalized reaction scheme summarizing this approach is provided herein as figure 1 and the conditions under which the reaction or series of reactions is carried out are provided in example 15.

Storage of polymer alkanal reagents

Preferably, the polymer alkanals of the invention are stored under an inert atmosphere, such as under argon or under nitrogen, since the aldehyde function is capable of reacting with atmospheric oxygen to produce the corresponding carboxylic acid. It is also preferred to minimize exposure of the polymer alkanals of the invention to moisture due to the potential for reaction of the aldehyde portion of the molecule with water (e.g., exposure to moisture to form the corresponding hydrate). Thus, preferred storage conditions are under dry argon or other dry inert gas at temperatures below about-15 ℃. Storage under low temperature conditions will reduce the rate of hydrolysis of the polymer aldehyde to the corresponding hydrate form. In addition, where the polymer segment of the polymer alkanal is PEG, the PEG portion of the alkanal reacts slowly with oxygen to form peroxide along the PEG portion of the molecule. Peroxide formation can ultimately lead to chain scission, thus increasing the polydispersity of the PEG alkanal reagent. In view of the above, it is further preferred that the PEG alkanals of the invention are stored in the dark.

Bioactive conjugates

Chemical nature of coupling

Conjugation to proteins-random and N-terminal selectivity

The above-described polymer alkanals can be used to attach to a biologically active agent or surface bearing at least one amino group for reaction. Typically, the PEG aldehydes of the present invention are coupled to amino groups by reductive amination, resulting in the formation of secondary amine linkages between the polymer segment and the surface or bioactive agent. In the context of the conjugation of a polymer alkanal of the invention to an amino group-containing bioactive agent or surface, the polymer alkanal reacts with the target amino group-containing molecule in a suitable solvent to form the corresponding imine-linked intermediate, which is then reduced to form a secondary amine linkage between the polymer and the bioactive agent or surface. The reduction of the imine to the corresponding amine is achieved by addition of a reducing agent. Exemplary reducing agents include sodium cyanoborohydride, sodium borohydride, lithium aluminum hydride, and the like.

In general, certain polymer alkanals of the invention can be used to selectively target modifications at the N-terminus, which conditions can differentiate the reactivity of α amine at the N-terminal amino acid.

To facilitate N-terminal modification, a pH of about 5 to 5.5 is most preferred, since selective N-terminal modification is believed to be facilitated due to the difference in pKa values of the amino group of the N-terminal amino acid and the amino group of lysine. Generally, conditions favorable for N-terminal selectivity include a pH of less than 7, and typically not less than about 4. The most advantageous pH to promote N-terminal selectivity can be determined by one skilled in the art and depends on the particular protein to be modified. Suitable buffers for conjugation include sodium phosphate, sodium acetate, sodium carbonate, and Phosphate Buffered Saline (PBS). Typically, the polymer alkanal is added to the protein-containing solution in an equimolar amount or molar excess relative to the target protein. Polymer alkanals are added to the target protein in a molar ratio of about 1: 1 (polymer alkanal: protein), 1.5: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 8: 1, or 10: 1. The molar excess of PEG-alkanal relative to the target protein is typically between about 2 and 5. The reductive amination reaction is typically carried out at a temperature at or below about room temperature (25 c), although the temperature may be about-15 c to 100 c, more preferably about 4 c to 37 c, for about-one to twenty-four hours. The reducing agent is also typically added in excess, that is, in an amount of about 2-fold to 30-fold molar excess relative to the polymer-protein conjugate. It is preferred to add the reducing agent in a 10-fold to 20-fold molar excess over the polymer-protein conjugate. The exact reaction time is determined by detecting the progress of the reaction over time. The progress of the reaction is typically detected by taking aliquots from the reaction mixture at various time points and analyzing by SDS-PAGE or MALDI-TOF mass spectrometry or any other suitable analytical method. The resulting pegylated conjugates are further characterized by using analytical methods such as MALDI, capillary electrophoresis, gel electrophoresis, and/or chromatography.

More specifically, for coupling aldehyde polymer derivatives to proteins or peptides, a number of different methods may be used. One method (i.e., the random pegylation method) is to covalently attach PEG to any number of lysine residues that are available on a surface. To carry out such reactions, proteins or peptides (such as biomolecules of those exemplary nature provided below) are typically reacted with a polymer alkanal of the invention in an amine-free buffer at a mild pH of generally about 5-8. (amine-free buffers are preferred because the amino groups in the buffer compete with the protein amino groups for coupling to the polymer alkanal). Suitable amine-free buffers are selected to have a suitable pK so that the desired pH range is available for the conjugation chemistry. The coupling reaction is typically carried out between minutes and hours (e.g., from 5 minutes to 24 hours or more), with coupling taking place on average between about 0.2 and 4 hours to form an imine-coupled conjugate. Any of a number of suitable reducing agents, as described above (e.g., sodium cyanoborohydride), are then added to the reaction mixture. The resulting mixture is then subjected to low to ambient temperature conditions, e.g., 4 ℃ to 37 ℃ for about one hour to 48 hours. Preferably, the reduction reaction is completed in less than about 24 hours. Random coupling is promoted at a pH of around 7 to 7.5, while coupling at the N-terminus is promoted at a low pH (e.g., around 5.5).

To increase the degree of modification, i.e., to facilitate an increase in the amount of PEG covalently attached to the target molecule at the effective site, any one or more of the above conditions (e.g., molar ratio of polymer alkanal to protein or peptide, temperature, reaction time, pH, etc.) can be increased, either alone or in combination. Regardless of the molecular weight of the PEG alkanal used, the resulting product mixture is preferably, but must be, purified to isolate excess reagent, non-PEGylated protein (or any target molecule), multi-PEGylated conjugate, and free or unreacted PEG alkanal.

Random pegylation of exemplary proteins has been provided in examples 4 and 6. Site-selective pegylation of exemplary proteins is described in examples 7 to 13.

Characterization/optional isolation of PEG-MER (MER)

Optionally, conjugates produced by reacting the PEG aldehydes of the present invention with a bioactive agent are purified to obtain/isolate different pegylated species. Alternatively, for lower molecular weight PEGs, for example having a molecular weight of less than about 20 kilodaltons, preferably less than or equal to about 10 kilodaltons, more preferably, the product mixture can be purified to obtain a distribution around some number of PEG/protein molecules. For example, the product mixture can be purified to obtain an average of one to five PEG/protein values, typically about 3 PEG/protein values. The strategy for purification of the final conjugation reaction mixture will depend on a number of factors-the molecular weight of the polymer used, the specific protein, the desired dosage regimen, and the residual activity and in vivo performance of each conjugation material.

If desired, PEG conjugates having different molecular weights can be separated using gel filtration chromatography. That is, gel filtration chromatography is used to fractionate different PEG-blocks (1-block, 2-block, 3-block, etc.) based on their different molecular weights (where the difference mainly corresponds to the average molecular weight of the PEG chain). For example, in an exemplary reaction in which a 100kDa protein is randomly conjugated to a PEG alkanal having a molecular weight of about 20kDa, the resulting reaction mixture is likely to contain unmodified protein (Mw 100kDa), mono-PEGylated protein (Mw 120kDa), di-PEGylated protein (Mw 140kDa), and the like. While this method can be used to separate PEG conjugates having different molecular weights, this method is generally inefficient for separating positional isomers having different pegylation sites within the protein. For example, gel filtration chromatography can be used to separate one from another from a mixture of PEG 1-mer, 2-mer, 3-mer, etc., although each of the recovered PEG-mer compositions can contain PEG attached to a different reactive amino group (e.g., lysine residue) within the protein.

Gel filtration columns suitable for performing this type of separation include Superdex, commercially available from Amersham biosciences^TMAnd Sephadex^TMAnd (3) a column. The selection of a particular column will depend on the desired fractionation range. Elution is generally carried out by using a non-amine type buffer such as phosphate, acetate and the like. The collected fractions can be analyzed by a number of different methods, such as (i) OD at 280nm for protein content, (ii) BSA protein analysis, (iii) iodine assay for PEG content (Sims g.e.c. et al, anal. biochem, 107, 60-63, 1980), or (iv) by running SDS PAGE gels followed by staining with barium iodide.

The separation of positional isomers is carried out by reversed phase chromatography using an RP-HPLC C18 column (Amersham Biosciences or Vydac) or by ion exchange chromatography using an ion exchange column such as a Sepharose ion exchange column commercially available from Amersham Biosciences. Either method can be used to separate PEG-biomolecule isomers (positional isomers) having the same molecular weight.

Storage of

Depending on the intended use of the formed PEG-conjugate, after conjugation and optional additional separation steps, the conjugate mixture may be concentrated, sterile filtered, and stored at low temperatures of about-20 ℃ to about-80 ℃. Alternatively, the conjugate may be lyophilized, with or without residual buffer, and stored as a lyophilized powder. In some cases, it is preferred to exchange the buffer used for conjugation, such as sodium acetate, for a volatile buffer, such as ammonium carbonate or ammonium acetate, which is easily removed during the freeze-drying process, so that the lyophilized protein conjugate powder formulation is free of residual buffer. Alternatively, the buffer exchange step may be applied using the formulation buffer, and thus the lyophilized conjugate is in a form suitable for reconstitution into the formulation buffer and eventual administration to a mammal.

