the methods of the invention relate generally to the formation of polymers containing at least one terminal amine thereon . in most aspects of the invention , the polymers which can be modified using the processes described herein are substantially non - antigenic polymers . within this genus of polymers , polyalkylene oxides are preferred and polyethylene glycols ( peg ) are most preferred . for purposes of ease of description rather than limitation , the process is sometimes described using peg as the prototypical polymer . it should be understood , however , that the process is applicable to a wide variety of polymers which can be linear , substantially linear , branched , etc . one of the only requirements is that the polymer contains the means for covalently attaching an azide thereon and can withstand the processing required to transform the azide to the amine under the conditions described herein . in accordance with the foregoing , one preferred aspect of the invention for preparing a polymer having a terminal amine , includes : r 1 is a capping group or n 3 ; and r 2 is a substantially non - antigenic polymer ; with a phosphine - based reducing agent or an alkali metal borohydride reducing agent . as stated above , r 1 is a capping group . for purposes of the present invention , capping groups shall be understood to mean a group which is found on the terminal of the polymer . in some aspects , it can be selected from any of co 2 h , c 1 - 6 alkyls ( ch 3 preferred ), oh , etc . or other terminal groups as they are understood by those of ordinary skill . r 2 is also preferably a polymer that is water soluble at room temperature such as a polyalkylene oxide ( pao ) and more preferably a polyethylene glycol such as mpeg or bis - activated peg . a non - limiting list of such polymers therefore includes polyalkylene oxide homopolymers such as polyethylene glycol ( peg ) or polypropylene glycols , polyoxyethylenated polyols , copolymers thereof and block copolymers thereof , provided that the water solubility of the block copolymers is maintained . for purposes of illustration and not limitation , the polyethylene glycol ( peg ) residue portion of r 2 can be — ch 2 ch 2 — o —( ch 2 ch 2 o ) x — ch 2 ch 2 — x is the degree of polymerization , i . e . from about 10 to about 2 , 300 . in alternative aspects of the invention , when bis - activated polymers are desired , r 1 is n 3 , and the resultant reactant is used in making the bis - amine - terminated polymer compounds . such bis - activated polymers can be of formula ( ia ): 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 substantially non - antigenic polymers , pao &# 39 ; s and peg &# 39 ; s 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 daltons in most aspects of the invention . more preferably , the substantially non - antigenic polymer has a weight average molecular weight from about 2 , 000 to about 48 , 000 daltons . r 2 can also be a “ star - peg ” or multi - armed peg &# 39 ; s such as those described in nof corp . drug delivery system catalog , 2005 , 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 , r 2 can be of the formula : n is an integer from about 10 to about 340 , to preferably provide polymers having a total molecular weight of from about 12 , 000 to about 40 , 000 ; and at least 1 , but up to 3 , of the terminal portion a of the residue is / are capped with a methyl or other lower alkyl . see also the aforementioned nektar catalog , page 26 “ 4 - arm peg .” such compounds prior to terminal amination preferably include : also contemplated within the scope of the invention , is the formation of a terminal amine on various other peg - based compounds , including those branched polymer residues described in commonly assigned u . s . pat . nos . 5 , 605 , 976 , 5 , 643 , 575 , 5 , 919 , 455 and 6 , 113 , 906 , the disclosure of each being incorporated herein by reference . a representative list of some specific compounds includes : l 1 and l 3 are independently selected bifunctional linking groups such as one of the following non - limiting compounds : r 9 - 13 are independently selected from the same group as c 1 - 6 alkyls , etc . and preferably h or ch 3 ; r 6 is selected from the same group as that which defines r 9 - 13 , no 2 , c 1 - 6 halo - alkyl and halogen ; and q , t and y are each independently selected positive integers such as from 1 to about 12 ; and l 2 is a branched linking group such as a diamino alkyl or lysine residue . see , for example , the aforementioned u . s . pat . no . 