MX2008004375A - Methods of preparing polymers having terminal amine groups using protected amine salts - Google Patents

Abstract

The present invention is directed to methods of preparing linear polymers such as polyalkylene oxides containing a terminal amine in high purity. One preferred method includes reacting a polyalkylene oxide such as polyethylene glycol containing a terminal tosylate with a protected amine salt to form a polymer containing a terminal protected amine and thereafter deprotecting the polymer containing the terminal protected amine to form the polymer having a terminal amine. The resultant polymer-amines are of sufficient purity so that expensive and time consuming purification steps required for pharmaceutical grade polymers are avoided.

Description

METHODS FOR PREPARING POLYMERS THAT HAVE AMINA TERMINAL GROUPS USING AMINA SALTS PROTECTED Field of the Invention The present invention relates to methods for preparing activated polymers such as polyalkylene oxides. Particularly, the invention relates to methods for preparing linear polymers containing a terminal amine at a high purity. Background of the Invention The conjugation of water-soluble polyalkylene oxides to therapeutic portions such as proteins and polypeptides is known. See, for example, U.S. Patent No. 4,179,337, the disclosure of which is incorporated herein by reference. The '337 patent discloses that physiologically active polypeptides modified with PEG, circulate for prolonged periods in vivo, and that they have reduced immunogenicity and antigenicity. To conjugate the polyalkylene oxides, the terminal hydroxide groups of the polymer must first be converted into reactive functional groups. This process is often referred to as "activation" and the product is called "activated polyalkylene oxide". Other polymers are similarly activated.

