METHOD OF MAKING RELEASABLE POLYMERIC REAGENTS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 ET.S.C. §119(e) to ET.S.

Provisional Patent Application No. 62/744,512, filed on October 11, 2018, the disclosure of which is incorporated herein by reference in its entirety.

FIELD

[0002] The instant application relates to (among other things) improved methods of preparing fluorenyl-based polymeric reagents, methods of recovering and purifying such polymeric reagents, methods of reducing unwanted impurities in a fluorenyl-based polymeric reagent, fluorenyl-based polymeric reagents prepared by the methods described herein, and conjugates prepared by reaction with fluorenyl-based polymeric reagents prepared by the methods described herein.

BACKGROUND

[0003] Modification of bioactive molecules by covalent attachment of polyethylene glycol can enhance the pharmacological and pharmaceutical properties of such molecules and has been used successfully in several approved drugs. For example, PEGylation has been used to create marketed drugs in which a biopharmaceutical agent is covalently attached to polyethylene glycol with a stable bond, such as, for example, CIMZIA® (PEGylated tumor necrosis factor (TNF)), NEULASTA® (PEGylated granulocyte-colony stimulating factor (GCSF)), PEGASYS® (PEGylated interferon a-2a), and ADYNOVATE® (PEGylated Factor VIII). In many cases, stable covalent attachment of one or more polyethylene glycol chains to an active agent results in PEG conjugates having reduced functional activity when compared to the unmodified molecule. When an active drug is covalently attached to a polymer via a stable linkage, the polymer-bound drug may retain the properties of the unbound drug, although its efficacy per gram of drug generally differs from the unmodified drug, since the covalently attached polymer can change, among other things, the steric and electronic environment

surrounding the drug molecule, and can render the drug less effective (on a per gram basis of drug). However, this effect can be offset in certain instances, by, for example, a longer circulation time such that enhanced drug efficacy may be achieved.

[0004] PEGylation technologies also exist in which a PEG reagent is covalently attached to a therapeutic agent via a releasable linkage. Releasable PEGylation (in some instances also referred to as“reversible PEGylation”), is a technology in which an active agent conjugate (which includes a drug molecule that is releasably chemically bonded to one or more water-soluble polymer moieties such as polyethylene glycol), following administration, releases the one or more polymer moieties from the drug over time, through a chemical process that occurs in vivo. Such releasably-polymer modified drugs are sometimes referred to as pro-drugs, since in theory, the polymer(s) is/are released over time in the circulation and the activity of the parent drug molecule can be recovered. The efficacy of releasable drug delivery systems can be affected by, e.g., the release rate of one or more covalently attached polymer moieties from a drug conjugate.

[0005] One class of PEGylation reagents that may be used to form drug conjugates capable of undergoing reversible (releasable) PEGylation is based on the

fluorenylmethyloxycarbonyl (FMOC) amine protecting group (see, e.g., Wuts, P.G., Greene, T.W., Protective Groups in Organic Synthesis , Fourth Ed., 2007, John Wiley & Sons, Inc., New Jersey; Bentley, M.D., et al., ET.S. Patent No. 8,252,275; Shechter Y., et al., Eur J Pharm Biopharm^ 2008, 70, 19-28). A generalized structure of one particular class of these reagents, referred to herein as polymeric, or in some particular instances, PEGylated FMOC reagents, possessing a branched architecture, is shown in Structure X below (as described, for example, in Bentley, M.D., et al., ET.S. Patent No. 8,252,275, incorporated herein by reference in its entirety, where descriptions for Ll and L2, and Reland Re2 are provided therein). In this illustrative structure, the polymeric reagent is activated as an N-hydroxyl succinimidyl carbonate (NHS) ester, i.e., comprises a succinimidyl carbonate ester leaving group.

[0006] The PEG2 FMOC reagents may be reacted with a therapeutic agent comprising a nucleophilic atom capable of reacting with an FMOC-type reagent to form, for example, a carbamate adduct. This reaction scheme is illustrated below, showing, as an example, reaction of a protein lysine group with a generalized form of an exemplary PEG2-FMOC reagent comprising a reactive N-succinimidyl carbonate group (Scheme I).

Scheme I.

[0007] During the manufacturing of various FMOC reagents by previously described synthetic approaches, the Applicants have experienced unwanted side reactions. These side reactions can result in the formation of undesirable side products that (i) can potentially adversely impact the quality of the final polymer-therapeutic agent conjugate, and (ii) may alter the mechanism of release of the FMOC polymer moiety (or moieties) from the therapeutic agent. The instant disclosure describes challenges associated with previously described methods of preparing the subject polymeric FMOC reagents, and provides several process improvements aimed at overcoming such problems, to thereby provide, for example, improved methods of making the polymeric FMOC reagents, improved methods for making intermediates useful in preparing the polymeric FMOC reagents, improved methods for activating an FMOC

intermediate, methods for removing one or more undesirable polymeric FMOC-derived side-products or impurities, and methods for stabilizing certain polymeric FMOC reagents, among other things. Thus, the present disclosure seeks to address these and other challenges related to the preparation of the subject polymeric FMOC reagents and their conjugates.

SUMMARY

[0008] In a first aspect, provided herein is a method, i.e., an improved method, for preparing a reactive polymeric reagent. The method comprises (i) reacting a water-soluble 9-hydroxymethyl fluorene polymer having a structure (I):

wherein POLYa is a first water-soluble, non-peptidic polymer; POLYb is a second, water-soluble non-peptidic polymer; Rel, when present, is a first electron-altering group; and Re2, when present, is a second electron-altering group; Li is a first linking moiety; and L2 is a second linking moiety;

with dibenzotriazolyl carbonate (BTC) in an aprotic organic solvent in the presence of a base under anhydrous conditions to provide a reaction mixture comprising a water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer having a structure:

wherein POLYa, POLYb, Rel, Re2, Li, and L2 each have values as described in step (i), and (ii) recovering the water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer (II) by precipitation with an anhydrous solvent effective to promote precipitation of the water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer.

[0009] In one or more embodiments related to the method, in step (i) the water-soluble 9-hydroxymethyl fluorene polymer of structure (I) is reacted with less than about 30 equivalents of dibenzotriazolyl carbonate (di-BTC).

[0010] In some embodiments, the base is an amine. In some further embodiments, the base is a non-nucleophilic amine or is a weakly nucleophilic amine. Bases, include, for example, pyridine, 4-dimethylaminopyridine, N,N-diisopropylethylamine, 2,6-di-tert-butylpyridine, N-methylimidazole, N-methylmorpholine, 2,6-lutidine, 2,4,6-collidine, N,N,2,6-tetramethylpyridine-4-amine, and insoluble-polymer-bound forms of any of the foregoing. The amine may also be a polyamine such as, for example, N,N,N’,N’-tetramethyl-l,6-hexamethyldiamine, N,N’, N’, N”,N”-pentamethyldiethylenetriamine, and

hexamethy 1 enetetramine .

[0011] In one or more embodiments of the method, in step (ii), the anhydrous solvent effective to promote precipitation further comprises an acid.

[0012] In yet some further embodiments, the method further comprises, prior to the reacting step, dissolving the water-soluble 9-hydroxymethyl fluorene polymer of structure (I) in the aprotic organic solvent to form a polymer solution, and drying the polymer solution by azeotropic distillation to provide a polymer solution having a water content of less than about 500 ppm.

[0013] In some additional embodiments, the recovered water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer from step (ii) comprises less than 10 mole percent of a water-soluble fulvene polymer.

[0014] In yet some other embodiments of the method, the recovering step comprises filtering the reaction mixture from step (i) to remove solids to provide a solution comprising the water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer, followed by adding an amount of an anhydrous solvent effective to precipitate the water-soluble 9-methyl

benzotriazolyl carbonate fluorene polymer from the solution.

[0015] In yet some additional embodiments, the anhydrous solvent effective to promote precipitation of the water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer comprises a small amount of acid.

[0016] Yet in some further embodiments, the method comprises washing the recovered water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer with an anhydrous solvent in which the water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer is insoluble or is substantially insoluble, the solvent comprising from about 0.0001 to about 0.5 mole percent acid.

[0017] In some further embodiments, the method further comprises (iii) purifying the recovered water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer.

[0018] In a second aspect, provided herein is a method comprising converting a recovered or purified water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer as described above to a different reactive carbonate, such as, e.g., a water-soluble 9-methyl N-hydroxy succinimidyl carbonate fluorene polymer, under conditions effective to carry out such transformation. In one or more illustrative embodiments, such conversion is carried out in the presence of dimethylaminopyridine. In yet one or more embodiments, the conversion reaction is carried out in a solvent, such as for example, dichloromethane.

[0019] Also provided, in a third aspect, is a method for preparing an N-hydroxyl succinimidyl carbonate ester-activated polymeric reagent. The method comprises (i) reacting a water-soluble 9-hydroxymethyl fluorene polymer having a structure:

wherein POLYa is a first water-soluble, non-peptidic polymer; POLYb is a second, water-soluble non-peptidic polymer; Rel, when present, is a first electron-altering group; Re2, when present, is a second electron-altering group; Li is a first linking moiety; L2 is a second linking moiety; Rel, which may or may not be present, is a first electron-altering group; and Re2, which may or may not be present, is a second electron-altering group,

with from about 1 to 20 equivalents of disuccinimidyl carbonate in an anhydrous aprotic organic solvent in the presence of base to provide a reaction mixture comprising a water-soluble 9-methyl N-succinimidyl carbonate fluorene polymer having a structure:

wherein POLYa, POLYb, Rel, Re2, Li, and L2 each have values as described in step (i); and (ii) recovering the water-soluble 9-methyl N-hydroxysuccinimidyl carbonate fluorene polymer of structure (III) from the reaction mixture.

