WO2011005980A1

WO2011005980A1 - Novel heterobifunctional polyethylene glycol reagents, their preparation and uses thereof

Info

Publication number: WO2011005980A1
Application number: PCT/US2010/041394
Authority: WO
Inventors: Daniel E. Levy; Samuel Zalipsky
Original assignee: Intradigm Corporation
Priority date: 2009-07-09
Filing date: 2010-07-08
Publication date: 2011-01-13

Abstract

The invention relates to novel heterobifunctional polyethylene glycol reagents, methods of producing them and methods of using them.

Description

NOVEL HETEROBIFUNCTIONAL POLYETHYLENE GLYCOL REAGENTS, THEIR PREPARATION AND USES THEREOF

Field of the Invention

[0001] The invention relates to novel heterobifunctional polyethylene glycol reagents, methods of producing them and methods of using them.

Background of the Invention

[0002] Polyethylene glycol (PEG) is an inert, non-toxic water-soluble polymer. As a result of these and other desirable properties, derivatives of PEG have found use as reagents in a variety of pharmaceutical, biomedical and biotechnical applications.

[0003] However, the low reactivity of the terminal hydro xyl groups limits the uses of unmodified PEG as a reagent and requires that the terminal hydroxyl groups be converted to more reactive moieties. According to prior art methods, more reactive PEG analogues have been generated by conversion of one of the terminal PEG hydroxyl groups to an activated moiety followed by isolation of the

monofunctionalized PEG from a mixture of unmodified PEG, monofunctionalized PEG and bifunctionalized PEG.

[0004] Alternatively, prior art methods have also been employed to first protect one of the terminal hydroxyl groups as an alkyl ether (or another non-reactive

functionality) followed by conversion of the second, unblocked terminal hydroxy group to an electrophilic center (e.g., an activated carboxylate) to generate a bifunctionalized PEG. Such bifunctional PEG reagents contain reactive groups on both ends of the PEG reagent. These bifunctional reagents may contain the same reactive group on both terminal ends of the PEG (i.e., homobifunctional PEG reagents) or different groups (i.e., heterobifunctional PEG reagents). Heterobifunctional PEG reagents provide advantages over homobifunctional PEG reagents in that each functional group of the heterobifunctional PEG reagent can form a covalent attachment with a different molecule on each terminus. In such a reaction, the two separate molecules become linked by the PEG polymer. (Zalipsky et al., J. Bioactive and Compatible Polymers, 5, 227-231, (1990); Zalipsky, Bioconjugate Chem., 6, 150-165 (1995); Thompson et al., Polymer, 49, 345-373 (2008)).

Additional heterobifunctional PEG reagents comprising terminal vinyl sulfone and ester functionalities are described in WO 2009/064459. AU references mentioned herein are expressly incorporated by reference.

[0005] The synthesis of modified PEG reagents, however, can be complicated and lengthy, particularly when different functionalities are desired on each end of the PEG reagent (i.e., heterobifunctional PEG reagents). Further, the coupling of the PEG reagent to biologically relevant target molecules can proceed with less than desirable levels of efficiency and target specificity.

[0006] Thus, there remains a significant need to develop new heterobifunctional PEG reagents, and methods for producing such reagents.

Summary of the Invention

[0007] In one aspect, the present invention provides a PEG reagent comprising a compound of Formula (I):

wherein:

each Ri is independently selected from the group consisting of branched or straight-chain C₁-C₆ alkyl, branched or straight-chain C₂-C₆ alkenyl, and branched or straight-chain C₂-C₆ alkynyl,

wherein both Ri can be taken together to form a 5- or 6-membered cyclic acetal or thioacetal, and

wherein each Ri is independently, optionally substituted with one or more R₂, or when both Ri are taken together to form a 5- or 6-membered cyclic acetal or thioacetal, the cyclic acetal or thioacetal is optionally substituted with one or more

R₂; each R₂ is independently selected from the group consisting of branched or straight-chain Ci-Cβ alkyl, branched or straight-chain C2-C6 alkenyl, branched or straight-chain C₂-C₆ alkynyl, hydroxy, Ci-C₆ alkoxy, -CO₂H, -CO₂(RJi -CONH₂, -CONH(RJ] -CON(RJi, carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl, nitro, cyano, and halo,

wherein one or more hydrogens in the alkyl, alkenyl or alkynyl chain may be replaced by one or more halogens, and

wherein each carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl may be optionally substituted with one or more R5;

R3 is CO-CI₄ aryl, C5-C14 heteroaryl or C5-C14 heterocyclyl,

wherein said heteroaryl or heterocyclyl contains one or more heteroatoms selected from the group consisting of N, N(R₄), O, S, S(O), and S(O)₂, and

wherein R3 is optionally substituted with one or more R5;

R4 is selected from the group consisting of hydrogen, branched or straight- chain Ci-C₆ alkyl, branched or straight-chain C₂-Ce alkenyl, branched or straight- chain C₂-C₆ alkynyl, wherein one or more hydrogens in the alkyl, alkenyl or alkynyl chain may be replaced by one or more halogens;

R₅ is selected from the group consisting of branched or straight-chain Ci-C₆ alkyl, branched or straight-chain C₂-Ce alkenyl, branched or straight-chain C₂-Ce alkynyl, -CF₃, -Rg-ORg-OH, -SH, -SRøprotected OH (e.g., acyloxy), -NO₂, -CN, - NH₂, -NHRp-N(RJi, -NHCORg-NHCONH₂, -NHCONHRg-NHCON(RJi, - NRCORg-NHCO₂H, -NHCO₂Rg-CO₂Rg-CO₂H, -CORg-CONH₂, -CONHRg- CON(RJi, -S(O)₂H, -S(O)₂Rg-S(O)₃H, -S(O)₃Rg-S(O)₂NH₂, -S(O)H, -S(O)Rg- S(O)₂NHRg-S(O)₂N(RJi, -NHS(O)₂H, -NHS(O)₂Rg-F, -Cl, -Br, and =O, where chemically feasible;

RQs selected from the group consisting of hydrogen, branched or straight- chain Ci-C₆ alkyl, branched or straight-chain C₂-Ce alkenyl, branched or straight- chain C₂-C₆ alkynyl, carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, or heteroaralkyl and each RQs optionally substituted with one or more halogen, nitro, cyano, amino, -NH-(unsubstituted aliphatic), -N-(unsubstituted aliphatic)2, carboxy, carbamoyl, hydroxy, -O-(unsubstituted aliphatic), -SH, -S-(unsubstituted aliphatic), - CF₃, -S(O)₂NH₂ ainsubstituted aliphatic, unsubstituted carbocyclyl, unsubstituted heterocyclyl, unsubstituted aryl, unsubstituted aralkyl, unsubstituted heteroaryl, or unsubstituted heteroaralkyl;

A is selected from the group consisting of Ci-C₆ alkyl, C₂-C₆ alkenyl, and C₂- C_O alkynyl;

wherein A is optionally substituted with branched or straight-chain C₁-

C₆ alkyl, branched or straight-chain C₂-C₆ alkenyl, branched or straight-chain C₂-C₆ alkynyl, hydroxy, Ci-C₆ alkoxy, CO₂H, CO₂(Ci-C₆ alkyl), CONH₂, CONH(Ci-C₆ alkyl), CON(Ci-C₆ alkyl)₂, nitro, cyano, or halo;

each X is independently O or S;

j is an integer from O to 10; and

n is an integer from 1 to 1,500.

[0008] In a particular embodiment of the present invention, Formula (I) has the following formula:

[0009] While any PEG moiety can be used in the compounds of Formula (I), preferred PEGs include PEG3400 and PEG8000.

[0010] In another aspect, the present invention provides a PEG reagent comprising a compound of Formula (II):

wherein:

each Ri is independently selected from the group consisting of branched or straight-chain Ci-C₆ alkyl, branched or straight-chain C₂-C₆ alkenyl, and branched or straight-chain C₂-C₆ alkynyl,

R₂;

each R₂ is independently selected from the group consisting of branched or straight-chain Ci -C_O alkyl, branched or straight-chain C2-C6 alkenyl, branched or straight-chain C₂-C₆ alkynyl, hydroxy, Ci-C₆ alkoxy, -CO₂H, -CO₂(RJi -CONH₂, -CONH(RJ] -CON(RJi, carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl, nitro, cyano, and halo,

wherein each carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl may be optionally substituted with one or more R₇;

R₄ is selected from the group consisting of hydrogen, branched or straight- chain Ci-C₆ alkyl, branched or straight-chain C₂-Ce alkenyl, branched or straight- chain C₂-C₆ alkynyl, wherein one or more hydrogens in the alkyl, alkenyl or alkynyl chain may be replaced by one or more halogens;

R₆ is C₆-Ci₄ aryl, C5-C14 heteroaryl or C5-C 14 heterocyclyl,

wherein Re is optionally substituted with one or more R7; and

R₇ is selected from the group consisting of branched or straight-chain Ci-C₆ alkyl, branched or straight-chain C₂-C₆ alkenyl, branched or straight-chain C₂-C₆ alkynyl, -CF₃, -Rg-ORg-OH, -SH, -SRPprotected OH (e.g., acyloxy), -NO₂, -CN, - NH₂, -NHRp-N(RJl, -NHCOR 0-NHCONH₂, -NHCONHRg-NHCON(RJ₂, - NRCORf]-NHCO₂H, -NHCO₂Rf]-CO₂Rg-CO₂H, -CORg-CONH₂, -CONHRg- CON(R[β, -S(O)₂H, -S(O)₂RO-S(O)₃H, -S(O)₃Rg-S(O)₂NH₂, -S(O)H, -S(O)Rg- S(O)₂NHRg-S(O)₂N(RJi, -NHS(O)₂H, -NHS(O)₂Rg-F, -Cl, -Br, and =O, where chemically feasible;

wherein R[Is selected from the group consisting of hydrogen, branched or straight-chain Ci-C₆ alkyl, branched or straight-chain C₂-C₆ alkenyl, branched or straight-chain C₂-C₆ alkynyl, carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, or heteroaralkyl and each RQs optionally substituted with one or more halogen, nitro, cyano, amino, -NH-(unsubstituted aliphatic), -N-(unsubstituted aliphatic)₂, carboxy, carbamoyl, hydroxy, -O-(unsubstituted aliphatic), -SH, -S-(unsubstituted aliphatic), - CF₃, -S(O)₂NH₂ Qinsubstituted aliphatic, unsubstituted carbocyclyl, unsubstituted heterocyclyl, unsubstituted aryl, unsubstituted aralkyl, unsubstituted heteroaryl, or unsubstituted heteroaralkyl;

A is selected from the group consisting of Ci -C_O alkyl, C₂-C_O alkenyl, and C₂- C_O alkynyl;

wherein A is optionally substituted with branched or straight-chain C₁- C_δ alkyl, branched or straight-chain C₂-C₆ alkenyl, branched or straight-chain C₂-C₆ alkynyl, hydroxy, Ci-C₆ alkoxy, CO₂H, CO₂R₆, CO₂(Ci-C₆ alkyl), CONH₂,

CONH(Ci-C₆ alkyl), CON(C₁-C₆ alkyl)₂, nitro, cyano, or halo;

each X is independently O or S;

j is an integer from O to 10; and

n is an integer from 1 to 1,500.

[0011] In a particular embodiment of the present invention, Formula (II) has the following formula:

[0012] While any PEG moiety can be used in the compounds of Formula (II), preferred PEGs include PEG3400 and PEG8000.

[0013] In another aspect, the present invention provides a PEG reagent comprising a compound of Formula (III):

wherein:

Ri, R2, R4, R₆, R7, R0X, j, and n are defined as in Formula (II);

each B is any natural amino acid, or an unnatural alpha or beta amino acid, wherein each is independently optionally substituted with one or more -CO₂Re or R₇;

wherein any -CO₂H present in B is optionally substituted with R₆ to yield a -CO₂R₆ moiety; and k is ε in integer from 1 to 5.

[0014] In < i particular embodiment of the present invention, Formula (III) has the following formula:

[0015] While any PEG moiety can be used in the compounds of Formula (III), preferred PEGs include PEG3400 and PEG8000.

[0016] In another aspect of the present invention, methods for the synthesis of functionalized PEG reagents of the present invention are provided. Such methods are more efficient compared to prior art methods, reduce the amount of synthetic steps, and increase the overall synthetic yield of the PEG reagent.

[0017] In one aspect, the present invention provides a method for producing a heterobifunctional PEG according to Scheme I:

Scheme I

In Scheme I:

Ri, R₂, R₃, R₄, R₅, A, X, j and n are defined as in Formula (I);

Rs is selected from the group consisting of branched or straight-chain CI-C_Θ alkyl, branched or straight-chain C₂-Ce alkenyl, branched or straight-chain C₂-C₆ alkynyl, C₆-Cu aryl, C_O-C_I4 carbocycle, Cs-Ci₄ heteroaryl, and C5-C14 heterocycle, wherein each alkyl, alkenyl and alkynyl is optionally substituted with one or more R₂,

wherein each heteroaryl or heterocycle contains one or more heteroatoms selected from the group consisting of N, N(R₄), O, S, S(O), and S(O)2, and

wherein each aryl, carbocycle, heteroaryl, or heterocycle is optionally substituted with one or more R5; and

Rg, taken with the -OC(O)O- it is attached to, is any synthetically useful carbonate.

[0018] In Scheme I, (A) is converted to the corresponding ester (B) by any esterifϊcation method known in the art (e.g., reaction with alcohol and catalytic acid such as HCl). The free hydroxy 1 group on (B) is then converted to carbonate (C) by treatment with, for example, the appropriate carbonate or chloroformate. (C) is then ,

reacted with amine to yield carbamate (D). The ester of (D) is

hydrolyzed (e.g., under basic conditions) to yield carboxylic acid (E). Finally, carboxylic acid (E) is converted to the heterobifunctional PEG of Formula (I) using standard techniques in the art.

[0019] In another aspect, the present invention provides a method for producing a heterobifunctional PEG according to Scheme II:

Scheme II

R₁, R₂, R₄, R_O, R₇, A, X, j, and n are defined as in Formula (II); and

R₈ is defined as in Scheme I.

XR₁

[0020] In Scheme II, (F) is first reacted with amine and then with amine NH2 ACOORs to generate a mixture of bifuncti in (G) is the

major component (when A is CH₂CH₂) of the product mixture. The PEG mixture containing (G) is hydrolyzed (e.g., under basic conditions) and then subjected to ion exchange chromatography to yield the pure carboxylic acid (H). Carboxylic acid (H) is then converted the heterobifunctional PEG of Formula (II) using standard techniques in the art.

[0021] In another aspect, the present invention provides a method for producing a heterobifunctional PEG according to Scheme III: Scheme III

In Scheme III:

R₁, R₂, R₄, Re, R₇, B, X, j, k, and n are defined as in Formula (III); and

R₈ is defined as in Scheme I. 2

[0022] In Scheme III, (I) is first reacted with amine and then with amine (B)k-Rs to generate a mixture of bifunctional P

is the major component (when B is beta-alanine and k = 1) of the product mixture. In

embodiments wherein (B )k contains more than one carboxylic acid (i.e., when k is greater than 1 , and/or one or more B S are substituted with or naturally comprise a carboxylic acid), certain carboxylic acids may be protected as, e.g., -CO₂Rs esters, where appropriate. The PEG mixture containing (J) is hydrolyzed (e.g., under basic conditions) and then subjected to ion exchange chromatography to yield the pure carboxylic acid (K). Carboxylic acid (K) is then converted the heterobifunctional PEG of Formula (III) using standard techniques in the art. In embodiments wherein (B)k contains more than one carboxylic acid (Le., when k is greater than 1, and/or one or more BΘare substituted with or naturally comprise a carboxylic acid), some or all of the carboxylic acids may be activated as, e.g., -CO2R6 esters, where appropriate.

[0023] The methods illustrated in Schemes (I), (II) and (III) advantageously provide efficient synthetic protocols for the generation of novel heterobifunctional PEGs bearing versatile reactive end groups that are useful where existing heterobifunctional PEGs are not. In particular, the acetal functionality provides a masked aldehyde which, when unmasked, is useful for the formation of imines and oximes through direct condensation. Additionally, the unmasked aldehyde is useful for the formation of amines via reductive amination, olefins via Wittig, aldol, Horner-Emmons or Julia- Lithgoe reactions or peptides via Ugi reactions. One skilled in the art will recognize that any process capable of forming a chemical bond through reaction with an aldehyde will be applicable to the PEGs of the present invention.

[0024] In another aspect, the present invention provides methods of using a heterobifunctional PEG to generate a vector of Formula (IV):

comprising the ste

p of reacting a compound of Formula (I) with (X) and (Y), wherein (X) and (Y) can be a small molecule, protein, polypeptide, peptide, monosaccharide, polysaccharide, antibody, polynucleotide, oligonucleotide, other polymeric species, polypeptide side chain or a biologically relevant targeting moiety, or a fragment, dimer, trimer or oligomer thereof. (X) is typically conjugated via an imine, oxime or amine linkage (represented by a squiggly bond).

[0025] In some embodiments, (X) and (Y) are simultaneously reacted with a heterobifunctional PEG. In other embodiments, (X) and (Y) are sequentially reacted with a heterobifunctional PEG. In yet other embodiments, (Y) is reacted with a heterobifunctional PEG and the acetal end of the resulting product is then hydro lyzed to an aldehyde prior to reacting with (X).

[0026] In some embodiments, (X) is a cationic polymer. In some embodiments, (Y) is a targeting moiety. In one embodiment, (X) is a cationic polymer and (Y) is a targeting moiety.

[0027] In another aspect, the present invention provides methods of using a heterobifunctional PEG to generate a vector of Formula (V):

comprising the step of reacting a compound of Formula (II) with (X) and (Y), wherein (X) and (Y) can be a small molecule, protein, polypeptide, peptide, monosaccharide, polysaccharide, antibody, polynucleotide, oligonucleotide, other polymeric species, polypeptide side chain or a biologically relevant targeting moiety, or a fragment, dimer, trimer or oligomer thereof. (X) is typically conjugated via an imine, oxime or amine linkage (represented by a squiggly bond).

[0028] In some embodiments, (X) and (Y) are simultaneously reacted with a heterobifunctional PEG. In other embodiments, (X) and (Y) are sequentially reacted with a heterobifunctional PEG. In yet other embodiments, (Y) is reacted with a heterobifunctional PEG and the acetal end of the resulting product is then hydro lyzed to an aldehyde prior to reacting with (X).

[0029] In some embodiments, (X) is a cationic polymer. In some embodiments, (Y) is a targeting moiety. In one embodiment, (X) is a cationic polymer and (Y) is a targeting moiety.

[0030] In another aspect, the present invention provides methods of using a heterobifunctional PEG to generate a vector of Formula (VI):

O H V^ comprising the s

tep of re nd (Y), wherein (X) and (Y) can be a small molecule, protein, polypeptide, peptide, monosaccharide, polysaccharide, antibody, polynucleotide, oligonucleotide, other polymeric species, polypeptide side chain or a biologically relevant targeting moiety, or a fragment, dimer, trimer or oligomer thereof. (X) is typically conjugated via an imine, oxime or amine linkage (represented by a squiggly bond). In some

embodiments (Y) is attached via a PEG or aminoethoxyalkyl moiety. In other embodiments (Y) is attached via an aminoethoxy ethyl moiety.