6 , 113 , 906 , for example . in a further embodiment , and as an alternative to pao - based polymers , r 2 is optionally selected from among one or more effectively non - antigenic materials such as dextran , polyvinyl alcohols , carbohydrate - based polymers , hydroxypropylmethacryl - amide ( hpma ), polyalkylene oxides , and / or copolymers thereof . see also commonly - assigned u . s . pat . no . 6 , 153 , 655 , the contents of which are incorporated herein by reference . it will be understood by those of ordinary skill that the same type of activation is employed as described herein as for pao &# 39 ; s such as peg . those of ordinary skill in the art will further realize that the foregoing 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 polymer can be functionalized for attachment to the azide group ( s ) if required without undue experimentation prior to amination . one of the reactants required for the process of preparing the desired amine - terminated polymer is the azide ( n 3 ) terminated starting material . the preparation of such azide modified polymers has been reported in the literature , see for example the aforementioned zalipsky , eur . polym . j . ( 1983 ). alternatively , the azide can be made by reacting a compound of formula ( ii ): wherein r 3 is selected from the group consisting of br , cl , tosylate or mesylate , brosylate , tresylate , nosylate ; and with nan 3 in the presence of a solvent such as ethanol and / or dmf . in one aspect of the invention , the conversion of the peg - azide to the corresponding amine can be accomplished when the azide and phosphine - based reducing agent are reacted in a solvent and the reactants are allowed to reflux for a time sufficient to cause formation of the terminal amine on the polymer . there are a number of suitable phosphine - based reducing agents which can be used in the process of the invention . a non - limiting list includes reducing agents such as triphenylphosphine , tritolylphosphine , tributylphosphine , tert - butyldiphenylphosphine , diphenyl ( p - tolyl ) phosphine , tris ( 2 , 4 , 6 - trimethoxyphenyl ) phosphine , tris ( 2 , 6 - dimethoxy - phenyl ) phosphine , tris ( 2 - methoxyphenyl ) phosphine , tris ( 3 - chlorophenyl ) phosphine , tris ( 3 - methoxyphenyl ) phosphine , tris ( 4 - fluorophenyl ) phosphine , tris ( 4 - methoxyphenyl )- phosphine , tris ( p - chlorophenyl ) phosphine , and tris ( pentafluorophenyl ) phosphine . preferably , the phosphine - based reducing agent is triphenylphosphine . there are also a number of suitable solvents which can be used in the process of the present invention . suitable solvents include those which are polar solvent such as consisting of methanol , ethanol , butanol , isopropanol , tetrahydrofuran ( thf ), dimethylformamide ( dmf ) and dioxane . methanol is the preferred solvent . in an alternative aspect of the invention , an alkali metal borohydride reducing agent is used for the conversion of the peg - azide to the peg - amine . suitable alkali metal borohydride reducing agents reagents include but are not limited to sodium borohydride , ( preferred ), lithium borohydride and potassium borohydride . others will be apparent to those of ordinary skill . when this route is selected for amination , the reduction of the azide using the alkali metal borohydride reducing agent is carried out in a c 1 - 6 alcohol , with isopropyl alcohol , methanol or ethanol being preferred . the methods of the present invention are preferably carried out using at least about an equimolar amount of the reactants . more preferably , the phosphine - based reducing agent is present in a molar excess with respect to the compound of formula ( i ) when the polymer has only one azide . a preferred ratio of triphenylphosphine to mpeg is about 5 to about 1 . more preferably , the ratio is about 3 to 1 . when delta - peg ( bis - peg or activated on each terminal ) is used , the ratio is about 10 to about 2 , preferably 6 to 2 . similar ratios are used per amine group to be added if branched polymers are used . the amount of the phosphine - based reducing agent present in the reaction is preferably about at least about a two - fold to molar excess with respect to the compound of formula ( i ) when bis - azide polymer is used . it will be understood that when terminally branched polymers are used , the molar excess of phosphine - based reducing agent preferably used is at least equal to the number of azide moieties found on the polymer . in the case of the alkali