GB2270766A

GB2270766A - Electrophoresis method

Info

Publication number: GB2270766A
Application number: GB9322874A
Authority: GB
Inventors: Branko Kozulic; Urs Heimgartner
Original assignee: Individual
Current assignee: Individual
Priority date: 1990-04-20
Filing date: 1993-11-05
Publication date: 1994-03-23
Anticipated expiration: 2010-04-20
Also published as: JP3184234B2; GB9008873D0; GB9322874D0; JPH04227612A; GB2246127B; GB2246127A; GB2270766B

Description

Title: Improvements in or relating to electrophoresis Field of the invention This invention concerns electrophoresis. Background to the invention The specification of British Patent Application No. 9008873.3 (Publication No. 2246127) from which the present application is divided describes and claims novel acrylic monomers of the formula:

where RI is H or (CHOH) mCH2OH, m being 0,1 or 2; R, is monohydroxyalkyl, polyhydroxyalkyl or hydrocarbon radical having from 2 to 30 carbon atoms; R3 is H or CH3 ; and n is an integer in the range 1-4.

Polymers comprising the monomer and gels comprising the polymer and suitable for use in electrophoretic separation are also described and claimed.

Where R2 is a hydrophobic residue it is possible to obtain a series of monomers with increasing hydrophobicity. Such monomers are still water soluble due to the hydrophilic sugar alcohol part. Having a hydrophobic and a hydrophilic portion, these compounds constitute amphiphatic molecules. As shown in this invention, many homogenous gels containing hydrophobic residues can be prepared from such amphiphatic monomers.

Electrophoretic migration of charged molecules through such gels is influenced also by hydrophobicity of the migrating molecule, as shown in this invention, in a new

technique we have named hydrophobic electrophoresis, which is the subject of the 41 present application.

Summary of the invention In one aspect the present invention thus provides a method of separating molecules on the basis of their hydrophobicity, comprising subjecting the molecules to an electric field in an electrophoretic gel comprising immobilized amphiphatic molecules.

The invention also provides a method of separating molecules, comprising contacting a mixture of said molecules with a separation medium while being subjected to an electric field, wherein said separation medium comprises a polymer containing hydrophilic and hydrophobic residues, said hydrophobic residues being incorporated into the polymer by a free-radical polymerisation reaction including a monomer having the hydrophobic residue, wherein separation is effected by hydrophpbic interactions between said molecules and said hydrophobic residues of the polymer.

In a further aspect of the invention there is provided a method for gel-electrophoretic separation of molecules characterised by electrophoretic mobilities dependent on the hydrophobicity of the gel and the separating molecules using a gel with incorporated immobilized amphiphatic molecules.

The methods of the invention are conveniently performed using an electrophoretic gel or separation medium based on the monomer of GB 2246127, in which R2 is hydrophobic.

The term monohydroxyalkyl as used here includes aliphatic alcohols having one hydroxy group. The following are illustrative of monohydroxyalkyl radicals within the scope of this invention: 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl, 2-hydroxy-2-methyl-propyl,

2-hydroxypentyl, 3-hydroxypentyl, 4-hydroxypentyl, 5-hydroxypentyl, 2-hydroxy-2-methyl-butyl, 3-hydroxy-3methyl-butyl.