[0031] As described in the context of Formula (III), B can be any natural amino acid, or an unnatural alpha or beta amino acid, wherein each B is independently optionally substituted with one or more -CO₂R_O or R₇. Further, any -CO2H present in B is optionally substituted with R^ to yield a -CO2R₆ moiety. Accordingly, a vector of Formula (VI) may comprise one or more (Y) moieties (attached by, for example, coupling via a -CO2R6 moiety). In some embodiments, the vector of Formula (VI) comprises 1-10 (Y) moieties. In some embodiments, the vector of Formula (VI) comprises 1-5 (Y) moieties. In other embodiments, the vector of Formula (VI) comprises 2-4 (Y) moieties. In yet other embodiments, the vector of Formula (VI) comprises 3 (Y) moieties.

[0032] In some embodiments, (X) and (Y) are simultaneously reacted with a heterobifunctional PEG. In other embodiments, (X) and (Y) are sequentially reacted with a heterobifunctional PEG. In yet other embodiments, (Y) is reacted with a heterobifunctional PEG and the acetal end of the resulting product is then hydro lyzed to an aldehyde prior to reacting with (X).

[0033] In some embodiments, (X) is a cationic polymer. In some embodiments, (Y) is a targeting moiety. In one embodiment, (X) is a cationic polymer and (Y) is a targeting moiety. In some embodiments, (Y) comprises a monosaccharide or polysaccharide optionally attached via a PEG or aminoethoxyalkyl moiety. In some embodiments, (Y) comprises N-acetyl galactosamine or an N-acetyl galactosamine beta amino ethoxy ethyl glycoside.

[0034] In another aspect, the present invention provides a method of using a heterobifunctional PEG to generate a vehicle for targeted nucleic acid delivery of Formula (VII):

comprising the steps of (a) reac f Formula (I) with (X) and (Y); and (b) mixing

with a nucleic acid. njugated via an imine, oxime or amine linkage (represented by a squiggly bond). In some embodiments, (X) and (Y) are as described in the context of Formula (IV). In some embodiments, (X) is a cationic polymer. In some embodiments, (Y) is a targeting moiety.

[0035] In some embodiments, (X) and (Y) are simultaneously reacted with heterobifunctional PEG. In other embodiments, (X) and (Y) are sequentially reacted with heterobifunctional PEG. In yet other embodiments, (Y) is reacted with a heterobifunctional PEG and the acetal end of the resulting product is then hydro lyzed to an aldehyde prior to reacting with (X).

[0036] In another aspect, the present invention provides a method of using a heterobifunctional PEG to generate a vehicle for targeted nucleic acid delivery of Formula (VIII):

compris

ing the step Formula (II) with (X) and (Y); and (b) mixing with a nucleic acid. (X) is typically conjugated via an imine, oxime or amine linkage (represented by a squiggly bond). In some embodiments, (X) and (Y) are as described in the context of Formula (IV). In some embodiments, (X) is a cationic polymer. In some embodiments, (Y) is a targeting moiety.

[0037] In some embodiments, (X) and (Y) are simultaneously reacted with heterobifunctional PEG. In other embodiments, (X) and (Y) are sequentially reacted with heterobifunctional PEG. In yet other embodiments, (Y) is reacted with a heterobifunctional PEG and the acetal end of the resulting product is then hydro lyzed to an aldehyde prior to reacting with (X).

[0038] In another aspect, the present invention provides a method of using a heterobifunctional PEG to generate a vehicle for targeted nucleic acid delivery of

Formula (IX):

comprising the steps of (a) reacting a compound of Formula (III) with (X) and (Y); and (b) mixing with a nucleic acid. (X) is typically conjugated via an inline, oxime or amine linkage (represented by a squiggly bond). In some embodiments, (X) and (Y) are as described in the context of Formula (IV). In some embodiments, (X) is a cationic polymer. In some embodiments, (Y) is a targeting moiety. In some embodiments (Y) is attached via a PEG or aminoethoxyalkyl moiety. In other embodiments (Y) is attached via an aminoethoxy ethyl moiety.

[0039] As described in the context of Formula (III), B can be any natural amino acid, or an unnatural alpha or beta amino acid, wherein each B is independently optionally substituted with one or more -CO2R0 or R₇. Further, any -CO2H present in B is optionally substituted with R^ to yield a -CO2R6 moiety. Accordingly, a vehicle of Formula (IX) may comprise one or more (Y) moieties (attached by, for example, coupling via a -CO₂R_O moiety). In some embodiments, the vehicle of Formula (IX) comprises 1-10 (Y) moieties. In some embodiments, the vehicle of Formula (IX) comprises 1-5 (Y) moieties. In other embodiments, the vehicle of Formula (IX) comprises 2-4 (Y) moieties. In yet other embodiments, the vehicle of Formula (IX) comprises 3 (Y) moieties.

[0040] In some embodiments, (X) and (Y) are simultaneously reacted with heterobifunctional PEG. In other embodiments, (X) and (Y) are sequentially reacted with heterobifunctional PEG. In yet other embodiments, (Y) is reacted with a heterobifunctional PEG and the acetal end of the resulting product is then hydro lyzed to an aldehyde prior to reacting with (X).

[0041] In some embodiments, (Y) comprises a monosaccharide or polysaccharide optionally attached via a PEG or aminoethoxyalkyl moiety. In other embodiments, (Y) comprises N-acetyl galactosamine or an N-acetyl galactosamine beta

aminoethoxyethyl glycoside.

Detailed Description of the Invention

[0042] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. These materials, methods and examples are illustrative only, and are not intended to be limiting. All publications, patents and other documents mentioned herein are incorporated by reference in their entirety.

[0043] Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Definitions

[0044] In order to further define the invention, the following terms and definitions are provided. As used above, and throughout the specification, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0045] "Aliphatic" refers to straight chain or branched hydrocarbons that are completely saturated or that contain one or more units of unsaturation. For example, aliphatic groups include substituted or unsubstituted linear or branched alkyl, alkenyl and alkynyl groups. Unless indicated otherwise, the term "aliphatic" encompasses both substituted and unsubstituted hydrocarbons.

[0046] "Alkyl" refers to both straight and branched saturated chains containing, for example, 1-3, 1-6, 1-9, or 1-12 carbon atoms.

[0047] "Alkenyl" refers to both straight and branched saturated chains containing, for example, 1-3, 1-6, 1-9, or 1-12 carbon atoms, and at least one carbon-carbon double bond.

[0048] "Alkynyl" refers to both straight and branched saturated chains containing, for example, 1-3, 1-6, 1-9, or 1-12 carbon atoms, and at least one carbon-carbon triple bond.

[0049] An "activating agent" refers to an agent which, for example, facilitates a reaction at a carboxylic acid or, for example, allows for more facile nucleophilic substitution on a carbon atom adjacent to a hydroxy group. Activating agents are generally known in the art and are routinely used to convert, for example, carboxylic acids to active ester and hydroxyl groups to leaving groups. Examples of carboxylic acid activating agents include, but are not limited to, N,NEcarbonyldiimidazole (CDI), N^NEHicyclohexylcarbodiimide (DCC), l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (EDCI), l-[bis(dimethylamino)methylene]-lH- l,2,3-triazolo[4,5-b]pyridinium-3-oxid- e hexafluorophosphate (HATU), 1-hydroxy- 1,2,3-benzotriazole (HOBT), O-benzotriazol-l-yl-N,N,NCNaetramethyluronium tetrafluoroborate (TBTU), disuccinimidyl carbonate (DSC), O-(N-Succinimidyl)- 1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU) and 4- nitrophenyltrifluoroacetate. Examples of hydroxy activating agents include, but are not limited to, methane sulfonyl chloride, trifluoroacetic anhydride, bis(4-nitrophenyl) carbonate, 4-nitrophenylchloroformate and disuccinimidylcarbonate (DSC).

[0050] "Aryl" refers to monocyclic or polycyclic aromatic carbon ring systems having five to fourteen members. Examples of aryl groups include, but are not limited to, phenyl (Ph), 1-naphthyl, 2-naphthyl, 1-anthracyl and 2-anthracyl. The term "aralkyl" refers to an alkyl group substituted by an aryl. Also explicitly included within the scope of the term "aralkyl" are alkenyl or alkynyl groups substituted by an aryl. Examples of aralkyl groups include benzyl and phenethyl. The term "aryl", "aryl group" or "aryl ring" also refers to rings that are optionally substituted, unless otherwise indicated.

[0051] "Carbocyclyl" or "carbocyclic," refers to monocyclic or polycyclic non- aromatic carbon ring systems, which may contain a specified number of carbon atoms, for example from 3 to 12 carbon atoms, which are completely saturated or which contain one or more units of unsaturation. A carbocyclic ring system may be monocyclic, bicyclic or tricyclic. A carbocyclyl ring may be fused to another ring, such as an aryl ring or another carbocyclic ring. Examples of carbocyclic rings could include cyclohexyl, cyclopentyl, cyclobutyl, cyclopropyl, cyclohexenyl,

cyclopentenyl, indanyl, tetrahydronaphthyl and the like. The term "carbocyclic" or "carbocyclyl," whether saturated or unsaturated, also refers to rings that are optionally substituted unless indicated. The term "carbocyclic" or "carbocylyl" also

encompasses hybrids of aliphatic and carbocyclic groups, such as

(cycloalkyl) alkyl, (cycloalkenyl)alkyl and (cycloalkyl)alkenyl.

[0052] "Halo" refers to a fluorine, chlorine, bromine or iodine substituent.

[0053] "Heteroaryl" refers to monocyclic or polycyclic aromatic ring systems having five to fourteen members and one or more heteroatoms. One having ordinary skill in the art will recognize that the maximum number of heteroatoms in a stable, chemically feasible heteroaryl ring is determined by the size of the ring and valence. The term "heteroaralkyl" refers to an alkyl group substituted by a heteroaryl. Also explicitly included within the scope of the term "heteroaralkyl" are alkenyl or alkynyl groups substituted by a heteroaryl. In general, a heteroaryl ring may have one to four heteroatoms. Heteroaryl groups include, without limitation, 2-furanyl, 3-furanyl, N- imidazolyl, 2imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-isoxazolyl, 4isoxazolyl, 5- isoxazolyl, 2-oxadiazolyl, 5-oxadiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 2- pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5- pyriniidyl, 3-pyridazinyl, 2-thiazolyl, 4thiazolyl, 5-thiazolyl, 5-tetrazolyl, 2-triazolyl, 5-triazolyl, 2-thienyl, and 3-thienyl. The term "heteroaryl ring", "heteroaryl group", or "heteroaralkyl" also refers to rings that are optionally substituted. Examples of fused polycyclic heteroaryl and aryl ring systems in which a carbocyclic aromatic ring or heteroaryl ring is fused to one or more other rings include, tetrahydronaphthyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl, quinolinyl, benzothiazolyl, benzoxazolyl, benzimidazolyl, isoquinolinyl, isoindolyl, acridinyl, benzoisoxazolyl, and the like.

[0054] "Heterocyclic" or "heterocyclyl" refers to non-aromatic saturated or unsaturated monocyclic or polycyclic ring systems containing one or more heteroatoms and with a ring size of three to fourteen. One having ordinary skill in the art will recognize that the maximum number of heteroatoms in a stable, chemically feasible heterocyclic ring is determined by the size of the ring, degree of unsaturation, and valence. In general, a heterocyclic ring may have one to four heteroatoms so long as the heterocyclic ring is chemically feasible and stable and may be fused to another ring, such as a carbocyclic, aryl or heteroaryl ring, or to another heterocyclic ring. A heterocyclic ring system may be monocyclic, bicyclic or tricyclic. Also included within the scope of within the scope of the term "heterocyclic" or "heterocyclyl", as used herein, is a group in which one or more carbocyclic rings are fused to a heteroaryl.

[0055] Examples of heterocyclic rings include, but are not limited to, 3- IH- benzimidazol-2-one, 3-lH-alkyl-benzimidazol-2-one, 2-tetrahydrofuranyl, 3- tetrahydrofuranyl, 2-tetrahydrothiophenyl, 3-tetrahydrothiophenyl, 2-morpholino, 3- morpholino, 4-morpholino, 2-thiomorpholino, 3-thiomorpholino, 4-thiomorpholino, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl, 1-piperazinyl, 2-piperazinyl, 1- piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 4-thiazolidinyl, diazolonyl, N- substituted diazolonyl, 1 -phthalimidinyl, benzoxane, benzotriazol-1-yl,

benzopyrrolidine, benzopiperidine, benzoxolane, benzothiolane, benzothiane, aziranyl, oxiranyl, azetidinyl, pyrrolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, pyrazolinyl, pyrazolidinyl, pyranyl, dioxanyl, dithianyl, trithianyl, quinuclidinyl, oxepanyl, succinimidyl and thiepanyl. The term "heterocyclic" ring, whether saturated or unsaturated, also refers to rings that are optionally substituted, unless otherwise indicated.

[0056] An aryl, aralkyl, heteroaryl, or heteroaralkyl group may contain one or more independently selected substituents. Examples of suitable substituents on the unsaturated carbon atom of an aryl or heteroaryl group include halogen, -CF₃ , -Rp -ORp-OH, -SH, -SRøprotected OH (such as acyloxy), -NO₂, -CN, -NH₂, -NHRO -N(RIJl, -NHCORO-NHCONH2, -NHCONHRO-NHCON^ft, -NRCORO-NHCO2H, -NHCO₂RO-CO₂RO-CO₂H, -CORO-CONH₂, -CONHRp-CON(R^, -S(O)₂H, -S(O)₂RO-S(O)₃H, -S(O)₃R0-S(O)₂NH2 QS(O)H, -S(O)RO-S(O)₂NHRO- S(O)₂N(R[Ji, -NHS(O)₂H, or -NHS(O)₂R Owhere RQs selected from H, aliphatic, carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, or heteroaralkyl and each RQs optionally substituted with one or more halogen, nitro, cyano, amino, -NH- (unsubstituted aliphatic), -N-(unsubstituted aliphatic)₂, carboxy, carbamoyl, hydroxy, -O-(unsubstituted aliphatic), -SH, -S-(unsubstituted aliphatic), CF₃, -S(O)₂NH₂Q unsubstituted aliphatic, unsubstituted carbocyclyl, unsubstituted heterocyclyl, unsubstituted aryl, unsubstituted aralkyl, unsubstituted heteroaryl, or unsubstituted heteroaralkyl.

[0057] An aliphatic group or a non-aromatic heterocyclic ring may contain one or more substituents. Examples of suitable substituents on the saturated carbon of an aliphatic group or of a non-aromatic heterocyclic ring include those listed above for the unsaturated carbon as well as the following: =O, =S, =NNHR O=NN(R ft, =N-ORp =NNHCOR0=NNHCO₂R0=NNHSO₂R0=N-CN, or =NROwherein RQs as defined above. Guided by this specification, the selection of suitable substituents is within the knowledge of one skilled in the art.

[0058] "Natural amino acid" refers to any of the 20 amino acids found in nature. These amino acids are alanine, asparagine, aspartic acid, arginine, cysteine, glutamine, glycine, glutamic acid, histidine, isoleucine, lysine, leucine, phenylalanine, methionine, serine, proline, tryptophan, threonine, tyrosine, and valine. The following amino acid abbreviations are commonly used in the art:

A = AIa = Alanine T = Thr = Threonine

[0059] "Protecting group" refers to a group used in organic synthesis to temporarily mask the characteristic chemistry of a select functional group. Suitable protecting groups for the methods and compounds described herein include, but are not limited to, those described in standard textbooks, such as Greene, T. W. et al., Protective Groups in Organic Synthesis, Wiley, N. Y. (1999).

[0060] "Unnatural amino acid" refers to any amino acid other than the 20 amino acids found in nature (listed above). Examples of such amino acids include, but are not limited to, β-alanine, α-aminobutyric acid, α-amino-α-methylbutyrate,

aminocyclopropane-carboxylate, amino isobutyric acid, amino norbornyl-carboxylate, L-N-methylglutamic acid, cyclohexylalanine, cyclopentylalanine, D-alanine, D- arginine, D-aspartic acid, D-cysteine, D-glutamine, D-glutamic acid, D-histidine, D- iso leucine, D-leucine, D-lysine, D-methionine, D-ornithine, D-phenylalanine, D- proline, D-serine, D-threonine, D-tryptophan, D-tyrosine, D-valine, D-α- methylalanine, D-α-methylarginine, D-α-methylasparagine, D-α-methylaspartate, D- α-methylcysteine, D-α-methylglutamine, D-α-methylhistidine, D-α-methylisoleucine, D-α-methylleucine, D-α-methyllysine, D-α-methylmethionine, D-α-methylornithine, D-α-methylphenylalanine, D-α-methylproline, D-α-methylserine, D-α- methylthreonine, D-oc-methyltryptophan, D-α-methyltyrosine, D-α-methylvaline, D- N-methylalanine, D-N-methylarginine, D-N-methylasparagine, D-N-methylaspartate, D-N-methylcysteine, D-N-methylglutamine, D-N-methylglutamate, D-N- methylhistidine, D-N-methyliso leucine, D-N-methylleucine, D-N-methyllysine, N- methylcyclohexylalanine, D-N-methylornithine, N-methylglycine, N- methylaminoisobutyrate, N-(l-methylpropyl)glycine, N-(2-methylpropyl)glycine, D- N-methyltryptophan, D-N-methyltyrosine, D-N-methylvaline, γ-aminobutyric acid, L- t-butylglycine, L-ethylglycine, L-homophenylalanine, L-α-methylarginine, L-α- methylaspartate, L-α-methylcysteine, L-α-methylglutamine, L-α-methylhistidine, L-α- methylisoleucine, L-α-methylleucine, L-α-methylmethionine, L-α-methylnorvaline, L-α-methylphenylalanine, L-α-methylserine, L-α-methyltryptophan, L-α- methylvaline, N-CN-(2,2-diphenylethyl)carbamylniethyl)glycine, l-carboxy-l-(2,2- diphenylethylamino)cyclopropane, L-norvaline, L-N-methylalanine, L-N- methylarginine, L-N-niethylasparagine, L-N-methylaspartic acid, L-N- methylcysteine, L-N-methylglutamine, L-N-methylhistidine, L-N-methylisolleucine, L-N-methylleucine, L-N-methyllysine, L-N-methylmethionine, L-N- methylnorleucine, L-N-methylnorvaline, L-N-niethylornithine, L-N- methylphenylalanine, L-N-methylproline, L-N-methylserine, L-N-methylthreonine, L-N-methyltryptophan, L-N-methyltyrosine, L-N-methylvaline, L-N- methylethylglycine, L-N-methyl-t-butylglycine, L-norleucine, α-methyl- aminoisobutyrate, α-methyl-γ-aminobutyrate, α-methylcyclohexylalanine, α- methyleyleopentylalanine, α-methyl-α-napthylalanine, α-methylpenieillamine, N-(4- aminobutyl)glycine, N-(2-aminoethyl)glycine, N-(3-aminopropyl)glycine, N-amino- α-methylbutyrate, α-napthylalanine, N-benzylglycine, N-(2-earbamylethyl)glycine, N- (earbamylmethyl)glycine, N-(2-earboxyethyl)glycine, N-(earboxyniethyl)glycine, N- cyclobutylglycine, N-cycloheptylglycine, N-cyclohexylglycine, N-cyclodecylglycine, N-cyclododecylglycine, N-cyclooctylglycine, N-cyclopropylglycine, N- cycloundecylglycine, N-(2,2-diphenylethyl)glycine, N-(3,3-diphenylpropyl)glycine, N-(3-guanidinopropyl)glycine, N-(l-hydroxyethyl)glycine, N-(hydroxyethyl)glycine, N-(imidazolylethyl)glycine, N-(3-indolylyethyl)glycine, N-methyl-γ-aminobutyrate, D-N-methylmethionine, N-methylcyelopentylalanine, D-N-methylphenylalanine, D- N-methylproline, D-N-methylserine, D-N-methylthreonine, N-(l-methylethyl)glycine, N-methyl-α-napthylalanine, N-methylpenicillamine, N-(p-hydroxyphenyl)glycine, N- (thiomethyl)glycine, penicillamine, L-α-methylalanine, L-α-methylasparagine, L-α- methyl-t-butylglycine, L-methylethylglycine, L-α-methylglutamate, L-α- methylhomophenylalanine, N-(2-methylthioethyl)glycine, L-α-methyllysine, L-α- methylnorleucine, L-α-methylornithine, L-α-methylproline, L-α-methylthreonine, L- α-methyltyrosine, L-N-niethylhomophenylalanine, N-(N-(3,3- diphenylpropyl)carbamylniethyl)glycine, amino-malonic acid, amino-succinic acid, α- amino-glutaric acid, β-amino-glutaric acid, α-amino-adipic acid, β-amino-adipic acid, α-amino-pimelic acid, β-amino-pimelic acid, γ-amino-pimelic acid, α-amino-suberic acid, β-amino- suberic-acid, γ-amino- suberic acid, α-amino-azelaic acid, β-amino- azelaic acid, γ-amino-azelaic acid, δ-amino-azelaic acid, α-amino-sebacic acid, β- amino-sebacic acid, γ-amino-sebacic acid, and δ-amino-sebacic acid. [0061] In order that the invention herein described may be fully understood, the following detailed description is set forth. The present invention provides

heterobifunctional PEG reagents containing terminal protected (or "masked") aldehyde and ester functionalities. These heterobifunctional PEGs are useful for a variety of reasons described above. Advantageously, the protected aldehyde and ester functionalities provide different reactivities such that a reaction, e.g., with a cationic polymer, can occur selectively at one terminus over the other.