Small molecule conjugation

Conjugation of PEG-alkanals of the invention to small molecules such as amphotericin B is generally performed as described in example 14, although the exact reaction conditions will vary depending on the small molecule that is desired to be modified. Typically, the conjugation is performed by using a slight molar excess of the PEG reagent relative to the small molecule, e.g., about 1.2-1.5-fold to about 5-10-fold molar excess. In some cases, depending on the molecule, the small molecule drug may actually be used in excess, such as when the PEG-small molecule conjugate precipitates in a reaction solvent, such as an ether, while unreacted drug remains in solution.

Target molecules and surfaces

The reactive polymer alkanals of the invention can be covalently or non-covalently attached to a variety of entities including membranes, chemical separation and purification surfaces, solid supports, metal/metal oxide surfaces such as gold, titanium, tantalum, niobium, aluminum, steel, and their oxides, silica, macromolecules, and small molecules. In addition, the polymers of the present invention are also useful in biochemical sensors, bioelectronic switches, and gates. The polymer alkanals of the invention are also useful as supports for peptide synthesis, in the preparation of polymer-coated surfaces and polymer grafts, in the preparation of polymer-ligand conjugates for affinity assignment, in the preparation of crosslinked or uncrosslinked hydrogels, and in the preparation of polymer-cofactor adducts for use in bioreactors.

The bioactive agent used to couple to the polymer of the present invention may be any one or more of the following. Suitable agents may be selected from, for example, hypnotics and sedatives, psychostimulants, tranquilizers, respiratory drugs, anticonvulsants, muscle relaxants, anti-parkinson agents (dopamine antagonists), analgesics, anti-inflammatories, anxiolytics (anxiolytics), appetite suppressants, anti-migraine agents, muscle contractants, anti-infective agents (antibiotics, antivirals, antifungals, vaccines), antiarthritics, antimalarials, antiemetics, antiepileptics (anepileptics), bronchodilators, cytokines, growth factors, anticancer agents, antithrombotic agents, antihypertensives, cardiovascular agents, antiarrhythmics, anthelmintics (antiasthmatics), antiasthmatic agents, hormonal agents including contraceptives, sympathomimetics, diuretics, modulators, antiandrogens, antiparasitics, anticoagulants, antineoplastic agents, agents for treating hypoglycemia, nutritional agents and supplements, growth supplements, anti-inflammatory agents, vaccines, antibodies, diagnostic agents, and contrast agents.

More specifically, the activator can fall into one of a number of structural classes, including but notlimited to small molecules (preferably insoluble small molecules), peptides, polypeptides, proteins, polysaccharides, steroids, nucleotides, oligonucleotides, polynucleotides, fats, electrolytes, and the like. Preferably, the activator coupled to the polymer alkanal of the invention has a natural amino group, or is modified to contain at least one reactive amino group suitable for coupling to the polymer alkanal of the invention.

Specific examples of active agents suitable for covalent linkage to the polymers of the invention include but are not limited to asparaginase, amdoxovivir (DAPD), anti-oviridin, Becapelin (becaplerin), calcitonin, cyanobacterial antiviral protein (cyanovirin), dineskin-toxin linker (denileukin difittox), Erythropoietin (EPO), EPO agonists (e.g., peptides of about 10-40 amino acids in length and including specific core sequences as described in WO 96/40749), streptococcal DNase α, erythropoiesis stimulating protein (NESP), clotting factors such as coagulation factor V, coagulation factor VII, factor VIIa, factor VIII, factor IX, factor X, factor XII, factor KG, vascular helium (thymosin Willebrand) factor, ceredase, cerezyme, α -glucosidase, collagen, cyclophilin α, interferon 630, interleukin 1, interleukin 2, growth factor receptor ligand (VEGF), interferon receptor ligand, interleukin-receptor ligand (VEGF), growth hormone receptor ligand, interleukin-receptor ligand (VEGF), growth factor receptor ligand, interleukin-receptor ligand (VEGF), interferon-2), growth hormone receptor ligand (VEGF-macrophage), growth factor receptor ligand (VEGF-2), growth factor receptor ligand (VEGF-FGF), growth factor receptor ligand (VEGF), growth factor receptor ligand (VEGF-fibroblast growth factor receptor ligand), growth factor receptor ligand (VEGF-2), growth factor receptor ligand (VEGF-factor receptor ligand), interferon-fibroblast growth factor receptor ligand (VEGF-fibroblast-factor receptor ligand), interferon-factor receptor ligand(VEGF-2), growth factor receptor ligand (VEGF-fibroblast growth factor receptor ligand), growth hormone (VEGF-fibroblast growth factor receptor ligand), interferon-fibroblast growth factor receptor ligand (VEGF-fibroblast growth factor receptor ligand), fibroblast growth factor receptor ligand (VEGF-2), fibroblast growth factor receptor ligand (VEGF-fibroblast growth factor receptor ligand), fibroblast growth factor receptor ligand (VEGF-fibroblast growth factor receptor ligand), fibroblast growth factor receptor (VEGF-fibroblast growth factor receptor ligand), fibroblast growth factor receptor ligand (VEGF-fibroblast growth factor receptor ligand), fibroblast growth factor receptor ligand (VEGF-fibroblast growth factor receptor ligand), fibroblast growth factor receptor (VEGF-fibroblast growth factor receptor), fibroblast growth factor receptor ligand (VEGF-fibroblast growth factor receptor ligand), fibroblast growth factor receptor ligand), fibroblast growth factor receptor (VEGF-fibroblast growth factor receptor ligand), fibroblast growth factor receptor (VEGF-fibroblast growth factor receptor, fibroblast growth factor receptor), fibroblast growth factor receptor ligand), fibroblast growth factor receptor (VEGF-fibroblast growth factor receptor, fibroblast growth factor receptor), fibroblast growth factor receptor, fibroblast growth factor), polypeptide-fibroblast growth factor receptor, fibroblast growth factor), polypeptide-fibroblast growth factor receptor, fibroblast growth factor), fibroblast growth factor receptor, fibroblast growth factor), fibroblast growth factor receptor, fibroblast.

Additional agents suitable for covalent linkage to the polymers of the present invention include, but are not limited to amifostine, amiodarone, aminocaproic acid, amikauricine sodium, aminoglutethimide, aminolevulinic acid, aminosalicylic acid, amsacrine, anagrelide, anthracyclines, asparaginase, anthracycyclines, bexarotene (bexarotene), bicalutamide (bicalutamide), bleomycin, buserelin, cabergoline, capecitabine, carboplatin, carmustine, chlorambucil sodium, cisplatin, clarithropine, clodronate, cyclophosphamide, cyproterone, arabinoside, cytarabine, camptothecin, 13-trans-retinoic acid, all-trans-retinoic acid, dacarbazine, dactinomycin, daunorubicin, desferrioxamine, dexamethasone, diclofenac, hexenol, docetaxel, doxorubicin, epirubicin, azacine, cefalotin, cefaloxime, cefalotin.

Preferred small molecules coupled to the polymer alkanals of the invention are those having at least one amino group. Preferred molecules include Aminomaurite, amphotericin B, doxorubicin, aminocaproic acid, aminolevulinic acid, aminosalicylic acid, meta-hydroxylamine tartrate, disodium pamidronate, daunorubicin, thyroxine sodium, lisinopril, cilastatin sodium, mexiletine, cephalexin, desferrioxamine, and amifostine.

Preferred peptides or proteins for coupling to the polymer alkanals of the invention include EPO, IFN- α - β -gamma, consensus IFN, factor VII, factor VIII, factor IX, IL-2, reminisce (infliximab), rituximab (rituximab), Enbrel (etanercept), Synagis (palivizumab), reopro (abciximab), Herceptin (trastuzimab), tPA, Cerizyme (imaglucerase), Hepatitus-B vaccine, rDNase,&ltttttttranslation =α&&l&/t&g-1 protease inhibitor, GCSF, GMCSF, hGH, insulin, FSH and PTH.

The above exemplary bioactive agents are intended to include, if applicable, analogs, agonists, antagonists, inhibitors, isomers, and pharmaceutically acceptable salt forms thereof. With respect to peptides and proteins, the invention is intended to include synthetic, recombinant, natural, glycosylated, and non-glycosylated forms, as well as biologically active fragments thereof. The above biologically active proteins are additionally intended to include variants with one or more amino acid substitutions, deletions, and the like, so long as the resulting variant protein possesses at least some of the activity of the parent (native) protein.

Pharmaceutical composition

The invention also includes pharmaceutical formulations comprising the conjugates provided herein and a pharmaceutical excipient. Typically, the conjugate is itself in a solid form (e.g., a precipitate) that can be mixed with a suitable pharmaceutical excipient in either a solid or liquid form.

Exemplary excipients include, but are not limited to, those selected from the group consisting of carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof.

Carbohydrates such as sugars, derivatised sugars such as sugar alcohols, aldonic acids, esterified sugars, and/or sugar polymers may be listed as excipients. Specific carbohydrate excipients include, for example: monosaccharides, if sugars, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, and the like; and sugar alcohols such as mannitol, xylitol, maltobionic acid, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, inositol, and the like.

The excipient may also include inorganic salts or buffers such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, monosodium phosphate, disodium phosphate, and combinations thereof.

The formulation may also include an antimicrobial agent for preventing or arresting the growth of microorganisms. Non-limiting examples of antimicrobial agents suitable for the present invention include alkylammonium chlorides, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimerosal, and combinations thereof.

Antioxidants can also be present in the formulation. Antioxidants are used to prevent oxidation, thereby preventing degradation of the conjugate or other components of the formulation. Suitable antioxidants for use in the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.