The term polyhydroxyalkyl as used in this invention includes aliphatic alcohols having more than one hydroxy group. The following serve as illustration: 2,3 dihydroxypropyl, 2,3-dihydroxybutyl, 2,4-dihydroxybutyl, 3,4-dihydroxybutyl, 2,3, 4 trihydroxybutyl, 2,3-dihydroxpentyl, 2,4-dihydroxypentyl, 2,5-dihydroxypentyl, 3,4dihydroxypentyl, 3,5-dihydroxypentyl, 4,5-dihydroxypentyl, 2,3, 4-trihydroxypentyl, 2,4, 5-trihydroxypentyl, 2,3, 5-trihydroxypentyl, 3,4, 5-trihydroxypentyl, 2,3, 4,5-tetrahyroxypentyl, 2,3-dihydroxyhexyl, 2,4-dihydroxyhexyl, 2,5-dihydroxyhexyl, 2,6-dihydroxyhexyl, 3,4-dihydroxyhexyl, 3,5-dihydroxyhexyl, 3,6-dihydroxyhexyl, 4,5-dihydroxylhexyl, 4,6-dihydroxyhexyl, 5,6-dihydroxyhexyl, 2,3, 4-trihydroxyhexyl, 2,3, 5-trihydroxyhexyl, 2,3, 6-trihydroxyhexyl, 2,4, 5-trihydroxyhexyl, 2,4, 6-trihydroxyhexyl, 2,5, 6-trihydroxyhexyl, 3,4, 5-trihydroxyhexyl, 3,4, 6-trihydroxyhexyl, 3,5, 6-trihydroxyhexyl, 4,5, 6-trihydroxyhexyl, 2,3, 4,5-tetrahydroxyhexyl, 2,4, 5,6-tetrahydroxyhexyl, 2,3, 5,6-tetrahydroxyhexyl, 2,3, 4,6-tetrahydroxyhexyl, 3,4, 5,6-tetrahydroxyhexl, 2,3, 4,5, 6-pentahydroxhexyl.

The term hydrocarbon radical as used herein includes aliphatic, cycloaliphatic and aromatic (including aliphatic-and cycloaliphatic-substituted aromatic and aromatic substituted aliphatic and cycloaliphatic) radicals. Where a named radical has several isomeric, including stereoisomeric, forms, all such forms are included. The following are illustrative of hydrocarbon radicals: ethyl, propyl, butyl, pentyl, hexyl, benzyl, phenylethyl, cyclopentyl, cyclohexyl, methylcyclopentyl, octyl, decyl, dodecyl, octadecyl, naphthyl.

The monomers of the present invention can be synthesised by reacting the Nsubstituted amino group of a sugar alcohol with an activated derivative of acrylic or methacrylic acid, such as acryloyl chloride or methacryloyl chloride. Other activated derivatives, such as acrylic acid anhydride or methacrylic acid anhydride or Nhydroxysuccinimide ester of acrylic or methacrylic acid may be advantageous in some

instances. The N-substituted amino sugar alcohols can be prepared in several ways but the most widely used procedure includes reductive amination of the reducing monosaccharide with an amine. Many N-alkyl sugar aminio alcohols are known and have been used for different applications (references 6-8). Di-alditylamines have also been described (reference 9). The N-substituted alditol amines served as the starting material for the preparation of new monomers disclosed in this invention.

Acylation of the amines with an activated derivative of acrylic or methacrylic acid can be done in different solvents. The choice is determined by solubility of the amine, reactivity of the activated derivative of acrylic or methacrylic acid and possible side reactions. For example, alcohols are not suitable if acryloyl or methacryloyl chloride are to be used, due to the predominant reaction between the solvent and reagent. If the amine is soluble other solvents, such as for example dioxane or acetonitrile, are suitable. A possible side reaction in such solvents is esterification of one or several hydroxy groups. Water can also be used as solvent. If an acid is liberated during the acylation reaction it is advantageous to neutralize it by a base. It is possible to use inorganic and organic bases and particularly useful are LiOH, NaOH, KOH, Ca (OH) 2 and tertiary amines. Alternatively, two moles of the amine can be taken for one mole of the activated acrylic or methacrylic acid derivative and then one mole of the amine serves to neutralize the acid which is liberated.