Heterobifunctional PEGs

[0062] In one aspect, the present invention provides a PEG reagent comprising a compound of Formula (I):

wherein:

each Ri is independently selected from the group consisting of branched or straight-chain C₁-Ce alkyl, branched or straight-chain C₂-C_O alkenyl, and branched or straight-chain C2-C6 alkynyl,

R₂;

each R₂ is independently selected from the group consisting of branched or straight-chain Q-C₆ alkyl, branched or straight-chain C2-C6 alkenyl, branched or straight-chain C₂-C₆ alkynyl, hydroxy, Ci-C₆ alkoxy, -CO₂H, -CO₂(RJl -CONH₂, -CONH(RJ] -CON(RJi, carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl, nitro, cyano, and halo,

wherein one or more hydrogens in the alkyl, alkenyl or alkynyl chain may be replaced by one or more halogens, and wherein each carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl may be optionally substituted with one or more Rs;

R₃ is C_O-C_I4 aryl, C₅-C14 heteroaryl or C₅-C14 heterocyclyl,

wherein R₃ is optionally substituted with one or more R₅; R₄ is selected from the group consisting of hydrogen, branched or straight- chain C₁-Ce alkyl, branched or straight-chain C₂-Ce alkenyl, branched or straight- chain C2-C6 alkynyl, wherein one or more hydrogens in the alkyl, alkenyl or alkynyl chain may be replaced by one or more halogens;

R5 is selected from the group consisting of branched or straight-chain Ci-C₆ alkyl, branched or straight-chain C₂-Ce alkenyl, branched or straight-chain C₂-Ce alkynyl, -CF₃, -R0-OR0-OH, -SH, -SRøprotected OH (e.g., acyloxy), -NO₂, -CN, - NH₂, -NHRO-N(RJl, -NHCORO-NHCONH2, -NHCONHR 0-NHCON(RJi, -

NRCORO-NHCO2H, -NHCO₂RO-CO₂RO-CO₂H, -CORO-CONH₂, -CONHRO- CON(RJi, -S(O)₂H, -S(O)₂RO-S(O)₃H, -S(O)₃RO-S(O)₂NH₂, -S(O)H, -S(O)RO- S(O)₂NHRO-S(O)₂N(RJi, -NHS(O)₂H, -NHS(O)₂RO-F, -Cl, -Br, and =O, where chemically feasible;

RQs selected from the group consisting of hydrogen, branched or straight- chain C₁-Ce alkyl, branched or straight-chain C₂-Ce alkenyl, branched or straight- chain C₂-Ce alkynyl, carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, or heteroaralkyl and each RQs optionally substituted with one or more halogen, nitro, cyano, amino, -NH-(unsubstituted aliphatic), -N-(unsubstituted aliphatic)₂, carboxy, carbamoyl, hydroxy, -O-(unsubstituted aliphatic), -SH, -S-(unsubstituted aliphatic), - CF3, -S(O)₂NH₂, unsubstituted aliphatic, unsubstituted carbocyclyl, unsubstituted heterocyclyl, unsubstituted aryl, unsubstituted aralkyl, unsubstituted heteroaryl, or unsubstituted heteroaralkyl;

A is selected from the group consisting of Ci-C₆ alkyl, C₂-C₆ alkenyl, and C₂- C₆ alkynyl;

wherein A is optionally substituted with branched or straight-chain Ci- C₆ alkyl, branched or straight-chain C₂-C₆ alkenyl, branched or straight-chain C₂-C₆ alkynyl, hydroxy, Ci-C₆ alkoxy, CO₂H, CO₂(Ci-C₆ alkyl), CONH₂, CONH(Ci-C₆ alkyl), CON(Ci-C₆ alkyl)₂, nitro, cyano, or halo; each X is independently O or S;

j is an integer from 0 to 10; and

n is an integer from 1 to 1,500.

[0063] In certain embodiments of Formula (I), each Ri is independently a branched or straight-chain Ci-C₆ alkyl, or both Ri are taken together to form a 5- or 6- membered cyclic acetal, optionally substituted as discussed above. In other embodiments, Ri is methyl, ethyl, isopropyl or both Ri are taken together to form a 1,3-dioxolane ring or a 1,3-dioxane ring. In another embodiment, Ri is ethyl.

[0064] In certain embodiments of Formula (I), each R₂ is independently selected from the group consisting of branched or straight- chain Ci-Cβ alkyl, branched or straight-chain C₂-C₆ alkenyl, and branched or straight-chain C₂-C₆ alkynyl optionally substituted as discussed above. In another embodiment, each R₂ is independently selected from the group consisting of branched or straight-chain Ci-C₆ alkyl, branched or straight-chain C₂-C₆ alkenyl, and branched or straight-chain C₂-C₆ alkynyl.

[0065] In certain embodiments of Formula (I), R₃ is Cβ-Ci4 aryl, C₅-C14 heteroaryl optionally substituted as discussed above. In certain embodiments of Formula (I), R3 is a C₆-CiO aryl or a C5-C10 heteroaryl optionally substituted as discussed above. In other embodiments of Formula (I), R3 is a C₆ aryl, or C5 heteroaryl optionally substituted as discussed above. In other embodiments, R₃ is phenyl, pyridyl, pyrimidinyl, or naphthyl optionally substituted as discussed above. In other embodiments, R3 is phenyl optionally substituted as discussed above. In yet other embodiments, R₃ is phenyl optionally substituted with one or more nitro or one or more halo. In some embodiments, R₃ is trichlorophenyl, trifluorophenyl,

pentafluorophenyl, ortho- or para-nitrophenyl. In other embodiments, R₃ is para- nitrophenyl.

[0066] In certain embodiments of Formula (I), R5 is selected from the group consisting of -CF₃, -Rp-ORp-OH, -SH, -SRPprotected OH {e.g., acyloxy), -NO₂, - CN, -NH₂, -NHRp-N(RJi, -NHCORg-NHCONH₂, -NHCONHR 0-NHCON(R %, - NRCOR0-NHCO₂H, -NHCO₂Rf]-CO₂Rp-CO₂H, -CORp-CONH₂, -CONHRp- CON(RJi, -S(O)₂H, -S(O)₂Rp-S(O)₃H, -S(O)₃Rp-S(O)₂NH₂, -S(O)H, -S(O)Rp- S(O)₂NHRp-S(O)₂N(RJi, -NHS(O)₂H, -NHS(O)₂RP-F, -Cl, -Br, and =O, where chemically feasible. In other embodiments R5 is selected from the group consisting of -CF₃, -NO₂, -CN, -CO₂Rp-CO₂H, -CORp-CONH₂, -CONHRp-CON(RJi, -S(O)₂H, -S(O)₂RP-S(O)₃H, -S(O)₃RP-S(O)₂NH₂, -S(O)H, -S(O)Rp-S(O)₂NHRp-S(O)₂N(RJi, -NHS(O)₂H, -NHS(O)₂RP-F, -Cl, -Br, and =O, where chemically feasible. In other embodiments R5 is selected from the group consisting of -CO2R 0-CORO-CON(RJi₅ - S(O)RO-S(O)₂RO-S(O)₃Rp-CF₃, nitro, cyano, and halo. In other embodiments R₅ is selected from the group consisting of -CF3, nitro, cyano, and halo. In yet other embodiments R5 is ortho- or para-nitro. In another embodiment, R5 is para-nitro.

[0067] In certain embodiments of Formula (I), R3s selected from the group consisting of hydrogen, branched or straight-chain C1-C₆ alkyl, branched or straight- chain C₂-C₆ alkenyl, or branched or straight-chain C₂-C₆ alkynyl optionally substituted as discussed above.

[0068] In certain embodiments of Formula (I), A is Ci-Cβ alkyl optionally substituted as discussed above. In other embodiments of Formula (I), A is C1-C₃ alkyl optionally substituted as discussed above. In another embodiment, A is CH₂.

[0069] In some embodiments of Formula (I), X is O.

[0070] In some embodiments of Formula (I), j is an integer from 0 to 6. In other embodiments, ] is an integer from 1 to 3. In a particular embodiment, j is 1.

[0071] In some embodiments, n is an integer from 5 to 1,000. In other

embodiments, n is an integer from 20 to 500. In a particular embodiment, n is an integer from 50 to 250.

[0072] While any PEG moiety can be used in the compounds of Formula (I), preferred PEGs include PEG3400 and PEG8000.

NO₂

Compound 6 [0073] In another aspect, the present invention provides a PEG reagent comprising a compound of Formula (II):

O

H

H -AA

Formula (II) wherein:

each Ri is independently selected from the group consisting of branched or straight-chain C₁-C₆ alkyl, branched or straight-chain C₂-C₆ alkenyl, and branched or straight-chain C2-C6 alkynyl,

wherein each Ri is independently, optionally substituted with one or more R₂, or when both Ri are taken together to form a 5- or 6-membered cyclic acetal or thioacetal, the cyclic acetal or thioacetal is optionally substituted with one or more R₂;

each R₂ is independently selected from the group consisting of branched or straight-chain Ci -C_O alkyl, branched or straight-chain C2-C6 alkenyl, branched or straight-chain C₂-C₆ alkynyl, hydroxy, Ci-C₆ alkoxy, -CO₂H, -CO₂(RJi -CONH₂, -CONH(RJ] -CON(RJ₂, carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl, nitro, cyano, and halo,

R₄ is selected from the group consisting of hydrogen, branched or straight- chain Ci-C₆ alkyl, branched or straight-chain C2-C6 alkenyl, branched or straight- chain C₂-C₆ alkynyl, wherein one or more hydrogens in the alkyl, alkenyl or alkynyl chain may be replaced by one or more halogens;

R₆ is C₆-Ci₄ aryl, C5-C14 heteroaryl or C5-C 14 heterocyclyl,

wherein R₆ is optionally substituted with one or more R7; and

R₇ is selected from the group consisting of branched or straight-chain Ci-C₆ alkyl, branched or straight-chain C₂-C₆ alkenyl, branched or straight-chain C₂-C₆ alkynyl, -CF₃, -Rp-ORP-OH, -SH, -SRPprotected OH (e.g., acyloxy), -NO₂, -CN, - NH₂, -NHRp-N(RJi, -NHCORg-NHCONH₂, -NHCONHR0-NHCON(Rpi, - NRCORg-NHCO₂H, -NHCO₂R 0-CO₂Rp-CO₂H, -CORp-CONH₂, -CONHRp- CON(RJi, -S(O)₂H, -S(O)₂RP-S(O)₃H, -S(O)₃RP-S(O)₂NH₂, -S(O)H, -S(O)RP- S(O)₂NHRg-S(O)₂N(RJi, -NHS(O)₂H, -NHS(O)₂R0-F, -Cl, -Br, and =O, where chemically feasible;

wherein RQs selected from the group consisting of hydrogen, branched or straight-chain Ci -C_O alkyl, branched or straight-chain C2-C6 alkenyl, branched or straight-chain C₂-C₆ alkynyl, carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, or heteroaralkyl and each RQs optionally substituted with one or more halogen, nitro, cyano, amino, -NH-(unsubstituted aliphatic), -N-(unsubstituted aliphatic)2, carboxy, carbamoyl, hydroxy, -O-(unsubstituted aliphatic), -SH, -S-(unsubstituted aliphatic), - CF3, -S(O)₂NH₂ Qinsubstituted aliphatic, unsubstituted carbocyclyl, unsubstituted heterocyclyl, unsubstituted aryl, unsubstituted aralkyl, unsubstituted heteroaryl, or unsubstituted heteroaralkyl;

A is selected from the group consisting of Ci -C_O alkyl, C₂-C_O alkenyl, and C₂- C₆ alkynyl;

wherein A is optionally substituted with branched or straight-chain C₁- C₆ alkyl, branched or straight-chain C₂-C₆ alkenyl, branched or straight-chain C₂-C₆ alkynyl, hydroxy, Ci-C₆ alkoxy, CO₂H, CO₂R₆, CO₂(Ci-C₆ alkyl), CONH₂,

CONH(Ci-C₆ alkyl), CON(C₁-C₆ alkyl)₂, nitro, cyano, or halo;

each X is independently O or S;

j is an integer from O to 10; and

n is an integer from 1 to 1,500.

[0074] In certain embodiments of Formula (II), each Ri is independently a branched or straight-chain Ci-C₆ alkyl, or both Ri are taken together to form a 5- or 6- membered cyclic acetal, optionally substituted as discussed above. In other embodiments, Ri is methyl, ethyl, isopropyl or both Ri are taken together to form a 1,3-dioxolane ring or a 1,3-dioxane ring. In a particular embodiment, Ri is ethyl.

[0075] In certain embodiments of Formula (II), each R2 is independently selected from the group consisting of branched or straight- chain Ci-C₆ alkyl, branched or straight-chain C₂-C₆ alkenyl, and branched or straight-chain C₂-C₆ alkynyl optionally substituted as discussed above. In another embodiment, each R₂ is independently selected from the group consisting of branched or straight-chain Ci-C₆ alkyl, branched or straight-chain C₂-C₆ alkenyl, and branched or straight-chain C₂-C₆ alkynyl.

[0076] In other embodiments of Formula (II), R₆ is a Cs-C₆ aryl, a Cs-C₆ heteroaryl or a C₃-C₆ heterocyclyl optionally substituted as discussed above. In another embodiment, R₆ is Cs-C₆ heterocyclyl optionally substituted as discussed above. In another embodiment, Re is a C3-C6 heterocyclyl optionally substituted as discussed above. In yet another embodiment, Re is C5 heterocyclyl optionally substituted as discussed above. In some embodiments, R_O is a phthalimidyl, glutarimidyl, tetrahyrophthalimidyl, morbornene-2,3-dicarboximidyl, or succinimidyl optionally substituted as discussed above. In other embodiments, R₆ is an optionally sulfated succinimidyl. In yet other embodiments, Re is succinimidyl. In other embodiments of Formula (II), Re is a Ce aryl, or C₅ heteroaryl optionally substituted as discussed above. In other embodiments, R₆ is phenyl, pyridyl, pyrimidinyl, benzotriazolyl or naphthyl optionally substituted as discussed above. In some embodiments, R₆ is phenyl optionally substituted as discussed above. In another embodiment, Re is phenyl optionally substituted with one or more nitro or one or more halo. In other embodiments, Re is trichlorophenyl, trifluorophenyl, pentafluorophenyl, ortho- or para-nitrophenyl. In yet other embodiments, Re is para-nitrophenyl.

[0077] In certain embodiments of Formula (II), R₇ is selected from the group consisting of -CF₃, -R0-OR0-OH, -SH, -SRPprotected OH (e.g., acyloxy), -NO₂, - CN, -NH₂, -NHRp-N(RJi, -NHCORg-NHCONH₂, -NHCONHR P-NHCON(R %, - NRCORg-NHCO₂H, -NHCO₂Rg-CO₂Rp-CO₂H, -CORp-CONH₂, -CONHRp- CON(RJi, -S(O)₂H, -S(O)₂RP-S(O)₃H, -S(O)₃Rp-S(O)₂NH₂, -S(O)H, -S(O)Rp- S(O)₂NHRp-S(O)₂N(RJi, -NHS(O)₂H, -NHS(O)₂Rp-F, -Cl, -Br, and =O, where chemically feasible. In other embodiments of Formula (II), R₇ is selected from the group consisting of -CF₃, -NO₂, -CN, -CO₂Rp-CO₂H, -CORp-CONH₂, -CONHRp- CON(RJi, -S(O)₂H, -S(O)₂Rp-S(O)₃H, -S(O)₃Rp-S(O)₂NH₂, -S(O)H, -S(O)Rp- S(O)₂NHRp-S(O)₂N(RJi, -NHS(O)₂H, -NHS(O)₂RP-F, -Cl, -Br, and =O, where chemically feasible. In some embodiments of Formula (II) wherein R₆ is phenyl, R₇ is selected from the group consisting Of-CO₂Rp-CORp-CON(RJi, -S(O)Rp-S(O)₂Rp -S(O)₃RP-CF₃, nitro, cyano, and halo. In other embodiments of Formula (II) wherein Re is phenyl, R₇ is selected from the group consisting Of-CF₃, nitro, cyano, and halo. In some embodiments of Formula (II) wherein Rg is phenyl, R₇ is ortho- or para-nitro. In other embodiments of Formula (II) wherein Re is phenyl, R₇ is para-nitro.

[0078] In certain embodiments of Formula (II), RCIs selected from the group consisting of hydrogen, branched or straight-chain Ci-Ce alkyl, branched or straight- chain C₂-C₆ alkenyl, or branched or straight-chain C₂-C₆ alkynyl optionally substituted as discussed above. [0079] In certain embodiments of Formula (II), A is Ci-C₆ alkyl optionally substituted as discussed above. In other embodiments of Formula (I), A is C1-C3 alkyl optionally substituted as discussed above. In a particular embodiment, A is CH2CH2.

[0080] In some embodiments of Formula (II), X is O.

[0081] In some embodiments of Formula (II), j is an integer from 0 to 6. In other embodiments, j is an integer from 1 to 3. In a particular embodiment, j is 1.

[0082] In some embodiments, n is an integer from 5 to 1,000. In other

[0083] While any PEG moiety can be used in the compounds of Formula (II), preferred PEGs include PEG3400 and PEG8000.