The surfactant may be present as an excipient. Exemplary surfactants include: polysorbates (polysorbates), such as "Tween 20" and "Tween 80", and pluronics such as F68 and F88 (both commercially available from BASF, Mount Olive, New Jersey); sorbitan esters; lipids, such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably not in liposomal form), fatty acids and fatty acid esters; steroids, such as cholesterol; and chelating agents such as EDTA, zinc and other such suitable cations.

An acid or base may be present in the formulation as an excipient. Non-limiting examples of acids that can be used include those selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric 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, sodium citrate, sodium formate, sodium sulfate, potassium fumarate, and combinations thereof.

Thepharmaceutical preparations include all types of formulations and those especially suitable for injection, such as powders and suspensions and solutions which are capable of reconstitution. The amount of conjugate (i.e., the conjugate formed between the active agent and the polymer described herein) in the composition will vary depending on a number of factors, but is optimally a therapeutically effective amount when the composition is stored in a unit dose container (e.g., a vial).

The amount of any individual excipient in the composition will vary depending on the activity of the excipient and the specific needs of the composition. Typically, the optimum amount of any single excipient is determined by routine experimentation, i.e., by preparing compositions containing varying amounts of excipient (from low to high), investigating stability and other parameters, and then determining the range of amounts that achieve optimum performance without significant adverse effects.

In general, however, the excipient is present in the composition in an amount of about 1% to about 99% by weight, preferably about 5% to 98% by weight, more preferably about 15% to 95% by weight excipient, with concentrations below 30% by weight being most preferred.

These aforementioned pharmaceutical excipients have been described in "Remington: the Science&Practice of Pharmacy, nineteenth edition, Williams&Williams (1995), "Physician's Desk Reference", 52 th edition, Medical Economics, Montvale, NJ (1998), and Kibbe, A.H., handbook of Pharmaceutical Excipients, third edition, American Pharmaceutical Association, Washington, D.C., 2000.

The pharmaceutical formulations of the present invention are typically, although not necessarily, administered by injection routes and are therefore generally liquid solutions or suspensions prior to administration. The pharmaceutical preparation may also take other forms such as syrups, creams, ointments, tablets, powders, and the like. Other modes of administration are also contemplated, such as pulmonary, rectal, transdermal, transmucosal, oral, intrathecal, subcutaneous, intraarterial, and the like.

As previously mentioned, the conjugate can be injected by parenteral route by intravenous injection, or less preferably by intramuscular or subcutaneous injection. Suitable types of formulations for parenteral administration include ready-to-use injection solutions, dry powders for mixing with solvents prior to use, suspensions for injection, dry insoluble compositions for mixing with vehicles prior to use, and emulsions and liquid concentrates which require dilution prior to administration, and the like.

Method of administration

The invention also provides methods of administering a conjugate provided herein to a patient suffering from a condition responsive to treatment with the conjugate. The method comprises administering, typically via injection, a therapeutically effective amount of the conjugate (preferably provided as part of a pharmaceutical formulation). The method of administration may be used to treat any condition that may be remedied or prevented by the administration of the particular conjugate. One skilled in the art will recognize which symptoms a particular conjugate is effective in treating. The actual dose administered will vary depending on the age, weight and general condition of the subject and the severity of the symptoms being treated, the judgment of the care professional, and the conjugate being administered. Therapeutically effective amounts are known to those skilled in the art and/or described in the relevant reference texts and literature. Generally, a therapeutically effective amount is about 0.001mg-100mg, preferably at a dosage of 0.01 mg/day to 75 mg/day, and more preferably at a dosage of 0.10 mg/day to 50 mg/day.

The unit dose of any given conjugate (again preferably provided as part of a pharmaceutical formulation) can be administered in a variety of dosing procedures, depending on the judgment of the clinician, the needs of the patient, and the like. Specific dosing procedures are known to those skilled in the art or determined experimentally using routine methods. Exemplary dosing procedures include, but are not limited to, administration five times a day, four times a day, three times a day, twice a day, once a day, three times a week, two times a week, once a week, twice a month, once a month, and any combination thereof. Dosing of the composition is stopped once the clinical endpoint effect has been achieved.

One advantage of the conjugates administered according to the present invention is that the individual water-soluble polymer moieties can be cleaved. This result is desirable when clearance from the body is a potential issue because of the size of the polymer. Optimally, cleavage of each water-soluble polymer moiety can be facilitated by the use of physiologically cleavable and/or enzymatically degradable linkages such as urethane, amide, carbonate or ester-containing linkages. In this way, the clearance of the conjugate (via cleavage of the individual water-soluble polymer moieties) can be tailored by the choice of polymer molecule size and type of functional group that provides the desired clearance properties. One skilled in the art can determine the appropriate molecular size of the polymer and the functional groups that can be cleaved. For example, one skilled in the art, using routine experimentation, can determine the appropriate molecular size and cleavable functional groups by first preparing various polymer derivatives having different polymer molecular weights and cleavable functional groups, and then obtaining a clearance curve (e.g., by periodic sampling of blood or urine) by administering the polymer derivatives to a patient and taking periodic blood and/or urine samples. Once a series of clearance curves for each tested conjugate were obtained, the appropriate conjugate could be determined.

All articles, books, patents, patent publications and other publications mentioned herein are incorporated by reference in their entirety.

Examples

It is to be understood that while the invention has been described in conjunction with certain preferred specific embodiments thereof, the foregoing description, as well as the examples which follow, are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention relates.

Raw materials and methods.

All PEG reagents in the accompanying examples are commercially available unless otherwise indicated. All NMR data were obtained from a 300MHz NMR spectrometer manufactured by Bruker.

Lysozyme was obtained from Sigma.

Example 1

_MSynthesis of PEG (2K) -butyraldehyde

Preparation of mPEG (2K Da) -butyraldehyde, diethyl acetal

A mixture of mPEG (2K Da) (2.0g) and toluene (30ml) was azeotropically dried under reduced pressure by distilling off the toluene. Dried mPEG (molecular weight, 2kilodaltons) was dissolved in anhydrous toluene (15ml), to which was added a 1.0M solution of potassium tert-butoxide in tert-butanol (4.0ml, 0.004 mole) and 4-chlorobutyraldehyde diethylacetal (0.5g, 0.00277 mole) (AlfaAesar). The mixture was stirred overnight at 100 ℃ and 105 ℃ under an argon atmosphere. After cooling to room temperature, the mixture was filtered and added to 150ml of ethyl ether at 0-5 ℃. The precipitated product was filtered off and dried under reduced pressure. Yield: 1.6 g. The reaction was carried out in essentially quantitative yield. That is, substantially all of the mPEG starting material is converted to the corresponding diethyl acetal, based on the absence¹H NMR measured hydroxyl protons corresponding to the mPEG-OH starting material.

NMR(d₆-DMSO)：1.09ppm(t，CH₃-C-)1.52ppm(m，C-CH₂-CH₂-)，3.24ppm(s，-OCH₃) 3.51ppm (s, PEG backbone), 4.46ppm (t, -CH, acetal).

Preparation of mPEG (2KD) -butyraldehyde

A mixture of mPEG (2K Da) butyraldehyde from a. above, diethyl acetal (1.0g), deionized water (20ml), and the amount of 5% phosphoric acid to adjust the pH to 3.0 was stirred at room temperature for 3 hours. To the mixture was added sodium chloride (1.0g) and the pH was adjusted to 6.8 by the addition of 0.1M sodium hydroxide. The product, mPEG (2D) butyraldehyde, was extracted with dichloromethane (3X 20 ml). The extract was dried over anhydrous magnesium sulfate and the solvent was distilled off under reduced pressure to give the isolated form of mPEG butyraldehyde product. Yield: 0.72 g.

The reaction was carried out in essentially quantitative yield. That is, substantially all of the mPEG butyraldehyde diethyl acetal is converted to the corresponding aldehyde based on the absence of the hydroxyl protons corresponding to the mPEG-OH starting material and the hydroxyl protons corresponding to the diethyl acetal, as determined by 1H NMR.

NMR(d₆-DMSO)：1.75ppm(p，-CH₂-CH₂-CHO-)₂.44ppm(dt，-CH₂-CHO)，3.24ppm(s，-OCH₃) 3.51ppm (s, PEG backbone), 9.66ppm (t, -CHO).

Example 2

Synthesis of mPEG (30kD) -butyraldehyde

Preparation of mPEG (30KDa) -butyraldehyde, diethyl acetal

mPEG_30K-O-CH₂(CH₂)₂-CH(OCH₂CH₃)₂

A mixture of mPEG (30kD) (60% solution in toluene, 3.30g), toluene (30ml) and BHT (butylated hydroxytoluene, 0.004g) was azeotropically dried by distilling off the solvent under reduced pressure. Dried mPEG 30K was dissolved in anhydrous toluene (15ml), to which was added a 1.0M solution of potassium tert-butoxide in tert-butanol (4.0ml, 0.004 mole), 4-chlorobutyraldehyde diethylacetal (0.5g, 0.00277 mole) (Alfa Aesar) and potassium bromide (0.05 g). The resulting mixture was stirred at 105 ℃ overnight under argon. The mixture is filtered, concentrated to dryness under reduced pressure, and the crude product is dissolved in 20ml of dichloromethane.

The solution containing the product was added to ethyl ether (300ml) at room temperature to precipitate the product. The precipitated product was isolated by filtration and dried under reduced pressure. Yield: 1.92 g.