Water is a preferential solvent for acylation of many alditol amines because they are little soluble in other solvents and because the esterification of the hydroxy groups is less favorable in water. A disadvantage is hydrolysis of the activated acrylic and methacrvlic acid derivatives. By carefully controlling the reaction condition, as shown in reference 1, it is possible to achieve high yields of the alditol acrylamides and methacrylamides. Monomers for use in the present invention were synthesised substantially by the same procedure. The acylation reaction is preferentially carried out at low temperature (0-15 C), at slightly alkaline pH (7.5-11) in a two-phase (mostly water-methylene chloride) system. The amides are purified from the by-products and unreacted starting material preferentially by a combination of an anionic and cationic ion exchanger. The ion exchangers can be used in sequence or as a mixture. The

combination of a strong cationic ion exchanger, such as Dowex 50 or Amberlite IR- 120, and a weak anion exchanger, such as Amberlite IRA-68, was found particularly suitable (Dowex and Amberlite are Trade Marks). The resulting water solution of pure monomer is poured into crystallisation dishes or first partially concentrated by rotary evaporation. It is advantageous to add a small quantity of a polymerisation inhibitor (p-methoxy phenol, phenothiazine, sodium nitrite, etc) prior to water evaporation.

Some monomers crystallized after the water evaporated, some solidified into a hard mass and some remained as viscous liquids. The solid ones were usually recrystallized and those which remained in solution were treated with mixed ion exchangers and charcoal prior to polymerization.

The monomers for use in the present invention can be polymerized by a free radical polymerization using the usual initiators. Examples of such initiators include peroxides, 2, 2'-azo-bis-isobutyronitrile and N, N, N', N'-tetra-methylethylenediamine plus ammonium or alkali metal persulfate. The polymerization may be a block polymerization or an emulsion polymerization. For block polymerization the monomer solution containing an initiator is polymerized in a homogenous phase. For emulsion polymerization the monomer solution is dispersed and polymerized in the form of droplets in another phase which is not a good solvent for the monomer.

The monomers may be polymerized either alone or with other compounds and materials having polymerizable double bonds. When monomers are polymerized alone or with another monomer having only one double bond, soluble polymers may be formed.

Many different monomer combinations were polymerized. The water solubility of the polymers was mainly dependent on four parameters. The hydrophobicity of the amphiphatic monomer, the ratio between a hydrophilic and amphiphatic monomer, type of the hydrophilic monomer and total monomer concentration. Examples of water soluble polymers include the copolymer of acrylamide and N-acryloyl-N-hexyl-l-amino-l-deoxy-D-glucitol (NHAGU), polymerized at 0.43 total monomer concentration and 7 : 1 molar ratio of acrylamide to the amphiphatic monomer.

The second one is the copolymer composed of N-acryloyl-N-hexyl-1-amino-1-deoxy-Dglucitol (NHAGU) and N-acryloyl-l-amino-1-deoxy-D-glucitol (1-NAGA) polymerized

at 0.43 total monomer concentration and 5: 1 molar ratio of the hydrophilic to amphiphatic monomer. Some of the monomers when polymerized alone formed water insoluble polymers. Illustrative of these polymers are poly (N-acryloyl-N-butyl-1-amino-1-deoxy-D-galactitol) and poly (Nacryloyl-N-hexyl-1-amino-1-deoxy-D-glucitol). The soluble copolymers containing amphiphatic monomers with more hydrophobic residues, especially those with hexyl and octyl groups, behaved as surfactants. When their water solutions were agitated, the foam on the surface remained stable for several hours in many cases. Polymeric surfactants have been previously described (references 10,11), but they did not comprise amphiphatic monomers.

Cross-linked polymers are formed when the monomers were copolymerized with other monomers having at least two double bonds. The crosslinked polymers were usually in form of gels. The appearance of these gels was transparent or slightly to fully opaque.

Transparent, and therefore homogeneous, gels were formed when the concentration of the cross-linker was relatively low. In addition, the transparency of gels containing amphiphatic monomers was dependent on the concentration and hydrophobicity of these monomers. Many different combinations were polymerized, as described below.