[0084] In a particular embodiment of the present invention, Formula (II) has the following formula:

[0085] In anoth eagent comprising a

compound of Formula (III):

X XRK₁₁ O

^ KV

wherein:

R₁, R2, R4, Re, R7, RpX, j, and n are defined as in Formula (II);

each B is any natural amino acid, or an unnatural alpha or beta amino acid, wherein each is independently optionally substituted with one or more -CO2R₆ or R₇;

wherein any -CO₂H present in B is optionally substituted with R₆ to yield a -CO₂RO moiety; and

k is an integer from 1 to 5.

[0086] In certain embodiments of Formula (III) wherein more than one B is present (i.e., (B)k wherein k is greater than 1), the (B)k moiety comprises at least one natural amino acid and at least one unnatural alpha or beta amino acid, wherein each is independently and optionally substituted with one or more -CO2R6, -CO2H, or R₇. In other embodiments of Formula (III) wherein more than one B is present (Le., (B)k wherein k is greater than 1), the (B)_k moiety comprises at least two natural amino acids, wherein each is independently and optionally substituted with one or more

-CO2R6, -CO2H, or R₇. In another embodiment, B is a natural amino acid optionally substituted with one or more -CO2R₆, -CO2H, or R₇. In other embodiments, B is an unnatural alpha or beta amino acid optionally substituted with one or more -CO₂R_O, - CO₂H, or R₇. In yet other embodiments, B is an unnatural alpha or beta amino acid comprising at least two carboxylic acid moieties. In other embodiments, B is selected from the group consisting of amino-malonic acid, amino-succinic acid, α-amino- glutaric acid, β-amino-glutaric acid, α-amino-adipic acid, β-amino-adipic acid, α- amino-pimelic acid, β-amino-pimelic acid, α-amino-suberic acid, β-amino-suberic- acid, α-amino-azelaic acid, β-amino-azelaic acid, α-amino-sebacic acid, β-amino- sebacic acid, aspartic acid, glutamic acid, and Glu-Glu (Le., a glutamic acid dimer). In yet other embodiments, B is amino-malonic acid, aspartic acid, glutamic acid, or Glu-Glu.

[0087] In certain embodiments of Formula (III), k is an integer from 1 to 3. In other embodiments, k is either 1 or 2. In yet other embodiments, k is 1.

[0088] While any PEG moiety can be used in the compounds of Formula (III), preferred PEGs include PEG3400 and PEG8000.

[0089] In a particular embodiment of the present invention, Formula (III) has the following formula:

[0090] W ula (II) and Formula (II

) p q g p eviously reported in the art, heterobifunctional PEG reagents of Formula (II) and Formula (III) provide additional advantages. Among these are the ability to incorporate unique amino acids (such as beta-alanine) and the ability to incorporate termini bearing multiple carboxylate groups. The incorporation of unique amino acids (such as beta- alanine) is useful for the quantification of the degree of PEG conjugation to peptides using amino acid analysis. The incorporation of termini bearing multiple carboxylate groups is useful for improving the efficiency of purification by ion-exchange chromatography (particularly when larger PEGs are incorporated), and for presentation of clustered binding ligands to take advantage of multivalency opportunities.

[0091] This invention specifically contemplates the use of any chemically feasible salts of the heterobifunctional PEGs of the present invention. Non-limiting examples of such salts include alkali metal (e.g., sodium and potassium) and alkaline earth metal (e.g., magnesium) salts of, for example, -OH, -SH, -CO₂H, and -SO₃H. Synthesis of Heterobifunctional PEGs

[0092] The heterobifunctional PEG reagents of Formulas (I), (II), (III) and their precursors can be synthesized from the appropriate sized, unfunctionalized or partially functionalized, PEG. The particular reactions may be carried out according to methods well-known in the literature. Thus, in some aspects, the present invention provides a method for producing a heterobifunctional PEG according to Scheme I, II or III.

[0093] In general, compounds of Formula (I) can be synthesized using any synthetic techniques known in the art. For example, some compounds of Formula (I) can be synthesized using Scheme I:

Scheme I

Ri, R₂, R₃, R₄, R₅, A, X, j and n are defined as in Formula (I);

[0094] In Scheme I, (A) is converted to the corresponding ester (B) by any esterifϊcation method known in the art (e.g., reaction with alcohol and catalytic acid such as HCl). The free hydroxy 1 group on (B) is then converted to carbonate (C) by treatment with, for example, the appropriate carbonate or chloroformate. (C) is then

XR₁

R₁X NH,

reacted with amine to yield carbamate (D). The ester of (D) is

[0095] In certain embodiments of Scheme I, Rs is selected from the group consisting of branched or straight-chain C₁-Ce alkyl, C₆-C_H aryl, or C₅-C₁₄ heteroaryl optionally substituted as discussed above. In another embodiment, Rs is branched or straight-chain Ci-Ce alkyl optionally substituted as discussed above. In another embodiment, Rg is branched or straight-chain Ci-C₆ alkyl. In a particular

embodiment, Rs is methyl.

[0096] In one embodiment of Scheme I, Rg is phenyl, aryloxy, or succinimidyl, each of which is optionally substituted with R₅. In other embodiments, Rg is phenyl, succinimidyl, or aryloxy, each of which is optionally substituted with one or more electron withdrawing groups such as nitro, fluoro, chloro, cyano, sulpho, carboxy, amido, trifluoromethyl, or combinations thereof. In other embodiments, R₉ is sulpho- succinimidyl, 1 -oxybenzotriazolyl, nitrophenyl, or halo-substituted phenyl. In yet other embodiments, R9 is phenyl substituted with one or more fluorines, chlorines or nitro groups. In another embodiment of Scheme I, R9 is para-nitrophenyl, ortho- nitrophenyl, fluorophenyl or chlorophenyl. In another embodiment, Rg is para- nitrophenyl. In another embodiment, R₉ is succinimidyl.

[0097] While any PEG moiety can be used in Scheme I, preferred PEGs include PEG3400 and PEG8000.

[0098] Scheme I ^Illustrates the synthesis of Compound 6. (See Examples 1-5 for synthetic details).

Scheme in

H

[0

099] In Scheme I P(I) is converted to the corresponding methyl ester (2) with methanol and catalytic HCl. The free hydroxyl group on (2) is then converted to a succinimidyl carbonate (3) by treatment with disuccinimidyl carbonate. Succinimidyl carbonate (3) is then reacted with diethoxypropylamine to yield carbamate (4). The methyl ester of (4) is hydro lyzed with NaOH to yield carboxylic acid (5). Finally, carboxylic acid (5) is converted to a nitrophenyl ester by treatment with nitrophenyl trifiuoroacetate to yield Compound 6.

[0100] While any PEG moiety can be used in the compounds of Scheme I P preferred PEGs include PEG3400 and PEG8000.

[0101] In general, compounds of Formula (II) can be synthesized using any synthetic techniques known in the art. For example, some compounds of Formula (II) can be synthesized using Scheme II:

Scheme II

In Scheme II:

R₁, R₂, R₄, Re, R₇, A, X, j, and n are defined as in Formula (II); and

R₈ is defined as in Scheme I. 2

[0102] In Scheme II, (F) is first reacted with amine and then with amine NH₂ ACOORs to generate a mixture of bifuncti

ein (G) is the major component (when A is CH₂CH₂) of the product mixture. The PEG mixture containing (G) is hydro lyzed (e.g., under basic conditions) and then subjected to ion exchange chromatography to yield the pure carboxylic acid (H). Carboxylic acid (H) is then converted the heterobifunctional PEG of Formula (II) using standard techniques in the art.

[0103] In certain embodiments of Scheme II, Rs is selected from the group consisting of branched or straight-chain C₁-C_O alkyl, C₆-Ci₄ aryl, or Cs-Ci₄ heteroaryl optionally substituted as discussed above. In another embodiment, Rg is branched or straight-chain Ci-C₆ alkyl optionally substituted as discussed above. In another embodiment, R₈ is branched or straight-chain Ci-C₆ alkyl. In a particular

embodiment, Rg is ethyl.

[0104] While any PEG moiety can be used in the compounds of Scheme II, preferred PEGs include PEG3400 and PEG8000.

[0105] Scheme IIQUustrates the synthesis of Compound 10. (See Examples 6-8 for synthetic details). Scheme II π

[0106] In Scheme IIP(7) is first reacted with diethoxypropylamine and then with beta-alanine ethyl ester to generate a mixture of bifunctional PEGs wherein (8) is the major component of the product mixture. The PEG mixture containing (8) is hydro lyzed under basic conditions and then subjected to ion exchange

chromatography to yield the pure carboxylic acid (9). Carboxylic acid (9) is then activated with disuccinimidyl carbonate to yield the heterobifunctional PEG (10).

[0107] While any PEG moiety can be used in the compounds of Scheme II P preferred PEGs include PEG3400 and PEG8000.

[0108] In general, compounds of Formula (III) can be synthesized using any synthetic techniques known in the art. For example, some compounds of Formula (III) can be synthesized using Scheme III:

Scheme III

XR₁ O

Λ ( V^{(B) R}

In Scheme III:

Ri, R.2, R₄, Re, R₇, B, X, j, k, and n are defined as in Formula (III); and

R₈ is defined as in Scheme I.

[0109] In Scheme III, (I) is first reacted with amine and then with

amine (B)_k-R₈ to generate a mixture of bifunctional PEGs wherein (J) is the major component (when B is beta-alanine and k = 1) of the product mixture. In

embodiments wherein (B)_k contains more than one carboxylic acid (i.e., when k is greater than 1, and/or one or more B S are substituted with or naturally comprise a carboxylic acid), certain carboxylic acids may be protected as, e.g., -CO₂Rs esters, where appropriate. The PEG mixture containing (J) is hydrolyzed (e.g., under basic conditions) and then subjected to ion exchange chromatography to yield the pure carboxylic acid (K). Carboxylic acid (K) is then converted the heterobifunctional PEG of Formula (III) using standard techniques in the art. In embodiments wherein (B)k contains more than one carboxylic acid (i.e., when k is greater than 1, and/or one or more BS are substituted with or naturally comprise a carboxylic acid), some or all of the carboxylic acids may be activated as, e.g., -CO₂R0 esters, where appropriate.

[0110] While any PEG moiety can be used in the compounds of Scheme III, preferred PEGs include PEG3400 and PEG8000.

[0111] Scheme III Qllustrates the synthesis of Compound 10a. (See Examples 13- 15 for synthetic details).

Scheme HID

OEt CO₂Me

[0

GLU-GLU trimethyl ester hydrochloride to generate a mixture of bifunctional PEGs wherein (8a) is the major component of the product mixture. The PEG mixture containing (8a) is hydrolyzed under basic conditions and then subjected to ion exchange chromatography to yield the pure tri-carboxylic acid (9a). Carboxylic acid (9a) is then activated with disuccinimidyl carbonate to yield the heterobifunctional PEG (10a).

[0113] While any PEG moiety can be used in the compounds of Scheme III P preferred PEGs include PEG3400 and PEG8000.

[0114] The methods illustrated in Schemes I, IpH, II Dili and III advantageously provide efficient synthetic protocols for the generation of novel heterobifunctional PEGs bearing versatile reactive end groups that are useful where existing

heterobifunctional PEGs are not. In particular, the acetal functionality provides a masked aldehyde which, when unmasked, is useful for the formation of imines and oximes through direct condensation. Additionally, the unmasked aldehyde is useful for the formation of amines via reductive animation, olefins via Wittig, aldol, Horner- Emmons or Julia-Lithgoe reactions or peptides via Ugi reactions. One skilled in the art will recognize that any process capable of forming a chemical bond through reaction with an aldehyde will be applicable to the PEGs of the present invention.

[0115] While the methods illustrated in Schemes 1, 1011, IipiII, and IHD

advantageously provide efficient synthetic protocols for the generation of novel heterobifunctional PEGs bearing versatile reactive end groups that are useful where existing heterobifunctional PEGs are not, the methods illustrated in Schemes II, II□ III, and III Epro vide additional advantages. Among these are the ability to incorporate unique amino acids (such as beta-alanine) and the ability to incorporate termini bearing multiple carboxylate groups. The incorporation of unique amino acids (such as beta-alanine) is useful for the quantification of the degree of PEG conjugation to peptides using amino acid analysis. The incorporation of termini bearing multiple carboxylate groups is useful for improving the efficiency of purification by ion- exchange chromatography (particularly when larger PEGs are incorporated), and for presentation of clustered binding ligands to take advantage of multivalency opportunities.

Uses of Heterobifunctional PEGs

[0116] In one aspect, the present invention provides novel classes of

heterobifunctional PEG reagents. Such heterobifunctional PEG reagents are advantageous in that they provide alternative methods of linking the PEG reagents to target biological molecules (e.g., via an aldehyde or activated ester moiety). In addition, such PEG reagents provide more efficient and specific means of coupling the PEG reagents to target biological molecules.

[0117] The heterobifunctional PEG reagents of the present invention provide two functional groups that can have different relative reactivities. In some embodiments, one functional group may be more reactive than the other. In other embodiments, one functional group may prefer, or even be selective, for a particular target molecule. In other embodiments, one functional group may selectively form a covalent bond with a target under certain reaction conditions.

[0118] The difference in reactivity between the functional groups facilitates the attachment of the PEG reagent to two different target molecules. Bifunctional derivatization of the PEG reagent typically proceeds by coupling of the first (and usually the more reactive) functional group with a first target molecule, even in the presence of the second (and usually less reactive) functional group. The second functional group can then be subsequently coupled to a second target molecule. In some embodiments, the PEG functional groups can be coupled to target molecules without activation. In other embodiments, the PEG functional groups are activated and then coupled to the target molecules.

[0119] The heterobifunctional PEGs of Formulas (I), (II) and (III) comprise functionalities particularly useful for coupling to target molecules (e.g., biologically relevant molecules). For example, Compound 6, Compound 10 and Compound 10a comprise a protected aldehyde moiety and an activated ester moiety (either a nitrophenyl ester in Compound 6 or a succinimidyl ester in Compounds 10 and 10a). Accordingly, the activated ester portion of the heterobifunctional PEGs can first be reacted with a free amine on a first target molecule (see, e.g., Examples 9 and 11).

While reaction with a free amine is favored, the activated ester can also react with free thiols or alcohols on the first target molecule under e.g., basic conditions. Next, the protected aldehyde moiety can be deprotected to yield a reactive aldehyde which serves as a site for coupling to a second target molecule. For example, the free aldehyde can be conjugated to amino (to yield an imine linkage) or aminooxy groups (to yield an oxime linkage) by direct condensation. Alternatively, the aldehyde can be conjugated to an amino group by reductive amination to yield an amine linkage (see, e.g., Examples 10 and 12). Such amino and aminooxy groups can be present on, but not limited to, lipids, polymers, pre-formed liposomes and pre-formed nanoparticles, peptides, proteins, enzymes, antibodies, aminoglycosides, proteoglycans,

glycosaminoglycans, and the like. The skilled artisan would recognize that additional reactions are useful for conjugation with aldehydes. Such reactions include, but are not limited to, Wittig, Horner-Emmons, Julia-Lithgoe, Ugi, aldol reactions, and the like.

[0120] The aldehyde mediated coupling reactions are advantageous in that the deprotected aldehyde is able to selectively react with amine groups. For example, the aldehyde portion of the heterobifunctional PEGs of the present invention can selectively react with lysine residues present in polymers or polypeptides. Such specificity is complementary to that seen in previously disclosed heterobifunctional PEGs comprising a vinyl sulfone which targets, for example, cysteine and lysine residues.

[0121] The heterobifunctional PEGs of the present invention can also be reacted with amine scaffolds in order to generate a "handle" for fatty acid attachment. While any amine scaffold may be used, amino-alcohols (such as l-amino-2,3-propanediol) are particularly useful. In such embodiments, the active ester component of the heterobifunctional PEG can be reacted with an amino scaffold. The free hydroxyl groups on the scaffold portion of the resulting heterobifunctional PEG derivative can then be acylated with fatty acids. The protected aldehyde can then be deprotected and coupled to a target molecule as described above.

[0122] Any biologically relevant targeting moiety/molecule may be used in the vectors of the present invention. In certain embodiments, the targeting moiety is a small molecule, polypeptide, peptide, protein, antibody, or a fragment, dimer, trimer or oligomer thereof. Suitable biologically relevant small molecule targeting moieties include, but are not limited to, vascular endothelial cell growth factor for targeting endothelial cells; FGF2 for targeting vascular lesions and tumors; transferrin for targeting tumors; melanotropin (alpha MSH) peptides for tumor targeting; ApoE and peptides for LDL receptor targeting; von Willebrand Θ Factor and peptides for targeting Coxsackie-adeno viral receptor (CAR) expressing cells; PDl and peptides for targeting Neuropilin 1 ; EGF and peptides for targeting EGF receptors expressing cells; folic acid and ligands for targeting folate receptors; RGD peptides for targeting integrin expressing cells and any other suitable targeting moiety. Additional examples of suitable target molecules include, but are not limited to, RGD, folate, LHRH, somatostatin, YIGSR, bombesin, hyaluronic acid, SLX, antibody fragments, N-acetyl galactosamine, mannose-6-phosphate, vitamin B 12, any cell penetrating peptide and any extracellular binding ligand. Further, any fragments or peptides of the above moieties having the same or similar targeting properties may be used as for their respective targets.

[0123] Further, compounds of Formulas (II) and (III) can advantageously comprise a unique amino acid such as beta-alanine. Such an amino acid is useful in

determining the composition of the synthesized conjugates using amino acid analysis (e.g., beta-alanine is a unique amino acid that can be specifically detected during analysis). In addition, the introduction of amino acids during the synthesis of the compounds of Formulas (II) and (III) provides the carboxylic acid handle necessary for the ion-exchange chromatography step (see Scheme II, conversion of (H) to (I); Scheme II ^conversion of (8) to (9); Scheme III, conversion of (J) to (K); and Scheme Ilipconversion of (8a) to (9a).

[0124] In another aspect, the present invention provides methods of using a heterobifunctional PEG to generate a vector of Formula (IV):

comprising the ste

[0125] In some embodiments, (X) and (Y) are simultaneously reacted with a heterobifunctional PEG. In other embodiments, (X) and (Y) are sequentially reacted with a heterobifunctional PEG. In yet other embodiments, (Y) is reacted with a heterobifunctional PEG and the acetal end of the resulting product is then hydro lyzed to an aldehyde prior to reacting with (X). [0126] In some embodiments, (X) is a cationic polymer. In some embodiments, (Y) is a targeting moiety. In one embodiment, (X) is a cationic polymer and (Y) is a targeting moiety.

[0127] In another aspect, the present invention provides methods of using a heterobifunctional PEG to generate a vector of Formula (V):

>

comprising the step of reacting a compound of Formula (II) with (X) and (Y), wherein

(X) and (Y) can be a small molecule, protein, polypeptide, peptide, monosaccharide, polysaccharide, antibody, polynucleotide, oligonucleotide, other polymeric species, polypeptide side chain or a biologically relevant targeting moiety, or a fragment, dimer, trimer or oligomer thereof. (X) is typically conjugated via an imine, oxime or amine linkage (represented by a squiggly bond).

[0128] In some embodiments, (X) and (Y) are simultaneously reacted with a heterobifunctional PEG. In other embodiments, (X) and (Y) are sequentially reacted with a heterobifunctional PEG. In yet other embodiments, (Y) is reacted with a heterobifunctional PEG and the acetal end of the resulting product is then hydro lyzed to an aldehyde prior to reacting with (X).