NMR(d₆-DMSO)：1.09ppm(t，CH₃-C-)1.52ppm(m，C-CH₂-CH₂-)，3.24ppm(s，-OCH₃) 3.51ppm (s, PEG backbone), 4.46(t, -CH, acetal).

The substitution of the acetal reagent on the hydroxyl end of the mPEG-OH reagent proceeds with very high efficiency, that is to say essentially 100% substitution, based on the yield and analysis of the product. The product was produced in high purity (without further purification) with no detectable or significant amount of unreacted mPEG-OH. Typically, the alkanal or acetal polymer of the invention is produced in high purity-that is, typically, the desired alkanal product is present in the final composition in at least 85% purity, preferably at least 90% purity, and more preferably at least 95% purity.

Preparation of mPEG (30K Da) -butyraldehyde

mPEG_30K-O-CH₂(CH₂)₂-C(O)H

A mixture of mPEG (30K Da) butyraldehyde from above a, diethyl acetal (1.0g), deionized water (20ml), and the amount of 5% phosphoric acid to adjust the pH to 3.0 was stirred at room temperature for 3 hours. To the mixture was added sodium chloride (1.0g) and the pH was adjusted to 6.8 by the addition of 0.1M sodium hydroxide. The product was extracted with dichloromethane (3X 20 ml). The extract was dried over anhydrous magnesium sulfate and the solvent was distilled off. The wet product was dried under reduced pressure. Yield: 0.82 g.

NMR(d₆-DMSO)：1.75ppm(p，-CH₂-CH₂-CHO-)₂.44DDm(dt，-CH₂-CHO)，3.24ppm(s，-OCH₃) 3.51ppm (s, PEG backbone), 9.66ppm (t, -CHO).

Degree of substitution: -100%.

The conversion into the corresponding aldehyde is carried out in essentially quantitative yields.

Example 3

Comparative stability of mPEG-propionaldehyde and mPEG-butyraldehydeat basic pH methoxy-PEG-propionaldehyde and mPEG-butyraldehyde were each exposed to high pH conditions for longer periods of time to compare the relative stability of each polymer at basic pH conditions. As illustrated below, larger amounts of propionaldehyde PEG reacted under these conditions to form mPEG-OH and liberate acrolein (due to the retro-Michael type reaction) but no loss of PEG butyraldehyde compound was detected. Details of this experiment are provided below.

A. Stability of mPEG (2K Da) -butyraldehyde at basic pH

mPEG (2K Da) -butyraldehyde (from example 1) (0.5g) was dissolved in 10ml of 5mM phosphate buffer (pH 8.0) and the resulting solution was stirred at room temperature for 24 h. NaCl (0.5g) was added and the product extracted with dichloromethane (3X 10 ml). The extract was dried over anhydrous magnesium sulfate and the solvent was distilled off under reduced pressure at 25 ℃.

To be provided with¹Based on H NMR analysis, the product was unchanged. That is, no decomposition of PEG-butyraldehyde was detected, even after a prolonged period of time under these basic pH conditions.

B. Stability of mPEG (5K Da) -propanal at basic pH

mPEG (5KDa) -propionaldehyde (Shearwater Corporation, degree of aldehyde substitution 82%) (0.5g) was dissolved in 10ml of 5mM phosphate buffer (pH 8.0). The resulting solution was stirred at room temperature for 24 h. Gas chromatography headspace gas analysis indicated that the solution contained acrolein (CH) produced from the elimination reaction under basic conditions (such as those typically used for protein conjugation)₂CH-CHO). NaCl (0.5g) was added and the product was extracted with dichloromethane (3X 10m 1). The extract was dried over anhydrous magnesium sulfate and the solvent was distilled off under reduced pressure at 25 ℃.

Based on 1H NMR analysis, the degree of substitution of mPEG (5k Da) propionaldehyde was reduced to 62% (that is, 38% of the PEG-propionaldehyde had decomposed), and the product contained a relatively large amount of mPEG-OH, derived from the loss of the C-3 segment corresponding to the propionaldehyde portion of PEG-propionaldehyde.

Example 4

PEGylation of lysozyme

("Lysozyme-NH)₂"refers to a lysozyme molecule having one of the indicated reactive amino groups).

A. Random PEGylation with 2KD PEG alkanals.

Model proteins, lysozyme, 129 amino acid secretase, are used to illustrate the coupling of alkanal polymers of the invention to exemplary proteins. Lysozyme contains six lysine residues as PEG potential sites.

Lysozyme (2.1mg) was dissolved in 50mM phosphate buffer (pH7.6) at 1m l, to which mPEG (2kD) -butyraldehyde (from example 1, 1.5mg) was added. To this solution was added the reducing agent NaCNBH₃(sodium cyanoborohydride), the solution was stirred at room temperature for 24 hours. The lysozyme conjugates formed have a central- (CH)₂)₄-a PEG chain with a chain coupled to the amino group of lysozyme.

The reaction products were analyzed by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry and showed peaks corresponding to three PEG species pairs of lysozyme at 16208Da, 18422Da and 20520Da (corresponding to the size of the PEG butyraldehyde reagent) with masses that differ by about 2000 Da. The mass of unmodified (natural) lysozyme was 14153 as determined by MALDI-TOF. Thus, the conjugates formed are in fact mixtures of mono-, di-, and tri-PEGylated proteins (1-, 2-, and 3-mer).

The random pegylation of exemplary proteins of the invention was described above, resulting in a distribution of pegylated products. If desired, the reaction mixture can be further separated to isolate each PEG conjugate, i.e., lysozyme linked to one PEG molecule, lysozyme linked to two PEG molecules, and lysozyme linked to three PEG molecules. Within each of the conjugate compositions described above (1-mer, 2-mer, 3-mer), the PEG molecule may be attached to a different reactive amino site within the lysozyme molecule.

B. PEGylation with 5KD PEG alkanal, mPEG-2-methylbutyraldehyde.

Conjugation of model proteins (lysozyme) by Using α branched PEG reagent, mPEG_5kD-2-methylbutanal. Lysozyme (3mg) was dissolved in approximately 1mL of sodium phosphate buffer at a pH of about 5.5 to 7.5. Two to five-fold molar excess of PEG reagent, mPEG_5kD-2-methylbutanal added to the lysozyme solution, the solution formed being placed on a rotary mixer and reacted at room temperature. After about 15 minutes, a 20-fold molar excess of NaCNBH was added₃And allowing the reaction to proceed. Aliquots were taken at various time intervals (4 hours, 8 hours, 12 hours, 16 hours, etc.) and the progress of the reaction was monitored by SDS-PAGE and MALDI-TOF mass spectrometry.

After completion of the reaction, the resulting conjugate mixture was concentrated, sterile filtered, and stored at low temperature (-20 ℃ to-80 ℃) until further use.

Example 5

Preparation of branched PEG2(40.3KDA) -butyraldehyde

The following provides a method for preparing exemplary polymeric alkanals having branched PEG segments.

Overview. The overall synthesis involves first coupling a tetra (ethylene glycol) spacer to a reactive alkanal precursor, 4-chlorobutyraldehyde diethylacetal. The introduction of the oligomeric ethylene glycol spacer may provide greater stability to the formed product by extending the chain length between the reactive aldehyde (or acetal) group and the reactive group contained in the linker moiety X', thereby minimizing the occurrence of potential side reactions and improving yield.

The use of an oligomeric spacer such as tetraethylene glycol also provides a reactive functional group (in this case a hydroxyl group) that can be converted, if desired, for coupling to a polymer segment that has been purified by chromatographic separation to remove polymeric impurities such as PEG-diol, mPEG-OH, and the like. In this manner, undesirable functionalized polymeric impurities such as PEG-dialdehyde (resulting from PEG diol) and the like are removed from the PEG segment before the coupling of the PEG segment to the alkanal precursor results in the final product, i.e., a water-soluble polymer alkanal that is substantially free of polymeric impurities.

Turning now to a specific reaction, the coupling of a tetra (ethylene glycol) spacer to 4-chlorobutyraldehyde diethylacetal results in the formation of the desired mono-alkanal product contaminated with the dialkanal product and the starting tetra (ethylene glycol). However, direct work-up by virtue of solubility differences of all components in the reaction mixture makes it possible to achieve easy preparation of high-purity mono-alkanal products, i.e. alkanal (acetal) functions having substitution on only one of the reactive hydroxyl groups of the tetra (ethylene glycol) molecule. (reaction A.). The product from reaction a is then converted to the corresponding methanesulfonate ester by reaction with methanesulfonyl chloride, i.e., the free hydroxyl group of the tetra (ethylene glycol) is first converted to the methanesulfonate ester (reaction B), followed by conversion of the methanesulfonate ester to the primary amino group (reaction C). The reactive amino group of the acetal reagent is then coupled to an exemplary branched polymer backbone segment having a reactive carbonyl carbon suitable for reaction with the amino group of the acetal reagent. The precursor of the PEG reactant, mPEG-disubstituted lysine, was purified by ion exchange chromatography to remove polymeric impurities prior to conversion to the corresponding activated ester. The N-hydroxysuccinimidyl ester of mPEG-disubstituted lysine is then reacted with an aminoalkanal acetal to form a branched PEG-spacer-alkanal acetal via formation of an amide bond. The acetal is then readily hydrolyzed in an acid-catalyzed reaction to form the corresponding alkanal, especially 2-branched PEG (40kDa) butyraldehyde.

This synthetic method, i.e., not limited to branched polymer segments, may be used for polymer segments having any of the geometries described herein.