Illustrative of transparent gels are poly (N-acryloyl-N- (2-hydroxyethyl)-l-amino- 1-deoxy-D-glucitol-co-N, N'-methylene-bis-acrylamide) containing 6. 790 g of monomer and 0.210 g of the cross-linker in 100 ml, poly (N-acryloyl-N-ethyl- 1-amino-1-deoxy-D-galactitol-co-N, N'-methylene-bis-acrylamide) containing 5.820 g monomer and 0.180 g cross-linker in 100 ml and poly (N-acryloyl-N-

hexyl-1-amino-1-deoxy-D-glucitol-co-acrylamide-co-N, N'-methylene-bis-acrylamide) containing 1.2 g of the amphiphatic monomer, 2.7 g of acrylamide and 0.04 g of the cross-linker in 100 ml. Unexpectedly, transparent or slightly opaque gels were obtained from some monomers or combinations that formed insoluble polymers. For example, N-acryloyl-N-butyl-1-amino-1-deoxy-D-galactitol when polymerized alone gave a water insoluble polymer but when polymerized in the presence of a cross-linker it gave an almost transparent gel.

An important application of the new gels was found in their use as separation media.

The transparent and slightly opaque gels were useful as a matrix for electrophoresis.

Three types of molecules were electrophoresed in the gels containing the monomers.

The first one is bromphenol blue (3', 3", 5', 5"-tetrabromophenol sulfonphtalein), a relatively small molecule having three substituted benzene groups and two negative charges (above pH 5). The second type is DNA, represented by a series of fragments with sizes from 75 to 23 000 base pairs. Proteins are the third type of molecules electrophoresed in the new gels.

The electrophoretic migration of bromphenol blue and DNA fragments was compared gin several gels, each one being made of different monomer. All gels were polymerized from solutions containing 5.820 g monomer and 0.180 g of N, N'-methylene-- bis-acrylamide in 100 ml. The monomers include

N-acryloyl-N-methyl-1-amino-1-deoxy-D-galactitol, N-acryloyl-N-ethyl-1-amino-l-deoxy-D-galactitol, N-acryloyl-N-propyl-1- amino-1-deoxy-D-galactitol and N-acryloyl-N-butyl-l-amino-deoxy-D-galactitol. Thus the only difference between the monomers was substitution on the amide nitrogen, ranging from methyl to butyl. Accordingly, the gels differed in their hydrophobicity.

The migration rate of bromphenol blue was similar in the gels made of methyl and ethyl monomers. In the first gel the dye migrated slightly behind the 123 base pairs (bp) DNA fragment and in the second gel slightly ahead of the 123 bp fragment. In the gel with propyl groups the dye migrated approximately as the 246 bp fragment. More importantly, in the gel with butyl groups bromphenol blue migrated approximately as the 1000 bp fragment. In addition, at the beginning of electrophoresis the dye zone was concentrated as it entered the gel and a slight change in color (to pale blue) of bromphenol blue could be observed in this gel. Such effects were not noticed in other three gels. These findings indicate that electrophoretic migration of bromphenol blue is decreased due to hydrophobic binding to the butyl groups in the gel. It is important to note that the electrophoretic migration of DNA fragments from 123 to 6000 bp was comparable in all gels, indicating a similar effective porosity of these four gels. The bands were sharper and resolution was better in gels with methyl and propyl than with gels with ethyl and butyl groups. In all gels the 506 and 516 bands (from the 1 kbp standard mixture, BRL) were resolved and at least 10 bands (from the 123 bp standard

mixture, BRL) were distinguishable.

The above findings demonstrate that by introducing a new separation principle in gel electrophoresis, that is hydrophobicity, it is possible to resolve molecules having a similar mobility in a hydrophilic gel. For example, it would be difficult to resolve bromphenol blue and the 123 bp DNA fragment solely on basis of their mobility in the gels composed of monomers containing methyl or ethyl groups. The resolution is, however, very efficient in the gel with butyl groups because due to the hydrophobic interaction in that gel bromphenol blue migrates as a 1000 bp DNA fragment.

Many gels with hydrophobic residues were used for electrophoresis of proteins. The ggels were usually composed of acrylamide, amphiphatic monomer and N, N'methylene-bis-acrylamide. The relative ratios of the three components were chosen to give essentially transparent gels. The composition of gels is conveniently expressed in terms of total monomer concentration (T) in g/100 ml, cross-linker concentration (C) in g of cross-linker/g total monomer X 100 and the molar ratio of the amphiphatic monomer to acrylamide. When the conversion of monomers to polymers is 100%, the polymer composition will reflect the initial composition of monomers. Although in

practice the conversion yield is never 100%, it is convenient to define the gels in terms of the initial monomer compositions. A typical series of gels was characterized by T = 12, C = 1 and the ratio of N-acryloyl-N-hexyl-1-amino-1-deoxy-D-glucitol to acrylamide from 1: 20 to 1: 160. The gel having no amphiphatic monomer (the blank) contained Nacryloyl-1-amino-1-deoxy-D-glucitol in the molar ratio to acrylamide 1: 20.

Bovine serum albumin (BSA) was used as a model protein. When electrophoresed in these gels under standard conditions (reference 12), BSA was not retarded although bromphenol blue was. Actually the protein migrated somewhat longer than in the gel with no hexyl groups. Likewise, in gels with octyl groups (molar ratio to acrylamide from 1: 40, gel is slightly opaque, to 1: 80, transparent gel) no retardation of BSA could be observed.

One common way to make hydrophobic interactions favorable is to increase the salt concentration. This is an accepted practice in hydrophobic chromatography. However,

buffers of high ionic strength are not suitable in electrophoresis because they cause excessive heating and reduce the electrophoretic mobility of proteins. Therefore, another way was looked for to increase hydrophobic interactions.

Gel electrophoresis of proteins is sometimes performed in the presence of detergents.

Nonionic and zwitterionic detergents are generally used to improve water solubility of hydrophobic proteins, whereas strong anionic or cationic detergents are utilized to unfold proteins. Of them, sodium dodecyl sulphate is the most widely used. It binds to proteins (1.4 g/g protein) through its hydrocarbon part and makes them essentially uniformly charged by converting all proteins into a rod-like shape. During gel electrophoresis larger protein-SDS complexes are more retarded due to the sieving effect of the gel. Since proteins migrate as a function of their size, SDS electrophoresis is often used to estimate the size of an unknown protein.

Detergents are known to decrease or prevent hydrophobic interactions between proteins and hydrophobic molecules in solution and between proteins and hydrophobic surfaces.

For that purpose they are routinely added into protein solutions that are used in many assays, including immunoassays. Based on prior art it was therefore reasonable to assume that detergents will decrease hydrophobic interactions also during electrophoresis in a gel containing hydrophobic residues. Nevertheless, we have tested the influence of SDS on electrophoretic migration of proteins in the gels with hydrophobic residues. It was surprisingly found that already at 0. 02% SDS concentration the migration of BSA was retarded in the gels having 1: 40 and 1: 20 molar ratio of N-acryloyl-N-hexyl-1-amino-l-deoxy-D-glucitol to acrylamide. When the

concentration of SDS was increased to 0. 05% the retardation was observed in the gel with 1 : 160 molar ratio and the protein migrated only slightly in the 1 : 20 gel. At 0. 1% SDS in the 1: 90 gel BSA migrated approximately half the distance it migrated in the control gel. It hardly entered the 1: 20 gel. At 0. 2% SDS in the 1: 160 gel BSA migrated less than half the distance in the control gel and it remained on top of the 1: 40 and 1: 20 gels.

The above findings demonstrate that addition of a detergent may be beneficial for

hydrophobic interactions between some large molecules and hydrophobic residues in the gel. We propose the following mechanism to account for this finding. Addition of SDS to the gel causes a change in gel structure. The hydrocarbon chain of SDS binds to the hydrocarbon chain of the monomer and makes it negatively charged. The charged complex is better available to the molecules migrating through the gel for two reasons.

First, the hydrated sulfate group prevents"hiding"of the hydrocarbon chain in the polymer structure. Second, the electrophoretic force acting on SDS stretches the whole complex away from the polymer backbone. The incoming protein-SDS complexes have hydrophobic amino acid residues, normally hidden in the interior of the native molecule, on the surface. When one SDS molecule dissociates (or is electroeluted) from the protein amino acid residues and in its vicinity also one from the matrix, a new complex is formed between the amino acid residues and the hydrophobic group from the matrix. The protein is subsequently either electroeluted or dissociated by SDS from the matrix, but the protein-matrix complex exists long enough to dramatically change the migration rate of a protein.

The increasing retardation effect seen with higher SDS concentrations indicates that the primary role of SDS is to make available the hydrophobic groups from the matrix.

Once the protein is hydrophobically bound through multiple points, it may be difficult to release it, as demonstrated by the finding that in the 1: 40 and 1: 20 gels a 0.2% SDS the protein remained on the top of the gel.

The above findings clearly show that hydrophobic properties of molecules can be utilized for their electrophoretic resolution. Hydrophobic electrophoresis of some small molecules can be achieved without addition of a detergent. This is probably so because small molecules can penetrate better the gel matrix and because their hydrophobic residues are more exposed than they are in large molecules. Other molecules may require the presence of a detergent. A charged detergent may be advantageous also for hydrophobic electrophoresis of uncharged molecules. It is known that uncharged molecules can be resolved by capillary electrophoresis in the presence of anionic or cationic detergents (reference 13). The selectivity in hydrophobic electrophoresis introduced herein may be changed by utilizing different concentrations of various

detergents. More importantly, the strength of hydrophobic interactions can be changed by using different hydrophobic residues in the matrix, as demonstrated in this invention.

The invention will be further described, by way of illustration, in the following Examples and with reference to the drawings, in which: Figure 1 is a grid of circles representing schematically the solubility of a range of acrylamide/NHAGU copolymers at different molar concentrations, as described in Example 10; Figure 2 is similar to Figure 1 for 1-NAGA/NHAGU copolymers, as described in Example 11; Figure 3 is a grid of circles representing schematically the transparency of gels of a range of acrylamide/NHAGU/N, N'-methylene-bis-acrylamide copolymers, at different molar concentrations, as described in Example 12; Figure 4 is similar to Figure 3 for gels of l-NAGA/NHAGU/N. N'-methylene-bisacrylamide copolymers, as described in Example 13; and Figures 5 to 8 illustrate schematically the results of electrophoresis in various different gels, as described in Examples 20 to 23.

Detailed description of the invention Example 1. Synthesis of N-acrvlovl-N-ethyl-l-amino-l-deoxv-D-galactitol.

The N-ethyl-l-amino-1-deoxy-D-galactitol (63 g) is mixed with about 300 ml of water and 1 g of sodium nitrite. Then 100-150 ml of methylene chloride are added, and the solution is cooled in an ice bath. Acryloyl chloride (10% molar excess over the amine) is mixed with the same volume of methylene chloride. Potassium hydroxide, equal to twice the molar amount of acryloyl chloride, is dissolved in water and cooled. To the

well stirred two-phase reaction mixture, portions of acryloyl chloride and KOH solutions were added in such a way that the pH remained between 7.5-9. 5 (as checked frequently by a narrow range pH paper). After the last additions of acryloyl chloride and KOH solutions, the reaction mixture was further stirred for about one hour (the pH was periodically checked and corrected, if necessary). The two phases were allowed to separate in a separating funnel and the lower organic phase was discarded. The aqueous phase was treated with charcoal and filtered. The filtrate was treated with a combination of ion exchangers either in solution or by passing the filtrate through columns packed with ion exchangers. Thus, to the stirred solution, portions of Amberlite IR-120, Hui ana IRA-68 (free base) were added (pH being kept neutral or slightly acid) until the silver reaction for chloride was negative. The resin was then removed, the filtrate treated with activated charcoal and the solution poured into crystallization dishes. Alternatively, the monomer solution was passed through 600 ml of IR-120 and 600 ml of IRA-68, preferentially packed in more than two columns. The solution passed always first through the cationic ion exchanger. A small quantity (several hundred mg) of p-methoxy phenol or sodium nitrite (polymerization inhibitors) were added to the monomer solution. The water evaporated within 1-2 weeks. The monomer appeared as a white solid. The yield was 72%. The monomer was recrystallized from ethanol-acetone, m. p. 80-84 C (polymerization).