[0129] In some embodiments, (X) is a cationic polymer. In some embodiments, (Y) is a targeting moiety. In one embodiment, (X) is a cationic polymer and (Y) is a targeting moiety.

[0130] In another aspect, the present invention provides methods of using a heterobifunctional PEG to generate a vector of Formula (VI):

comprising the step of reacting a compound of Formula (III) with (X) and (Y), wherein (X) and (Y) can be a small molecule, protein, polypeptide, peptide, monosaccharide, polysaccharide, antibody, polynucleotide, oligonucleotide, other polymeric species, polypeptide side chain or a biologically relevant targeting moiety, or a fragment, dimer, trimer or oligomer thereof. (X) is typically conjugated via an imine, oxime or amine linkage (represented by a squiggly bond). In some

[0131] As described in the context of Formula (III), B can be any natural amino acid, or an unnatural alpha or beta amino acid, wherein each B is independently optionally substituted with one or more -CO₂R^ or R₇. Further, any -CO2H present in B is optionally substituted with R^ to yield a -CO2R6 moiety. Accordingly, a vector of Formula (VI) may comprise one or more (Y) moieties (attached by, for example, coupling via a -CO₂Re moiety). In some embodiments, the vector of Formula (VI) comprises 1-10 (Y) moieties. In some embodiments, the vector of Formula (VI) comprises 1-5 (Y) moieties. In other embodiments, the vector of Formula (VI) comprises 2-4 (Y) moieties. In yet other embodiments, the vector of Formula (VI) comprises 3 (Y) moieties.

[0132] In some embodiments, (X) and (Y) are simultaneously reacted with a heterobifunctional PEG. In other embodiments, (X) and (Y) are sequentially reacted with a heterobifunctional PEG. In yet other embodiments, (Y) is reacted with a heterobifunctional PEG and the acetal end of the resulting product is then hydro lyzed to an aldehyde prior to reacting with (X).

[0133] In some embodiments, (X) is a cationic polymer. In some embodiments, (Y) is a targeting moiety. In one embodiment, (X) is a cationic polymer and (Y) is a targeting moiety. In some embodiments, (Y) comprises a monosaccharide or polysaccharide optionally attached via a PEG or aminoethoxyalkyl moiety. In some embodiments, (Y) comprises N-acetyl galactosamine or an N-acetyl galactosamine beta aminoethoxy ethyl glycoside.

[0134] In embodiments wherein (Y) is a targeting moiety, (Y) can be used to direct a vector of Formula (VI) to, for example, a particular area of the body, a particular tissue or tissue type, a particular organ, etc. Accordingly, the presence of more than one (Y) group may increase the targeting efficiency of the vector (i.e., the ability of the vector to reach a target). [0135] In another aspect, the present invention provides a method of using a heterobifunctional PEG to generate a vehicle for targeted nucleic acid delivery of Formula (VII):

comprising

the steps of (a) reacting a compound of Formula (I) with (X) and (Y); and (b) mixing with a nucleic acid. (X) is typically conjugated via an imine, oxime or amine linkage (represented by a squiggly bond). In some embodiments, (X) and (Y) are as described in the context of Formula (IV). In some embodiments, (X) is a cationic polymer. In some embodiments, (Y) is a targeting moiety.

[0136] In some embodiments, (X) and (Y) are simultaneously reacted with heterobifunctional PEG. In other embodiments, (X) and (Y) are sequentially reacted with heterobifunctional PEG. In yet other embodiments, (Y) is reacted with a heterobifunctional PEG and the acetal end of the resulting product is then hydro lyzed to an aldehyde prior to reacting with (X).

[0137] In another aspect, the present invention provides a method of using a heterobifunctional PEG to generate a vehicle for targeted nucleic acid delivery of Formula (VIII):

comprisi d (Y); and (b) mixin

g with a nucleic acid. (X) is typically conjugated via an imine, oxime or amine linkage (represented by a squiggly bond). In some embodiments, (X) and (Y) are as described in the context of Formula (IV). In some embodiments, (X) is a cationic polymer. In some embodiments, (Y) is a targeting moiety.

[0138] In some embodiments, (X) and (Y) are simultaneously reacted with heterobifunctional PEG. In other embodiments, (X) and (Y) are sequentially reacted with heterobifunctional PEG. In yet other embodiments, (Y) is reacted with a heterobifunctional PEG and the acetal end of the resulting product is then hydro lyzed to an aldehyde prior to reacting with (X).

[0139] In another aspect, the present invention provides a method of using a heterobifunctional PEG to generate a vehicle for targeted nucleic acid delivery of

Formula (IX):

comprisin

g the step h (X) and (Y); and (b) mixing with a nucleic acid. (X) is typically conjugated via an imine, oxime or amine linkage (represented by a squiggly bond). In some embodiments, (X) and (Y) are as described in the context of Formula (IV). In some embodiments, (X) is a cationic polymer. In some embodiments, (Y) is a targeting moiety. In some embodiments (Y) is attached via a PEG or aminoethoxyalkyl moiety. In other embodiments (Y) is attached via an aminoethoxyethyl moiety.

[0140] As described in the context of Formula (III), B can be any natural amino acid, or an unnatural alpha or beta amino acid, wherein each B is independently optionally substituted with one or more -CO2R₆ or R₇. Further, any -CO2H present in B is optionally substituted with Re to yield a -CO₂Re moiety. Accordingly, a vehicle of Formula (IX) may comprise one or more (Y) moieties (attached by, for example, coupling via a -CO2R0 moiety). In some embodiments, the vehicle of Formula (IX) comprises 1-10 (Y) moieties. In some embodiments, the vehicle of Formula (IX) comprises 1-5 (Y) moieties. In other embodiments, the vehicle of Formula (IX) comprises 2-4 (Y) moieties. In yet other embodiments, the vehicle of Formula (IX) comprises 3 (Y) moieties.

[0141] In some embodiments, (X) and (Y) are simultaneously reacted with heterobifunctional PEG. In other embodiments, (X) and (Y) are sequentially reacted with heterobifunctional PEG. In yet other embodiments, (Y) is reacted with a heterobifunctional PEG and the acetal end of the resulting product is then hydro lyzed to an aldehyde prior to reacting with (X).

[0142] In embodiments wherein (Y) is a targeting moiety, (Y) can be used to direct a vehicle of Formula (XI) to, for example, a particular area of the body, a particular tissue or tissue type, a particular organ, etc. Accordingly, the presence of more than one (Y) group may increase the targeting efficiency of the vehicle (Le., the ability of the vehicle to reach a target).

[0143] In some embodiments, (Y) comprises a monosaccharide or polysaccharide optionally attached via a PEG or amino ethoxy alky 1 moiety. In other embodiments, (Y) comprises N-acetyl galactosamine or an N-acetyl galactosamine beta

aminoethoxyethyl glycoside.

[0144] In some embodiments, the heterobifunctional PEG regent used in the vectors above is a compound of Formula (I). In other embodiments, the heterobifunctional PEG reagent used in the vectors above is a compound of Formula (II). In other embodiments, the heterobifunctional PEG reagent used in the vectors above is a compound of Formula (III). In other embodiments, the heterobifunctional PEG reagent used in the vectors above is Compound 6. In other embodiments, the heterobifunctional PEG reagent used in the vectors above is Compound 10. In other embodiments, the heterobifunctional PEG reagent used in the vectors above is

Compound 10a.

[0145] Suitable nucleic acids include, but are not limited to, a recombinant plasmid; a replication-deficient plasmid; a mini-plasmid lacking bacterial sequences; a recombinant viral genome; a linear nucleic acid fragment encoding a therapeutic peptide or protein; a hybrid DNA/RNA double strand; double stranded DNA; an antisense DNA or chemical analogue thereof; a blunt, double blunt and overhanging double stranded DNA or RNA fragment comprising 5-200 base pairs; an antisense RNA or chemical analogue thereof; a linear polynucleotide that is transcribed as an antisense RNA or a ribozyme; a ribozyme; and a viral genome. [0146] In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 15-30 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 15 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 16 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 17 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 18 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 19 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 20 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 21 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 22 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 23 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 24 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 25 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 26 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 27 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 28 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 29 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 30 base pairs.

[0147] Chemical modification may be useful in some embodiments of the invention to increase stability of the nucleic acid molecule used or to reduce cytokine production. Incorporation of non-naturally occurring chemical analogues, such as 2 B O-Methyl ribose analogues of RNA, DNA, LNA and RNA chimeric oligonucleotides, and other chemical analogues of nucleic acid oligonucleotides, is one type of possible chemical modification. Possible modifications also include the addition of flanking sequences at the 5 and/or 3Qnds; the use of phosphorothioate, methylphosphonate, sulfone, sulfate, ketyl, phosphorodithioate, phosphoramidate, phosphate esters, or 2 D O-methyl rather than phosphodiester linkages in the backbone; and/or the inclusion of non-traditional bases, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine, and uridine. Non-traditional nucleic acid bases that can be introduced into nucleic acids include, for example, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3- methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5- methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5- bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine, λvybutosine, wybutoxosine, A- acetyltidine, 5-(carboxyhydroxymethyl)uridine, 5'-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1- methyladenosine, 1-niethy lino sine, 2,2-dimethylguanosine, 3-methylcytidine, 2- methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5- methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5- methylcarbonyhnethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2- methylthio-N6-isopentenyladenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine, threonine derivatives and others (see, for example, Molecular Therapy, 2007;15: 1663-1669, incorporated herein by reference in its entirety). These polynucleotide variants may be modified such that the activity of the nucleic acid molecule is not substantially decreased.

[0148] In certain embodiments, oligonucleotides of the invention may be 2ED- substituted oligonucleotides, as described in U.S. Patent Nos. 5,623,065, 5,856,455, 5,955,589, 6,146,829, and 6,326,199, herein incorporated by reference in their entirety, in which 2 Substituted nucleotides are introduced within an oligonucleotide to induce increased binding of the oligonucleotide to a complementary target strand while allowing expression of RNase H activity to destroy the targeted strand. See also, Sproat, B. S., et al., Nucleic Acids Research, 1990;18:41, incorporated herein by reference in its entirety. Nucleic acid molecules comprising 2ED-methyl and ethyl nucleotides are also encompassed by the invention.

[0149] A number of groups have taught the preparation of other 2 ED-alkyl guanosines. Gladkaya, et al., Khim. Prir. Soedin., 1989;4:568, incorporated herein by reference in its entirety, discloses N₁-methyl-2ED-(tetrahydropyran-2-yl) and 2ED- methyl guanosine and Hansske, et al., Tetrahedron, 1984;40:125, incorporated herein by reference in its entirety, discloses a 2ED-methylthiomethylguanosine. The 2ED- methylthiomethyl derivative of 2,6-diaminopurine riboside has also been reported. Sproat, et al., Nucleic Acids Research, 1991;19:733, incorporated herein by reference in its entirety, teaches the preparation of 2ED-allyl-guanosine. Iribarren, et al., Proc. Natl. Acad. ScL, 1990;87:7747, incorporated herein by reference in its entirety, also studied 2 ED-allyl oligoribonucleotides. In certain embodiments, the nucleic acid molecules of the invention comprise 2ED-methyl-, 2ED-allyl-, and 2 ED-dimethylallyl- substituted nucleotides.

[0150] In certain embodiments, at least one of the 2EHeoxyribofuranosyl moiety of at least one of the nucleosides of an oligonucleotide is modified. A halo, alkoxy, aminoalkoxy, alkyl, azido, or amino group may be added. For example, F, CN, CF3, OCF₃, OCN, O-alkyl, S-alkyl, SMe, SO₂ Me, ONO₂, NO₂, NH₃, NH₂, NH-alkyl, OCH₂ CH=CH₂ (allyloxy), OCH₃=CH₂, OCCH, where alkyl is a straight or branched chain of Ci to C₂o, with unsaturation within the carbon chain.

PCT/US91/00243, application Ser. No. 463,358, and application Ser. No. 566,977, disclose that incorporation of, for example, a 2ED-methyl, 2ED-ethyl, 2ED-propyl, 2 B O-allyl, 2 ED-aminoalkyl or 2 EUeoxy-2 Efluoro groups on the nucleosides of an oligonucleotide enhance the hybridization properties of the oligonucleotide. The nucleic acid molecules of the invention can be augmented to further include either or both a phosphorothioate backbone or a 2BO-Ci C₂o-alkyl (e.g., 2ED-methyl, 2ED- ethyl, 2 ED-propyl), 2 ED-C₂ C₂₀-alkenyl (e.g. , 2 ED-allyl), 2 ED-C₂ C₂₀-alkynyl, 2 ES- Ci C₂₀-alkyl, 2ES-C₂ C₂₀-alkenyl, 2ES-C₂ C₂₀-alkynyl, 2ENH-Ci C₂₀-alkyl (2 ED- aminoalkyl), 2ENH-C₂ C₂₀-alkenyl, 2ENH-C₂ C₂₀-alkynyl or 2 Edeoxy-2 Efluoro group for increased stability. See, e.g., U.S. Patent No 5,506,351, herein incorporated by reference in its entirety.

[0151] Exemplary modified nucleotides can be found in U.S. Patent Nos. 7,101,993, 7,056,896, 6,911,540, 7,015,315, 5,872,232, and 5,587,469, herein incorporated by reference in their entirety.

[0152] Suitable cationic polymers useful for forming a vector include, but are not limited to, linear or branched HK copolymers (copolymers of histidine and lysine), linear or branched polyethyleneimine (PEI), polylysine, linear or non-linear polyamidoamine, protamine sulfate, polybrine, chitosan, polymethacrylate, polyamines, spermine analogues and any other suitable polymer.

[0153] A preferred cationic polymer is an HK copolymer. In certain embodiments, the HK copolymer is synthesized from any appropriate combination of polyhistidine, polylysine, histidine and/or lysine. In certain embodiments, the HK copolymer is linear. In certain preferred embodiments, the HK copolymer is branched.

[0154] In certain preferred embodiments, the branched HK copolymer comprises a polypeptide backbone. Preferably, the polypeptide backbone comprises 1-10 amino acid residues, and more preferably 2-5 amino acid residues.

[0155] In certain preferred embodiments, the polypeptide backbone consists of lysine amino acid residues.

[0156] In certain preferred embodiments, the number of branches on the branched HK copolymer is one greater than the number of backbone amino acid residues. In certain preferred embodiments, the branched HK copolymer contains 1-11 branches. In certain more preferred embodiments, the branched HK copolymer contains 2-5 branches. In certain even more preferred embodiments, the branched HK copolymer contains 4 branches.

[0157] In some embodiments, the branch of the branched HK copolymer comprises 10-100 amino acid residues. In certain preferred embodiments, the branch comprises 10-50 amino acid residues. In certain more preferred embodiments, the branch comprises 15-25 amino acid residues. In certain embodiments, the branch of the branched HK copolymer comprises at least 3 histidine amino acid residues in every subsegment of 5 amino acid residues. In certain other embodiments, the branch comprises at least 3 histidine amino acid residues in every subsegment of 4 amino acid residues. In certain other embodiments, the branch comprises at least 2 histidine amino acid residues in every subsegment of 3 amino acid residues. In certain other embodiments, the branch comprises at least 1 histidine amino acid residues in every subsegment of 2 amino acid residues.

[0158] In certain embodiments, at least 50% of the branch of the HK copolymer comprises units of the sequence KHHH. In certain preferred embodiments, at least 75% of the branch comprises units of the sequence KHHH.

[0159] In certain embodiments, the HK copolymer branch comprises an amino acid residue other than histidine or lysine. In certain preferred embodiments, the branch comprises a cysteine amino acid residue, wherein the cysteine is a N-terminal amino acid residue.

[0160] In certain embodiments, some or all of the lysine residues of the HK copolymer branch are replaced with alternative amino acids bearing positive charges. Example of suitable amino acids include, but are not limited to, arginine and ornithine.

[0161] In certain embodiments, suitable HK copolymers include, but are not limited to those found in U.S. Patent Nos. 6,692,911, 7,070,807 and 7,163,695, all of which are incorporated herein by reference.

[0162] As discussed above, any biologically relevant targeting moiety/molecule may be used in the vectors of the present invention. In certain embodiments, the targeting moiety is a small molecule, polypeptide, peptide, protein, antibody, or a fragment, dimer, trimer or oligomer thereof. Suitable biologically relevant small molecule targeting moieties include, but are not limited to, vascular endothelial cell growth factor for targeting endothelial cells; FGF2 for targeting vascular lesions and tumors; transferrin for targeting tumors; melanotropin (alpha MSH) peptides for tumor targeting; ApoE and peptides for LDL receptor targeting; von WillebrandS Factor and peptides for targeting Coxsackie-adenoviral receptor (CAR) expressing cells; PDl and peptides for targeting Neuropilin 1; EGF and peptides for targeting EGF receptors expressing cells; folic acid and ligands for targeting folate receptors; RGD peptides for targeting integrin expressing cells and any other suitable targeting moiety. Additional examples of suitable target molecules include, but are not limited to, RGD, folate, LHRH, somatostatin, YIGSR, bombesin, hyaluronic acid, SLX, antibody fragments, N-acetyl galactosamine, mannose-6-phosphate, vitamin B 12, any cell penetrating peptide and any extracellular binding ligand. Further, any fragments or peptides of the above moieties having the same or similar targeting properties may be used as for their respective targets.

[0163] While we have hereinbefore presented a number of embodiments of this invention, it is apparent that our basic construction can be altered to provide other embodiments which utilize the methods of this invention.

Examples

Example 1: Preparation of PEG-3400-Methyl Ester (2)

[0164] Acetyl chloride (2.50 mL, 35.16 mmol) was added to 250 mL of methanol. This mixture was stirred for 5 min and then added to compound (1) (20.01 g, 5.81 mmol) as prepared in Zalipsky et al. (J. Bioactive and Compatible Polymers, Vol. 5, 227-231, (1990)). After stirring at room temperature for 24 hours, the resulting solution was concentrated to dryness. The residue was dissolved in methylene chloride (15 mL) and the resulting solution was added to ether (300 mL) with vigorous stirring. The resulting solids were filtered, washed with ether and dried under vacuum yielding the desired PEG-3400 methyl ester compound (2) (18.93 g, 94% yield) as a white powder. Example 2: Preparation of PEG-3400 Methyl Ester Succinimidyl Carbonate (3)

[0165] Compound (2) (1.01 g, 0.29 mmol) was dissolved in a mixture of methylene chloride (0.64 mL), acetonitrile (0.26 mL) and pyridine (0.14 mL). Disuccinimidyl carbonate (181.1 mg, 0.71 mmol) was added and the reaction was stirred at room temperature for 3 days. The reaction was concentrated to dryness and the residue was recrystallized from isopropanol (10 mL) giving compound (3) (0.97 g, 92% yield) as a white solid.

Example 3: Preparation of PEG-3400 Methyl Ester Diethoxypropylamine

Carbamate (4)

[0166] Compound (3) (923.0 mg, 0.26 mmol) was dissolved in methylene chloride (10 mL) and 3, 3 -diethoxypropylamine (62 μL, 0.38 mmol) was added. The reaction was stirred at room temperature for 23 hours after which, it was concentrated to dryness. The residue was recrystallized from isopropanol (10 mL) giving compound (4) (771.6 mg, 83% yield) as a white solid.