Overview of the overall synthesis:

A. tetra (ethylene glycol) mono-butyraldehyde, diethyl acetal

HO-(CH₂CH₂O)₄CH₂CH₂O-CH₂(CH₂)₂-CH(OCH₂CH₂)₂

A mixture of tetra (ethylene glycol) (97.1g, 0.500 mol) and toluene (200ml) was azeotropically dried by distilling off toluene under reduced pressure (rotary evaporator). Dried tetra (ethylene glycol) was dissolved in dry toluene (180ml) and 4-chlorobutyraldehyde diethylacetal (18.1g, 0.100 moles) (AlfaAesar) was neutralized by the addition of a 1.0M solution of potassium tert-butoxide in tert-butanol (120.0ml, 0.120 moles). The mixture was stirred at 95-100 ℃ overnight under argon. After cooling to room temperature, the mixture was filtered and the solvent was distilled off under reduced pressure. The crude product was dissolved in 1000ml of deionized water and the resulting solution was filtered through activated carbon. Sodium chloride (10g) was added and the product was extracted with dichloromethane (250, 200 and 150 ml). The extract was dried (over MgSO)₄Above) and the solvent is distilled off under reduced pressure (by rotary evaporation).

The crude product was dissolved in 300ml of 10% phosphate buffer (pH 7.5) and the impurities were extracted with ethyl acetate (2 × 50 ml). The product was extracted with dichloromethane (200, 150, and 100 ml). The extract was dried (over MgSO)₄Above) and the solvent is distilled off under reduced pressure (by rotary evaporation).

Yield: 20.3 g. NMR (d)₆-DMSO)：1.10ppm(t，CH₃-C-)1.51ppm(m，C-CH₂-CH₂-)，3.49ppm(bm，-OCH₂CH₂O-), 4.46ppm (t, -CH, acetal), 4.58ppm (t, -OH).

Purity: 100% (no evidence of unreacted starting material).

B. Tetra (ethylene glycol) - α -methanesulfonate-omega-butyraldehyde, diethyl acetal, CH₃-S(O)₂-O-(CH₂CH₂O)₄CH₂CH₂O-CH₂(CH₂)₂-CH(OCH₂CH₂)₂A mixture of tetra (ethylene glycol) mono-butyraldehyde, diethyl acetal (12.5g, 0.037 mol) and toluene (120ml) was azeotropically dried by distilling off the toluene under reduced pressure (rotary evaporator). The dried tetra (ethylene glycol) mono-butyraldehyde, diethyl acetal was dissolved in dry toluene (100 ml). To the direction ofTo the solution were added 20ml of anhydrous dichloromethane and 5.7ml of triethylamine (0.041 mol). 4.5g of methanesulfonyl chloride (0.039 mol) were then added dropwise. The solution was stirred at room temperature overnight under a nitrogen atmosphere. Sodium carbonate (5g) was then added and the mixture stirred for 1 hour. The solution is then filtered and the solvent is distilled off under reduced pressure (rotary evaporator).

1.10ppm(t，CH₃-C-)1.51ppm(m，C-CH₂-CH₂-)，3.17ppm(s，CH₃-methanesulfonate ester), 3.49ppm (bm, -OCH)₂CH₂O-)，4.30ppm(m，-CH₂-methane sulphonate), 4.46ppm (t, -CH, acetal).

Purity: -100%.

C. Tetra (ethylene glycol) - α -amino-omega-butyraldehyde, diethyl acetal H₂N-(CH₂CH₂O)₄CH₂CH₂O-CH₂(CH₂)₂-CH(OCH₂CH₂)₂

A mixture of tetra (ethylene glycol) - α -methanesulfonate-omega-butyraldehyde, diethyl acetal (14.0g), concentrated ammonium hydroxide (650ml), and ethanol (60ml) was stirred at room temperature for 42 hours, then all volatiles were distilled off under reduced pressure the crude product was dissolved in 150ml of deionized water, the pH of the solution was adjusted to 12 with 1.0M NaOH, and the product was extracted with dichloromethane (3X 100 ml).The extract was dried (over MgSO)₄Above) and the solvent is distilled off under reduced pressure (rotary evaporator).

Yield: 10.6 g. NMR (D)₂O)：1.09ppm(t，CH₃-C-)1.56ppm(m，C-CH₂-CH₂-)，2.69ppm(t，CH₂-N)，3.56ppm(bm，-OCH₂CH₂O-), 4.56ppm (t, -CH, acetal). Purity: -100%.

D. Branched PEG2(40.3kDa) -butyraldehyde, diethyl acetal

Preparation of PEG2(40kDa) -N-hydroxysuccinimide (as described in U.S. Pat. No.5,932,462(Harris, J. et al.) precursor PEG-2 lysine, a branched PEG with ionizable carboxyl groups, from the corresponding PEG 2-lysine was purified by ion exchange chromatography, as also described in U.S. Pat. No.5,932,462.

To a solution of PEG2(40kDa) -N-hydroxysuccinimide (5.0g, 0.000125 mol) (Shearwater Corporation) in dichloromethane (100ml) was added tetra (ethylene glycol) - α -amino- ω -butyraldehyde, diethyl acetal (0.50g, 0.000148 mol) and triethylamine (0.035ml), and the reaction mixture was stirred at room temperature overnight under argon atmosphere.

NMR(d₆-DMSO)：1.10ppm(t，CH₃-C)，1.51ppm(m，C-CH₂-CH₂-)，3.24ppm(s，-OCH₃) 3.51ppm (s, PEG backbone), 4.46ppm (t, -CH-, acetal). Degree of substitution: -100%.

E. Branched PEG2(40.3KDa) -butyraldehyde

Branched PEG2(40.3KDa) -butyraldehyde, diethyl acetal (4.8g) was dissolved in 100ml of water and the pH of the solution was adjusted to 3 with dilute phosphoric acid. The solution was stirred at room temperature for 3 hours, followed by the addition of 0.5M sodium hydroxide sufficient to adjust the pH of the solution to about 7. The product was extracted with dichloromethane, and the extract was dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure.

Yield: 4.2 g. NMR (d)₆-DMSO)：1.75ppm(p，-CH₂-CH₂-CHO-)，2.44ppm(dt，-CH₂-CHO)，3.24ppm(s，-OCH₃) 3.51ppm (s, PEG backbone), 9.66ppm (t, -CHO).

Degree of substitution: -100%.

The foregoing illustrates yet another embodiment of the invention-the preparation of representative branched PEG alkanals. The foregoing also illustrates embodiments of the invention in which the polymer alkanal contains short oligomeric groups, in this case tetraethylene glycol interposed between the polymer backbone and the alkanal segment of the molecule. Furthermore, this example illustrates the use of precursor polymer segments containing ionizable groups, enabling the polymer segments to be purified by ion exchange chromatography prior to coupling to the alkanal-acetal reagent, thereby effectively removing polymer impurities early in the reaction scheme. In this case, the resulting product is free of bifunctional polymeric impurities such as those resulting from the reaction of PEG-diol, or mPEG, or monopegylated lysine, etc., which may be present in mPEG 2-lysine precursor, but are removed by chromatographic separation.

Example 6

Preparation of Polymer-alkanal protein conjugates

Random PEGylation of EPO

Recombinant EPO (produced in e.coli, mammalian cells (e.g., CHO cells) or another microbial source) is conjugated to mPEG-butyraldehyde 30kDa (prepared as described in example 2).

EPO (. about.2 mg) was dissolved in 1ml of 50mM phosphate buffer (pH7.6) and mPEG (30kDa) -butyraldehyde was added in an amount of 5 times the molar concentration of EPO. Adding reducing agent NaCNBH₃The solution was stirred at room temperature for 24 hours to get PEG-butyraldehydeThe agent is coupled to the protein via an amine linkage.

The reaction mixture was analyzed by SDS-PAGE to determine the extent of PEGylation. Confirmation of the degree of PEGylation, 1-mer, 2-mer, etc., was performed by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. The peaks shown for the native and mono-PEGylated species differ by about 30,000 Da. The reaction mixture formed contains a mixture of native and mono-pegylated proteins. Increasing the ratio of PEG reagent to protein can increase the degree of multi-pegylation, i.e., the formation of 2-mer, 3-mer, etc.

The use of high molecular weight PEG alkanals having a molecular weight greater than about 20kDa facilitates the formation of mono-PEGylated species. Lower molecular weight PEG alkanals, when coupled to proteins, are more prone to the formation of multi-PEGylated species under these conditions.

The random pegylation of exemplary proteins of the invention was described above, resulting in a distribution of pegylated EPO products. The reaction mixture can be further separated, if desired, to isolate individual isomers, as described below.

PEG conjugates with different molecular weights were then separated by gel filtration chromatography. The different PEG conjugates (1-mer, 2-mer, 3-mer, etc.) are ranked on the basis of their different molecular weights (in this case, varying by approximately 30 kDa). In particular, the separation is carried out by using a series of column systems suitable for efficiently separating the products in the molecular weight range observed, for example Superdex^TM200 columns (Amersham Biosciences). The product was eluted with 10ml of acetate buffer at a flow rate of 1.5 ml/min. The collected fractions (1ml) were analyzed for protein content by OD at 280nm and PEG content using the iodine assay(Sims G.E.C. et al, anal. biochem, 107, 60-63, 1980). Alternatively, the results were visualized by running the SDS PAGE gel followed by staining with barium iodide. Fractions corresponding to the elution peaks were collected, concentrated by using a 10-30kD cut-off membrane, and lyophilized. This method results in the isolation/purification of conjugates having the same molecular weight, but fails to achieve conjugates having the same molecular weight but different pegylation sites (i.e., positional isomers)) Separation of (4).