[0020] Exemplary water-soluble 9-methyl N-hydroxysuccinimidyl carbonate fluorene polymers include the following, structures XI - XIV, wherein mPEGO- is shorthand for methoxypolyethylene glycol or CEbO^EbCEbO^CEbCEbO-, (although any of a number of reactive groups suitable for reaction with a functional group of a target drug molecule can also suitably be envisioned, such as benzotriazolyl carbonate, as can additional water-soluble polymeric chains substituted onto the fluorene core other than polyethylene glycol), wherein each (n) is in a range from about 3 to 2273, including each and every one of the subranges and particular values for (n) described elsewhere herein,

Structure (XI): 9-hydroxymethyl-4-(mPEG-carboxyamide)-7-(3-(mPEGcarbamoyl-propyl)- fluorene-N-hydroxysuccinimidyl carbonate (“CAC-PEG2-FMOC-NHS”)

Structure (XII): 9-hydroxymethyl-2,7-di(mPEG-amidoglutaric amide)fluorene-N- hydroxysuccinimidyl carbonate (“G2-PEG2-FMOC-NHS”)

Structure (XIII): 9-Hydroxymethyl-4-(mPEG-carboxyamide)-7-(mPEG amidoglutaric amide)fluorene-N-hydroxysuccinimidyl carbonate (“CG-PEG2-FMOC-NHS”)

and

Structure XIV: 9-hydroxymethyl-2,7-(bis-mPEG-carboxyamide)-fluorene-N- hydroxysuccinimidyl carbonate (“C2-PEG2-FMOC-NHS”).

[0021] In some embodiments of the foregoing method, prior to reacting step (i), the water-soluble 9-hydroxymethyl fluorene polymer is dissolved in the anhydrous aprotic organic solvent to provide a polymer solution, followed by drying the polymer solution to remove water that may be present to provide a dried polymer solution having a water content of less than 500 ppm.

[0022] In some further embodiments of the method, the drying is repeated until a dried polymer solution having a water content of less than 200 ppm is attained.

[0023] In some additional particular embodiments, the drying step comprises

azeotropically distilling the polymer solution.

[0024] In some further embodiments related to the foregoing, the drying is repeated until the water content of the polymer solution remains constant.

[0025] In one or more additional embodiments, the method further comprises, prior to the recovering step, adding an acid to the reaction mixture from step (i) in an amount effective to neutralize the base.

[0026] In some embodiments, the method is effective to produce a recovered water-soluble 9-methyl N-hydroxysuccinimidyl carbonate fluorene polymer comprising 15 mole percent or less of a water-soluble fulvene polymer.

[0027] In one or more embodiments of the method, the precipitating solvent is at a temperature above its freezing point and below room temperature.

[0028] In yet some further embodiments, the precipitating solvent comprises a small amount of acid.

[0029] In some embodiments, the method further comprises washing the recovered water-soluble 9-methyl N-hydroxysuccinimidyl carbonate fluorene polymer with an acidified precipitating solvent.

[0030] In one or more embodiments, the method further comprises purifying the recovered water-soluble 9-methyl N-hydroxysuccinimidyl carbonate fluorene polymer.

[0031] In yet some additional embodiments, the purifying step comprises dissolving the recovered water-soluble 9-methyl N-hydroxysuccinimidyl carbonate fluorene polymer in a solvent to provide a solution, passing the solution through a thiol-containing resin to remove any water-soluble fulvene polymer to thereby provide a purified solution, and removing solvent from the purified solution to recover purified water soluble 9-methyl N-hydroxysuccinimidyl carbonate fluorene polymer.

[0032] In yet one or more further embodiments, a recovered or purified water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer or other reactive carbonate prepared by a method as described herein, is reacted with an amine-containing biologically active agent to provide a conjugate.

[0033] In another aspect, provided is a brominated water-soluble fluorene polymer. In one or more embodiments, the brominated water-soluble fluorene polymer has a structure selected from:

wherein POLYa, POLYb, Li, and L2 have values as described elsewhere herein.

[0034] Additional aspects and embodiments are set forth in the following description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] This section intentionally left blank as a placeholder.

DETAILED DESCRIPTION

Definitions

[0036] In describing and claiming certain features of this disclosure, the following terminology will be used in accordance with the definitions described below unless indicated otherwise.

[0037] 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 as well as two or more of the same or different polymers, reference to a "conjugate" refers to a single conjugate as well as two or more of the same or different conjugates, reference to an "excipient" includes a single excipient as well as two or more of the same or different excipients, and the like.

[0038] "Water soluble, non-peptidic polymer" refers to a polymer that is at least 35% (by weight) soluble in water at room temperature. Preferred water soluble, non-peptidic polymers are however preferably greater than 70% (by weight), and more preferably greater than 95% (by weight) soluble in water. Typically, an unfiltered aqueous preparation of a "water-soluble" polymer transmits at least 75% of the amount of light transmitted by the same solution after filtering. Preferably, such unfiltered aqueous preparation transmits at least 95% of the amount of light transmitted by the same solution after filtering. Most preferred are water-soluble polymers that are at least 95% (by weight) soluble in water or completely soluble in water. With respect to being "non-peptidic," a polymer is non-peptidic when it contains less than 35% (by weight) of amino acid residues.

[0039] The terms "monomer," "monomeric subunit" and "monomeric unit" are used interchangeably herein and refer to one of the basic structural units of a polymer. In the case of a homo-polymer, a single repeating structural unit forms the polymer. In the case of a co-polymer, two or more structural units are repeated— either in a pattern or randomly— to form the polymer. Preferred polymers used in connection with the present invention are homo-polymers. The water-soluble, non-peptidic polymer comprises three or more monomers serially attached to form a chain of monomers.

[0040] "PEG" or "polyethylene glycol," as used herein, is meant to encompass any water-soluble polyethylene oxide). Unless otherwise indicated, a "PEG polymer" or a polyethylene glycol is one in which substantially all (preferably all) monomeric subunits are ethylene oxide subunits, though, the polymer may contain distinct end capping moieties or functional groups, e.g., for conjugation. PEG polymers will generally comprise one of the two following structures: "-(CEbCEbO)n-" or "-(CEbCEbO)n-iCEbCEb-," depending upon whether or not the terminal oxygen(s) has been displaced, e.g., during a synthetic transformation. As stated above, for the PEG polymers, the variable (n) ranges from about 3 to 2273, and the terminal groups and architecture of the overall PEG can vary. Additional sub-ranges for“n” are described herein. PEG polymers in connection with the present disclosure are typically end-capped, where a preferred end-capping group is a lower alkyl group, with a most preferred end-capping group being methyl.

[0041] Molecular weight in the context of a water-soluble polymer, such as PEG, can be expressed as either a number average molecular weight or a weight average molecular weight. Unless otherwise indicated, all references to molecular weight herein refer to the weight average molecular weight. Both molecular weight determinations, number average and weight average, can be measured using gel permeation chromatography or other liquid chromatography techniques (e.g. gel filtration chromatography). Most commonly employed are gel permeation chromatography and gel filtration chromatography. Other methods for determining molecular weight include end-group analysis or the measurement of colligative properties (e.g., freezing-point depression, boiling-point elevation, or osmotic pressure) to determine number average molecular weight or the use of light scattering techniques, ultracentrifugation, MALDI TOF, or viscometry to determine weight average molecular weight. PEG polymers are typically polydisperse (i.e., the number average molecular weight and the weight average molecular weight of the polymers are not equal), possessing low polydispersity values of preferably less than about 1.2, more preferably less than about 1.15, still more preferably less than about 1.10, yet still more preferably less than about 1.05, and most preferably less than about 1.03.

[0042] "Branched," in reference to the geometry or overall structure of a polymer, refers to a polymer having two or more polymer "arms" or“chains” extending from a branch point or central structural feature. Examples of some preferred branched polymers are those having one or more of the following features: having two or more polymer arms, having two polymer arms, comprised of polymer chains having the same structure (for example, comprised of the same monomer subunits), and comprised of polymer arms having the same weight average molecular weight.

[0043] A "stable" linkage or bond refers to a chemical bond that is substantially stable in water, that is to say, does not undergo hydrolysis or degradation under physiological conditions to any appreciable extent over an extended period of time. Examples of hydrolytically stable linkages generally include but are not limited to the following: carbon-carbon bonds (e.g., in aliphatic chains), ether linkages, amide linkages, amine linkages, and the like. It is to be understood however, that the stability of any given chemical bond may be affected by the particular structural features of the molecule in which the bond is positioned as well as the placement of the subject linkage within a given molecule, adjacent and neighboring atoms, and the like, as will be understood by one of skill in the chemical arts. One of ordinary skill in the art can determine whether a given linkage is stable or releasable in a given context by, for example, placing a linkage-containing molecule of interest under conditions of interest (e.g., under

physiological conditions) and testing for evidence of release over a suitable time period.