Example 4: Preparation of PEG-3400 Carboxylic Acid Diethoxypropylamine Carbamate (5)

[0167] Sodium hydroxide (1 M in water, 0.31 mL) was diluted into water (15 mL) and compound (4) (750.9 mg, 0.21 mmol) was added. The reaction was stirred at room temperature for 17 minutes after which, sodium chloride (4.2 g) was dissolved into the mixture. After adjusting the pH to 5-6 with acetic acid, the reaction was washed with methylene chloride (3 X 5 mL). The combined extracts were dried over anhydrous magnesium sulfate, filtered and concentrated to a volume of approximately 3 mL. The resulting solution was poured into ether (150 mL) with rapid stirring. The resulting solids were filtered, washed with ether and dried under vacuum. The crude isolated solids were purified by ion exchange chromatography using a DEAE- Sephadex (7 g) column prepared in potassium tetraborate solution (50 g/500 mL water) and washed with water until neutral pH. The column was eluted sequentially with water (200 mL), 6 mM ammonium bicarbonate (100 mL), 8 mM ammonium bicarbonate (100 mL), 10 mM ammonium bicarbonate (100 mL) and 48 niM ammonium bicarbonate (100 mL). All product fractions were combined and sodium chloride (20 g) was dissolved into the combined fractions. The pH was adjusted to 5- 6 with acetic acid and the product was extracted with methylene chloride (3 X 30 mL). The combined extracts were dried over anhydrous magnesium sulfate, filtered and concentrated to a volume of 3 mL. This residue was poured into ether (150 mL) with rapid stirring. The resulting solids were filtered, washed with ether and dried under vacuum giving compound (5) (322.9 mg, 43% yield) as a white solid.

Example 5: Preparation of PEG-3400 p-Nitrophenyl Ester Diethoxypropylamine Carbamate (6; Compound 6)

[0168] Compound (5) (295.4 mg, 0.081 mmol) was dissolved in pyridine (1.1 mL) and nitrophenyl-4-trifluoroacetate (86.1 mg, 0.37 mmol) was added. The reaction was stirred at room temperature for 1 hour after which additional nitrophenyl-4- trifluoroacetate (20 mg) was added. After stirring at room temperature for an additional 1 hour, the reaction was poured into ether (150 mL) with rapid stirring.

The resulting solids were filtered, washed with ether and dried under vacuum giving compound (6; Compound 6) (265.5 mg, 87% yield) as a white solid.

Example 6: Preparation of PEG-3400 Mono-Diethoxypropylamine Carbamate M ono-Beta-Alanine Carbamate (8)

[0169] Compound (7) (7.82 g, 2.12 mmol, derived from PEG-3400) was dissolved in methylene chloride (75 mL) and 3,3-diethoxypropylamine (0.34 mL, 2.12 mmol) was added drop wise. After stirring at room temperature for 1 hour, beta-alanine ethyl ester hydrochloride (0.65 g, 4.25 mmol) was added followed by

diisopropylethylamine (1.48 mL, 8.50 mmol). The reaction was stirred at room temperature for 19 hours after which, it was concentrated to dryness. The residue was recrystallized from isopropanol (70 mL) giving compound (8) (7.35 g, 93% yield) as a mixture of homo-bifunctional (undesired) and hetero-bifunctional (desired) products. Example 7: Preparation of PEG-3400 Mono-Diethoxypropylamine Carbamate Mono-Beta-Alanine Carboxylic Acid (9)

[0170] The compound (8) product mixture (7.35 g, 1.98 mmol) was dissolved in water (65 mL) and sodium hydroxide (1 M in water, 5.93 mL, 5.93 mmol) was added. The reaction was stirred at room temperature for 3 hours after which sodium chloride (13 g) was dissolved into the mixture. The resulting solution was adjusted to pH 5-6 with acetic acid and the product was extracted with methylene chloride (3 X 40 mL). The combined extracts were dried over anhydrous magnesium sulfate, filtered and concentrated to a volume of 10 mL. The residue was poured into ether (300 mL) with rapid stirring. The resulting solids were filtered, washed with ether and dried under vacuum. The crude isolated solids were purified by ion exchange chromatography using a DEAE-Sephadex (15 g) column prepared in potassium tetraborate solution (50 g/500 mL water) and washed with water until neutral pH. The column was eluted sequentially with water (300 mL), 6 mM ammonium bicarbonate (400 mL) and 48 mM ammonium bicarbonate (200 mL). All product fractions were combined and sodium chloride (40 g) was dissolved into the combined fractions. The pH was adjusted to 5-6 with acetic acid and the product was extracted with methylene chloride (3 X 30 mL). The combined extracts were dried over anhydrous magnesium sulfate, filtered and concentrated to a volume of 10 mL. This residue was poured into ether (200 mL) with rapid stirring. The resulting solids were filtered, washed with ether and dried under vacuum giving compound (9) (2.68 g, 36% yield from compound (7)) as a white solid.

Example 8: Preparation of PEG-3400 Mono-Diethoxypropylamine Carbamate Mono-Beta-Alanine Succinimidyl Ester (10; Compound 10)

[0171] Compound (9) (499.4 mg, 0.14 mmol) was dissolved in anhydrous acetonitrile (5 mL). Anhydrous pyridine (32.9 μL, 0.41 mmol) was added followed by disuccinimidyl carbonate (104.1 mg, 0.41 mmol). The reaction was stirred at room temperature for 22 hours after which, it was poured into ether (250 mL) with rapid stirring. The solids were filtered, washed with ether and combined with warm isopropanol (50⁰C, 5 mL). The resulting mixture was filtered and the solids were washed with warm isopropanol (50⁰C, 2 X 2 mL). The combined isopropanol filtrates were warmed to dissolve all solids. The product was allowed to crystallize. The crystals were filtered, washed with isopropanol, washed with ether and dried under vacuum giving compound (10; Compound 10) (458.8 mg, 90% yield) as a white solid. Example 9: Conjugation of Heterobifunctional PEGs with RGD

[0172] Coupling of the heterobifunctional PEGs of the present invention to peptides can generally be accomplished using the following protocol.

[0173] A heterobifunctional PEG can be coupled to RGD to yield, e.g., compound (12). In this synthesis, Compound 6 is added to a solution of compound (11) in DMSO. The mixture is then made basic and additional compound (11) is added in several portions over 4-24 hours. The reaction is monitored, preferably by RP-HPLC. When the reaction is complete, the reaction is diluted with phosphate buffer (20 mM, 2 ml, pH 8) and is dialyzed against, for example, phosphate buffer (20 mM, 1L-24H, IL-1.5H) followed by dialysis against DI water (2X, 1L-2H) using a 3500 MWCO membrane. The dialyzed solution is then lyophilized to give compound 12.

[0174] Synthesis of Compound (13): Compound (11) (19 mg, 22.8 μmol) was dissolved in DMSO (1 mL) and Compound 10 (100 mg, 27.8 μmol, from 3400k PEG) was added. The mixture was made basic (pH -8-9) with DIEA (10 μL).

Additional compound (11) (15 mg) was added in three portions over 8 hrs and the reaction was monitored by RP-HPLC. When the reaction was complete (determined by 2 HPLC injections without significant reduction of the peak corresponding to compound (H)), the reaction was diluted with phosphate buffer (20 mM, 2 ml, pH 8). The diluted solution was dialyzed against phosphate buffer (20 mM, 1L-24H, IL- 1.5H) followed by dialysis against DI water (2X, 1L-2H) using a 3500 MWCO membrane. The dialyzed solution was lyophilized to give compound 13 (108 mg) as a white powder.

CH

13 (From Compound 10) -

Example

10: Coupling of Aldehyde Protected Heterobifunctional PEG/RGD Conjugate To Target Molecule

[0175] The heterobifunctional PEGs of the present invention, once conjugated to e.g., RGD, can be further conjugated to, for example, lipids, polymers, liposomes or nanoparticles. This can be accomplished in a one-pot synthesis where a) the aldehyde moiety on the heterobifunctional PEG is "liberated"; and b) the liberated aldehyde moiety is conjugated to a target molecule via reductive amination.

[0176] The aldehyde moiety on the heterobifunctional PEGs conjugated to proteins (e.g., RGD) can generally be "liberated" or deprotected using standard conditions in the art {e.g., with aqueous acid). [0177] Generally, hydrolysis of the acetal of compound (12) or (13) is carried out under aqueous acid conditions (e.g., aqueous HCl or aqueous TFA). The hydrolysis is monitored, preferably by RP-HPLC. When the reaction is complete, the reaction is diluted with phosphate buffer (20 mM, 2 ml, pH 8) and is dialyzed against, for example, phosphate buffer (20 mM, 1L-24H, IL- 1.5H) followed by dialysis against DI water (2X, 1L-2H) using a 3500 MWCO membrane. The dialyzed solution is then lyophilized to give compound (14) or (15), respectively.

[0178] Synthesis of Compound (17): Compound (13) (20 mg) is dissolved in aqueous TFA (0.2%, 1 mL). After 16 hrs, the pH of the resulting solution (736 μL, containing compound (15)) is adjusted to 5 with NaH₂PO₄ (0.2 M in water, 662 μL) and Na₂HPO₄ (0.2 M in water, 70 μL) and cooled to 2-8°C. An HK polymer (of the type described by Leng et al., Drug News Perspect 20(2), March 2007, 50 mg, 1 eq) is then added followed by NaCNBH3 (1.1 mg, 5 eq). The reaction is monitored by RP- HPLC is considered finished after 2.5 hrs. The reaction mixture is dialyzed against DI water (3X, 900 mL-2H) and then DI water (900 mL-16H) using a 3500 MWCO dialysis membrane. The dialyzed solution is purified using weak cation exchange on HIprep 16/10 CM FF resin (carboxymethyl sepharose fast flow) with a linear gradient of 20-50% MPB over 30 column volumes (MPA = 5OmM phosphate pH 7. MPB = MPA + 2 M NaCl.). The fractions containing compound (17) are combined and concentrated to < 10 mL using a 200 ml stir cell with a 5000 MWCO membrane. The resulting solution is diluted with aqueous acetic acid (0.05%, 70 mL) and again concentrated to < 10 mL using a 200 mL stir cell with at 5000 MWCO membrane. The dilution and concentration steps are repeated an additional three times and the resulting solution is lyophilized giving pure compound (17) (19.9 mg) as a white powder.

NH

NH₂

O

H^

R

H

Example 11: Conjugation of Heterobifunctional PEGs with N-acetyl- galactosamine-beta-aminoethyl glycoside

[0179] Using the protocol described in Example 9, heterobifunctional PEGs of the present invention can be directly coupled with N-acetyl-galactosamine-beta- aminoethyl glycoside through the active ester terminus.

Example 12: Reductive Animation of Amines with the Aldehyde Group of Heterobifunctional PEGs Conjugated to N-acetyl-galactosamine-beta-aminoethyl glycoside

[0180] Using the protocol described in Example 10, heterobifunctional PEGs of the present invention, conjugated to N-acetyl-galactosamine-beta-aminoethyl glycoside, can be conjugated to an HK polymer (of the type described by Leng et al., Drug News Perspect 20(2), March 2007) through the acetal/aldehyde terminus. Example 13: Preparation of PEG-8000 Mono-Diethoxypropylamine Carbamate GLU-GLU Carbamate Trimethyl Ester (8a)

[0181] Compound (7) (9.05 g, 1.09 mmol, derived from PEG8000) was dissolved in methylene chloride (75 niL) and 3,3-diethoxypropylamine (0.18 mL, 1.09 mmol) was added drop wise. After stirring at room temperature for 1 hour, GLU-GLU trimethyl ester hydrochloride (0.58 g, 1.64 mmol) was added followed by

diisopropylethylamine (0.57 mL, 3.28 mmol). The reaction was stirred at room temperature for 19 hours after which, it was concentrated to dryness. The residue was recrystallized from isopropanol (90 mL) giving compound (8a) (8.88 g, 95% yield) as a mixture of homo-bifunctional (undesired) and hetero-bifunctional (desired) products.

Example 14: Preparation of PEG-8000 Mono-Diethoxypropylamine Carbamate GLU-GLU Carbamate Tricarboxylic Acid (9a)

[0182] The compound (8a) product mixture (8.88 g, 1.04 mmol) was dissolved in water (35 mL) and sodium hydroxide (1 M in water, 9.34 mL, 9.34 mmol) was added. The reaction was stirred at room temperature for 3 hours after which sodium chloride (30 g) was dissolved into the mixture. The resulting solution was adjusted to pH 5-6 with acetic acid and the product was extracted with methylene chloride (3 X 20 mL). The combined extracts were dried over anhydrous magnesium sulfate, filtered and concentrated to a volume of 20 niL. The residue was poured into ether (300 mL) with rapid stirring. The resulting solids were filtered, washed with ether and dried under vacuum. The crude isolated solids were purified by ion exchange chromatography using a DEAE-Sephadex (15 g) column prepared in potassium tetraborate solution (50 g/500 mL water) and washed with water until neutral pH. The column was eluted sequentially with water (500 mL), 6 mM ammonium bicarbonate (200 mL), 8 mM ammonium bicarbonate (200 mL), 10 mM ammonium bicarbonate (200 mL), 12 mM ammonium bicarbonate (200 mL), 14 mM ammonium bicarbonate (200 mL), 16 mM ammonium bicarbonate (200 mL) and brine (200 mL). All product fractions were combined and sodium chloride (75 g) was dissolved into the combined fractions. The pH was adjusted to 5 with acetic acid and the product was extracted with methylene chloride (5 X 50 mL). The combined extracts were dried over anhydrous magnesium sulfate, filtered and concentrated to a volume of 10 mL. This residue was poured into ether (300 mL) with rapid stirring. The resulting solids were filtered, washed with ether and dried under vacuum giving compound (9a) (2.73 g, 29% yield from compound (7)) as a white solid.

Example 15: Preparation of PEG-8000 Mono-Diethoxypropylamine Carbamate GLU-GLU Carbamate Trisuccinimidyl Ester (10a)

[0183] Compound (9a) (0.95 g, 0.11 mmol) was dissolved in anhydrous acetonitrile (3.6 mL). Anhydrous pyridine (83.1 μL, 1.03 mmol) was added followed by disuccinimidyl carbonate (263.1 mg, 1.03 mmol). The reaction was stirred at room temperature for 5 days after which, it was concentrated to dryness. Isopropanol (10 mL) was added and the mixture was warmed to dissolve all solids. The product was allowed to crystallize after which, the solids were collected, washed with isopropanol, washed with ether and dried under vacuum. The dried solids were recrystallized from isopropanol (10 mL). The resulting solids were filtered, washed with isopropanol, washed with ether and dried under vacuum giving the desired compound (10a)

(0.86 g, 88% yield) as a white solid.

Example 16: Conjugation of Heterobifunctional PEGs with multiple N-acetyl- galactosamine-beta-aminoethoxyethyl glycoside units (Compound 19, carboxylic acid coupling) [0184] Compound (9a) (203.4 mg, 0.024 mmol) and tetra-acetyl-galactosamine- beta-aminoethoxyethyl glycoside hydrochloride salt (18) (33.7 mg, 0.072 mmol) were dissolved in anhydrous dichloromethane (1 mL). Diisopropylethylamine (37.5 μL, 0.215 mmol) was added followed by HBTU (37.2 mg, 0.072 mmol). The reaction was stirred at room temperature for 19 hours after which, it was poured into ether (100 mL) with rapid stirring. The resulting solids were filtered, washed with ether and recrystallized from isopropanol (2 mL). The recrystallized product was filtered, washed with isopropanol, washed with ether and dried under vacuum giving the desired compound (19) (198.5 mg, 85% yield) as a white solid.

^⁰V

c

E

xample 17: Conjugation of Heterobifunctional PEGs with multiple

aminoethoxyethanol units (Compound 21, carboxylic acid coupling)

[0185] Compound (9a) ( 152.9 mg, 0.018 mmol) and aminoethoxyethanol (20) (5.37 μL, 0.054 mmol) were dissolved in anhydrous dichloromethane (1 mL).

Diisopropylethylamine (18.8 μL, 0.108 mmol) was added followed by HBTU (20.4 mg, 0.054 mmol). The reaction was stirred at room temperature for 28 hours after which, it was poured into ether (100 mL) with rapid stirring. The resulting solids were filtered, washed with ether and recrystallized from isopropanol (1.5 mL). The recrystallized product was filtered, washed with isopropanol, washed with ether and dried under vacuum giving the desired compound (21) (110.8 mg, 70% yield) as a white solid.

Example 18: Conjugation of Heterobifunctional PEGs with multiple

aminoethoxyethanol units (Compound 21, active ester coupling)

[0186] Using the protocol described in Example 9, heterobifUnctional PEGs of the present invention (Compound 10a) can be directly coupled with three

aminoethoxyethanol units through the three active ester termini giving the desired compound (21).

O M

E tyl- g

alactosamine-beta-aminoethoxyethyl glycoside units (active ester coupling)

[0187] Using the protocol described in Example 9, heterobifunctional PEGs of the present invention (e.g., Compound 10a) can be directly coupled to three N-acetyl- galactosamine-beta-aminoethoxyethyl glycoside units through the three active ester termini giving the desired compound e.g., compound (19).

Example 20: Full Deprotection and Conjugation of Compound (19) with an Amine via Reductive Amination (Compound 23)

[0188] The heterobifunctional PEGs of the present invention, once conjugated to proteins or sugars (e.g., RGD, N-acetyl galactosamine), can be further conjugated to, for example, lipids, polymers, liposomes or nanoparticles. This can be accomplished in a one-pot synthesis where a) the hydroxyl moieties on the protected N-acetyl galactosamine are "liberated"; and b) the aldehyde moiety on the heterobifunctional PEG is "liberated"; and c) the liberated aldehyde moiety is conjugated to a target molecule via reductive amination.

[0189] The hydroxyl moieties on the heterobifunctional PEGs conjugated to sugars (e.g., N-acetyl galactosamine) can generally be "liberated" or deprotected using standard conditions in the art (e.g., with aqueous base).

[0190] Generally, hydrolysis of the acetoxy groups of compound (19) is carried out under aqueous basic conditions (e.g., aqueous sodium hydroxide). The hydrolysis is monitored, preferably by RP-HPLC.

[0191] The aldehyde moiety on the heterobifunctional PEGs conjugated to proteins or sugars (e.g., RGD, N-acetyl galactosamine) can generally be "liberated" or deprotected using standard conditions in the art (e.g., with aqueous acid).

[0192] Generally, hydrolysis of the acetal of compound (19) or (21) is carried out under aqueous acid conditions (e.g., aqueous HCl or aqueous TFA). The hydrolysis is monitored, preferably by RP-HPLC.

[0193] Synthesis of Compound (23): Compound (19) (80 mg, 8.2 μmol) was dissolved in deionized water (1.78 mL) and sodium hydroxide (1 M in deionized water, 221 μL, 27 eq) was added. Hydrolysis of the acetoxy groups was followed by RP-HPLC and was complete within 30 min. The basic reaction mixture was acidified with trifluoroacetic acid (1 M in deionized water) to pH 1.4. The hydrolysis of the acetal was monitored by RP-HPLC and was complete after 2 hrs. The pH of the reaction mixture (now containing compound (22)) was adjusted to 5 using NaHaPO₄ (0.2 M in deionized water, 2 mL) and sodium hydroxide (1 M in deionized water, 65 μL). An HK polymer (of the type described by Leng et al., Drug News Perspect 20(2), March 2007, 119 mg, 8.2 μmol) was then added followed by NaCNBH₃ (3 nig). The reaction was monitored by RP-HPLC and was considered finished after 1.5 hrs. The reaction mixture was dialyzed with a 3500 MWCO dialysis membrane against deionized water (I L, 2 x 1.5 hr, 1 x 16 hr). The dialyzed solution is purified using weak cation exchange on HIprepl6/10 CM FF resin with a linear gradient of 20-50% MPB over 30 column volumes (MPA = 5OmM phosphate pH 7. MPB =

MPA + 2 M NaCl.). The fractions containing mainly monoPEGylated product were combined and concentrated using a 200 mL stir-cell with 5000 MWCO membrane to < 10 ml. The retentate was washed and concentrated to < 10 ml four times with acetic acid (0.05% in deionized water, 80 mL) using the stir-cell. The final wash was lyopholized giving the desired compound (23) (53 mg) as a white powder.