Polymers terminated with amine such as PEG-NH2 are known. See Zalipsqui et al. EUR. Polym. J. Vol.19 No.12., Pp1177-1183. They can be used in their "original state" for direct conjugation to COOH groups found in some biologically active compounds. More frequently, PEG-NH2 (or PEG-amine) is used as an intermediate that additionally functionalizes when other polymeric delivery systems are desired. For example, certain systems of the polymer-based drug delivery platform containing benzyl elimination systems, trimethyl blocking systems, etc., may include PEG-NH2 as an essential intermediate in the synthesis process. See Greenwald et al. J. Med. Chem. Vol. 42, No. 18, 3657-3667; Greenwald et al. J. Med. Chem. Vol. 47, No. 3, 726-734; Greenwald et al. J. Med. Chem. Vol. 43, No. 3, 475-487. The content of each of the above citations is incorporated herein by reference. PEG-amines are also useful for conjugation (via reductive amination) with biologically active small molecules and polypeptides having available aldehyde groups. See also the 2005-2006 Nektar Advanced PEGylation catalog, page 24, the content of which is incorporated herein by reference. In the past, it was generally known that PEG-amines could be prepared by preparing PEG-halide, mesylate or tosylate from PEG-OH and then by carrying out a nucleophilic displacement reaction with aqueous ammonia (Hoffmann reaction), sodium azide or potassium phthalimide (Gabriel's reagent). The reaction of PEG-halide with ammonia directly forms the PEG-amine. More significantly, a major disadvantage is that a significant percentage of PEG-halide is hydrolyzed to form PEG-OH during the treatment with concentrated aqueous ammonia. This is a particular concern when forming the highest molecular weight PEG amines. The higher the molecular weight of PEG, the more PEG-OH is formed. For example, in the case of PEG5 ooo the amount is about 5% and with higher molecular weight PEG such as PEG 0, or the amount can be up to 20%. Therefore, the purity of the desired final product can be considerably reduced. Even when PEG-azide is used as the intermediate to make the PRG-amine, certain problems have been observed when using metal-catalyzed hydrogenation. In addition, the reaction with potassium phthalimide provides a substantially protected amine that is deprotected with hydrazine in ethanol under reflux. This is also associated with the disadvantages. The severe conditions required for the elimination of the phthaloyl group and the need for intensive purification of the final product add significantly to the desired product cost. Due to the above, it would be desirable to provide the improved methods for preparing PEG-amines and related polymers having terminal amines that address the problems and disadvantages of the prior art. The present invention addresses this need. Brief Description of the Invention In a preferred aspect of the invention, improved methods for preparing polymers having terminal amines are provided. The methods include reacting a substantially non-antigenic polymer of formula (I) (I) R3-R2-R? wherein R ^ is a terminal group of reactive polymer such as a leaving group; R2 is a substantially non-antigenic polymer; and R3 is a terminal group or an R ^ with a protected amine salt to form a polymer containing a protected terminal amine; and then reacting the polymer containing the protected terminal amine resulting from the above with an acid, to remove the protecting group and form the polymer having a terminal amine. Examples of the preferred reactive polymer terminal groups include the leaving groups such as tosylate, mesylate, brosylate, tresylate, nosylate, Br, Cl, etc. The reaction of the polymer of formula (I) with the protected amine salt is preferably carried out in a solvent such as dimethylformamide and the reactants are reacted under reaction conditions and for a period that is sufficient to substantially complete each of the reaction steps that ultimately cause the formation of the terminal amine in the polymer. In the most preferred aspects of the invention, the polymer that is converted to the amine derivative is a PEG-tosylate and the tosylate group may be in at least one or more of the PEG terminals. In an alternative aspect of the invention, the preferred protected amine salt is the potassium salt of methyl tert-butyl imidodicarbonate (KNMeBoc) or the potassium salt of di-tert-butyl imidodicarbonate (KNBOC2). The purity of the polymer containing the terminal amine formed by the process described herein is greater than about 95%, preferably greater than 98% and more preferably greater than 99%. One of the main advantages of the present invention is that the resulting terminal amine-containing polymers such as the polyalkylene oxide derivatives thereof are prepared in high purity. Thus, the product contaminants, i.e., the starting materials, such as mPEG-OH are not in appreciable amounts, i.e., they are in amounts of less than about 5%, more preferably less than about 2% and very preferably less than about 1%. When the preferred PEG-amines are formed more