Generally, a stable linkage is one that exhibits a rate of hydrolysis of less than about 1-2% per day under physiological conditions. Hydrolysis rates of representative chemical bonds can be found in most standard organic chemistry textbooks.

[0044] A covalent“releasable” linkage, for example, in the context of a water-soluble polymer such as a polymeric FMOC reagent that is covalently attached to a target molecule, such as for example, an active moiety or other molecule, is one that, under physiological conditions, releases or detaches one or more water-soluble polymers from the active moiety. The release may occur, for example, by any suitable mechanism, and at a rate that is clinically useful. A releasable linkage may also be referred to as a physiologically cleavable bond or linkage.

[0045] "Alkyl" refers to a hydrocarbon chain, typically ranging from about 1 to 15 atoms in length. 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, 3-methylpentyl, and the like.

[0046] "Lower alkyl" refers to an alkyl group containing from 1 to 6 carbon atoms, and may be straight chain or branched, as exemplified by methyl, ethyl, «-butyl, /-butyl, and /-butyl.

[0047] "Alkoxy" refers to an -OR group, wherein R is alkyl or substituted alkyl, preferably Ci-6 alkyl (e.g., methoxy, ethoxy, propyloxy, and so forth).

[0048] The term "substituted" as in, for example, "substituted alkyl," refers to a moiety

(e.g., an alkyl group) substituted with one or more noninterfering substituents, such as, but not limited to: alkyl, C3-8 cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g., fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl; substituted phenyl; and the like.

"Substituted aryl" is aryl having one or more noninterfering groups as a substituent. For substitutions on a phenyl ring, the substituents may be in any orientation (i.e., ortho, meta, or para). Substituents on aryl moieties that are a part of a more complex system, such as a naphthalene or fluorene core, may occupy any aryl ring position not otherwise occupied in the structure.

[0049] "Noninterfering substituents" are those groups that, when present in a molecule, are typically nonreactive with other functional groups contained within the molecule.

[0050] "Aryl" means one or more aromatic rings, each of 5 or 6 core carbon atoms. Aryl includes multiple aryl rings that may be fused, as in naphthyl or unfused, as in biphenyl. Aryl rings may also be fused or unfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclic rings. As used herein, "aryl" includes heteroaryl. An aromatic moiety (e.g., Ar1, Ar2, and so forth), means a structure containing aryl.

[0051] “Heteroaryl" is an aryl group containing from one to four heteroatoms, preferably sulfur, oxygen, or nitrogen, or a combination thereof. Heteroaryl rings may also be fused with one or more cyclic hydrocarbon, heterocyclic, aryl, or heteroaryl rings.

[0052] "Heterocycle" or "heterocyclic" means one or more rings of 5-12 atoms, preferably 5-7 atoms, with or without unsaturation or aromatic character and having at least one ring atom that is not a carbon. Preferred heteroatoms include sulfur, oxygen, and nitrogen.

[0053] Substituted heteroaryl" is a heteroaryl having one or more noninterfering groups as substituents.

[0054] Substituted heterocycle" is a heterocycle having one or more side chains formed from noninterfering substituents.

[0055] An "organic radical" as used herein shall include alkyl, substituted alkyl, aryl, and substituted aryl.

[0056] An“anhydrous” substance is one that contains 500 parts per million (ppm) water or less. Preferably, an anhydrous substance or condition is one that contains 450 ppm water or less, or 400 ppm water or less. More preferably, an anhydrous substance of condition contains 200 ppm water or less. Most preferably, an anhydrous substance contains less water than is measurable by modem analytical methods, which currently is less than 100 ppm. An illustrative range of water content for an anhydrous substance or condition described herein is from about 80 ppm to about 200 ppm.

[0057] “Anhydrous conditions”, for example, in reference to reaction conditions, refers to conditions in which care has been taken to exclude moisture from reactants, solvents, glassware, the atmosphere, and the like. Anhydrous conditions typically include the use of dried solvents (using any suitable drying technique well-known to those of skill in the chemical arts), dried reagents, an inert atmosphere, dried reaction equipment, and the like. Typical methods for drying solvents and measuring the residual water in such solvents may be found in works such as by Pangborn, A. B., et al Organometallics, 1996, 15, 1518-1520; and Williams, D. B. G., et al J Org. Chem. 2010, 75, 8351-8354.

[0058] "Substantially" or "essentially" means nearly totally or completely, for instance,

95% or greater of a given quantity.

[0059] Similarly,“about” or“approximately” as used herein means within plus or minus

5% of a given quantity.

[0060] "Optional" or "optionally" means that the subsequently described circumstance may but need not necessarily occur, so that the description includes instances where the circumstance occurs and instances where it does not.

[0061] "Pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to a component that may be included in the compositions described herein and causes no significant adverse toxicological effects to a subject.

[0062] The term "patient," or“subject” as used herein refers to a living organism suffering from or prone to a condition that can be prevented or treated by administration of a compound or composition or combination as provided herein, such as a cancer, and includes both humans and animals. Subjects include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and preferably are human.

Overview

[0063] The polymeric FMOC reagents and some of the intermediates leading to the final polymeric reagent are susceptible to base-catalyzed loss of the carbonate functionality. The basic portion of the substance catalyzing the reagent’s decomposition may be negatively charged or uncharged. This process is the same type of process that provides the PEG2-FMOC-therapeutic agent conjugates their efficacy. (Note that references to PEG-containing reagents or conjugates is also meant herein to apply equally to other water-soluble polymers such as those described herein). More particularly, a PEG2-FMOC conjugate releases a covalently attached therapeutic agent (“drug”) by reaction in vivo with any substance capable of abstracting the ionizable benzylic proton at the 9-position of the fluorene ring. The drug release process is illustrated in Scheme II in relation to a particular illustrative PEG2-FMOC conjugate structure.

+ other drug j
co2

Scheme II.

[0064] Following a similar mechanistic pathway to the pathway illustrated in Scheme II, the PEG2-FMOC-NHS reagent (or any other suitably activated PEG2-FMOC reagent) may react with a basic substance that is charged or uncharged. This is shown in Scheme III, illustrating the mechanism of the undesirable base-catalyzed elimination reaction of a PEG2-FMOC

intermediate or reagent to provide a PEG2 fulvene derivative.

PEG2 Fulvene

Scheme III.

[0065] The PEG2-FMOC-BTC intermediates are degraded following a similar base-catalyzed process as shown for the NHS reagent in Scheme III, where the leaving group is 1-hydroxybenzotriazolyl rather than N-hydroxysuccinimide.

[0066] Thus, in order to provide an advantageous polymeric reagent, synthetic processes to provide these and other similar ester-like reagents should ideally be carried out under conditions effective to minimize losses due to the interaction of basic substances with the intermediates and final reagents. Such methodologies are particularly preferred when the fluorene ring is substituted with one or more electron withdrawing groups, such as Rel and Re2 in Structure X. The reaction methods, conditions, intermediates, product recovery and purification procedures described herein illustrate that handling and care, for example, to exclude basic substances during processing or workup steps can also be important, for example, when the linker(s), i.e. Li or L2, connecting the polymer become more electron withdrawing due to the presence of certain atoms near or directly attached to the fluorene core. For example, the presence of electron withdrawing groups attached to the fluorenyl core can make the elimination reaction (e.g., Schemes II and III) much more facile, since electron withdrawing influences can affect the proton at the 9 position, making it more acidic and thus more susceptible to removal by a basic species. Thus, base catalyzed decomposition can be particularly problematic, and while typically not severe with the PEG2-G2-FMOC reagent class, it can be very severe with the

PEG2-C2-FMOC reagents and reactive intermediates. As a result, the processes previously described for manufacture of the less reactive PEG-FMOC reagents have been discovered to be less preferred for providing good quality, highly reactive PEG-FMOC reagents (such as, e.g., the NHS reagents), and may not be successfully applied to the preparation of PEG-FMOC reagents of all reactivity types.

[0067] One consequence of the presence of the PEG2 fulvene in the reagent is the potential for an undesirable side reaction between targeted therapeutic agents and the PEG2 fulvene impurity. This side reaction is shown in illustrative Scheme IV below (where the exemplary water-soluble FMOC reagent shown is meant to be illustrative, and not limiting with respect to the structure of the FMOC polymeric fulvene).

Protein-S'

Scheme IV.

[0068] In this reaction, a thiol group of a therapeutic agent, for example, a protein, may react with the PEG2 fulvene (see for example, Culbertson, S., et al., ET.S. Patent No. 8,905,235) ETpon formation of a polymer conjugate as shown in Scheme IV, rather than of the intended conjugate formed by reaction between the PEG2 FMOC active carbonate reagent and, for example, a protein (see, e.g., Scheme I), release of the protein may occur by a totally different mechanism, and under very different environmental conditions. Thus, in one or more aspects, one object of the methods provided herein is to minimize and/or prevent the process shown in Scheme IV. A similar reaction may occur with any reactive nucleophile, such as, for example, an amine, but such reactions are not typically anticipated under the conjugation conditions. [0069] It should be noted that the PEG2-fulvene is not removed during customary purification of a PEG-FMOC-NHS reagent, since a typical purification process is a re-precipitation procedure that removes most small molecules, but does not remove other PEG polymeric impurities. This represents a challenge that has not previously been addressed, but is addressed, among other challenges, by the instant disclosure.