metal borohydride , it is preferred that there is at least about a ten - fold molar excess of the borohydride to the polymer . more preferably , there is about a 15 - fold molar excess . the molar excess can be as high as 50 - or 100 - fold , if desired . the methods of the present invention are preferably carried out under reflux conditions , i . e . at a temperature of about the boiling point or slightly above of the solvent . the high - purity peg - amine can then be used in any art - recognized way . for example , and without limitation , it can be used for direct conjugation with co 2 h groups or other suitable reactive groups found on biologically active targets of interest using techniques well known to those of ordinary skill . alternatively , the peg - amine can be used as a highly pure intermediate in more complex polymer linking systems such as the aforementioned benzyl - elimination ( rnl ) platforms or even as part of peg - liposome systems . for the sake of illustration , the rnl systems can be made by reacting the peg - amine with a suitable protected benzyl alcohol followed by deprotection and activation using techniques known to those of ordinary skill . see also the last example below . in many aspects of the invention , a polyalkylene oxide ( pao ) such as uncapped peg - oh ( di - peg oh ) or mpeg is converted into a compound of formula ( iia ) or ( iib ): after the polymer containing the azide is formed , it is converted as described herein to form the desired peg - amine derivatives in high purity . the high purity peg - amine can then be used in any art - recognized way . the following examples serve to provide further appreciation of the invention but are not meant in any way to restrict the effective scope of the invention . method a . a solution of 20kda δpeg - oh ( i . e . bis - peg - oh ) ( 2 . 8 g , 0 . 14 mmol ) was azeotroped for 2 hours in toluene . the toluene was removed under vacuum and the solid residue was redissolved in 50 ml of anhydrous dichloromethane ( dcm ). to this solution was added triethylamine ( 195 μl , 1 . 4 mmol ) and 4 - dimethylaminopyridine ( dmap ) ( 165 mg , 1 . 35 mmol ). the mixture was cooled in an ice bath and p - toluenesulfonyl chloride ( 267 mg , 1 . 4 mmol ) in dcm was then added dropwise . the reaction mixture was gradually warmed to room temperature and stirred overnight . the reaction mixture was washed twice with a 0 . 1 n hcl solution . the organic phase was evaporated under vacuum . the resulting solid was dissolved in the minimum amount of ch 2 cl 2 and then , precipitated by addition of ethyl ether . after filtration the resulting solid was recrystallized with 2 - isopropanol ( ipa ). 13 c nmr ( 67 . 8 mhz , cdcl 3 ) δ143 . 92 , 132 . 38 , 129 . 1 , 127 . 29 , 67 . 99 - 70 . 22 , 21 . 09 . method b . to a solution of 30kda mpeg - oh ( 25 g , 0 . 83 mmol , 1 eq ) in 200 ml of dcm were added 150 ml of a 30 % aqueous naoh solution and bnet 3 nci ( 76 mg , 0 . 33 mmol , 0 . 4 eq ). then , to the vigorously stirred mixture a solution of p - toluenesulfonyl chloride ( 475 mg , 2 . 5 mmol , 3 eq ) in 150 ml of dcm was added dropwise via addition funnel , and the reaction mixture was stirred at room temperature overnight . after addition of 200 ml of dcm and 200 ml of a saturated nacl solution , the organic phase was separated and washed twice with 200 ml of a saturated nacl solution . the organic phase was dried over anhydrous mgso 4 , filtered and evaporated under vacuum to give a solid that was dissolved in a minimum amount of dcm and precipitated with ethyl ether . filtration provided 24 g of 30kda mpeg - ots ( 96 % yield ). 13 c nmr : 21 . 56 , 58 . 86 , 68 . 45 , 69 . 07 , 69 . 96 - 71 . 71 ( peg ), 127 . 64 , 129 . 53 , 132 . 70 , 144 . 43 . gpc : 100 % peg - azide ( peg - n 3 ). to a solution of 30kda mpeg - ots ( 2 g , 0 . 066 mmol , 1 eq ) in 20 ml anhydrous dmf was added nan 3 ( 13 mg , 0 . 2 mmol , 3 eq ). the reaction mixture was heated to 80 ° c . overnight and then cooled and evaporated under vacuum . the resulting solid was recrystallized with ch 3 cn / ipa to give 1 . 7 g of 30kda mpeg - n 3 ( 85 % yield ). 13 c nmr ( ppm ): 50 . 49 , 58 . 84 , 69 . 37 - 71 . 72 ( peg ). gpc : 99 . 3 %. peg - amine ( peg - nh 2 ). to a solution of 30kda mpeg - n 3 ( 10 . 64 g , 0 . 35 mmol , 1 eq ) in 120 ml anhydrous meoh was added triphenylphosphine ( 279 mg , 1 . 