economically high purity, the expert can elaborate the final products that incorporate the PEG-amine more efficiently and at a lower cost. The reaction to make the PEG-amine can be forced to complete and the excess small molecule reagent can be removed by re-crystallization. The efficiencies result, in part, because the separation of the final polymer with the desired amine from the starting alcohol or the reactive intermediate (eg, tosylate) is not required. In addition, routine ion exchange or column or CLAR techniques are not required to provide the desired PEG-amine. Thus, the present invention provides the highly pure PEG-amine without the expensive column purification. Another advantage is the fact that the amine made from the processes described herein will not change the structure of PEG at all. Therefore it will be compatible with all current and future applications of PEG-amines. Detailed Description of the Invention The methods of the invention are generally related to the formation of polymers containing at least one terminal amine therein. In most aspects of the invention, polymers that can be modified using the processes described herein are substantially non-antigenic polymers. Within this polymer genre, polyalkylene oxides are preferred and polyethylene glycols (PEG) are most preferred. With the purpose to facilitate description rather than limiting it, the process is occasionally described using PEG as the prototypical polymer. It should be understood, however, that the process is applicable to a wide variety of polymers that may be linear, substantially linear, branched, etc. One of the only requirements is that the polymer contains the means to covalently attach the terminal group of the desired reactive polymer thereto and that can withstand the processing required to transform the tosyl or other intermediate to the amine under the conditions described herein. According to the above, a preferred aspect of the invention for preparing a polymer having a terminal amine, includes: a) reacting a substantially non-antigenic polymer of formula (I) wherein RT is a terminal group of reactive polymer; R2 is a substantially non-antigenic polymer; and R3 is a terminal group or R- ,; with a protected amine salt to form a polymer containing a protected terminal amine; and b) reacting the polymer containing the protected terminal amine resulting from the above with an acid, to remove the protecting group and form the polymer having an terminal amine. For the purposes of the present invention, it will be understood that the term "terminal group of the reactive polymer" when used in the context with R (, means a leaving group such as those known in the relevant art.) A non-limiting list of groups Suitable projections include tosylate, mesylate, brosylate, tresylate, nosylate, Br, Cl, etc. As indicated above, in one embodiment, R3 may be a terminal group For the purposes of the present invention, terminal groups shall be understood to be they mean a group that is in the polymer terminal, in some aspects, they can be selected from either CO2H, alkyls of 1 to 6 carbon atoms (CH3 is preferred), OH, etc. or other terminal groups as understood by the experts R2 is also preferably a polymer that is soluble in water at room temperature such as a polyalkylene oxide (PAO) and preferably a poly ethylene glycol such as mPEG or bis-activated PEG. A non-limiting list of such polymers therefore includes the polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycol, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, with the proviso that the water solubility of the block copolymers is maintained. For purposes of illustration and not limitation, the portion of the polyethylene glycol residue (PEG) of R2 can be selected from: -CH2CH2-O- (CH2CH2O) x-CH2CH2- and -S-CH2CH2-O- (CH2CH2O) x -CH2CH2S- where: x is the degree of polymerization, ie from about 10 to about 2,300. In the alternative aspects of the invention, when the bis-activated polymers are desired, R3 is the same as Ri, and the resulting reactant is used in the preparation of the finished polymer compounds with bis-amine. Such bis-activated polymers can be of formula (Ia): (the) R ^ CHzCH-O-tCHzCHzOh-CHzCHa-R! wherein R is preferably tosylate and x is the same as above. The degree of polymerization for the polymer represents the number of repeating units in the polymer chain and is dependent on the molecular weight of the polymer. Although the substantially non-antigenic polymers, PAOs and PEGs, can vary substantially in weight average molecular weight, preferably, R 2 has a weight average molecular weight of from about 200 to about 100,000 DA, in most aspects of the invention. More preferably, the substantially non-antigenic polymer has a weight average molecular weight of about 2,000 to about 48,000 Daltons. R2 may also be a "star type PEG" or PEG with multiple arms such as those described in the 2001 catalog "Polyethylene Glycol and Derivatives for Biomedical Application" of Shearwater Corporation, the disclosure of which is incorporated herein by reference.