[0070] Among the numerous process improvements and improved materials described herein, the present disclosure provides but is not limited to the following:

(i) improvements to methods for manufacturing reactive water-soluble polymeric FMOC reagents, such as, for example, C2-PEG2-FMOC-NHS;

(ii) methods in which the extremely toxic reagent, phosgene (or its precursors (e.g.

triphosgene)), is replaced with safer reagents effective to form intermediates that can readily be converted into a reactive polymeric FMOC N-hydroxysuccinimide (NHS) reagent (i.e., methods that are absent phosgene or a phosgene precursor);

(iii) methods for the direct activation of 9-hydroxylmethyl fluorene polymers such as C2-PEG2-FMOC-OH with disuccinimidyl carbonate (DSC);

(iv) methods for removing the reactive PEG fulvene impurity prior to activation of polymeric hydroxymethylfluorene;

(v) methods for stabilization of the polymer FMOC active carbonate reagents by acidic additives; and

(vi) methods where a typical polymer-FMOC intermediate, e.g. the chloroformate (ClC(O)O-) or a BTC derivative, is used as a reagent in the formation of a polymer-FMOC-therapeutic agent conjugate to thereby minimize the production of polymer-substituted fulvene, and to improve the yield of the polymer-FMOC therapeutic agent conjugate.

These and other aspects and embodiments are described in greater detail in the sections which follow.

Methods

[0071] As described above, process improvements are provided herein that allow for the manufacture of particularly reactive PEG-FMOC reagents. These discoveries were arrived at, at least in part, by first identifying the root causes of the reactive paths that can lead to the destruction of such reagents. While hydrolysis of a polymer reagent is generally a pathway for destruction of active ester or active carbonate reagents, this pathway has already been addressed (i.e., minimized) in previous descriptions of methods for making and recovering PEG-FMOC reagents (see, e.g., Bentley, M., et al, ET.S. Patent No. 8,252,275). In the methods described herein, attention is directed to the elimination process that leads to formation of the water soluble polymeric fulvene side product that can form as a primary impurity in the water-soluble polymer-FMOC active carbonate product. The fulvene is formed from the reagent as shown in illustrative Scheme III above. As the polymeric fulvene side product is an undesirable impurity in the reagent - because it is a reactive rather than an inert impurity - one of the aims of the methods provided herein is to remove or minimize formation of the fulvene. To that end, several process reactive substances that can lead to formation of the fulvene impurity have been identified, and modified reaction processes have in turn been developed (i) that eliminate or remove those substances, or (ii) in which substances have been added that are effective to neutralize undesirable reactive substances, so that they may not catalyze or otherwise cause the polymeric fulvene to form. A generalized reaction scheme is shown below as Scheme Va.

fulvene

wherein, in each of the structures provided above, POLYa is a first water-soluble, non-peptidic polymer; POLYb is a second, water-soluble non-peptidic polymer; Rel, when present, is a first electron-altering group; Re2, when present, is a second electron-altering group; Li is a first linking moiety; and L2 is a second linking moiety, where the features of each of POLYa, POLYb, Rel, Re2, Li, and L2 are provided in greater detail below.

[0072] Specifically, the undesirable substances that most often lead to formation of the fulvene species from the polymeric reagent, or from reactive intermediates leading to it, are basic substances that are ordinarily present in the reaction process. Such substances can be any chemical species that bears an atom that has a tendency to act as a base. For example, water may act as an acid or a base, and the tendency to act as one or the other is generally dependent on the pH of the medium. In the method of making a desired active carbonate polymer FMOC reagent, the most commonly definable substance that may act as a base is pyridine or a similar type basic substance. This chemical species acts to promote formation of the final active carbonate reagent. So its inclusion, or the inclusion of similar species, is typically utilized to provide favorable product yields. To prevent or minimize the reaction shown in Scheme IV, one of the approaches described herein is to either remove (or substantially remove) pyridine (or a similar species) during product isolation, or, neutralize this or a similar base to minimize its reactivity as a base. Thus, in one or more aspects or embodiments, an improved method for preparing a water-soluble

polymeric FMOC reagent, such as an active carbonate ester, encompasses one or more steps for removing the basic species added to the process.

[0073] Also, in one or more additional aspects or embodiments of preparing a water-soluble polymeric FMOC reagent such as an active carbonate ester, or a precursor thereof, an acidic species is added to the reaction mixture following the reacting step, to thereby neutralize the pyridine or any other basic species that were added during the reaction process to facilitate product formation. In one or more embodiments, the acid is selected from, but is not limited to, acetic acid, triflouroacetic acid, citric acid, sodium dibasic phosphoric acid, potassium hydrogen phosphate, sulfuric acid, m-nitrobenzoic acid, trichloroacetic acid, phosphoric acid or any other inorganic or organic acidic species that does not cause undesirable effects in the reactive carbonate product. In one or more particular embodiments, the acid that is added to the reaction mixture is selected from acetic acid, citric acid, and phosphoric acid.

[0074] For example, in a method for preparing a reactive polymeric FMOC active carbonate reagent, a water-soluble 9-hydroxylmethyl fluorene polymer (e.g., of any one of structures (I), (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h)) is reacted with a reagent useful for forming an active carbonate of the water-soluble fluorene polymer in the presence of a base, followed by recovery of the water-soluble polymer 9-methyl fluorene active carbonate by precipitation. Suitable reactants for forming an active carbonate, or an active-carbonate precursor include, e.g., dibenzotriazolyl carbonate, N-hydroxy succinimide, chloroformates such as 4-nitrobenzyl chloroformate or 4-nitrophenyl chloroformate, and l-hydroxybenzotriazole.

[0075] Representative water-soluble 9-hydroxylmethyl fluorene polymer starting materials are shown below:

where POLYa is a first water-soluble, non-peptidic polymer; POLYb is a second, water-soluble non-peptidic polymer; Rel, when present, is a first electron-altering group; and Re2, when

present, is a second electron-altering group; Li is a first linking moiety; andL2 is a second linking moiety;

e Re2]

(I- a), where Rel and Re2, Li and L2 are as described above (and in greater detail herein), and each POLYa and POLYb are, in this case, mPEG where n independently is in a range from about 3 to about 2273;

are as described above (and in greater detail herein), and each POLYa and POLYb are, in this case, mPEG where n independently is in a range from about 3 to about 2273;

are as described above (and in greater detail herein), and each POLYa and POLYb are, in this case, mPEG where n independently is in a range from about 3 to about 2273;

each POLYa and POLYb are, in this case, mPEG where n independently is in a range from about 3 to about 2273;

POLYb are, in this case, mPEG where each n independently is in a range from about 3 to about 2273:

POLYa and POLYb are, in this case, mPEG and each n independently is in a range from about 3 to about 2273:

POLYb are, in this case, mPEG and each n independently is in a range from about 3 to about 2273, and

POLYb are, in this case, mPEG and each n independently is in a range from about 3 to about

2273.

[0076] Generally, the reaction is carried out under anhydrous conditions and in an anhydrous aprotic solvent. Exemplary aprotic solvents include, for example, halogenated aprotic solvents such as, for example, dichloromethane or trichloroethylene, or non-polar solvents such as benzene, chlorobenzene, nitrobenzene, xylene, cyclohexane, tetralin and toluene. In some embodiments, a mixture of two solvents may prove superior to either alone, and thus may be preferred. In some embodiments, the solvent is an aprotic polar solvent that is effective to dissolve the polymeric starting material. Other aprotic solvents that may be used include, e.g., dimethylformamide, acetone, acetonitrile, dioxane, tetrahydrofuran (THF), dimethylsulfoxide, HMPA (hexamethylphosphoramide), DMA (dimethylacetamide), and NMP

(A-methylpyrrolidinone). Again, in some embodiments, a mixture of two or more solvents may be preferred. Aprotic solvents lack an acidic hydrogen, i.e., they are not proton donors. Some aprotic solvents, however, tend to be mildly basic and may hence not be good choices. One skilled in the art of chemistry would be able to readily determine additional aprotic solvents suitable for use in the reactions described herein. Generally, the choice of a proper solvent depends on the solvent’s ability to dissolve all components of a reaction without reacting with any reaction component or reaction product. Alternatively, the ability of a solvent to be employed under anhydrous conditions is important, since in the reactions described herein, moisture should be minimized to protect moisture-sensitive reactants and products. Furthermore, a solvent may be used to co-distill with moisture that may be present. Some solvents may be better at removing moisture than others, as may be determined through routine experimentation using the guidance provided herein. In one or more embodiments, during the recovery step, the precipitating solvent comprises an acid (such as described above) in an amount effective to partially or completely neutralize excess base present in the reaction mixture.

[0077] As some processes that are suitable for a laboratory in which reactions are carried out by skilled and highly educated chemists are moved into production facilities (in which reactions may be carried out by skilled but less highly educated operators), safety of the technicians becomes a major concern. Thus, to address this concern, in yet one or more further aspects or embodiments, synthetic methods are provided herein in which certain undesirable or dangerous to handle reagents, such as phosgene or its precursors (e.g., triphosgene), are replaced with safer to handle reagents that ultimately lead to the reactive N-hydroxysuccinimide (NHS) reagent. That is to say, in one or more aspects or embodiments, provided herein is a method of preparing a water-soluble polymeric FMOC reagent such as an active carbonate or an

intermediate effective to form an active ester such as an active carbonate ester, that is carried out absent the use of phosgene or one of its precursors as shown generally in Scheme Va or Vb above. Additionally, in some embodiments, diBTC, which under certain circumstances is explosive, is eliminated as a direct route to the NHS carbonate is employed. Thus, in some further embodiments, neither a phosgene derivative nor diBTC is used to preparee the desired active carbonate.

[0078] One such method for making a water-soluble polymer FMOC active carbonate comprises as a reactant, dibenzotriazolyl carbonate (diBTC). DiBTC is an explosion hazard when handled as a dry powder, but is considered safe to handle as a suspension in certain halogenated solvents. Such suspensions are commercially available; moreover, diBTC does not have, like phosgene, the potential to release toxic gases. As a result, in one or more further aspects or embodiments, provided herein is a method that employs a safer-to-use reagent, such as diBTC, to effect the conversion of an intermediate water-soluble polymeric hydroxymethyl fluorene derivative (see, e.g., structure (I), or any one of structures (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h)) to the corresponding polymeric fluorene BTC active carbonate (see, for example, Scheme Va and b). Exemplary BTC carbonates are provided below.

are as described above (and in greater detail herein);

are as described above (and in greater detail herein), and POLYa and POLYb are, in this case, mPEG where each n independently is in a range from about 3 to about 2273;

are as described above (and in greater detail herein), and POLYa and POLYb are, in this case, mPEG where each n independently is in a range from about 3 to about 2273;

are as described above (and in greater detail herein), and POLYa and POLYb are, in this case, mPEG where each n independently is in a range from about 3 to about 2273;

wherein POLYa and POLYb are, in this case, mPEG and each n independently is in a range from about 3 to about 2273;

wherein POLYa and POLYb are, in this case, mPEG where each n independently is in a range from about 3 to about 2273;

wherein

POLYa and POLYb are, in this case, mPEG and each n independently is in a range from about 3 to about 2273;

wherein POLYa and POLYb are, in this case, mPEG and each n independently is in a range from about 3 to about 2273: and

wherein

POLYa and POLYb are, in this case, mPEG and each n independently is in a range from about 3 to about 2273.

[0079] In the method, a water-soluble 9-hydroxymethyl fluorene polymer having a structure such as structure (I) (or, for example, any one of structures (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h)) is reacted with dibenzotriazolyl carbonate in an anhydrous aprotic solvent in the presence of a base under anhydrous conditions to provide a reaction mixture comprising a water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer of structure (II), or in one or more embodiments, for example, any one of structures (Il-a), (Il-b), (II-c), (Il-d), (Il-e), (Il-f), (Il-g), (Il-h), followed by recovering the water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer by precipitation with an anhydrous solvent effective to promote precipitation of the water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer.

[0080] As described above, exemplary anhydrous aprotic solvents for carrying out the reaction include, for example, anhydrous halogenated aprotic solvents such as, for example,

dichloromethane or trichloroethylene, or non-polar solvents such as benzene, chlorobenzene, nitrobenzene, xylene, tetralin and toluene. Other anhydrous aprotic solvents that may be used include, e.g., dimethylformamide, acetone, acetonitrile, dioxane, tetrahydrofuran (THF), dimethylsulfoxide, HMPA (hexamethylphosphoramide), DMA (dimethylacetamide), and NMP (A'-rnethyl pyrrol idinone). Additional comments, found elsewhere in this application, related to choosing an acceptable solvent or mixture of solvents for a particular reaction, also apply here. One skilled in the art would be able to readily determine other suitable aprotic solvents for use in the reactions described herein. In some particular embodiments, the reaction solvent is an anhydrous chlorinated solvent such as dichloromethane or trichloroethylene. In yet some other embodiments, the reaction solvent is an anhydrous solvent selected from dimethylformamide, acetone, acetonitrile, and tetrahydrofuran. In some related embodiments, prior to the reaction, the water-soluble 9-hydroxymethyl fluorene polymer is dissolved in the aprotic organic solvent to form a solution, followed by azeotropic distillation of the solution to provide a solution and/or residue having a water content of less than 500 ppm. Additional approaches for preparing an anhydrous solvent, and/or anhydrous polymeric reactants, intermediates or reagents, and/or for providing anhydrous reaction conditions are described elsewhere herein.

[0081] The reaction comprises a sufficient amount of di-BTC to effect formation of the

BTC carbonate ester. Generally, a sufficient amount of di-BTC is less than about 30 equivalents. For example, in some embodiments of the method, the water-soluble 9-hydroxymethyl fluorene polymer is reacted with less than about 30 equivalents of di-BTC. For example, the water-soluble 9-hydroxymethyl fluorene polymer may be reacted with from about 1 equivalent to about 30 equivalents of di-BTC (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 1, 16, 17, 18, 19, 20, 21 22, 23, 24, 25, 26, 27, 28, 29, or 29.9 equivalents, inclusive of any and all ranges between any two of the preceding values). For example, the amount may be from about 1 to about 25 equivalents of di-BTC, or from about 1 to 20 equivalents of di-BTC, or from about 1 to 15 equivalents of di-BTC, or from about 1 to 10 equivalents of di-BTC. Additional suitable amounts of the di-BTC reagent relative to the water-soluble 9-hydroxymethyl fluorene polymer are from about 2 to about 20 equivalents, from about 5 to about 15 equivalents, or from about 10-20 equivalents.

[0082] Generally, the base used in the reaction is a non-nucleophilic amine or is a weakly nucleophilic amine. For example, illustrative bases that may be used include pyridine, 4-dimethylaminopyridine, N,N-diisopropylethylamine, 2,6-di-tert-butylpyridine, N-methylimidazole, N-methylmorpholine, 2,6-lutidine, 2,4,6-collidine, N,N,2,6-tetramethylpyridine-4-amine, and the like. Additionally, insoluble-polymer-bound forms of any of the foregoing bases may also be employed. For example, polymer bound 4-dimethylaminopyridine is available from Sigma-Aldrich (~3 mmol/g loading, matrix crosslinked with 2% divinylbenzene); also available from Sigma-Aldrich is polymer bound 2,6-di-tert-butylpyridine (-1.8 mmol/g loading, 1% crosslinked with divinyl benzene), along with a number of additional polymer-supported bases. The amine may also be a polyamine such as, for example, N,N,N’,N’-tetram ethyl- l,6-hexamethyldiamine, N,N’, N’, N”,N”-pentamethyldiethylenetriamine, and hexamethylenetetramine, or an insoluble polymer-bound form of any of the foregoing. In one or more embodiments of the method, the amount of base ranges from about 1 to about 30 equivalents, or from about 1 to about 10 equivalents. More particularly, the reaction may be carried out with about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 1, 16, 17, 18, 19, 20, 21 22, 23, 24, 25, 26, 27, 28, 29, or 30 equivalents of base, wherein the foregoing is inclusive of any and all ranges between any two of the preceding values. The optimum amount of base for any particular process is best determined by experiment. The minimum amount of base required to provide the highest reaction yield is preferred.

[0083] The reaction may be carried out with or without mechanical agitation. Typically, but not necessarily, the reaction is carried out with mechanical agitation. Mechanical agitation is especially recommended for large scale reactions to facilitate good mixing. Generally, the water-soluble 9-hydroxymethyl fluorene polymer is reacted with dibenzotriazolyl carbonate at a temperature in a range of from about -20 °C to about 35 °C. Additional exemplary temperature ranges include from about -10 °C to about 25 °C, or from about -5 °C to about 10 °C.

[0084] In turning now to the recovery of the water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer, in some embodiments, the water-soluble 9-hydroxymethyl fluorene polymer and the corresponding water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer product are both soluble in the aprotic organic solvent(s). Thus, in some embodiments, the resulting reaction mixture comprises solids, and recovery of the product comprises first

removing the solids comprised in the reaction mixture, followed by recovery of the benzotriazolyl carbonate product. For example, solids comprised in the reaction mixture may be removed by any suitable method using best practices. For example, the solids may be removed by filtration. Following removal of the solids, an anhydrous precipitating solvent is then typically added to the remaining solution (or filtrate in the instance of having removed solids by filtration) in an amount effective to precipitate the benzotriazolyl carbonate product.

Alternatively, following removal of the solids, the remaining benzotriazolyl carbonate product is added to the anhydrous precipitating solvent. That is to say, the anhydrous precipitating solvent may be added to the polymer product, benzotriazolyl carbonate, to effect precipitation, or the polymer product may be added to the anhydrous precipitating solvent. In some instances, the anhydrous precipitating solvent is miscible with the anhydrous aprotic organic solvent from the reacting step, and is also a solvent in which the 9-methyl benzotriazolyl carbonate fluorene product is insoluble or is substantially insoluble.