The separation of positional isomers was carried out by reverse phase chromatography using RP-HPLC C18 column (Amersham biosciences or Vydac). This procedure can effectively separate PEG-biomolecule isomers with the same molecular weight (positional isomers). The reverse phase chromatography was performed by using an RP-HPLC C18 preparative column and eluted with a gradient of water/0.05% TFA (eluent a) and acetonitrile/0.05% TFA (eluent B).

Fractions corresponding to the elution peaks were collected, acetonitrile and TFA were removed by evaporation, and the solvent was subsequently removed to isolate each PEG-positional isomer.

Example 7

Preparation of Polymer-alkanal protein conjugates

N-terminal PEGylation of EPO

Recombinant EPO (produced in e.coli, mammalian cells (e.g., CHO cells, but not limited to it) or another microbial source) is conjugated to mPEG-butyraldehyde 30kDa (example 2).

EPO (. about.2 mg) dissolved in 1ml of 0.1mM sodium acetate (pH5) and mPEG (30kDa) -butyraldehyde (from example 2) was added in an amount of 5 times the molar concentration of EPO. The reducing agent NaCNBH3 was added and the solution was stirred at 4 ℃ for 24 hours to couple the PEG-butyraldehyde reagent to the protein via an amine bond.

The reaction mixture was analyzed by SDS-PAGE to determine the extent of PEGylation. Confirmation of the degree of PEGylation, 1-mer, 2-mer, etc. was performed by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. The peaks shown for the native and mono-PEGylated species differ by about 30,000 Da. The reaction mixture formed contains predominantly a mixture of native and monopegylated proteins. The monopegylated material was purified by column chromatography to remove free EPO and higher molecular weight species.

Confirmation of N-terminal PEGylation was performed by peptide mapping. Increasing the ratio of PEG to protein increases the degree of PEGylation, resulting in a multi-PEGylated protein.

The pegylation of exemplary proteins of the invention was described above, resulting in a predominantly N-terminally monopegylated protein.

Example 8

N-terminal PEGylation of GCSF

Recombinant GCSF (produced in E.coli, mammalian cells (e.g., CHO cells) or other microbial sources) was coupled to mPEG-butyraldehyde(30 kDa).

GCSF (. about.2 mg) was dissolved in 1ml of 0.1mM sodium acetate (pH5) and mPEG (30kDa) -butyraldehyde (from example 2) was added in an amount of 5 times the molar concentration of GCSF. The reducing agent NaCNBH3 was added and the solution was stirred at 4 ℃ for 24 hours to couple the PEG-butyraldehyde reagent to the protein via an amine bond.

The reaction mixture was analyzed by SDS-PAGE to determine the extent of PEGylation. Confirmation of the degree of PEGylation, 1-mer, 2-mer, etc. was performed by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. The peaks shown for the native and mono-PEGylated species differ by about 30,000 Da. The reaction mixture formed contains mainly a mixture of native and monopegylated GCSF. The monopegylated material was purified by column chromatography to remove free GCSF and higher molecular weight species. Confirmation of N-terminal PEGylation was performed by peptide mapping. Increasing the ratio of PEG to protein increases the degree of PEGylation, resulting in a multi-PEGylated protein.

Example 9

N-terminal PEGylation of Interferon α

Recombinant IFN- α (produced in E.coli, mammalian cells (e.g., CHO cells, but not limited to it) or other microbial sources) was coupled to mPEG-butyraldehyde (30 kDa).

Interferon- α (. about.2 mg) was dissolved in 1ml of 0.1mM sodium acetate (pH5) and mPEG (30kDa) -butyraldehyde (from example 2) was added in an amount 5 times the molar concentration of IFN. NaCNBH was added as a reducing agent₃The solution was stirred at 4 ℃ for 24 hours to couple the PEG-butyraldehyde reagent to the protein via an amine linkage.

The reaction mixture was analyzed by SDS-PAGE to determine the degree of PEGylation, confirmation of 1-mer, 2-mer, etc. was performed by matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry, the peaks shown for native and monopegylated species differed by about 30,000 Da.

Conjugation of the proteins hGH, IFN- β, and FSH to another exemplary PEG-alkanal (mPEG-2-methylbutyraldehyde, 20kDa) was performed essentially as described in the examples above.

Example 10

N-terminal PEGylation of human growth hormone

Recombinant human growth hormone (produced in E.coli, mammalian cells (such as CHO, but not limited to it) or another microbial source) is coupled to mPEG-2-methylbutanal (20 kDa).

Human growth hormone (. about.2 mg) was dissolved in 1ml of 0.1mm sodium acetate (pH5) and mPEG-2-methylbutanal (20kDa) was added in an amount of 5 times the molar concentration of hGH. Adding reducing agent NaCNBH with 5-20 times molar excess₃The solution was stirred at 4 ℃ for 24 hours to allow the PEG- α methylbutanal reagent to couple to the protein via an amine linkage.

The progress of the reaction was analyzed by SDS-PAGE or MALDI-TOF mass spectrometry to determine the extent of PEGylation. Confirmation of the degree of PEGylation, 1-mer, 2-mer, etc. was performed by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. The peaks shown for native and mono-PEGylated and other species differ by about 20,000 Da. The reaction mixture formed contains predominantly a mixture of native and monopegylated proteins. The monopegylated material was purified by column chromatography to remove free hGH and higher molecular weight species. Confirmation of N-terminal PEGylation was performed by peptide mapping. Increasing the ratio of PEG aldehyde to protein increased the degree of PEGylation, resulting in a population of multi-PEGylated hGH.

Example 11

N-terminal PEGylation of Interferon- β

Recombinant interferon- β (produced in E.coli, mammalian cells (e.g., CHO cells, but not limited thereto) or another microbial source) was conjugated to mPEG-2-methylbutanal (20 kDa).

Interferon- β (. about.2 mg) was dissolved in 1ml of 0.1mM sodium acetate (pH5) and mPEG-2-methylbutyraldehyde (20kDa) was added in an amount 5 times the molar concentration of IFN- β.5 was added-20 times molar excess of reducing agent NaCNBH₃The solution was stirred at 4 ℃ for 24 hours to allow the PEG- α methylbutanal reagent to couple to the protein via an amine linkage.

The progress of the reaction is analyzed by SDS-PAGE or MALDI-TOF mass spectrometry to determine the degree of PEGylation the confirmation of 1-mer, 2-mer, etc. is performed by matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry for native and monopegylated and other species showing peaks that differ by about 20,000 Da.

Example 12

N-terminal PEGylation of FSH

Recombinant follicle stimulating hormone (produced in E.coli, mammalian cells (such as CHO, but not limited to it) or another microbial source) was conjugated to mPEG-2-methylbutanal (20 kDa).

Follicle stimulating hormone, FSH (. about.2 mg) was dissolved in 1ml of 0.1mM sodium acetate (pH5) and mPEG-2-methylbutyraldehyde (20kDa) was added in an amount 5 times the molar concentration of FSH. Adding reducing agent NaCNBH with 5-20 times molar excess₃The solution was stirred at 4 ℃ for 24 hours to allow the PEG- α methylbutanal reagent to couple to the protein via an amine linkage.

The progress of the reaction was analyzed by SDS-PAGE or MALDI-TOF mass spectrometry to determine the extent of PEGylation. Confirmation of the degree of PEGylation, 1-mer, 2-mer, etc. was performed by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. The peaks shown for native and mono-PEGylated and other species differ by about 20,000 Da. The reaction mixture formed contains predominantly a mixture of native and monopegylated proteins. The monopegylated material was purified by column chromatography to remove free FSH and higher molecular weight species. Confirmation of N-terminal PEGylation was performed by peptide mapping. Increasing the ratio of PEG aldehyde to protein increases the degree of pegylation, resulting in a population of multi-pegylated FSH.

Example 13

N-terminal PEGylation of human growth hormone

Recombinant hGH (produced in e.coli, mammalian cells (such as CHO, but not limited to it) or another microbial source) is covalently linked to branched PEG2(40.3kDa) -butyraldehyde (example 5E).

Human growth hormone (. about.2 mg) was dissolved in 1ml of 0.1mM sodium acetate (pH5) and branched PEG2(40.3KDa) -butyraldehyde was added in an amount of 5 times the molar concentration of hGH. Adding reducing agent NaCNBH with 5-20 times molar excess₃The solution was stirred at 4 ℃ for 24 hours to allow coupling of the branched PEG2(40.3KDa) -butyraldehyde reagent to the protein via an amine bond.

The progress of the reaction was analyzed by SDS-PAGE or MALDI-TOF mass spectrometry to determine the extent of PEGylation. Confirmation of the degree of PEGylation, 1-mer, 2-mer (if any), etc. was performed by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. The peaks shown for native and mono-PEGylated and other species differ byabout 40,000 Da. The resulting reaction mixture contains predominantly a mixture of native and monopegylated proteins, particularly due to the size and geometry of the branched PEG alkanal reagent. The monopegylated material was purified by column chromatography to remove free hGH and higher molecular weight species. Confirmation of N-terminal PEGylation was performed by peptide mapping.

Example 14

PEGylation of amphotericin B

The amino group of the small molecule amphotericin B is modified by attachment of a polymer alkanal.