06 mmol , 3 eq ). the reaction mixture was heated to reflux overnight , cooled and evaporated under vacuum . the resulting solid was dissolved in the minimum amount of dcm and then , precipitated by addition of ethyl ether . after filtration the solid was recrystallized with ch 3 cn / ipa to give 9 . 94 g of 30kda mpeg - nh 2 ( 95 % yield ). 13 c nmr ( ppm ): 41 . 61 , 58 . 83 , 69 . 42 - 73 . 23 ( peg ). gpc : 98 . 91 % the corresponding 20kda δpeg - azide and 20kda δpeg - amine are made from the 20kda δpeg - ots of method a . peg - chloride . a solution of 12kda mpeg - oh ( 12 g , 0 . 1 mmol ) was azeotroped for 2 hours in toluene . this mixture was cooled to 30 ° c . and thionyl chloride was then added . the reaction mixture was refluxed for 18 hours , followed by partial removal of the solvent , and precipitation of the product with ethyl ether . the solid was collected by filtration , washed with ethyl ether , and recrystallized from isopropanol to yield the product ( 10 . 8 g , 0 . 81 mmol ). 13 c nmr ( 67 . 8 mhz , cdcl 3 ) δ 69 . 64 - 70 . 9 , 42 . 5 . peg - azide ( peg - n 3 ). to a solution of 12kda mpeg - cl ( 10 . 6 g , 0 . 883 mmol ) in anhydrous dmf was added nan 3 ( 919 mg , 35 . 5 mmol ). the reaction was heated at 80 ° c . for 24 hours . after addition of ethyl ether , the solid was collected by filtration . the solid residue was dissolved in dcm and the solution was washed with water three times , dried over naso 4 , filtered and evaporated under vacuum . the resulting solid was recrystallized from dcm / ethyl ether to give the peg - azide ( 9 . 54 g , 0 . 795 mmol ). 13 c nmr ( 67 . 8 mhz , cdcl 3 ) δ 69 . 64 - 70 . 9 , 50 . 29 . peg - amine ( peg - nh 2 ). to a suspension of 12kda mpeg - n 3 ( 1 . 23 g , 0 . 1 mmol ) in ipa was added sodium borohydride ( 58 mg , 1 . 5 mmol ). the reaction mixture was refluxed overnight . the reaction mixture was cooled to room temperature and ethyl ether was added . the resulting solid was collected by filtration . the solid residue was dissolved in dcm and the solution was washed with water three times , dried over naso 4 , filtered , evaporated under vacuum and recrystallized from ipa to give peg - amine ( 1 . 13 g , 0 . 09 mmol ). 13 c nmr ( 67 . 8 mhz , cdcl 3 ) δ 68 . 42 - 72 . 89 , 41 . 53 . in this example , the 30kda mpeg - nh 2 of example 1 is converted into the activated peg linker according to the following reaction scheme . to a solution of alcohol , 22 ( 238 mg , 1 mmol , 6 eq ) in anhydrous ch 3 cl were 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 30kda mpeg - nh 2 ( hereinafter 21 ) ( 5 g , 0 . 17 mmol , 1 eq ) in 25 ml of anhydrous ch 3 cl . after stirring at room temperature for 3 days , the solvent was evaporated under vacuum . the resulting solid was dissolved in the minimum amount of dichloromethane and then , precipitated by addition of ether , filtered and recrystallized with ch 3 cn / ipa to give 4 . 85 g ( 94 % yield ). gpc : 98 . 39 %. 13 c nmr ( 75 . 4 mhz , cdcl 3 ) δ 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 . to a solution of 23 ( 4 . 85 g , 0 . 16 mmol ) in 20 ml ch 3 cn 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 ch 2 cl 2 . 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 the minimum amount of ch 2 cl 2 and then , precipitated by addition of ether to give 4 . 49 g ( 94 % yield ). gpc : 98 . 35 %. 13 c nmr ( 75 . 4 mhz , cdcl 3 ) δ 154 . 36 , 149 . 90 , 138 . 18 , 127 . 37 , 121 . 15 , 69 . 42 - 71 . 69 ( peg ), 63 . 93 , 58 . 80 , 40 . 83 . 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 the minimum amount of ch 2 cl 2 and then , precipitated by addition of ether , filtered and recrystallized with ch 3 cn / ipa to give 4 . 26 g ( 94 % yield ). gpc : 97 . 04 %. 13 c nmr ( 75 . 4 mhz , cdcl 3 ) δ 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 conjugation to any number of biologically active polypeptides , enzymes , proteins , small molecules , etc . having an available amine or hydroxyl thereon for conjugation . the procedures for such conjugation reactions have been described , for example , in commonly - assigned u . s . pat . no . 6 , 180 , 095 , the contents of which are incorporated herein by reference , or the aforementioned greenwald et al . j . med . chem . 1999 vol . 42 , no . 18 , 3657 - 3667 .
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 azide with a phosphine - based reducing agent such as triphenylphosphine or an alkali metal borohydride reducing agent such as sodium borohydride in a solvent to reflux . the resultant polymer - amines are of sufficient purity so that expensive and time consuming purification steps required for pharmaceutical grade polymers are avoided .