[0085] In one or more embodiments, the anhydrous solvent that is incorporated to promote precipitation of the BTC ester product may comprise an acid. In one or more embodiments, the acid is selected from, but is not limited to, acetic acid, triflouroacetic acid, citric acid, sodium dibasic phosphoric acid, potassium hydrogen phosphate, sulfuric acid, m-nitrobenzoic acid, chloroacetic acid, trichloroacetic acid, phosphoric acid or any other inorganic or organic acidic species that does not cause undesirable effects in the reactive carbonate product. In one or more particular embodiments, the acid that is added to the reaction mixture is selected from acetic acid, citric acid, and phosphoric acid. In some preferred embodiments, the acid is phosphoric acid. The acid is generally added in an amount that is sufficient to partially or completely neutralize the base that is contained in the reaction mixture. In some embodiments, the amount of acid that is added to the anhydrous solvent for promoting precipitation of the water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer comprises a small amount of acid. For example, the anhydrous solvent may comprise from about 0.0001 to about 0.5 mole percent acid. For example, the anhydrous precipitating solvent may comprise from about 0.0002 to about 0.4 mole percent acid, or from about 0.0010 to about 0.4 mole percent acid, or from about 0.0050 to about 0.3 mole percent acid. The exact amount of acid used in a particular

process is selected based on the anticipated amount of the base to be neutralized and is best determined by experiment.

[0086] Exemplary precipitating solvents can be determined by those of skill in the art and include aliphatic hydrocarbons and other non-reactive miscible solvents in which the BTC ester product is insoluble or substantially insoluble. Illustrative precipitating solvents include, for example, diethyl ether, isopropyl alcohol (IP A), methyl-t-butyl ether (MTBE), pentane, hexane and heptane, and mixtures of any two or more of the foregoing. One exemplary mixture is a mixture of isopropyl alcohol and methyl-t-butyl ether. One such preferred mixture is a 1 :1 mixture of isopropyl alcohol and methyl-t-butyl ether, although any combination of the two solvents may be employed.

[0087] Following recovery of the precipitated BTC ester product, e.g., by filtration, the recovered product may be further washed with an anhydrous precipitating solvent, i.e., a solvent in which the BTC ester is insoluble or substantially insoluble, where the solvent may comprise a small amount of acid, e.g., from about 0.0001 to about 0.5 mole percent acid. Washes of a recovered FMOC-polymeric product, with, for example, an anhydrous precipitating solvent (also referred to as a“non-solvent”), may further comprise an antioxidant, such as butylated hydroxyl toluene (BHT), to avoid oxidative degradation. The recovered product may then be further dried if desired, and/or further purified using standard art-known methods for purifying water-soluble polymeric reagents. One such particularly preferred method is chromatography, e.g., size exclusion chromatography.

[0088] Example 1 provides an illustration of the reaction and related processing steps described above. See, Example 1B, describing the preparation of G2-PEG2-FMOC-BTC-20kD from the corresponding G2-PEG2-FMOC-OH-20kD. As described therein, following dissolution of the G2-hydroxymethyl fluorene polymer in anhydrous dichloromethane and anhydrous toluene, the solvents were then removed by distillation to remove moisture, followed by dissolution in anhydrous acetonitrile. Following reaction of the G2 -hydroxymethyl fluorene polymer with di-BTC, the product was precipitated by addition of anhydrous isopropyl alcohol (containing butylated hydroxytoluene), recovered by filtration, and further washed with non solvents (e.g., isopropyl alcohol and diethyl ether), and dried under vacuum.

[0089] Example 3, Part 2, IB describes the preparation of C2-PEG2-FMOC-BTC-20kD from the corresponding C2-PEG2-FMOC-OH-20kD. Briefly, C2-PEG2-FMOC-OH-20kD, was dissolved in anhydrous solvent, toluene, and then dried by azeotropically distilling off the solvent under reduced pressure. This process was then repeated. The dried C2-PEG2-FMOC-OH-20kD was then dissolved in anhydrous acetonitrile, the solution cooled (e.g., to 5 °C), followed by addition of di-BTC and the base, pyridine. The mixture was then stirred for several hours. Following reaction, the mixture was added to a solution of cooled isopropyl alcohol containing the acid, phosphoric acid (0.005%). The mixture was then further mixed, and chilled MTBE containing phosphoric acid (0.005%) was added to the mixture followed by additional stirring to facilitate precipitation. The precipitated product was then recovered by filtration, and washed multiple times with a mixture of IPA/MTBE containing phosphoric acid, where subsequent washes contained slightly less phosphoric acid (e.g., 0.005% and 0.002 %). The product was then dried under vacuum at reduced temperature, 15 °C.

[0090] While a BTC derivative itself may be used to react with proteins or other suitable active agents to form polymer-active agent conjugates, the corresponding biotherapeutic agent conjugates are typically formed rapidly when a more active carbonate, e.g., the N-hydroxy succinimidyl (NHS) carbonate (sometimes called an active ester) is employed. Therefore, in yet one or more additional aspects or embodiments, provided is a method in which a polymeric FMOC-BTC derivative such as that of structure (II), or, for example, of any one of structures (II-a) through (Il-h), is used as an intermediate that may be converted into the desired NHS active carbonate ester (see, e.g., Scheme VI), e.g., having a generalized structure such as (X).

Particular PEG2 FMOC-NHS esters are shown in structures (XI), (XII), (XIII), and (XIV). A preferred NHS ester is shown in structure (XIV). The particular reaction conditions for converting a polymeric FMOC-BTC ester into the corresponding reactive NHS ester shown in illustrative Scheme Vb below (e.g., acetonitrile solvent, pyridine base, N-hydroxysuccinimide (NHS) reagent; or e.g., dichloromethane solvent, dimethylaminopyridine, NHS) are meant to be illustrative; suitable solvents, bases, coupling or other additional reagents, and reaction conditions can be readily determined by those skilled in the art for transforming a water-soluble polymer FMOC BTC carbonate (or other less reactive carbonates) into a corresponding NHS carbonate. For example, the conversion reaction may be carried out using from about 1 to about 30 equivalents of NHS. Additional illustrative amounts of the NHS reagent include, for example, from about 1 to about 25 equivalents, from about 2 to about 20 equivalents, from about 3 to about 15 equivalents, and from about 5 equivalents to about 15 equivalents. Typically, during addition of the NHS reagent, the reaction temperature is maintained at from about -20 °C to about 25°C. Illustrative reaction temperature ranges are selected from, for example, from about -15 °C to about 20 °C, from about -10 °C to about 10 °C, from about -10 °C to about 0 °C, from about -8 °C to about 5 °C , and from about -7 °C to about 0 °C. In some embodiments, the temperature is in a range between about -10 °C to about 10 °C. The optimum temperature for a specific reaction can be determined by experimentation. In some embodiments, conversion of the polymeric FMOC-BTC ester into the NHS ester by reaction with NHS is carried out in dichloromethane in the presence of dimethylaminopyridine. Suitable amounts of

dimethylaminopyridine to facilitate the conversion range, for example, from about 4 equivalents to about 0.10 equivalents, from about 3 equivalents to about 0.15 equivalents, from about 2 equivalents to about 0.20 equivalents, from about 1 equivalent to about 0.40 equivalents, and from about 0.75 equivalents to about 0.50 equivalents. In some embodiments, DMAP is present in an amount from about 2 equivalents to about 0.20 equivalents. In some other embodiments, DMAP is present in an amount from about 1 equivalent to about 0.4 equivalents. In some other embodiments, DMAP is present in an amount from about 0.75 equivalents to about 0.50 equivalents. All of the processes where active carbonates are prepared, isolated, purified, or otherwise handled, are preferably carried out in a very dry environment, preferably in a glove box under a blanket of dry nitrogen or argon or in a laboratory with very low humidity.

Furthermore, all solvents and reagents should preferably be of high quality and maintained in a dry environment prior to use. When drying techniques are used to remove moisture prior to a reaction, a moisture analysis is typically carried out to assure that the moisture levels are as low as can be attained using the specified procedure. In repeat experiments, the moisture analysis may be omitted if the moisture-removing process has been validated. Following preparation of a polymeric FMOC-NHS ester, the polymeric FMOC-NHS ester may be recovered and further processed as described below.

Illustrative PEG2 9-hydroxymethyl Fluorene

Exemplary PEG2 BTC Active Carbonate (e.g., an active ester)

Scheme Vb.

Illustrative PEG2 FMOC NHS Active Carbonate (Ester)

Scheme VI.

See, for example, Example 1C, in which an illustrative 9-methyl benzotriazolyl carbonate fluorene PEG polymer, G2-PEG2-FMOC-BTC-20kE), is converted to the corresponding

succinimidyl ester according to the method provided herein. As described in the example, G2-PEG2-FMOC-BTC-20kD was dissolved in an anhydrous solvent, dichloromethane, and the solution then cooled (to 8 °C). N-hydroxysuccinimide was then added to the cooled solution, and the reaction mixture stirred overnight at 8 °C. The resulting NHS ester product was then recovered by precipitation with isopropyl alcohol (containing citric acid and the antioxidant, BHT), isolated by filtration, and then further washed with non-solvents (first with anhydrous isopropyl alcohol (containing BHT), followed by anhydrous methyl-tert butyl ether containing citric acid and BHT), followed by vacuum drying. To determine percent substitution of the active carbonate, reaction with glycine was carried out, followed by analysis. Notably, the NHS ester prepared and processed as described above possessed 88.1 mole percent substitution;

following storage at 11 °C for 136 hours, the product exhibited a percent substitution of 86.6 mole % (a loss of about 1-2%). In contrast, a similar NHS ester product prepared but precipitated absent the addition of acid to the solvent, isopropyl alcohol, and also having no acid present in the methyl tert-butyl ether wash was found to have a degree of substitution of 86.2 mole percent following preparation (slightly less than by the present method). However, following storage at 11 °C for 136 hours, the stored product was determined to have a percent substitution of 75.3 mole % (that is, a loss of product of about 11 %). Thus, the process improvements described herein are effective to provide active fluorene polymer NHS ester reagents having greater stability upon storage, e.g., by using acidic additives during recovery and processing of both intermediates and active carbonate reagents.

[0091] This process is also further exemplified in Example 3, Part 2, IC, which describes the preparation of C2-PEG2-FMOC-NHS 20kD from C2-PEG2-FMOC-BTC 20kD.

[0092] Since the water-soluble polymer FMOC active NHS carbonate (ester) may be more highly desired as a reagent for conjugation with an active agent, such as, for example, a protein, to form a polymeric prodrug, in one or more further aspects or embodiments, also provided herein are methods for the direct activation of C2-PEG2-FMOC-OH (or any other water-soluble 9-hydroxymethyl fluorene polymer as described herein) with disuccinimidyl carbonate (DSC) to provide the corresponding NHS active carbonate. Illustrative polymeric starting materials include those described by structures (I), (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), and (I-h)), where the corresponding NHS active carbonates correspond to each of the

foregoing structures wherein the 9-methyl hydroxyl proton is replaced with ~C(0)0-succinimide. Similarly illustrative products, i.e., the NHS carbonates, include those having structures (III), (X), (XI), (XII), (XIII), (XIV). See generalized Schemes Via and VIb below, where Scheme Via illustrates the subject reaction for a generalized water soluble 9-hydroxymethyl fluorene polymer, while Scheme VIb shows the same reaction for a preferred water soluble PEG-2 9-hydroxymethyl fluorene polymer having a C2 core.

S CLAIMED:

1. A method for preparing a reactive polymeric reagent, said method comprising:

(i) reacting a water-soluble 9-hydroxymethyl fluorene polymer having a structure (I):

wherein

POLYa is a first water-soluble, non-peptidic polymer;

POLYb is a second, water-soluble non-peptidic polymer;

Rel, when present, is a first electron-altering group; and Re2, when present, is a second electron-altering group;

Li is a first linking moiety; and

L2 is a second linking moiety;

with dibenzotriazolyl carbonate in an aprotic organic solvent in the presence of a base under anhydrous conditions to provide a reaction mixture comprising a water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer having a structure:

, wherein POLYa, POLYb, Rei,

R , Li, and L2 each have values as described in step (i), and

(ii) recovering the water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer (II) by precipitation with an anhydrous solvent effective to promote precipitation of the water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer.

2 The method of claim 1, wherein step (i) comprises reacting the water-soluble 9- hydroxymethyl fluorene polymer of structure (I) with from about 1 to about 30 equivalents of dibenzotriazolyl carbonate.

3. The method of claim 1 or claim 2, wherein the base is an amine.

4. The method of claim 3, wherein the base is a non-nucleophilic amine or is a

weakly nucleophilic amine.

5. The method of claim 3 or claim 4, wherein the base is selected from the group consisting of pyridine, 4-dimethylaminopyridine, N,N-diisopropylethylamine, 2,6-di-tert- butylpyridine, N-methylimidazole, N-methylmorpholine, 2,6-lutidine, 2,4,6-collidine, N,N,2,6-tetramethylpyridine-4-amine, N,N,N’,N’-tetramethyl- 1 ,6-hexam ethyl diamine,

N, N’, N’,N”,N”-pentamethyldi ethyl enetri amine, hexamethylenetetramine, and insoluble- polymer-bound forms of any of the foregoing.

6. The method of any one of claims 1-5, wherein step (i) comprises from about 1 to about 30 equivalents of base.

7. The method of claim 6, wherein step (i) comprises from about 1 to about 10

equivalents of base.

8. The method of any one of claims 1-7, wherein step (i) is carried out under a dry and inert gas atmosphere.

9. The method of any one of claims 1-8, wherein step (i) is carried out with

mechanical agitation.

10. The method of any one of claims 1-9, wherein the reacting step is carried out at a temperature in a range of from about -20 °C to about 35 °C.

11 The method of claim 10, wherein the reacting step is carried out at a temperature in a range of from about -10 °C to about 25 °C.

12. The method of claim 10, wherein the reacting step is carried out at a temperature in a range of from about -5 °C to about 10 °C.

13. The method of any one of claims 1-12, where the water-soluble 9-hydroxymethyl fluorene polymer of step (i) is dissolved in the anhydrous, aprotic organic solvent.

14. The method of any one of claims 1-13, where in step (ii), the anhydrous solvent effective to promote precipitation further comprises an acid.

15. The method of claim 14, wherein the anhydrous solvent effective to promote precipitation of the water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer comprises from about 0.0001 to about 0.5 mole percent acid.

16. The method of claim 14 or 15, where the acid is selected from the group

consisting of acetic acid, phosphoric acid, citric acid, sodium dibasic phosphoric acid, potassium, hydrogen phosphate, sulfuric acid, meta-nitrobenzoic acid, trifluoroacetic acid, and trichloroacetic acid, p-toluenesulfonic acid.

17. The method of claim 16, wherein the acid is selected from acetic acid, citric acid and phosphoric acid.

18. The method of claim 17, wherein the acid is phosphoric acid.

19. The method of any one of claims 1-18, further comprising, prior to the reacting step, dissolving the water-soluble 9-hydroxymethyl fluorene polymer of structure (I) in the aprotic organic solvent to form a polymer solution, and drying the polymer solution by azeotropic distillation to provide a polymer solution having a water content of less than 500 ppm.

20. The method of any one of claims 1-19, wherein the recovered water-soluble 9- methyl benzotriazolyl carbonate fluorene polymer from step (ii) comprises less than 10 mole percent of a water-soluble fulvene polymer.

21. The method of any one of claims 1-20, wherein the dibenzotriazolyl carbonate in step (i) is in a halogenated solvent.

22. The method of claim 21, wherein the halogenated solvent is a chlorinated solvent that is either dichloromethane or trichloroethylene.

23. The method of any one of claims 1-20, wherein the aprotic organic solvent from step (i) is selected from dimethylformamide, acetone, acetonitrile, dioxane, and tetrahydrofuran.

24. The method of any one of claims 14-22, wherein the recovering step comprises filtering the reaction mixture from step (i) to remove solids to provide a solution comprising the water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer, followed by adding an amount of an anhydrous solvent effective to precipitate the water- soluble 9-methyl benzotriazolyl carbonate fluorene polymer from the solution.

25. The method of any one of claims 1-24, further comprising isolating the recovered precipitated water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer by filtration.

26. The method of any one of claims 1-25, wherein the anhydrous solvent in step (ii) is miscible with the aprotic organic solvent from step (i), and is a solvent in which the water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer is substantially insoluble.

27. The method of any one of claims 1-26, wherein the anhydrous solvent effective to promote precipitation of the water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer is selected from diethyl ether, isopropyl alcohol, methyl -t-butyl ether, pentane, hexane, heptane, and mixtures of the foregoing.

28. The method of any one of claims 1-27, further comprising washing the recovered water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer with an anhydrous solvent in which the water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer is substantially insoluble, the solvent comprising from about 0.0001 to about 0.5 mole percent acid.

29. The method of any one of claims 1-28, further comprising (iii) purifying the

recovered water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer.

30. The method of any one of the foregoing claims, comprising converting the

recovered or purified water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer to a different reactive carbonate.

31. The method of claim 30, wherein the different reactive carbonate is a water- soluble 9-methyl N-hydroxy succinimidyl carbonate fluorene polymer.

32. The method of claim 31, wherein the converting reaction is carried out by reacting the recovered or purified water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer with N-hydroxysuccinimide in dichloromethane.

33. The method of claim 32, wherein the converting reaction is carried out in the presence of dimethylaminopyridine.

34. The method of any one of claims 1-33, wherein each of POLYa and POLYb is a polyethylene glycol.

35. The method of claim 34, wherein each POLYa and POLYb is a polyethylene

glycol having a weight average molecular weight of from about 120 daltons to about

100,000 daltons, or from about 250 daltons to about 60,000 daltons, or from about 5,000 daltons to about 25,000 daltons.

36. The method of claim 35, wherein each POLYa and POLYb is a polyethylene

glycol having a weight average molecular weight of from about 250 daltons to about 60,000 daltons.