To a solution of amphotericin B.HCl in deionized water was added 2-fold molar excess of mPEG dissolved in 0.1M phosphate buffer pH6.5_2kButyraldehyde (example 1). Adding NaCNBH to the mixture₃(in a 1.5-10 fold molar excess) in pH6.5 phosphate buffer, the resulting solution being stirred overnight at room temperature under an argon atmosphere. Aliquots of the reaction mixture were removed at various time intervals and the progress of the reaction was monitored by 1H NMR. Once complete, the reaction mixture was further diluted by addition of water and saturated with NaCl. The product was then extracted with dichloromethane and the combined organic extracts were dried over anhydrous sodium sulfate, the drying agent was removed by filtration and the solvent was evaporated by rotary evaporation. The product was then precipitated by addition of diethyl ether and dried overnight under vacuum. The recovered product was analyzed by gel permeation chromatography to determine the extent of conjugation.

The crude product was purified by cation exchange chromatography using Poros 50HS cation exchange resin (PerSeptiveBioSystems, Framingham, MA). After washing the column with deionized water, the product was eluted with 1N NaCl solution. The conjugates containing the extracts were combined and the product extracted with dichloromethane. The organic solution was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated by rotary evaporation. The purified conjugate was purified by addition of diethyl ether.

The product was further purified by reverse phase HPLC chromatography using a Betasil C18 column (Keystone Scientific) if necessary.

Example 15

Method for preparing PEG (3500DA) - α -hydroxy-omega-butyraldehyde by anionic ring-opening polymerization of ethylene oxide directly onto anionic acetal precursor

In this example, an acetal precursor having sites suitable for initiating ring-opening polymerization of ethylene oxide was prepared by reacting 4-chlorobutyraldehyde diethylacetal with a diol (ethylene glycol). In this way, the halogen-acetal is converted into a hydroxy-terminated acetal. The hydroxyl group, when converted to the corresponding alkoxide anion, provides a site for initiating polymerization of Ethylene Oxide (EO), thereby forming a polymer alkanal precursor. The polymer alkanal precursor (acetal), after hydrolysis, is converted to the desired polymer alkanal.

A generalized reaction scheme for this synthetic method for preparing a polymer alkanal of the invention is shown in FIG. 1.

Preparation of 2- (4, 4-diethoxy-butoxy) ethanol (compound 15A).

A mixture of anhydrous ethylene glycol (120g, 1.93 moles), a 1.0M solution of potassium tert-butoxide in tert-butanol (70ml, 0.07 moles), and 4-chlorobutyraldehyde diethyl acetal (11g, 0.061 moles) was stirred at 100 ℃ overnight under an argon atmosphere. After cooling to room temperature, the reaction mixture was added to 600ml of distilled water. The product was extracted with dichloromethane (150, 125 and 125 ml). The combined extracts were then dried over anhydrous magnesium sulfate and the solvent was distilled off under reduced pressure at 25 ℃. The product was then subjected to vacuum distillation (kugelrohr, t 90-110 ℃, 0.10 mm hg). Yield 5.5 g.

NMR(d₆-DMSO)：1.11ppm(t，6H)，1.53ppm(m，-CH₂CH，2H)，1.64ppm(m，-CH₂CH₂CH，2H)，3.37ppm(t，-O-CH₂CH₂CH₂CH，2H)，3.53ppm(t，HO-CH₂CH₂O-，2H)，3.62ppm(q，-CH₂CH₃，4H)，3.70ppm(t，HO-CH₂-, 2H), 4.38ppm (t, -CH acetal, 1H), 4.54ppm (t, 1H, -OH).

Preparation of PEG (3,500Da) - α -hydroxy- ω -butyraldehyde diethyl acetal

Compound 15A (0.51g, 0.00247 mol), THF 200ml, and potassium naphthyl (0.3 mol/L-tetrahydrofuran solution, 20ml, 0.006 mol) were added to a glass reactor and stirred under argon atmosphere for 3 minutes. Ethylene oxide (8.8g 0.20 mol) was added to the solution and the reaction mixture was stirred at room temperature for 44 hours. Subsequently, the mixture was purged with argon, and 0.1M phosphate buffer (pH 8,100ml) was added. The THF layer was separated and discarded. Naphthalene was removed from the solution by ether extraction. The product was extracted from the residue with dichloromethane (3X 50 ml). The extract was dried over anhydrous sodium sulfate and concentrated to about 30 ml. Diethyl ether (250ml) was then added and the mixture was stirred at 0 ℃ for 15 minutes. The precipitated product was filtered off and dried under reduced pressure.

Yield 7.2 g.

NMR(d₆-DMSO)：1.09ppm(t，CH₃-，3H)1.52ppm(m，C-CH₂-CH₂-, 4H), 3.51ppm (s, polymer backbone), 4.46ppm (t, -CH, acetal, 1H), 4.57ppm (t-OH, 1H).

PEG (3,500Da) - α -hydroxy-omega-butyraldehyde

A mixture of PEG (3,500) - α -hydroxy- ω -butyraldehyde diethyl acetal (1.0g), deionized water (20ml), and the amount of 5% phosphoric acid to adjust the pH to 3.0 was stirred at room temperature for 3 hours, then, sodium chloride (1.0g) was added, the pH was adjusted to 6.8 by the addition of 0.1M sodium hydroxide, the product was extracted with dichloromethane (3X 20ml), the extract was dried over anhydrous magnesium sulfate and the solvent was distilled off, the wet product was dried under reduced pressure, yield: 0.82 g.

NMR(d₆-DMSO)：1.75ppm(p，-CH₂-CH₂-CHO-)，2.44ppm(dt，-CH₂-CHO), 3.51ppm (s, polymer backbone), 4.57ppm (t, -OH), 9.66ppm (t,-CHO)。

example 16

Preparation of methoxy-PEG (3500Da) butyraldehyde

This example illustrates the preparation of exemplary end-capped PEG-alkanals from PEG α -hydroxy-omega-alkanal acetals.

mPEG (3,500Da) -butyraldehyde diethyl acetal

PEG (3,500Da) - α -hydroxy- ω -butyraldehyde diethyl acetal (3.5g, 0.001 mol), toluene (50ml), a mixture of a 1.0M solution of potassium tert-butoxide in tert-butanol (5ml, 0.005 mol) and methyl p-toluenesulfonate (0.75g, 0.004 mol) was stirred at 45 ℃ overnight.

Yield: 3.1 g.

NMR(d₆-DMSO)：1.09ppm(t，CH₃-，3H)1.52ppm(m，C-CH₂-CH₂-, 4H), 3.24ppm (s, CH3O-, 3H), 3.51ppm (s, polymer backbone), 4.46ppm (t, -CH, acetal, 1H).

mPEG (3,500Da) -butyraldehyde

A mixture of mPEG (3,500) -butyraldehyde diethyl acetal (1.0g), deionized water (20ml), and 5% phosphoric acid adjusted to pH 3.0 was stirred at room temperature for 3 hours. Subsequently, sodium chloride (1.0g) was added and the pH was adjusted to 6.8 by the addition of 0.1M sodium hydroxide. The product was extracted with dichloromethane (3X 20 ml). The extract was dried over anhydrous magnesium sulfate and the solvent was distilled off. The wet product was dried under reduced pressure. Yield: 0.85 g.

NMR(d₆-DMSO)：1.75ppm(p，-CH₂-CH₂-CHO-，2H)₂.44ppm(dt，-CH₂-CHO，2H)，3.24ppm(s，-OCH₃3H), 3.51ppm (s, polymer backbone), 9.66ppm (t, -CHO, 1H).

Example 17

methoxy-PEG (2kDA)₂Preparation of (meth) butyraldehyde

This example describes the synthesis of an exemplary polymer 2-alkyl substituted alkanal of the invention. This polymer, instead of having a linear alkylene chain separating the aldehyde carbon from the linker, has a methyl substituent at the C-2 position.

For review: the protected aldolisation reagent, 17-a, was prepared from the commercially available starting material 2-methyl-4-chlorobutyrate by first reducing the butyrate ester carbon to the corresponding alcohol, followed by oxidation to butyraldehyde. This butyraldehyde is then protected as the corresponding acetal, yielding the protected acetal reagent for coupling to PEG. After coupling to PEG, the resulting polymer acetal is hydrolyzed in acid to provide the desired polymer alkanal.

Preparation of 4-chloro-2-methylbutanal diethylacetal

Preparation of 4-chloro-2-methylbutanol-1

A solution of 2-methyl-4-chlorobutyrate (TCI America) (22.0g, 0146 moles) in diethyl ether (80ml) was added dropwise over 30 minutes to a stirred solution of lithium aluminum hydride (4.55g, 0.12 moles) in diethyl ether (360ml) at 0 ℃ under an argon atmosphere. Methanol (12ml) was then added dropwise over 30 minutes, followed by ice-cold 2N HCl (420ml) over 20 minutes. The reaction mixture was transferred to a separatory funnel and the ether layer containing 4-chloro-2-methylbutanol-1 was separated. Additional product was extracted from the aqueous layer with ether (3X 200 ml). The ether extracts were combined, dried over anhydrous magnesium sulfate and the solvent was distilled off under reduced pressure. Yield 18.6 g.

NMR(d₆-DMSO)：0.84ppm(d，-CH₃，3H)，1.50ppm(m，-CH₂CH₂Cl，1H)，1.68ppm(m，-CH-，1H)，1.82ppm(m，-CH₂CH₂Cl，1H)3.26ppm(t，-CH₂OH，2H)，3.66ppm(m，-CH₂Cl，2H)，4.50ppm(t，-OH，1H)。

4-chloro-2-methylbutyraldehyde

Pyridinium chlorochromate (23.6g, 0.110g) was gradually added to a stirred solution of 4-chloro-2-methylbutanol-1 (8.80g, 0.078 moles) in anhydrous dichloromethane (470 ml). The mixture was stirred at room temperature overnight under argon. Dry diethyl ether (820ml) was added; the mixture was stirred for 20 minutes and then filtered to remove excess oxidant. The solution was then filtered through a column packed with 400g of Florisil, and the solvent was distilled off under reduced pressure. Yield 6.0 g.

NMR(d₆-DMSO)：1.06ppm(d，-CH₃，3H)，1.74ppm(m，-CH₂CH₂Cl，1H)，2.14ppm(m，-CH₂CH₂Cl，1H)，2.56ppm(m，-CH，1H)，3.69ppm(m，-CH₂Cl，2H)，9.60ppm(t，-CHO，1H)。

4-chloro-2-methylbutyraldehyde diethyl acetal

4-chloro-2-methylbutyraldehyde (4.8g, 0.040 mole), triethyl orthoformate (6.48g, 0.044 mole), ethanol (3.0g), and p-toluenesulfonic acid monohydrate(0.0144g，0.000757 moles) was stirred at 45 ℃ overnight under argon. Then, after cooling to room temperature, Na was added₂CO₃(0.40g) and the mixture was stirred for 15 minutes. The reaction mixture was filtered and ethanol and residual triethyl orthoformate were distilled off under reduced pressure. The residue was subjected to vacuum fractional distillation to obtain 3.2g of pure 4-chloro-2-methylbutanal diethylacetal.

NMR(d₆-DMSO)：0.85ppm(d，-CH₃，3H)1.13ppm(m，-CH₃，6H)，1.52ppm(m，-CH-，1H)，1.87ppm(m，-CH₂CH₂Cl，2H)，3.35-3.75ppm(bm，-OCH₂CH₃4H, and-CH₂Cl, 2H), 4.22ppm (d, -CH acetal, 1H).

mPEG (2K Da) -2-methylbutyraldehyde, diethyl acetal

A solution of mPEG (2K Da) (2.0g, 0.001 mol) and toluene (30ml) was azeotropically dried under reduced pressure by distilling off the toluene. Dried mPEG 2K was dissolved in anhydrous toluene (15ml) and a 1.0M solution of potassium tert-butoxide in tert-butanol (4.0ml, 0.004 moles) and 4-chloro-2-methylbutanal diethyl acetal from A above (0.5g, 0.00277 moles) were added. Next, lithium bromide (0.05g) was added and the mixture was stirred at 100 ℃ overnight under an argon atmosphere. After cooling to room temperature, the mixture was filtered and added to 150ml of ethyl ether at 0-5 ℃. The precipitated product was filtered off and dried under reduced pressure. Yield: 1.5 g.

NMR(d₆-DMSO)：0.83ppm(d，-CH₃，3H)1.10ppm(m，-CH₃O，6H)，1.24ppm(m，-CH-，1H)，1.72ppm(m，PEG-O-CH₂-CH₂-，2H)，3.24ppm(s，-OCH₃3H), 3.51ppm (s, polymer backbone), 4.18ppm (d, -CH acetal, 1H).

Degree of substitution: -100%.

mPEG (2K Da) -2-methylbutyraldehyde

A mixture containing mPEG (2K Da) -2-methylbutyraldehyde from B above, diethyl acetal (1.0g), deionized water (20ml), and the amount of 5% phosphoric acid to adjust the pH to 3.0 was stirred at room temperature for 3 hours. Next, sodium chloride (1.0g) was added and the pH was adjusted to 6.8 by the addition of 0.1M sodium hydroxide.

The product was extracted with dichloromethane (3X 20 ml). The extract was dried over anhydrous magnesium sulfate and the solvent was distilled off under reduced pressure. Yield: 0.83 g.

NMR(d₆-DMSO)：1.01ppm(d，-CH₃，3H)1.56ppm(m，-CH，1H)，1.90ppm(m，PEG-O-CH₂-CH₂-，1H)，2.45ppm(m，PEG-O-CH₂-CH₂-，1H)，3.24ppm(s，-OCH₃3H), 3.51ppm (s, polymer backbone), 9.56ppm (d, -CH aldehyde, 1H).

Degree of substitution: -100%.

Claims (51)

1. A compound having the structure:

wherein hGH represents human growth hormone comprising the N-terminal amino acid; and

wherein each mPEG represents a methoxy-polyethylene glycol group having a molecular weight of 18,000 and 22,000 daltons.

2. The compound of claim 1, wherein each mPEG has a nominal average molecular weight of 20,000 daltons.

3. The compound of claim 2, wherein the compound has a polydispersity other than hGH of less than 1.2.

4. The compound of claim 2, wherein the compound has a polydispersity of less than 1.1 other than hGH.

5. The compound of claim 2, wherein the compound has a polydispersity other than hGH of less than 1.05.

6. The compound of any one of claims 1, 2, 3, 4, or 5, wherein the hGH is linked to the N-terminal amino acid of the hGH by a secondary amine covalent bond.

7. The compound of claim 1, 2, 3, 4 or 5, wherein the compound is a mono-pegylated compound.

8. The compound of claim 7, wherein hGH is predominantly monopegylated at the N-terminal amino acid of said hGH.

9. The compound of any of claims 1-5, wherein the hGH is recombinant hGH.

10. The compound of claim 9, wherein the recombinant hGH is produced in e.

11. The compound of any one of claims 1-5, wherein the compound is a pure compound.

12. The compound of claim 11, wherein the compound is purified by chromatography.

13. The compound of claim 12, wherein the compound is purified by ion exchange chromatography.

14. A conjugate formed by reacting a polymer with human growth hormone (hGH) comprising an N-terminal amino acid, wherein the polymer has the formula:

wherein each mPEG represents a methoxy-polyethylene glycol group having a molecular weight of 18,000 and 22,000 daltons.

15. The conjugate of claim 14, wherein each mPEG has a nominal average molecular weight of 20,000 daltons.

16. The conjugate of claim 14, wherein the polymer has a nominal average molecular weight of 40,300 daltons.

17. The conjugate of any of claims 14, 15 or 16 wherein the polymer has a polydispersity of less than 1.2.

18. The conjugate of any of claims 14, 15 or 16 wherein the polymer has a polydispersity of less than 1.1.

19. The conjugate of any of claims 14, 15 or 16 wherein the polymer has a polydispersity of less than 1.05.

20. The conjugate of any one of claims 14-16 wherein the conjugate is a predominantly mono-pegylated conjugate.

21. The conjugate of claim 20, wherein said conjugate is predominantly monopegylated at the N-terminal amino acid of said hGH.

22. The conjugate of any of claims 14-16 wherein said hGH is recombinant hGH.

23. The conjugate of claim 23, wherein said recombinant hGH is produced in e.

24. The conjugate of any of claims 14-16, wherein said conjugate is formed by reacting said polymer with said hGH in a reaction mixture under reductive amination conditions.

25. The conjugate of claim 24, wherein the initial molar ratio of said polymer to said hGH in said reaction mixture is less than or equal to 5.

26. The conjugate of claim 25, wherein the conjugate is isolated from the reaction mixture.

27. The conjugate of claim 26, wherein the conjugate is chromatographically separated from the reaction mixture.

28. The conjugate of claim 25, wherein the chromatographic separation is effected by ion exchange.

29. A composition comprising a compound of any one of claims 1-13, and a pharmaceutical excipient.

30. The composition of claim 29, wherein the pharmaceutical excipient is selected from the group consisting of carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids and bases.

31. The composition of claim 29 or 30, wherein the composition is in liquid form.

32.The composition of claim 29 or 30, wherein the composition is in solid form.

33. The composition of claim 32, wherein the solid form is a lyophilized powder.

34. The composition of claim 29 or 30, wherein the composition is in unit dosage form.

35. The composition of claim 34, wherein the composition is stored in a unit dose container.

36. The composition of claim 34, wherein the container is a syringe.

37. Use of a compound according to any one of claims 1-13 for the manufacture of a medicament for the treatment of conditions treatable by administration of hGH.

38. A composition comprising a conjugate according to any of claims 14 to 28, and a pharmaceutical excipient.

39. The composition of claim 38, wherein the pharmaceutical excipient is selected from the group consisting of carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids and bases.

40. The composition of claim 38 or 39, wherein the composition is in liquid form.

41. The composition of claim 38 or 39, wherein the composition is in solid form.

42. The composition of claim 41, wherein the solid form is a lyophilized powder.

43. The composition of claim 38 or 39, wherein the composition is in unit dosage form.

44. The composition of claim 43, wherein the composition is stored in a unit dose container.

45. The composition of claim 44, wherein the container is a syringe.

46. Use of a conjugate according to any of claims 14-28 for the manufacture of a medicament for the treatment of a condition treatable by administration of hGH.

47. Use of a conjugate according to any of claims 14-28 for the treatment of a condition treatable by administration of hGH.

48. The use of a compound according to any one of claims 1-13 for the treatment of conditions treatable by administration of hGH.

49. A method of preparing a conjugate according to any of claims 14 to 28 comprising the steps of: reacting hGH with a polymer in a reaction mixture under reductive amination conditions; and optionally separating the conjugate from the reaction mixture.

50. The method of claim 49, wherein the reaction mixture has a pH of 5-8.

51. The method of claim 49, wherein the reaction mixture has a pH of 7-7.5.