In yet another preferred embodiment, R 2 is part of a branched polymer corresponding to the polymers of the invention. Specifically, R2 can be of formula: wherein: n is an integer from about 10 to about 340, to preferably provide polymers having a total molecular weight of about 12,000 to about 40,000; and at least 1, but up to 3, of the terminal portion a of the residue is terminated with a methyl or other lower alkyl and the remaining terminal groups are the R3 groups. See also the Nektar catalog already mentioned, page 26"4-arm PEG". Such compounds before the terminal amino include preferably: where Ri is wherein R7 is preferably methyl and all other variables are as previously defined herein. A specific polymer capable of undergoing the process of amination described herein is: Other aspects of this modality would of course be one or two of the terminal methyl groups substituted by the tosyl groups to correspond to the above R-i.

It is also contemplated within the scope of the invention, the formation of a terminal amine in other compounds based on PEG, including the branched polymer residues described in commonly assigned US Patents Nos. ,605,976, 5,643,575, 5,919,455 and 6,113,906, the description of each is incorporated herein by reference. A representative list of some specific compounds includes: (1a) or m.pEG_0_C II_ HN_ (cH ^ m.PEG-or-c-r -clH "(CH2CH2?) ~ CH2CH2" ~ "Rl II H or (1b) ( (2b) (3a) R3 - (CH2CH20) x L, L2 i-3 (CH2CH20) z CH2CH2 RT and (3b) wherein R7 is preferably methyl; z is an integer from 1 to about 120; Li and L3 are independently selected bi-functional linking groups such as one of the following non-limiting compounds: -NHíORuRn ?, { ) - (CR13R12) tOC (0 - in donae R9.13 are independently selected from the same group as defined previously R, and preferably H or CH3; R6 is selected from the same group as that which defines R, NO2, haloalkyl of 1 to 6 carbon atoms and halogen; and q, t and y are each the positive integers independently selected such as from 1 to about 12; and L2 is a branched linking group such as a diaminoalkyl residue or lysine. See, for example, the aforementioned US Patent No. 6,113,906. In a further embodiment, and as an alternative to the PAO-based polymers, R2 is optionally selected from one or more effectively non-antigenic materials such as dextran, polyvinyl alcohols, carbohydrate-based polymers, hydroxypropylmethacrylamide, polyalkylene oxides, and / or copolymers thereof. See also Commonly Assigned US Patent No. 6,153,655, the content of which is incorporated herein by reference. It will be understood by experts that the same type of activation is used as described herein in terms of such PAOs as PEG. Those skilled in the art will further understand that the above list is merely illustrative and that all polymeric materials having the qualities described herein are contemplated. It will also be understood that the water-soluble polymers described herein can be functionalized to bind to the R3 groups, for example tosylate, mesylate, if required without undue experimentation prior to amination. The conversion or activation of the polymer (for example PEG) terminal OH in a tosylate, etc. It has been reported in the art. See, for example, U.S. Patent No. 5,206,344 and U.S. Patent Publication No. 2003/0149307, the contents of which are incorporated herein by reference. In accordance with the methods of the present invention, the formation of the polymer containing a protected terminal amine requires reacting a substantially non-antigenic polymer of formula (i) containing one or more Ri groups for example the terminal groups of the reactive polymer such as tosylate, mesylate, brosylate, tresylate, nosylate, Br or Cl, with a protected amine salt. The reaction is carried out in a convenient solvent such as and without limitation dimethylformamide, tetrahydrofuran, dimethylacetamide or similar reagents and mixtures thereof. The preferred solvent is dimethylformamide. As will be appreciated by the experts, there are a number of other suitable solvents that can be used in the process of present invention. Suitable solvents include those which are polar solvents such as methanol, ethanol, butanol, isopropanol, dioxane, etc. This step can be carried out at about room temperature although the temperatures can range from about 0 ° C to reflux or boiling point of the solvent. A large variety of protected amine salts can be used in the processes of the invention. Generally, the protected amine salts may correspond to the formula: MNPiR4 wherein M is hydrogen or a metal chosen from lithium, sodium and potassium; PT is included but not limited to the following list C (O) H, C (O) OMe, C (O) OBzl (Z), C (O) OtBu (Boc), C (O) OCH2CCI3 (Troc), P (O) (OEt) 2, CPh3 (trityl); and R 4 is Pi or for example, alkyls of 1 to 6 carbon atoms, branched alkyls of 3 to 12 carbon atoms, cycloalkyls of 3 to 8 carbon atoms, substituted alkyls of 1 to 6 carbon atoms, substituted cycloalkyls of 3 to 8 carbon atoms, aryls, substituted aryls, aralkyls, heteroalkyls of 1 to 6 carbon atoms, substituted heteroalkyls of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms, phenoxy and heteroalkoxy of 1 to 6 carbon atoms carbon, etc. Within the previous group of possible reagents, a non-limiting list of some preferred convenient reagents includes no limitation, potassium salt of di-tert-butyl imidodicarbonate (KNBoC2), lithium salt of di-tert-butyl imidodicarbonate (LiNBoc2), sodium salt of di-tert-butyl imidodicarbonate, etc. In a preferred aspect of the invention, the protected amine salt is the potassium salt of di-tert-butyl imidodicarbonate (KNBoc2). In the alternative aspects of the aspect of the invention described above, the protected amine salt can be selected from potassium, sodium and lithium salts of NR4BoC, wherein R4 is as shown above, for example, alkyls of 1 to 6 carbon atoms. carbon, etc. and Boc is t-butylcarbonate. Within, of many aspects of the invention, the preferred protected amine salt is the potassium salt of methyl-r-butyl imidodicarbonate (KNMeBoc). After the polymer containing the protected amine is formed, the protecting group, (for example Boc) is removed via the hydrolyzation as mentioned above for the desired high purity PEG-amine derivatives. Protection groups other than Boc are eliminated using other techniques recognized in the art. The high purity PEG-amine can then be used in any manner recognized in the art. For example, and without limitation, it can be used for direct conjugation with the CO2H groups or other convenient reactive groups found in biologically active targets of interest using the techniques well known to the experts. Alternatively, PEG-amine can be used as a highly pure intermediate in more complex polymer binding systems such as the aforementioned benzyl elimination platforms (RNL) or even as part of PEG-liposome systems. For purposes of illustration, RNL systems can be made by reacting the PEG-amine with a convenient protected benzyl alcohol followed by deprotection and activation using techniques known to those skilled in the art. See also the last example later. After the polymer containing the protected terminal amine is formed, the polymer is deprotected, that is, the protecting group is removed, to form the final polymer with the desired amine. The deprotection can be performed using an acid such as and without limitation, HCl solution in Et 2 O (ether) or dioxane, acetic acid, dichloroacetic acid, formic acid, and trifluoroacetic acid in dichloromethane (or dichloroethane) (from 15% to 35% ), or another compatible solvent to dissolve the acid and allow deprotection under an organic environment. In many aspects of the invention, 20% of trifluoroacetic acid in dichloromethane is preferred. The methods of the present invention are preferably carried out using at least about an equimolar amount of the reagents. Preferably, the protected amine salt is present in a molar excess with respect to the compound of formula (I). Preferably, the protected amine salt is present in at least about a molar excess of 3-5 times that of mPEG which is from about 5 to about 1. When using delta-PEG (bis-PEG or activated in each terminal), the molar excess is about twice as high, as for example a molar excess of about 6-10 times. Similar ratios are used by the amine group that will be added if the branched polymers are used. It will be understood that when the terminally branched polymers are used, the molar excess of the protected amine salt used preferably is at least equal to the number of reactive terminal polymer portions (eg, tosylate, etc.) found in the polymer. In many aspects of the invention, a polyalkylene oxide (PAO) for example non-terminal PEG-OH (di-PEG OH) or mPEG is converted to a compound of formula (lia) or (llb): In many aspects of the invention , a polyalkylene oxide (PAO) eg uncovered PEG-OH (di-PEG OH) or mPEG is converted to a compound of the formula (Na) or (release): (lia) (llb) wherein R2 is a PAO such as PEG or mPEG; X is O, S, or NHR8, wherein R8 is, hydrogen, alkyls of 1 to 6 carbon atoms, branched alkyls of 3 to 12 carbon atoms, cycloalkyls of 3 to 8 carbon atoms, substituted alkyls of 1 to 6 carbon atoms, substituted cycloalkyls of 3 to 8 carbon atoms, aryls, substituted aryls, aralkyls, heteroalkyls of 1 to 6 carbon atoms, substituted heteroalkyls of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms, phenoxy and heteroalkoxy of 1 to 6 carbon atoms, etc; and R7 is for example a methyl, halogen, nitro, fluoromethyl, difluoromethyl, trifluoromethyl, substituted carboxyl, and a multi-halogen substituted benzenesulfonyl. CH3 is preferred. After the polymer containing the protected amine is formed, the protecting group, (for example Boc) is removed via hydrolyzation as mentioned above for the desired high purity PEG-amine derivatives.

The high purity PEG-amine can then be used in any manner recognized in the art. For example, it can be used without limitation EXAMPLES The following examples serve to provide a further understanding of the invention but in no way mean that they limit the effective scope of the invention.

Example 1 40KDabis-PEG amine TsCl, DCM BnEt3NCI (cat) Bo &jNK 20% TFA 40K? PEG-OH * - 40K? PEG-OTs »- 40K? PEG-NBor ^ * - ^? PEG-NH, TFA % NaOH / H2O D F, 45 ° C DC 1 2 3 4 a) PEG-tosylate (PEG-OTs). To a solution of 40KDa? PEG-OH (100 g, 2.5 mmol, 1.eq.) in 800 ml of DCM was added 400 ml of a solution of 30% aqueous NaOH and BnEt3NCI (228 mg, 1.0 mmol, 0.2 eq. ). Then, to the vigorously stirred mixture a solution of p-toluenesulfonyl chloride (2.86 g, 15 mmol, 3 eq.) In 500 ml of DCM was added dropwise via an addition funnel for 2 hours, and the reaction mixture it was stirred at room temperature overnight. After the addition of 500 ml of DCM and 200 ml of a saturated solution of NaCl, the organic phase was separated and washed twice with 200 ml of a saturated solution of NaCl. The organic phase was dried over anhydrous MgSO 4, filtered and evaporated under vacuum to give a solid which was dissolved in a minimum amount of DCM and precipitated with ethyl ether. Filtration provided 97 g of 40KDa? PEG-OTs (96% yield). 13C NMR: 21.57, 68.47, 69.07, 70.21-71.75 (PEG), 127.68, 129.55, 132.69, 144.44. b) PEG-NBoc2. To a solution of 40KDa? PEG-OTs (96 g, 2.38 mmol, 1 eq.) In 500 ml anhydrous DMF was added BoC2NK (3.65 g, 14.28 mmol, 3 eq.). The reaction mixture was heated at 45 ° C overnight. After cooling to room temperature, 1.2 l of ethyl ether was added. The resulting solid was filtered and washed with enough ether. The crude solid was dissolved in 1.2 L of DCM and washed twice with 200 mL of a saturated solution of NaCl. The organic layer was dried over MgSO 4, filtered and evaporated under vacuum. The resulting solid was recrystallized with DCM / ethyl ether to give 90 g of 0KDa? PEG-NBoc2 (94% yield). 13 C NMR (ppm): 27.93, 45.01, 69.05, 69.68-70.34 (PEG), 81.94, 152.20. c) PEG-amine salt (PEG-NH2TFA). To a 40KDa solution? PEG-NBoc2 (90 g, 2.23 mmol) in 900 ml DCM was added 225 ml of TFA. The reaction mixture was stirred at room temperature for 4 hours and then evaporated under vacuum. The resulting solid was dissolved in 300 ml of DCM and precipitated by the addition of 2 I of ethyl ether. After filtration, the solid was recrystallized again with DCM / ethyl ether to give 88 g of 40KDa? PEG-NH2-TFA (98% yield). 13 C NMR (ppm): 39.87, 66.77, 69.71-70.72 (PEG).

Example 2 20KDabis-PEG-amine TsCI, DCM BnEt3NCI (cat) Bocj K 20% TFA 20K? PEG-OH »- 20K? PEG-OTs * - ^? PEG-NBoCa» ~ "«? PEG-NH, TFA 30% NaOH / H2O DMF, 45" CDM 5 6 7 8 The corresponding 0KDa? PEG-amine is made from 20KDa? PEG-OTs prepared in the same manner except that PEG 20KDa is used in place of 40KDa PEG. Compound 6 is made under the same conditions as 2 in Example 1. The structure of 6 is confirmed by NMR. Compound 7 is made under the same conditions as 3 in Example 1. The structure of 7 is confirmed by NMR. Compound 8 is made under the same conditions as 4 in Example 1. The structure of 8 is confirmed by NMR. Example 3 40KDabis-PEG-N-methylamine TsCI. DCM BpEt3NCI (cat) BacNM? K 20% TFA 40K? PEG-OH »- ^? PEG-OTs» ~ ^? PEG-NMeBoc * - ««? PEG-NHM? TFA % Na? H / H2? DMF, 45 ° C DCM 1 9 10 11 a) PEG-tosylate (PEG-OTs). The procedure of the example 1 is repeated. b) PEG-NMeBoc. Compound 10 is made under the same conditions as in Example 1 except that an equivalent amount of BocMeNK is used in place of Boc2NK. The structure of 10 is confirmed by NMR. c) PEG-N-methylamine (PEG-NHMe). Compound 11 is made under the same conditions as in Example 1 except that an equal amount of compound 10 is used in place of compound 3. The structure is confirmed by NMR. Example 4 30KDamPEG-amine TsCI, DCM BnEtjNCI (cat) BoCjNK 20% TFA 8ViPEG-OH > - 30KmPEG-OTs »- 8 ^ mPEG-NBoC;,» * »< mPEG-NH., TFA 30% NaOH / H2O D F, 45 ° C DCM 12 13 14 15 a) 30KDamPEG-tosylate (30KDamPEG-OTs). To a solution of 30KDamPEG-OH (85 g, 2.83 mmol, 1 eq.) In 800 ml of DCM was added 400 ml of a solution of 30% aqueous NaOH and BnEt3NCI (257 mg, 1.13 mmol, 0.4 eq.). Then, to the vigorously stirred mixture a solution of p-toluenesulfonyl chloride (1.62 g, 8.49 mmol, 3 eq.) In 600 ml of DCM was added dropwise via an addition funnel for 2 hours, and the reaction mixture it was stirred at room temperature overnight. After the addition of 800 ml of DCM and 200 ml of a saturated solution of NaCl, the organic phase was separated and washed twice with 200 ml of a saturated solution of NaCl. The organic phase was dried over anhydrous MgSO, filtered and evaporated under vacuum to give a solid which was dissolved in a minimum amount of DCM and precipitated with ethyl ether. Filtration provided 80 g of 30KDamPEG-OTs (94% yield). 13C NMR: 21.57, 58.83, 68.41, 69.07, 69-69-72.95 (PEG), 127.62, 129.52, 132.68, 144. 40. b) 30KDamPEG-NBoc2. To a solution of 30KDamPEG-OTs (78 g, 2.59 mmol, 1 eq.) In 800 ml anhydrous DMF was added Boc2NK (1.99 g, 7.8 mmol, 3 eq.). The reaction mixture was heated at 45 ° C overnight. After cooling to room temperature, 3 I of ethyl ether was added. The resulting solid was filtered and washed with enough ether. The crude solid was dissolved in 1.2 ml of DCM and washed twice with 200 ml of a saturated solution of NaCl. The organic layer was dried over MgSO, filtered and evaporated under vacuum. The resulting solid was recrystallized with DCM / ethyl ether to give 67 g of 30KDamPEG-NBoc2 (86% yield). 13 C NMR (ppm): 27.93, 45.01, 58.83, 69.04, 69.66-71.70 (PEG), 81.94, 152.20. c) 30KDamPEG-amine salt (30KDamPEG-NH2 * TFA). To a solution of 30KDamPEG-NBoc2 (67 g, 2.22 mmol) in 670 ml of DCM was added 335 ml of TFA. The reaction mixture was stirred at room temperature for 4 hours and then evaporated under vacuum. The resulting solid was dissolved in 300 ml of DCM and precipitated by the addition of 2 l of ethyl ether. After filtration, the solid was recrystallized again with DCM / ethyl ether to give 64 g of 30KDamPEG-NH2TFA (98% yield). 13 C NMR (ppm): 39.81, 58.83, 66.90, 69.63-71.70 (PEG). Examples 5-8 The process of Examples 1-4 is repeated except that the corresponding PEG-CI derivative is used in place of the tosylate in equimolar amounts. Example 9 Link 930KDamPEG RNL In this example, 30KDamPEG-NH2 of Example 4 is converted to the activated PEG link according to the following reaction scheme. 22 23 24 25 a) 30KDamPEG RNL9 OTBDMS 23. To a solution of alcohol, 22 (238 mg, 1 mmol, 6 eq.) In anhydrous CH3CI was added DSC (235 mg, 0.92 mmol, and 5.5 eq.) And pyridine ( 88 μl, 1.08 mmol, 6.5 eq.). The resulting suspension was heated to reflux overnight, cooled to room temperature and added to a solution of 30KDamPEG-NH2 »TFA (hereinafter 21) (5 g, 0.17 mmol, 1 eq.) And pyridine (17 μl, 0.21 mmol, 1.25 eq.) in 25 ml of anhydrous CH3CI. After being stirred at room temperature for 3 days, the solvent was evaporated under vacuum. The resulting solid was dissolved in a minimum amount of dichloromethane and then precipitated by the addition of ether, filtered and recrystallized with CH3CN / IPA to give 4.85g (94% yield). GPC: 98.39%. 13C NMR (75.4 MHz, CDCI3) d 154.47, 149.59, 137. 90, 126.52, 121.02, 69.09-71.65 (PEG), 64.24, 58.83, 40.83, 25.84, 18.27, 5.28. b) 30KDamPEG RNL9OH 24. To a solution of 23 (4.85 g, 0.16 mmol) in 20 ml CH3CN and 10 ml of water was added 50 ml of glacial acetic acid. The reaction mixture was stirred at room temperature overnight and then evaporated under vacuum. The residue was dissolved in 75 ml of CH2Cl2. The organic phase was washed twice with 15 ml of water, dried over MgSO 4, filtered and evaporated under vacuum. The resulting solid was dissolved in a minimal amount of CH2Cl2 and then, precipitated by the addition of ether to give 4.49 g (94% yield). CPG: 98.35% .. 13C NMR (75.4 MHz, CDCI3) d 154.36, 149.90, 138.18, 127.37, 121.15, 69.42-71.69 (PEG), 63.93, 58.80, 40.83. 30KDa c) mPEG RNL9NHS 25. To a solution of 24 (4.49 g, 0. 15 mmol, 1 eq.) In 50 ml anhydrous CH 2 Cl 2 and 5 ml anhydrous DMF was added DSC (305 mg, 1.19 mmol, 8 eq.). The mixture was cooled to 0 ° C and then pyridine (87 μl, 1.07 mmol, 7.2 eq.) Was added. The reaction mixture was stirred at room temperature overnight and then evaporated under vacuum. The resulting solid was dissolved in a minimum amount of CH2Cl2 and then precipitated by addition of ether, filtered and recrystallized with CH3CN / IPA to give 4.26 g (94% yield). GPC: 97.04%. 13 C NMR (75.4 MHz, CDCl 3) d 168.33, 154.01, 151.51, 151.22, 129.80, 129.53, 121.68, 69.88-73.08 (PEG), 58.83, 40.89, 25.32.