E

Animation (Compound 25)

[0194] The heterobifunctional PEGs of the present invention, once conjugated to e.g., aminoethoxyethanol, can be further conjugated to, for example, lipids, polymers, liposomes or nanoparticles. This can be accomplished in a one-pot synthesis where a) the aldehyde moiety on the heterobifunctional PEG is "liberated"; and b) the liberated aldehyde moiety is conjugated to a target molecule via reductive animation.

[0195] The aldehyde moiety on the heterobifunctional PEGs conjugated to proteins (e.g., RGD) can generally be "liberated" or deprotected using standard conditions in the art (e.g., with aqueous acid).

[0196] Generally, hydrolysis of the acetal of compound (21) is carried out under aqueous acid conditions (e.g., aqueous HCl or aqueous TFA). The hydrolysis is monitored, preferably by RP-HPLC.

[0197] Synthesis of Compound (25): Compound (21) (60 mg, 6.84 μmol) was dissolved in aqueous trifluoroacetic acid (0.2%, 3 mL). Hydrolysis of the acetal to form the aldehyde was monitored by RP-HPLC and was complete within 1 hr. The pH of the resulting solution (now containing compound (24)) was adjusted to 5 using NaH₂PO₄ (0.2 M in deionized water, 2.8 mL) and Na₂HPO₄ (0.2 M in deionized water, 0.3 mL). An HK polymer (of the type described by Leng et al., Drug News Perspect 20(2), March 2007, 99 mg, 6.84 μmol) was then added followed by

NaCNBH₃ (2.5 mg). The reaction was monitored by RP-HPLC and was considered finished after 2 hrs. The reaction mixture was dialyzed with a 3500 MWCO dialysis membrane against deionized water (I L, 2 x 1.5 hr, 1 x 16 hr). The dialyzed solution was purified using weak cation exchange on HIprep 16/10 CM FF resin with a linear gradient of 20-50% MPB over 30 column volumes (MPA = 5OmM phosphate pH 7. MPB = MPA + 2 M NaCL). The fractions containing mainly monoPEGylated product were combined and concentrated using a 200 mL Stir cell with 5000 MWCO membrane to < 10 ml. The retentate was washed and concentrated down to < 10 ml four times with acetic acid (0.05% in deionized water, 75 mL) using the stir-cell. The final wash was lyopholized to give the desired compound (25) (56 mg) as a white powder.

Example 22: Preparation of GLU-GLU Trimethyl Ester Hydrochloride

(Compound 26)

[0198] Acetyl chloride (0.50 mL) was added to anhydrous methanol (50 mL) and stirred at room temperature for 5 minutes. Commercially available GLU-GLU (Aldrich, 518.9 nig, 1.88 mmol) was added and the reaction was stirred at room temperature for 18 hours. The resulting mixture was concentrated to dryness giving GLU-GLU trimethyl ester hydrochloride (26) (677.6 mg, 100% yield).

Example 23: Preparation of N-Benzylcarbamoyl GLU-GLU Trimethyl Ester (Compound 27)

[0199] GLU-GLU trimethyl ester hydrochloride (26) (677.6 mg, 1.91 mmol) was dissolved in ahydrous dichloromethane (15 mL). Diisopropylethylamine (1 mL, 5.74 mmol) and benzyl chloro formate (0.34 mL, 2.39 mmol) were added. The reaction was stirred at room temperature for 21 hours after which, it was diluted with dichloromethane (20 mL). The resulting solution was washed with aqueous hydrochloric acid (1 M, 3 X 10 mL), saturated aqueous sodium bicarbonate (2 X 10 mL) and brine (10 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated to dryness. The residue was recrystallized from ethyl acetate/hexane giving the desired product (27) (615.6 mg, 71% yield) in two isolated crops.

Example 24: Preparation of N-Beyzylcarbamoyl GLU-GLU Tricarboxylic Acid

(Compound 28)

[0200] N-Benzylcarbamoyl GLU-GLU trimethyl ester (27) (615.6 mg, 1.36 mmol) was dissolved in methanol (10 mL). Aqueous sodium hydroxide (6 M, 2.04 mL,

12.26 mmol) was added and the reaction was stirred at room temperature for 20 minutes. The reaction was neutralized using Dowex 50WX8 ion exchange resin. The resin was removed by filtration and the resulting solution was concentrated to dryness giving the desired product (28) (543.3 mg, 97% yield).

Example 25: Preparation of Pentaacetyl Galactosamine (Compound 29)

[0201] Pyridine (30 mL) and acetic anhydride (20 mL) were added to galactosamine hydrochloride (5.09 g, 23.6 mmol) and the resulting mixture was stirred at room temperature for 6 days. The reaction was diluted with chloroform (1 L) and washed sequentially with water (30 mL), saturated aqueous sodium bicarbonate (30 mL), saturated aqueous copper sulfate (10 X 30 mL), and water (3 X 30 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to dryness. The residue was recrystallized from ethanol (300 mL). The resulting solids were filtered, washed with ethanol and dried under vacuum giving the desired product (29) (7.01 g, 76% yield).

Example 26: Preparation of 5-(acetoxymethyl)-2-methyl-5,6,7,7a-tetrahydro- 3aH-pyrano[3,2-d]oxazole-6,7-diyl diacetate (Compound 30)

[0202] Pentaacetyl galactosamine (29) (7.01 g, 18.02 mmoles) was combined with anhydrous 1 ,2-dichloroethane (40 mL) and heated to 50⁰C. Trimethylsilyl triflate (3.74 mL, 20.72 mmoles) was added to the heterogeneous mixture and the reaction was stirred at 50⁰C for 17.5 hours. After cooling to room temperature under nitrogen, triethylamine (3.41 mL) as added and the resulting solution was concentrated to % its original volume. The product was purified from the remaining solution on silica gel (toluene/ethyl acetate/triethylamine 100/200/1) giving the desired product (30) (4.92 g, 83% yield). Example 27: Preparation of 5-acetamido-2-(acetoxymethyl)-6-(2-(2- (benzyloxycarbonylamino)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate (Compound 31)

[0203] 5-(acetoxymethyl)-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]oxazole- 6,7-diyl diacetate (30) (2.34 g, 7.11 mmoles), benzyl 2-(2- hydroxyethoxy)ethylcarbamate (2.55 g, 10.67 mmoles) and activated powdered 4A molecular sieves were combined with anhydrous dichloromethane (10 mL). The resulting mixture was stirred at room temperature for 3.5 hours after which, concentrated sulfuric acid (123.1 μL) was added. After stirring at room temperature for 20 hours, the reaction was diluted with dichloromethane (10 mL) and filtered through Celite. The filtrate was washed with saturated aqueous sodium bicarbonate (5 mL) and water (5 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to dryness. The residue was purified on silica gel (33% toluene/ethyl acetate then ethyl acetate) giving the desired product (31) (3.00 g, 74% yield).

Example 28: Preparation of 5-acetamido-2-(acetoxymethyl)-6-(2-(2- aminoethoxy)ethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate hydrochloride

(Compound 32)

[0204] 5-acetamido-2-(acetoxymethyl)-6-(2-(2-(benzyloxycarbonylamino)ethoxy)- ethoxy)-tetrahydro-2H-pyran-3,4-diyl diacetate (31) (486.2 mg, 0.86 mmole) and 10% palladium on carbon (190.2 mg) were combined and placed under vacuum. Methanol (10 mL) and chloroform (1 mL) were added. The resulting mixture was degassed under vacuum and stirred under hydrogen for 18 hours. The reaction was then filtered, concentrated and dried under vacuum giving the desired product (32) (408.9 mg, 100% yield).

Example 29: Preparation of N-Beyzylcarbamoyl GLU-GLU tri(5-acetamido-2- (acetoxymethyl)-6-(2-(2-aminoethoxy)ethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate)amide (Compound 33)

[0205] N-Beyzylcarbamoyl GLU-GLU tricarboxylic acid (28) (399.2 mg, 0.97 mmole) and 5-acetamido-2-(acetoxymethyl)-6-(2-(2-aminoethoxy)ethoxy)tetrahydro- 2H-pyran-3,4-diyl diacetate hydrochloride (32) (1.37 g, 2.92 mmole) were dissolved in anhydrous dichloromethane (40 mL). To the resulting mixture was added diisopropylethylamine (1.53 mL, 8.76 mmoles) and HBTU (1.11 g, 2.92 mmoles). The reaction was stirred at room temperature for 24 hours after which, it was concentrated to dryness. The residue was triturated with ethyl acetate (30 mL) and the resulting solids were collected by filtration and dried under vacuum giving the desired product (33) (697.4 mg, 43% yield).

Example 30: Preparation of N-Beyzylcarbamoyl GLU-GLU-(N-acetyl galactosamine beta aminoethoxyethyl glycoside)tri-amide (Compound 34)

[0206] N-Beyzylcarbamoyl GLU-GLU tri(5-acetamido-2-(acetoxymethyl)-6-(2-(2- aminoethoxy)ethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate)amide (33) (161.9 mg, 0.098 mmole) was dissolved in methanol (5 mL) and sodium hydroxide (6 M in water, 0.44 mL, 2.64 mmoles) was added. The reaction was stirred at room temperature for 1 hour after which, it was acidified with Dowex acidic ion exchange resin. The mixture was filtered, concentrated and dried under vacuum giving the desired product (34) (113.7 mg, 91 % yield). Example 31: Preparation of GLU-GLU-(N-acetyl galactosamine beta aminoethoxyethyl glycoside)tri-amide (Compound 35)

[0207] N-Beyzylcarbamoyl GLU-GLU-(N-acetyl galactosamine beta

aminoethoxyethyl glycoside)tri-amide (34) (98.4 mg, 0.077 mmole) and 10% palladium on carbon (33.8 mg) were combined and placed under vacuum. Methanol (10 mL) added. The resulting mixture was degassed under vacuum and stirred under hydrogen for 19 hours. The reaction was then filtered, concentrated and dried under vacuum giving the desired product (35) (76.0 mg, 86% yield).

l

E

[0208] An analogue of Compound 19 can be synthesized from utilizing Compound 10. In this strategy, the succinimidyl ester of Compound 10 is reacted with Compound 35 giving Compound 36. Subsequent treatment with trifluoroacetic acid followed by reductive amination using sodium cyanoborohydride (as in Example 20) provides Compound 37.

Claims

What is claimed is:

1. A reagent comprising a compound of Formula (I):

wherein:

each Ri is independently selected from the group consisting of branched or straight-chain Ci-Ce alkyl, branched or straight-chain C2-C₆ alkenyl, and branched or straight-chain C2-C6 alkynyl,

R₂;

each R₂ is independently selected from the group consisting of branched or straight-chain Ci-Ce alkyl, branched or straight-chain C2-C6 alkenyl, branched or straight-chain C₂-C₆ alkynyl, hydroxy, Ci-C₆ alkoxy, -CO₂H, -CO₂(RR -CONH₂, -CONH(RJ] -CON(RIJi, carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl, nitro, cyano, and halo,

wherein each carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl may be optionally substituted with one or more R₅;

R3 is C6-C14 aryl, C₅-Cu heteroaryl or C₅-C_H heterocyclyl,

wherein R3 is optionally substituted with one or more R₅;

R₄ is selected from the group consisting of hydrogen, branched or straight- chain Ci-Ce alkyl, branched or straight-chain C₂-Ce alkenyl, branched or straight- chain C₂-C₆ alkynyl, wherein one or more hydrogens in the alkyl, alkenyl or alkynyl chain may be replaced by one or more halogens;

R₅ is selected from the group consisting of branched or straight-chain Ci-C₆ alkyl, branched or straight-chain C2-C6 alkenyl, branched or straight-chain C₂-Ce alkynyl, -CF₃, -Rp-ORp-OH, -SH, -SRPprotected OH (e.g., acyloxy), -NO₂, -CN, - NH₂, -NHRp-N(RJl, -NHCORg-NHCONH₂, -NHCONHRp-NHCON^ft, - NRCORO-NHCO₂H, -NHCO₂RO-CO₂Rg-CO₂H, -CORp-CONH₂, -CONHRO- CON(R$>, -S(O)₂H, -S(O)₂RP-S(O)₃H, -S(O)₃Rp-S(O)₂NH₂, -S(O)H, -S(O)Rp- S(O)₂NHRp-S(O)₂N(RJi, -NHS(O)₂H, -NHS(O)₂Rp-F, -Cl, -Br, and =O, where chemically feasible;

RQs selected from the group consisting of hydrogen, branched or straight- chain Ci-C₆ alkyl, branched or straight-chain C₂-Ce alkenyl, branched or straight- chain C₂-C₆ alkynyl, carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, or heteroaralkyl and each RQs optionally substituted with one or more halogen, nitro, cyano, amino, -NH-(unsubstituted aliphatic), -N-(unsubstituted aliphatic)₂, carboxy, carbamoyl, hydroxy, -O-(unsubstituted aliphatic), -SH, -S-(unsubstituted aliphatic), - CF₃, -S(O)₂NH₂ ansubstituted aliphatic, unsubstituted carbocyclyl, unsubstituted heterocyclyl, unsubstituted aryl, unsubstituted aralkyl, unsubstituted heteroaryl, or unsubstituted heteroaralkyl;

A is selected from the group consisting OfCi-C₆ alkyl, C₂-C_O alkenyl, and C₂-

C₆ alkynyl;

wherein A is optionally substituted with branched or straight-chain Q- C₆ alkyl, branched or straight-chain C₂-C_O alkenyl, branched or straight-chain C₂-C₆ alkynyl, hydroxy, Ci-C₆ alkoxy, CO₂H, CO₂(Ci-C₆ alkyl), CONH₂, CONH(Ci-C₆ alkyl), CON(Ci-C₆ alkyl)₂, nitro, cyano, or halo;

each X is independently O or S;

j is an integer from O to 10; and

n is an integer from 1 to 1,500.

2. The reagent according to claim 1, wherein n is an integer from 5 to 1,000.

3. The reagent according to claim 2, wherein n is an integer from 20 to 500.

4. The reagent according to claim 3, wherein n is an integer from 50 to 250.

5. The reagent according to claim 1, wherein j is an integer from 0 to 10.

6. The reagent according to claim 5, wherein j is an integer from 1 to 6.

7. The reagent according to claim 6, wherein j is an integer from 1 to 3.

8. The reagent according to claim 7, wherein j is 1.

9. The reagent according to claim 1 , wherein each Ri is independently a branched or straight-chain Ci-C₆ alkyl, or both Ri are taken together to form a 5- or 6- membered cyclic acetal,

wherein each Ri is optionally substituted with one or more R₂.

10. The reagent according to claim 9, wherein each Ri is independently methyl, ethyl, isopropyl or both Ri are taken together to form a 1,3-dioxolane ring or a 1,3-dioxane ring.

11. The reagent according to claim 10, wherein each Ri is independently methyl or ethyl.

12. The reagent according to claim 1, wherein each R₂ is independently selected from the group consisting of branched or straight-chain Ci-C₆ alkyl, branched or straight-chain C₂-Ce alkenyl, and branched or straight-chain C₂-C₆ alkynyl,

wherein one or more hydrogens in the alkyl, alkenyl or alkynyl chain may be replaced by one or more halogens.

13. The reagent according to claim 12, wherein each R₂ is independently selected from the group consisting of branched or straight-chain Ci-C₆ alkyl, branched or straight-chain C₂-C₆ alkenyl, and branched or straight-chain C₂-C₆ alkynyl.

14. The reagent according to claim 1, wherein R₃ is a Ce-Cu aryl or C₅-C14 heteroaryl,

wherein said heteroaryl contains one or more heteroatoms selected from the group consisting of N, N(R₄), O, S, S(O), and S(O)₂, and

wherein R₃ is optionally substituted with one or more R₅.

15. The reagent according to claim 14, wherein R₃ is a Cβ-Cio aryl or a C5-C10 heteroaryl,

wherein said heteroaryl contains one or more heteroatoms selected from the group consisting of N, N(R₄), O, S, S(O), and S(O)2, and

wherein R3 is optionally substituted with one or more R5.

16. The reagent according to claim 15, wherein R₃ is a C₆ aryl or a C₅ heteroaryl,

wherein R3 is optionally substituted with one or more R5.

17. The reagent according to claim 16, wherein R₃ is phenyl, pyridyl, pyrimidinyl, or naphthyl, optionally substituted with one or more R5.

18. The reagent according to claim 17, wherein R₃ is phenyl optionally substituted with one or more nitro or one or more halo.

19. The reagent according to claim 16, wherein R₃ is ortho-nitrophenyl, para-nitrophenyl, trichlorophenyl, trifluorophenyl, or pentafluorophenyl.

20. The reagent according to claim 19, wherein R₃ is ortho-nitrophenyl or para-nitrophenyl.

21. The reagent according to claim 1, wherein R₅ is selected from the group consisting of -CF₃, -RO-ORO-OH, -SH, -SROprotected OH (e.g., acyloxy), -

NO₂, -CN, -NH₂, -NHRg-N(RJi, -NHCORp-NHCONH₂, -NHCONHRf]- NHCON(RJi, -NRCORO-NHCO2H, -NHCO₂RO-CO₂RO-CO₂H, -CORO-CONH₂, - CONHR 0-CON(R Ji, -S(O)₂H, -S(O)₂RO-S(O)₂NH₂, -S(O)₃H, -S(O)₃RO-S(O)H, - S(O)RO-S(O)₂NHRO-S(O)₂N(RJi, -NHS(O)₂H, -NHS(O)₂RO-F, -Cl, -Br, and =O, where chemically feasible.

22. The reagent according to claim 21, wherein R5 is selected from the group consisting of -CF₃, -NO₂, -CN, -CO₂RO-CO₂H, -CORO-CONH₂, -CONHRO- CON(RJi, -S(O)₂H, -S(O)₂RO-S(O)₃H, -S(O)₃RO-S(O)₂NH₂, -S(O)H, -S(O)RO- S(O)₂NHRO-S(O)₂N(RJi, -NHS(O)₂H, -NHS(O)₂R0and =O, where chemically feasible.

23. The reagent according to claim 22, wherein R₅ is selected from the group consisting of -CO₂R 0-CORO-CON(RJi, -S(O)RO-S(O)₂R 0-S(O)₃R 0-CF₃, nitro, cyano, and halo.

24. The reagent according to claim 23, wherein R₅ is selected from the group consisting Of-CF₃, nitro, cyano, and halo.

25. The reagent according to claim 24, wherein R₅ is ortho- or para-nitro.

26. The reagent according to claim 1, wherein RQs selected from the group consisting of hydrogen, branched or straight-chain Ci-C₆ alkyl, branched or straight-chain C2-C6 alkenyl, or branched or straight-chain C₂-Ce alkynyl.