37. The method of any one of claims 1-36, wherein the water-soluble 9- hydroxymethyl fluorene polymer of structure (I) has a structure:

wherein each n independently ranges from about 3 to about 2273, or from about 4 to about 1363, or from about 3 to about 136, or from about 136 to about 1818, or from about 113 to about 568, or from about 227 to about 568; and the water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer of structure (II) has a structure:

38. A method for preparing an N-hydroxyl succinimidyl carbonate ester-activated polymeric reagent, said method comprising:

(i) reacting a water-soluble 9-hydroxymethyl fluorene polymer having a

structure:

wherein

POLYa is a first water-soluble, non-peptidic polymer;

POLYb is a second, water-soluble non-peptidic polymer;

Rel, when present, is a first electron-altering group;

Re2, when present, is a second electron-altering group;

Li is a first linking moiety;

L2 is a second linking moiety;

Rel, which may or may not be present, is a first electron-altering group; and

Re2, which may or may not be present, is a second electron-altering group; with from about 1 to 20 equivalents of disuccinimidyl carbonate in an anhydrous aprotic organic solvent in the presence of a base to provide a reaction mixture comprising a water-soluble 9-methyl N-hydroxysuccinimidyl carbonate fluorene polymer having a structure:

(Ill), wherein POLYa, POLYb, Rel, Re2, Li, and L2 each have values as described in step (i);

and

(ii) recovering the water-soluble 9-methyl N-hydroxysuccinimidyl carbonate fluorene polymer of structure (III) from the reaction mixture.

39. The method of claim 38, wherein prior to reacting step (i), the water-soluble 9- hydroxymethyl fluorene polymer is dissolved in the anhydrous aprotic organic solvent to provide a polymer solution, followed by drying the polymer solution to remove water that may be present to provide a dried polymer solution having less than 500 ppm water content.

40. The method of claim 39, wherein the drying is repeated until a dried polymer solution having a water content of less than 200 ppm or less than 100 ppm is attained.

41. The method of claim 39 or 40, wherein the drying step comprises azeotropically distilling the polymer solution.

42. The method of any one of claims 39-41, wherein said drying is repeated until the water content of the polymer solution remains constant.

43. The method of any one of claims 38-42, wherein the base is a non-nucleophilic or a weakly nucleophilic amine.

44. The method of claim 43, wherein the base is selected from the group consisting of pyridine, 4-dimethylaminopyridine, N,N-diisopropylethylamine, 2,6-di-tert- butylpyridine, N-methylimidazole, N-methylmorpholine, 2,6-lutidine, 2,4,6-collidine, N,N,2,6-tetramethylpyridine-4-amine, N,N,N’,N’-tetramethyl-l,6-hexamethyldiamine,

N, N’, N’,N”,N”-pentamethyldi ethyl enetri amine, hexamethylenetetramine and insoluble- polymer-bound forms of any of the foregoing.

45. The method of any one of claims 38-44, wherein step (i) comprises from about 1 to about 15 equivalents of base.

46. The method of claim 45, wherein step (i) comprises from about 3 to about 10 equivalents of base.

47. The method of any one of claims 38-46, wherein step (i) is carried out under a dry and inert atmosphere.

48. The method of any one of claims 38-47, wherein the reacting step (i) comprises adding the disuccinimidyl carbonate to a solution of the water-soluble 9-methyl N- hydroxysuccinimidyl carbonate fluorene polymer in the anhydrous aprotic organic solvent while maintaining a temperature of from about 0 to about 30 °C.

49. The method of claim 48, wherein said reacting step (i) further comprises,

following the adding step, adjusting the temperature of the reaction mixture to between about 7.5 to about 18 °C, followed by addition of base.

50. The method of any one of claims 38-49, wherein step (i) is carried out with

mixing.

51. The method of claim 50, wherein during said mixing, the temperature of the

reaction mixture is maintained in a range between about 3 °C to about 21 °C.

52. The method of any one of claims 38-51, further comprising, prior to said

recovering step, adding an acid to the reaction mixture from step (i) in an amount effective to neutralize the base.

53. The method of claim 52, where the acid is selected from the group consisting of acetic acid, phosphoric acid, citric acid, sodium dibasic phosphoric acid, potassium hydrogen phosphate, sulfuric acid, meta-nitrobenzoic acid, trifluoroacetic acid, p- toluenesulfonic acid, and trichloroacetic acid.

54. The method of claim 53, wherein the acid is selected from acetic acid, citric acid and phosphoric acid.

55. The method of any one of claims 38-54, wherein the recovered water-soluble 9- methyl N-hydroxysuccinimidyl carbonate fluorene polymer comprises 15 mole percent or less of a water-soluble fulvene polymer.

56. The method of any one of claims 38-55, wherein said recovering step (ii) comprises (ii-a) filtering the reaction mixture to remove solids and provide a solution, followed by (ii-b) precipitating the water-soluble 9-methyl N-hydroxysuccinimidyl carbonate fluorene polymer from the solution.

57. The method of claim 56, wherein the precipitating step comprises addition of an anhydrous precipitating solvent in which the water-soluble 9-methyl N- hydroxysuccinimidyl carbonate fluorene polymer is substantially insoluble.

58. The method of claim 57, wherein the precipitating solvent is at a temperature above its freezing point and below room temperature.

59. The method of claim 57 or claim 58, wherein the precipitating solvent comprises a small amount of acid sufficient to essentially neutralize any remaining base.

60. The method of any one of claims 57, 58 or 59, wherein the precipitating solvent is selected from diethyl ether, isopropyl alcohol, methyl t-butyl ether, ethyl acetate, pentane, hexane, heptane and mixtures of the foregoing.

61. The method of any one of claims 38-60, further comprising washing the

recovered water-soluble 9-methyl N-hydroxysuccinimidyl carbonate fluorene polymer with an acidified precipitating solvent.

62. The method of any one of claims 38-61, further comprising (iv) purifying the recovered water-soluble 9-methyl N-hydroxysuccinimidyl carbonate fluorene polymer.

63. The method of claim 62, further comprising detecting water-soluble fulvene

polymer in the recovered water-soluble 9-methyl N-hydroxysuccinimidyl carbonate fluorene polymer, wherein the purifying step (iv) comprises dissolving the recovered water-soluble 9-methyl N-hydroxysuccinimidyl carbonate fluorene polymer in a solvent to provide a solution, passing the solution through a thiol-containing resin to remove any water-soluble fulvene polymer to thereby provide a purified solution, and removing solvent from the purified solution to recover purified water soluble 9-methyl N- hydroxysuccinimidyl carbonate fluorene polymer.

64. The method of any one of claims 1-63, wherein the water-soluble 9- hydroxymethyl fluorene polymer of step (i) has a structure:

wherein each n is independently from about 3 to 2273, or from about 4 to about 1363, or from about 3 to about 136, or from about 136 to about 1818, or from about 113 to about 568, or from about 227 to about 568.

65. The method of any one of claims 1-63, wherein the water soluble 9- hydroxymethyl fluorene polymer of step (i) has a structure:

wherein each n is independently from about 3 to 2273, or from about 4 to about 1363, or from about 3 to about 136, or from about 136 to about 1818, or from about 113 to about 568, or from about 227 to about 568.

66. The method of any one of claims 1-63, wherein the water soluble 9- hydroxymethyl fluorene polymer of step (i) has a structure:

wherein each n is independently from about 3 to 227.

67. The method of claim 65 or 66, wherein Rel and/or Re2 are each independently selected from halo, nitro, lower alkyl, lower alkoxy, trifluoromethyl, and -SCbH.

68. The method of any one of claims 65, 66 or 67, wherein Li and L2 each

independently has a length of from 1 to 25 atoms.

69. The method of claim 65, wherein Rel and Re2 are both located on the same

aromatic ring.

70. The method of claim 65, wherein Rel and Re2 are located on different aromatic rings.

71. The method of claim any one of claims 64-70, wherein Li and L2 are each

independently selected from the group consisting of -(CH2)i-6C(0)NH- and -NH-C(O), NH-C(0)-(CH2)I-6C(0)NH-.

72. The method of claim 71, wherein Li and L2 are each independently selected from the group consisting of -(CH2)C(0)NH-, -(CH2)3C(0)NH-, -NH-C(O), and NH-C(O)- (CH2)3C(0)NH-.

73. The method of any one of claims 64-72, wherein the weight average molecular weight of each polyethylene glycol in structure (I) is about the same (e.g., each“n” is about the same).

74. The method of claim 73, wherein each polyethylene glycol in structure (I) has a weight average molecular weight ranging from about 120 daltons to about 6,000 daltons.

75. The method of claim 73, wherein each polyethylene glycol in structure (I) has a weight average molecular weight ranging from about 6,000 daltons to about 80,000 daltons.

76. The method of claim 73, wherein each polyethylene glycol in structure (I) has a weight average molecular weight selected from the group consisting of about 5,000 daltons, 7500 daltons, 10,000 daltons, 15,000 daltons, 20,000 daltons, 30,000 daltons and 40,000 daltons.

77. The method of any one of claims 64-76, where in structures (I) and (II), Ll is attached to fluorene carbon-5 and L2 is attached to fluorene carbon-2.

78. The method of any one of claims 64-76, where in structures (I) and (II), Ll is attached to fluorene carbon-7 and L2 is attached to fluorene carbon-2.

79. The method of any one of claims 64-76, wherein structure (I) is selected from the group consisting of:

80. The method of any one of claims 1 to 37, further comprising reacting the

recovered or purified water-soluble 9-methyl benzotriazolyl carbonate fluorene polymer or other reactive carbonate with an amine-containing biologically active agent.

81. The method of any one of claims 38-79, further comprising reacting the recovered or purified water-soluble 9-methyl N-hydroxy succinimidyl carbonate fluorene polymer with an amine-containing biologically active agent.