The final product can be used for the conjugation of any number of biologically active polypeptides, enzymes, proteins, small molecules, etc. having an amine or hydroxyl available therein for conjugation. Methods for such conjugation reactions have been described, for example, in commonly assigned U.S. Patent No. 6,180,095, the content of which is incorporated herein by reference, or in the aforementioned reference Greenwald et al. J. Chem. Vol. 42, No. 18, 3657-3667.

Claims (22)

1. A method for preparing a polymer having a terminal amine, comprising: a) reacting a substantially non-antig polymer of formula (I) wherein RT is a terminal group of reactive polymer; R2 is a substantially non-antig polymer; and R3 is a terminal group or R-i; with a protected amine salt to form a polymer containing a protected terminal amine; and b) reacting the polymer containing the protected terminal amine resulting from step a) with an acid, to remove the protecting group and form the polymer having a terminal amine.

2. The method of claim 1, wherein R-, is a leaving group selected from the group consisting of tosylate, mesylate, brosylate, tresylate, nosylate, Br and Cl.

The method of claim 1, wherein the Reaction step a) is carried out in a solvent selected from the group consisting of dimethylformamide, tetrahydrofuran, dimethylacetamide and mixtures thereof.

4. The method of claim 2, wherein the group Outgoing is tosylate.

The method of claim 1, wherein the protected amine salt is selected from the group consisting of the potassium salt of di-tert-butyl imidodicarbonate (KNBoc2), lithium salt of di-tert-butyl imidodicarbonate (LiNBoc2 ), and di-tert-butyl imidodicarbonate sodium salt.

6. The method of claim 5, wherein the protected amine salt is the potassium salt of di-tert-butyl imidodicarbonate (KNBoc2).

The method of claim 1, wherein the protected amine salt is selected from the group consisting of potassium, sodium and lithium salts of NR Boc, wherein R4 is selected from the group consisting of hydrogen, alkyl of 1 to 6 carbon atoms, branched alkyls of 3 to 12 carbon atoms, cycloalkyls of 3 to 8 carbon atoms, substituted alkyls of 1 to 6 carbon atoms, substituted cycloalkyls of 3 to 8 carbon atoms, aryls, substituted aryls, aralkyls , heteroalkyls of 1 to 6 carbon atoms, substituted heteroalkyls of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms, phenoxy and heteroalkoxy of 1 to 6 carbon atoms; and Boc is f-butylcarbonate.

The method of claim 7, wherein the protected amine salt is potassium salt of methyl-tert-butyl imidocarbonate (KNMeBoc).

9. The method of claim 1, wherein the acid used to remove the protecting group is selected from the group consisting of hydrochloric acid, acetic acid, dichloroacetic acid, formic acid, and trifluoroacetic acid.

The method of claim 9, wherein the acid is trifluoroacetic acid.

11. The method of claim 1, wherein the terminal group R3 is CH3.

The method of claim 1, wherein R3 is RL 13.

The method of claim 1, wherein R2 is a polyalkylene oxide.

The method of claim 13, wherein the polyalkylene oxide is selected from the group consisting of polyethylene glycol and polypropylene glycol.

The method of claim 14, wherein the polyalkylene oxide is a polyethylene glycol of the formula: -O- (CH 2 CH 2 O) x- wherein x is an integer from about 10 to about 2,300.

The method of claim 1, wherein the substantially non-antig polymer has a weight average molecular weight of about 200 to about 100,000 Daltons.

The method of claim 16, wherein the substantially non-antig polymer has a weight average molecular weight of from about 2,000 to about 48,000 Daltons.

18. The method of claim 1, wherein the compound of formula (I) is: (lia) O (llb) wherein R 2 is a polyalkylene oxide: X is O, S, or NHR 8, wherein R 8 is selected from the group consisting of hydrogen, alkyls of 1 to 6 carbon atoms, branched alkyls of 3 to 12 carbon atoms, cycloalkyls from 3 to 8 carbon atoms, substituted alkyls of 1 to 6 carbon atoms, substituted cycloalkyls of 3 to 8 carbon atoms, aryls, substituted aryls, aralkyls, heteroalkyls of 1 to 6 carbon atoms, substituted heteroalkyls of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms, phenoxy and heteroalkoxy of 1 to 6 carbon atoms; and R7 is methyl, halogen, nitro, fluoromethyl, difluoromethyl, trifluoromethyl, substituted carboxyl, or benzenesulfonyl substituted with multi-halogen.

19. The method of claim 18, wherein R2 is a polyethylene glycol, X is O and R7 is methyl.

The method of claim 1, wherein the purity of the polymer containing the terminal amine formed by the process is greater than about 95%.

The method of claim 20, wherein the purity of the polymer containing the terminal amine formed by the process, said is greater than 98%.

22. The method of claim 21, wherein the purity of the polymer containing the terminal amine formed by the process is greater than 99%.