27. The reagent according to claim 1, wherein A is Ci-C₆ alkyl, wherein A is optionally substituted with branched or straight-chain Ci-C₆ alkyl, branched or straight-chain C₂-Ce alkenyl, branched or straight-chain C₂-C₆ alkynyl, hydroxy, Ci-C₆ alkoxy, CO₂H, CO₂(Ci-C₆ alkyl), CONH₂, CONH(Ci-C₆ alkyl), CON(C_I-C_O alkyl)₂, nitro, cyano, or halo.

28. The reagent according to claim 27, wherein A is Ci-C₃ alkyl, wherein A is optionally substituted with branched or straight-chain Ci-C₆ alkyl, branched or straight-chain C₂-C₆ alkenyl, branched or straight-chain C₂-C₆ alkynyl, hydroxy, Ci-C₆ alkoxy, CO₂H, CO₂(Ci-C₆ alkyl), CONH₂, CONH(Ci-C₆ alkyl), CON(Ci-Ce alkyl)₂, nitro, cyano, or halo.

29. The reagent according to claim 28, wherein A is Ci-C₃ alkyl.

30. The reagent according to claim 29, wherein A is CH₂.

31. The reagent according to claim 1, wherein X is O.

32. The reagent according to claim 1, wherein said reagent has the following formula:

,NO₂

33. A reagent comprising a compound of Formula (II):

wherein:

each Ri is independently selected from the group consisting of branched or straight-chain C₁-Ce alkyl, branched or straight-chain C2-C₆ alkenyl, and branched or straight-chain C2-C6 alkynyl,

R₂;

each R₂ is independently selected from the group consisting of branched or straight-chain Ci-Ce alkyl, branched or straight-chain C2-C6 alkenyl, branched or straight-chain C₂-C₆ alkynyl, hydroxy, Ci-C₆ alkoxy, -CO₂H, -CO₂(RtJl -CONH₂, -CONH(RJ] -CON(RIJi, carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl, nitro, cyano, and halo,

R₄ is selected from the group consisting of hydrogen, branched or straight - chain Ci-C₆ alkyl, branched or straight-chain C₂-C₆ alkenyl, branched or straight- chain C₂-C₆ alkynyl, wherein one or more hydrogens in the alkyl, alkenyl or alkynyl chain may be replaced by one or more halogens;

R₆ is C₆-Ci₄ aryl, C5-C14 heteroaryl or C5-C 14 heterocyclyl,

wherein R₆ is optionally substituted with one or more R7; and R₇ is selected from the group consisting of branched or straight-chain Ci-C₆ alkyl, branched or straight-chain C2-C6 alkenyl, branched or straight-chain C2-C6 alkynyl, -CF₃, -Rp-ORp-OH, -SH, -SRPprotected OH (e.g., acyloxy), -NO₂, -CN, - NH₂, -NHRp-N(R^, -NHCORg-NHCONH₂, -NHCONHRP-NHCON(R[B, - NRCOR0-NHCO₂H, -NHCO₂RP-CO₂RP-CO₂H, -CORp-CONH₂, -CONHRp- CON(RJl, -S(O)₂H, -S(O)₂RP-S(O)₃H, -S(O)₃Rp-S(O)₂NH₂, -S(O)H, -S(O)Rp- S(O)₂NHRp-S(O)₂N(RJi, -NHS(O)₂H, -NHS(O)₂RP-F, -Cl, -Br, and =O, where chemically feasible;

wherein RQs selected from the group consisting of hydrogen, branched or straight-chain Ci-C₆ alkyl, branched or straight-chain C2-C6 alkenyl, branched or straight-chain C₂-C₆ alkynyl, carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, or heteroaralkyl and each RQs optionally substituted with one or more halogen, nitro, cyano, amino, -NH-(unsubstituted aliphatic), -N-(unsubstituted aliphatic)₂, carboxy, carbamoyl, hydroxy, -O-(unsubstituted aliphatic), -SH, -S-(unsubstituted aliphatic), - CF₃, -S(O)2NH2θinsubstituted aliphatic, unsubstituted carbocyclyl, unsubstituted heterocyclyl, unsubstituted aryl, unsubstituted aralkyl, unsubstituted heteroaryl, or unsubstituted heteroaralkyl;

A is selected from the group consisting OfCi-C₆ alkyl, C2-C6 alkenyl, and C₂- C₆ alkynyl;

wherein A is optionally substituted with branched or straight-chain Ci-

C₆ alkyl, branched or straight-chain C₂-C₆ alkenyl, branched or straight-chain C₂-C₆ alkynyl, hydroxy, Ci-C₆ alkoxy, CO₂H, CO₂R₆, CO₂(Ci-C₆ alkyl), CONH₂,

CONH(Ci-C₆ alkyl), CON(Ci-C₆ alkyl)₂, nitro, cyano, or halo;

each X is independently O or S;

j is an integer from O to 10; and

n is an integer from 1 to 1,500.

34. The reagent according to claim 33, wherein n is an integer from 5 to 1,000.

35. The reagent according to claim 34, wherein n is an integer from 20 to 500.

36. The reagent according to claim 35, wherein n is an integer from 50 to 250.

37. The reagent according to claim 33, wherein j is an integer from 1 to 10.

38. The reagent according to claim 37, wherein j is an integer from 1 to 6.

39. The reagent according to claim 38, wherein j is an integer from 1 to 3.

40. The reagent according to claim 39, wherein j is 1.

41. The reagent according to claim 33, wherein each R₁ is independently a branched or straight-chain C_I-C_Θ alkyl, or both Ri are taken together to form a 5- or 6- membered cyclic acetal,

wherein each Ri is optionally substituted with one or more R₂.

42. The reagent according to claim 41, wherein each Ri is independently methyl, ethyl, isopropyl or both Ri are taken together to form a 1,3-dioxolane ring or a 1,3-dioxane ring.

43. The reagent according to claim 42, wherein each Ri is independently methyl or ethyl.

44. The reagent according to claim 33, wherein each R2 is independently selected from the group consisting of branched or straight-chain Ci-C₆ alkyl, branched or straight-chain C2-C₆ alkenyl, and branched or straight-chain C2-C₆ alkynyl,

45. The reagent according to claim 44, wherein each R2 is independently selected from the group consisting of branched or straight-chain Ci-C₆ alkyl, branched or straight-chain C2-C₆ alkenyl, and branched or straight-chain C₂-C₆ alkynyl.

46. The reagent according to claim 45, wherein R₆ is a Cs-C₆ aryl, a Cs-C₆ heteroaryl or a C₃-C₆ heterocyclyl,

wherein R₆ is optionally substituted with one or more R₇.

47. The reagent according to claim 46, wherein R^ is a Cs heterocyclyl or C₆ aryl, wherein said heterocyclyl contains one or more heteroatoms selected from the group consisting of N, N(R₄), O, S, S(O), and S(O)₂, and

wherein R^ is optionally substituted with one or more R₇.

48. The reagent according to claim 47, wherein R^ is succinimidyl, phthalimidyl, glutarimidyl, tetrahyrophthalimidyl, morbornene-2,3-dicarboximidyl, benzotriazolyl or phenyl optionally substituted with one or more nitro or one or more halo.

49. The reagent according to claim 48, wherein R^ is ortho-nitrophenyl or para-nitrophenyl.

50. The reagent according to claim 48, wherein R₆ is succinimidyl.

51. The reagent according to claim 33, wherein R₇ is selected from the group consisting of -CF₃, -RP-ORP-OH, -SH, -SRPprotected OH (e.g., acyloxy), - NO₂, -CN, -NH₂, -NHRp-N(RIJi, -NHCORf]-NHCONH₂, -NHCONHRp- NHCON(RJi, -NRCORP-NHCO₂H, -NHCO₂Rf]-CO₂Rg-CO₂H, -CORp-CONH₂, - CONHRp-CON(RJi, -S(O)₂H, -S(O)₂Rf]-S(O)₃H, -S(O)₃Rf]-S(O)₂NH₂, -S(O)H, - S(O)Rp-S(O)₂NHRf]-S(O)₂N(RJi, -NHS(O)₂H, -NHS(O)₂Rp-F, -Cl, -Br, and =O, where chemically feasible.

52. The reagent according to claim 51, wherein R₇ is selected from the group consisting of -CF₃, -NO₂, -CN, -CO₂Rp-CO₂H, -CORp-CONH₂, -CONHRp- CON(RJi, -S(O)₂H, -S(O)₂RP-S(O)₃H, -S(O)₃Rp-S(O)₂NH₂, -S(O)H, -S(O)Rp- S(O)₂NHRp-S(O)₂N(RJi, -NHS(O)₂H, -NHS(O)₂Rpand =O, where chemically feasible.

53. The reagent according to claim 52, wherein R₇ is selected from the group consisting Of-CO₂Rp-CORp-CON(RJi, -S(O)Rp-S(O)₂Rp-S(O)₃Rp-CF₃, nitro, cyano, and halo.

54. The reagent according to claim 53, wherein R₇ is selected from the group consisting Of-CF₃, nitro, cyano, halo, and =O.

55. The reagent according to claim 54, wherein R₇ is selected from the group consisting of =O, and ortho- or para-nitro.

56. The reagent according to claim 33, wherein RQs selected from the group consisting of hydrogen, branched or straight- chain C₁-C₆ alkyl, branched or straight-chain C₂-C₆ alkenyl, or branched or straight-chain C₂-C₆ alkynyl.

57. The reagent according to claim 33, wherein A is C\-Cβ alkyl, wherein A is optionally substituted with branched or straight-chain Ci-C₆ alkyl, branched or straight-chain C₂-C₆ alkenyl, branched or straight-chain C₂-C₆ alkynyl, hydroxy, C₁-C₆ alkoxy, CO₂H, CO₂(C₁-C₆ alkyl), CONH₂, CONH(C₁-C₆ alkyl), CON(C₁-C₆ alkyl)₂, nitro, cyano, or halo.

58. The reagent according to claim 57, wherein A is C₁-Ca alkyl, wherein A is optionally substituted with branched or straight-chain C₁-C₆ alkyl, branched or straight-chain C₂-C₆ alkenyl, branched or straight-chain C₂-C₆ alkynyl, hydroxy, C₁-C₆ alkoxy, CO₂H, CO₂(C₁-C₆ alkyl), CONH₂, CONH(C₁-C₆ alkyl), CON(C₁-C₆ alkyl)₂, nitro, cyano, or halo.

59. The reagent according to claim 58, wherein A is C₁-C₃ alkyl.

60. The reagent according to claim 59, wherein A is CH₂CH₂.

61. The reagent according to claim 33, wherein X is O.

62. The reagent according to claim 33, wherein said reagent has the following formula:

63. A

reagent comprising a compound of Formula (III):

XR₁ O

wherein:

R₁, R₂, R₄, R₆, R7, RpX, j, and n are defined as in Formula (II);

each B is any natural amino acid, or an unnatural alpha or beta amino acid, wherein each is independently optionally substituted with one or more -CO₂R_O or R₇;

wherein any -CO₂H present in B is optionally substituted with R₆ to yield a -CO₂RO moiety

k is an integer from 1 to 5.

64. The reagent according to claim 63, wherein each B is a natural amino acid optionally substituted with one or more -CO₂Re or R₇;

wherein any -CO₂H present in B is optionally substituted with R₆ to yield a -CO₂R₆ mo iety.

65. The reagent according to claim 63, wherein each B is an unnatural alpha or beta amino acid optionally substituted with one or more -CO₂R₆ or R₇;

wherein any -CO₂H present in B is optionally substituted with R₆ to yield a -CO₂R₆ moiety.

66. The reagent according to claim 63, wherein B is amino-malonic acid.

67. The reagent according to claim 63, wherein B is aspartic acid.

68. The reagent according to claim 63, wherein B is glutamic acid.

69. The reagent according to claim 63, wherein B is Glu-Glu.

70. The reagent according to claim 63, wherein k is 1, 2 or 3.

71. The reagent according to claim 70, wherein k is 3.

72. The reagent according to claim 70, wherein k is 1.

73. The reagent according to claim 63, wherein said reagent has the following formula:

Compound 10a.

74. A method of producing a compound of Formula (I), XR₁

comprising the

following steps according to Scheme I:

(a) converting carboxylic acid (A) to the corresponding carboxylic ester (B);

(b) converting the free hydroxyl group on (B) to yield carbonate (C);

XR₁

Ri₅ R2, R3, R₄, R5, A, X, j and n are defined as in Formula (I);

Rs is selected from the group consisting of branched or straight-chain Ci-C₆ alkyl, branched or straight-chain C2-C6 alkenyl, branched or straight-chain C2-C6 alkynyl, C₆-C_H aryl, C_O-C_I4 carbocycle, Cs-Ci₄ heteroaryl, and C5-Q4 heterocycle, wherein each alkyl, alkenyl and alkynyl is optionally substituted with one or more R2,

wherein each heteroaryl or heterocycle contains one or more heteroatoms selected from the group consisting of N, N(R₄), O, S, S(O), and S(O)₂, and

R9, taken with the -OC(O)O- it is attached to, is any synthetically useful carbonate.

75. The method according to claim 74, wherein Rg is selected from the group consisting of branched or straight-chain Ci-C₆ alkyl, Ce-Ci₄ aryl, or Cs-Q₄ heteroaryl,

wherein said alkyl is optionally substituted with one or more R₂,

wherein each aryl or heteroaryl is optionally substituted with one or more R₅.

76. The method according to claim 75, wherein Rg is branched or straight- chain Ci-Ce alkyl.

77. The method according to claim 74, wherein R₉ is phenyl, aryloxy, or succinimidyl, each of which is optionally substituted with R₅.

78. The method according to claim 77, wherein R₉ is phenyl, succinimidyl, or aryloxy, each of which is optionally substituted with one or more electron withdrawing groups selected from the group consisting of nitro, fluoro, chloro, cyano, sulpho, carboxy, amido, and trifluoromethyl.

79. The method according to claim 78, wherein R₉ is sulpho-succinimidyl, 1 -oxybenzotriazolyl, nitrophenyl, or halo-substituted phenyl.

80. The method according claim 79, wherein R₉ is phenyl substituted with one or more fluorines, chlorines or nitro groups.

81. The method according to claim 80, wherein R₉ is succinimidyl, para- nitrophenyl, ortho-nitrophenyl, fluorophenyl or chlorophenyl.

82. The method according to claim 81, wherein R₉ is succinimidyl or para- nitrophenyl.

83. The method according to claim 74, wherein the conversion of carboxylic acid (A) to the corresponding carboxylic ester (B) in step a), or the conversion of carboxylic acid (E) to the heterobifunctional PEG of Formula (I) in step e), comprises converting the carboxylic acid to an active ester by reacting said carboxylic acid with an activating agent and a base.

84. The method according to claim 83, wherein said activating agent is selected from the group consisting of disuccinimidyl carbonate, TSTU and 4- nitrophenyltrifluoroacetate.

85. The method according to claim 84, wherein said activating agent is 4- nitrophenyltrifluoroacetate.

86. The method according to claim 83, wherein said base is selected from the group consisting of triethylamine, diisopropylethylamine, pyridine and

dimethylaminopyridine.

87. The method according to claim 86, wherein said base is pyridine.

88. The method according to claim 74, wherein the conversion of (B) to (C) in step b) comprises activating the alcohol with an activating reagent.

89. The method according to claim 88, wherein said activating reagent is selected from the group consisting of bis(4-nitrophenyl) carbonate, 4- nitrophenylchloroformate and disuccinimidylcarbonate.

90. The method according to claim 88 or 89, wherein said activation of said alcohol further comprises using a base.

91. The method according to claim 90, wherein said base is selected from the group consisting of triethylamine, diisopropylethylamine, pyridine and dimethylaminopyridine.

92. A method of producing a compound of Formula (II),

XR₁

^ ^A ^ ^A

comprising th

e following steps according to Scheme II:

XR₁

R X T f ^{^}NH

(a) reacting (F) with and NH₂ACOOR₈ to generate a mixture of bifunctional PEGs inc

(b) hydrolyzing the m

ixture containing (G) to yield (H);

(c) subjecting the mixture of step b) to ion exchange chromatography to yield purified carboxylic acid (H);

(d) converting carboxylic acid (H) to the heterobifunctional PEG of Formula (II)

Scheme II

wherein:

Ri, R2, R4, Re, R₇, A, X, j, and n are defined as in Formula (II); and

R₈ is defined as in Scheme I.

93. The method according to claim 92, wherein (F) is first reacted with XR₁

R

amine and then with amine NH₂ACOORg in step a).

94. The method according to claim 92, wherein (F) is reacted with amines XR₁ and NH2ACOOR8 simultaneously in step a).

95. The method according to claim 92, wherein (G) is the major component of the product mixture produced in step a).

96. The method according to claim 92, wherein step b) is carried out under basic conditions.

97. The method according to claim 96, wherein said basic conditions comprise a base selected from the group consisting of sodium hydroxide, potassium hydroxide and lithium hydroxide.

98. The method according to claim 92, wherein the conversion of carboxylic acid (H) to the heterobifunctional PEG of Formula (II) in step d) comprises converting the carboxylic acid to an active ester by reacting said carboxylic acid with an activating agent and a base.

99. The method according to claim 98, wherein said activating agent is selected from the group consisting of disuccinimidyl carbonate, TSTU and 4- nitrophenyltrifluoroacetate.

100. The method according to claim 98, wherein said base is selected from the group consisting of triethylamine, diisopropylethylamine, pyridine and

dimethylaminopyridine.

101. The method according to claim 100, wherein said base is pyridine.

102. The method according to claim 92, wherein Compound 10 is prepared:

103. A m

p g p ( ),

XR₁

^ ^A ^V

comprising t

XR₁

R₁X M NH₂

(a) reacting (I) with and (B)k-Rs to generate a mixture of bifunctional PEGs including (J)

(b) hydrolyzing the m

ng (J) to yield (K);

(c) subjecting the mixture of step b) to ion exchange chromatography to yield purified arboxylic acid (K);

(d) converting carboxylic acid (K) to the heterobifunctional PEG of Formula (III) Scheme III

R₁, R₂, R₄, RO, R₇, B, X, j, k, and n are defined as in Formula (III); and

R8 is defined as in Scheme I.

104. The method according to claim 103, wherein (I) is first reacted with XR₁

R

amine and then with(B)k-Rs in step a).

105. The method according to claim 103, wherein (I) is reacted with amines XR₁

R X ^ ^{^}NH

and (B)k-Rs simultaneously in step a).

106. The method according to claim 103, wherein (J) is the major component of the product mixture produced in step a).

107. The method according to claim 103, wherein step b) is carried out under basic conditions.

108. The method according to claim 107, wherein any esters in (J) are hydro lyzed to carboxylic acids in step b).

109. The method according to claim 107, wherein said basic conditions comprise a base selected from the group consisting of sodium hydroxide, potassium hydroxide and lithium hydroxide.

110. The method according to claim 103, wherein the conversion of (K) to the heterobifunctional PEG of Formula (III) in step d) comprises converting all carboxylic acids to active esters by reacting said carboxylic acids with an activating agent and a base.

111. The method according to claim 101, wherein said activating agent is selected from the group consisting of disuccinimidyl carbonate, TSTU and 4- nitrophenyltrifluoroacetate.

112. The method according to claim 101, wherein said base is selected from the group consisting of triethylamine, diisopropylethylamine, pyridine and dimethylaminopyridine.

113. The method according to claim 103, wherein said base is pyridine.

114. The method according to claim 103, wherein Compound 10a is prepared: