Graft copolymers, their preparation and use in capillary electrophoresis

Abstract

The invention relates to graft copolymers, their preparation, and compositions, such as electrophoresis separation media, containing the same; also to ultra-high molecular weight poly(N,N-dimethylacrylamide) (“poly(DMA)”) polymers, their preparation, and compositions, such as electrophoresis separation media, containing the same; and more particularly to supports, such as capillaries, containing these polymers and methods for separating biomolecules, especially polynucleotides, using capillary electrophoresis. The graft copolymers can be prepared by, e.g., grafting polyacrylamide units onto a poly(DMA) backbone. Separation media comprising such graft copolymers or ultra-high molecular weight poly(DMA) polymers yield superior performance in the analysis and separation of biomolecules by capillary electrophoresis.

1 . A composition comprising a buffer and an effective amount of a poly(M 1 -g-M 2 ) or a salt thereof, wherein: 
 (a) each M 1  has the formula (I):                        wherein each A 1  is independently O, S or NX 1 ;    each of R 1 , R 2 , R 3  and R 4  is independently H, C 1 -C 20  alkyl, C 4 -C 12  cycloalkyl, C 5 -C 12  aryl, C 4 -C 12  heteroaryl, —(C 1 -C 20  alkyl)(C 5 -C 12  aryl) or —(C 1 -C 12  aryl)(C 1 -C 20  alkyl);    each R 5  is independently C 1 -C 20  alkyl, C 1 -C 20  heteroalkyl, C 4 -C 12  cycloalkyl, C 4 -C 12  heterocycloalkyl, C 5 -C 12  aryl, C 4 -C 12  heteroaryl, —(C 1 -C 20  alkyl)(C 4 -C 12  cycloalkyl), —(C 4 -C 12  cycloalkyl)(C 1 -C 20  alkyl), —(C 1 -C 20  heteroalkyl)(C 4 -C 12  cycloalkyl), —(C 4 -C 12  cycloalkyl)(C 1 -C 20  heteroalkyl), —(C 1 -C 20  alkyl)(C 4 -C 12  heterocycloalkyl), —(C 4 -C 12  heterocycloalkyl)(C 1 -C 20  alkyl), —(C 1 -C 20  heteroalkyl)(C 4 -C 12  heterocycloalkyl), —(C 4 -C 12  heterocycloalkyl)(C 1 -C 20  heteroalkyl), —(C 1 -C 20  alkyl)(C 5 -C 12  aryl), —(C 5 -C 12  aryl)(C 1 -C 20  alkyl), —(C 1 -C 20  heteroalkyl)(C 5 -C 12  aryl), —(C 5 -C 12  aryl)(C 1 -C 20  heteroalkyl), —(C 1 -C 20  alkyl)(C 4 -C 12  heteroaryl), —(C 4 -C 12  heteroaryl)(C 1 -C 20  alkyl), —(C 1 -C 20  heteroalkyl)(C 4 -C 12  heteroaryl), —(C 4 -C 12  heteroaryl)(C 1 -C 20  heteroalkyl), —(C 1 -C 4  alkyl) q NH 2 , —(C 1 -C 4  alkyl) q CONH 2 , —(C 1 -C 4  alkyl)NHCONH 2 , —(C 1 -C 4  alkyl)NHCOH or —(C 1 -C 4  alkyl) q NHCOCH 3 , where each q is 0 or 1; and    each X 1  is independently H, C 1 -C 20  alkyl, C 4 -C 12  cycloalkyl, C 5 -C 12  aryl, C 4 -C 12  heteroaryl, —(C 1 -C 20  alkyl)(C 5 -C 12  aryl), —(C 5 -C 12  aryl)(C 1 -C 20  alkyl), —(C 1 -C 4  alkyl) q NH 2 , —(C 1 -C 4  alkyl) q CONH 2 , —(C 1 -C 4  alkyl)NHCONH 2 , —(C 1 -C 4  alkyl) q NHCOH or —(C 1 -C 4  alkyl) q NHCOCH 3 , where each q is 0 or 1;      (b) each M 2  has the formula (II):                        wherein each A 2  is independently O, S or NX 2 ;    each of R 6 , R 7 , R 8  and R 9  is independently H, C 1 -C 20  alkyl, C 4 -C 12  cycloalkyl, C 1 -C 12  aryl, C 4 -C 12  heteroaryl, —(C 1 -C 20  alkyl)(C 5 -C 12  aryl) or —(C 5 -C 12  aryl)(C 1 -C 20  alkyl);    each R 10  is independently H, C 1 -C 20  alkyl, C 1 -C 20  heteroalkyl, C 4 -C 12  cycloalkyl, C 4 -C 12  heterocycloalkyl, C 5 -C 12  aryl, C 4 -C 12  heteroaryl, —(C 1 -C 20  alkyl)(C 4 -C 12  cycloalkyl), —(C 4 -C 12  cycloalkyl)(C 1 -C 20  alkyl), —(C 1 -C 20  heteroalkyl)(C 4 -C 12  cycloalkyl), —(C 4 -C 12  cycloalkyl)(C 1 -C 20  heteroalkyl), —(C 1 -C 20  alkyl)(C 4 -C 12  heterocycloalkyl), —(C 4 -C 12  heterocycloalkyl)(C 1 -C 20  alkyl), —(C 1 -C 20  heteroalkyl)(C 4 -C 12  heterocycloalkyl), —(C 4 -C 12  heterocycloalkyl)(C 1 -C 20  heteroalkyl), —(C 1 -C 20  alkyl)(C 5 -C 12  aryl), —(C 5 -C 12  aryl)(C 1 -C 20  alkyl), —(C 1 -C 20  heteroalkyl)(C 5 -C 12  aryl), —(C 5 -C 12  aryl)(C 1 -C 20  heteroalkyl), —(C 1 -C 20  alkyl)(C 4 -C 12  heteroaryl), —(C 4 -C 12  heteroaryl)(C 1 -C 20  alkyl), —(C 1 -C 20  heteroalkyl)(C 4 -C 12  heteroaryl), —(C 4 -C 12  heteroaryl)(C 1 -C 20  heteroalkyl), —(C 1 -C 4  alkyl) q NH 2 , —(C 1 -C 4  alkyl) q CONH 2 , —(C 1 -C 4  alkyl)NHCONH 2 , —(C 1 -C 4  alkyl)NHCOH or —(C 1 -C 4  alkyl) q NHCOCH 3 , where each q is 0 or 1; and      each X 2  is independently H, C 1 -C 20  alkyl, C 4 -C 12  cycloalkyl, C 5 -C 12  aryl, C 4 -C 12  heteroaryl, —(C 1 -C 20  alkyl)(C 5 -C 12  aryl), —(C 5 -C 12  aryl)(C 1 -C 20  alkyl), —(C 1 -C 4  alkyl) q NH 2 , —(C 1 -C 4  alkyl) q CONH 2 , —(C 1 -C 4  alkyl)NHCONH 2 , —(C 1 -C 4  alkyl) q NHCOH or —(C 1 -C 4  alkyl) q NHCOCH 3 , where each q is 0 or 1;    (c) provided that at least one M 1  is different from at least one M 2 .    
     
     
         2 . The composition of  claim 1 , which further comprises a sieve polymer, or a salt thereof, having a monomer unit that is acrylamide, N-acetyl-acrylamide, N-2-cyanoethyl-acrylamide, N,N-1,2-dihydroxyethylene-bis-acrylamide, N-4,4-dimethoxybutyl-acrylamide, N-2,2-dimethoxyethyl-acrylamide, N,N-dimethyl-acrylamide, N-2-glycolic acid methyl ester acrylamide, N-2-hydroxyethyl-acrylamide, N-hydroxymethyl-acrylamide, N-methoxymethyl-acrylamide, N-3-methoxypropyl-acrylamide, N-methyl-acrylamide, N-methyl-, N-2,2-dimethoxyethyl-acrylamide, N-morpholinoethyl-acrylamide, N-2,2,2-trichloro-1-hydroxyethyl-acrylamide, N-tri(hydroxymethyl)-methyl-acrylamide, methacrylamide, N-acetyl-methacrylamide, N-2-cyanoethyl-methacrylamide, N,N-1,2-dihydroxyethylene-bis-methacrylamide, N-4,4-dimethoxybutyl-methacrylamide, N-2,2-dimethoxyethyl-methacrylamide, N,N-dimethyl-methacrylamide, N-2-glycolic acid methyl ester methacrylamide, N-2-hydroxyethyl-methacrylamide, N-hydroxymethyl-methacrylamide, N-methoxymethyl-methacrylamide, N-3-methoxypropyl-methacrylamide, N-methyl-methacrylamide, N-methyl-, N-2,2-dimethoxyethyl-methacrylamide, N-morpholinoethyl-methacrylamide, N-2,2,2-trichloro-1-hydroxyethyl-methacrylamide, N-tri(hydroxymethyl)-methyl-methacrylamide, or a mixture thereof.  
     
     
         3 . The composition of  claim 2 , wherein the sieve polymer is poly(acrylamide).  
     
     
         4 . The composition of  claim 2 , wherein the sieve polymer is poly(N,N-dimethyl-acrylamide) and the sieve polymer has a weight-average molecular weight of at least about 3 MDaltons.  
     
     
         5 . A method for making poly(N,N-dimethylacrylamide), the method comprising polymerizing N,N-dimethylacrylamide in an inverse emulsion comprising an oil phase, an aqueous phase, a surfactant and an initiator to provide the poly(N,N-dimethylacrylamide), wherein the poly(N,N-dimethylacrylamide) has a weight-average molecular weight of at least about 3 MDaltons.  
     
     
         6 . The method of  claim 5 , wherein the oil phase comprises an aliphatic hydrocarbon having at least about 15 carbon atoms, an aliphatic hydrocarbon having a normal boiling point at or above about 270° C., a silicone oil, a fluorinated hydrocarbon, a liquid perfluoropolyether, or a mixture thereof.  
     
     
         7 . The poly(N,N-dimethylacrylamide) product of the method of  claim 5 .  
     
     
         8 . The composition of  claim 1 , which further comprises poly(hydroxymethylene), poly(oxyethylene), poly(oxypropylene), poly(oxyethylene-co-oxypropylene), poly(vinyl alcohol), poly(vinylpyrrolidone), poly(2-ethyl-2-oxazoline), poly(2-methyl-2-oxazoline), poly((2-ethyl-2-oxazoline)-co-(2-methyl-2-oxazoline)), poly(N-acetamidoacrylamide), poly(acryloxylurea), hydroxyethyl cellulose, hydroxymethyl cellulose, or a mixture thereof.  
     
     
         9 . The composition of  claim 1 , wherein the poly(M 1 -g-M 2 ) or a salt thereof has a weight-average molecular weight of from about 150,000 Daltons to about 20 MDaltons.  
     
     
         10 . The composition of  claim 9 , which further comprises a sieve polymer or a salt thereof having a weight-average molecular weight of from about 100,000 Daltons to about 5 MDaltons.  
     
     
         11 . The composition of  claim 10 , wherein the sieve polymer is substantially linear poly(acrylamide).  
     
     
         12 . The composition of  claim 1 , wherein M 1  is: 
 N-adamantyl-acrylamide, N-butoxymethyl-acrylamide, N-butyl-acrylamide, N-cyclohexyl-acrylamide, N,N-dibutyl-acrylamide, N-3-di(butyl)aminopropyl-acrylamide, N,N-diethyl-acrylamide, N-4,4-dimethoxybutyl-acrylamide, N,N-dimethyl-acrylamide, N-3-(dimethylamino)-propyl-acrylamide, N,N-dipropyl-acrylamide, N-dodecyl-acrylamide, N-2-ethylhexyl-acrylamide, N-isobornyl-acrylamide, N-methyl-acrylamide, N-methyl-, N-2,2-dimethoxyethyl-acrylamide, N-morpholinoethyl-acrylamide, N-octadecyl-acrylamide, N-propyl-acrylamide, N-3-(trimethylammonium)-propyl-acrylamide chloride, N-1,1,3-trimethylbutyl-acrylamide, N-adamantyl-methacrylamide, N-butoxymethyl-methacrylamide, N-butyl-methacrylamide, N-cyclohexyl-methacrylamide, N,N-dibutyl-methacrylamide, N-3-di(butyl)aminopropyl-methacrylamide, N,N-diethyl-methacrylamide, N-4,4-dimethoxybutyl-methacrylamide, N,N-dimethyl-methacrylamide, N-3-(dimethylamino)-propyl-methacrylamide, N,N-dipropyl-methacrylamide, N-dodecyl-methacrylamide, N-2-ethylhexyl-methacrylamide, N-isobornyl-methacrylamide, N-methyl-methacrylamide, N-methyl-, N-2,2-dimethoxyethyl-methacrylamide, N-morpholinoethyl-methacrylamide, N-octadecyl-methacrylamide, N-propyl-methacrylamide, N-3-(trimethylammonium)-propyl-methacrylamide chloride, N-1,1,3-trimethylbutyl-methacrylamide, or a mixture thereof.    
     
     
         13 . The composition of  claim 12 , wherein M 2  is: 
 acrylamide, N-acetyl-acrylamide, N-butoxymethyl-acrylamide, N-4,4-dimethoxybutyl-acrylamide, N-2,2-dimethoxyethyl-acrylamide, N-2-glycolic acid methyl ester acrylamide, N-2-hydroxyethyl-acrylamide, N-hydroxymethyl-acrylamide, N-methoxymethyl-acrylamide, N-3-methoxypropyl-acrylamide, N-methyl-acrylamide, N-methyl-, N-2,2-dimethoxyethyl-acrylamide, N-morpholinoethyl-acrylamide, N-2,2,2-trichloro-1-hydroxyethyl-acrylamide, N-tri(hydroxymethyl)-methyl-acrylamide, methacrylamide, N-acetyl-methacrylamide, N-butoxymethyl-methacrylamide, N-4,4-dimethoxybutyl-methacrylamide, N-2,2-dimethoxyethyl-methacrylamide, N-2-glycolic acid methyl ester methacrylamide, N-2-hydroxyethyl-methacrylamide, N-hydroxymethyl-methacrylamide, N-methoxymethyl-methacrylamide, N-3-methoxypropyl-methacrylamide, N-methyl-methacrylamide, N-methyl-, N-2,2-dimethoxyethyl-methacrylamide, N-morpholinoethyl-methacrylamide, N-2,2,2-trichloro-1-hydroxyethyl-methacrylamide, N-tri(hydroxymethyl)-methyl-methacrylamide, or a mixture thereof.    
     
     
         14 . The composition of  claim 12 , wherein M 2  is: 
 N-acetyl-acrylamide, N-butoxymethyl-acrylamide, N-4,4-dimethoxybutyl-acrylamide, N-2,2-dimethoxyethyl-acrylamide, N-2-glycolic acid methyl ester acrylamide, N-2-hydroxyethyl-acrylamide, N-hydroxymethyl-acrylamide, N-methoxymethyl-acrylamide, N-3-methoxypropyl-acrylamide, N-methyl-acrylamide, N-methyl-, N-2,2-dimethoxyethyl-acrylamide, N-morpholinoethyl-acrylamide, N-2,2,2-trichloro-1-hydroxyethyl-acrylamide, N-tri(hydroxymethyl)-methyl-acrylamide, N-acetyl-methacrylamide, N-butoxymethyl-methacrylamide, N-4,4-dimethoxybutyl-methacrylamide, N-2,2-dimethoxyethyl-methacrylamide, N-2-glycolic acid methyl ester methacrylamide, N-2-hydroxyethyl-methacrylamide, N-hydroxymethyl-methacrylamide, N-methoxymethyl-methacrylamide, N-3-methoxypropyl-methacrylamide, N-methyl-methacrylamide, N-methyl-, N-2,2-dimethoxyethyl-methacrylamide, N-morpholinoethyl-methacrylamide, N-2,2,2-trichloro-1-hydroxyethyl-methacrylamide, N-tri(hydroxymethyl)-methyl-methacrylamide, or a mixture thereof.    
     
     
         15 . The composition of  claim 1 , wherein the buffer is an aqueous buffer.  
     
     
         16 . The composition of  claim 15 , wherein the composition has a pH of from about 5 to about 11.  
     
     
         17 . The composition of  claim 15 , wherein the composition has a pH of from about 7 to about 10.  
     
     
         18 . The composition of  claim 15 , wherein M 1  is N,N-dimethylacrylamide and M 2  is acrylamide.  
     
     
         19 . The composition of  claim 16 , further comprising formamide, urea, pyrrolidone, N-methylpyrrolidone or a mixture thereof.  
     
     
         20 . The composition of  claim 16 , further comprising urea.  
     
     
         21 . The composition of  claim 16 , further comprising formamide.  
     
     
         22 . A capillary containing the composition of  claim 1 .  
     
     
         23 . The capillary of  claim 22 , wherein the capillary is a capillary tube.  
     
     
         24 . A method for separating a mixture of biomolecules, comprising: 
 (a) contacting the composition of  claim 1  with a mixture comprising a biomolecule; and    (b) applying an electric field to the composition in an amount sufficient to facilitate the separation of a biomolecule from the mixture.    
     
     
         25 . The method of  claim 24 , wherein the separation is performed within a capillary tube and two or more biomolecules are polynucleotides.  
     
     
         26 . The method of  claim 25 , wherein the separation has a crossover of at least 400 base pairs.  
     
     
         27 . Poly(N,N-dimethylacrylamide) having a weight-average molecular weight of at least about 3 MDaltons.