Example 2. Synthesis of N-acrvlovI-N-propvl-l-amino-l-deoxv-D-saIactitol.

The synthesis was carried out essentially as described in Example 1. The yield was 75%. The monomer was recrystallized from ethanol-acetone, m. p. 128-130 (polymerization).

Example 3. Synthesis ofN-acrvloyl-N-butvl-l-amino-l-deoxv-D-galactitol.

The synthesis was carried out substantially as described in Example 1. The yield was 66%. The monomer was recrystallized from acetonitrile, m. p. 120-123 C (polymerization).

The corresponding glucitol derivative remained as a viscous aqueous solution.

Example 4. Synthesis ofN-acryIoyI-N-hexvl-l-amino-l-deoxy-D-glucitoI (NHAGU).

Due to a low water solubility of N-hexyl-1-amino-l-deoxy-D-glucitol, the amine was suspended in water and mostly converted into its hydrochloride salt by addition of hydrochloric acid. The synthesis was then done essentially as described in Example 1, except that the amount of KOH needed was higher. The yield was 76%. The monomer was recrystallized from acetonitrile, m. p. 86-88 C (polymerization).

Example 5. Synthesis ofN-acrvlovl-N-octvl-l-amino-l-deoxv-D-slucitol.

The starting amine was first converted into its hydrochloride salt as described in Example 4. The synthesis was then carried out as described in Example 1. However, a larger portion of UV positive material was found in the methylene chloride than in the water phase. After evaporation of methylene chloride a slightly yellow solid mass was obtained. The crystallization attempts failed.

Example 6. Synthesis of N-acrvlovl-N-phenylethYI-l-amino-l-deoxy-D-galactitol.

The starting amine was first converted mostly into its hydrochloride salt and the synthesis was then continued as described in Example 1. The yield was 37% The monomer crystallized after evaporation of water, m. p. 52-56 C.

Example 7. Synthesis of N-acrvloyl-N- (2-hydroxyethyl) -1-amino-l-deoxy-D-galactito1.

The synthesis was done as described in Example 1. After evaporation of water the monomer appeared as a semi-solid white material. It was recrystallized from ethanol.

The crystals were filtered and washed with acetone. A large portion of solvent remained within the monomer. When evaporation of the remaining solvent was tried in the air, the monomer liquifled but became semi-solid again after several days. After drying in vacuo over phosphorous pentoxide, the monomer was obtained as a hard solid. It is hygroscopic.

The corresponding glucitol derivative remained as a viscous aqueous solution. When stored refrigerated as 40% solution, after several months a fungi-like semi-solid, almost transparent material appeared and grew to the size of over 3 cm.

Example 8. Synthesis of N-acrylovl-amino-N, N.-bis (I-deoxv-D-L-alactitoll.

The synthesis was done essentially as described in Example 1. Due to low water solubility the amine was partially converted into its hydrochloride salt. The monomer crystallized after evaporation of water. The yield was 62 %. The monomer is hardly soluble in methanol or ethanol and the crystallization attempts failed. The melting point was greatly dependent on the heating rate, being much higher (above 130 C) at a slow rate.

The corresponding glucitol derivative remained as an aqueous solution.

Example 9. Preparation of a water soluble polymer from N-acryloyl-N-ethyl-1- amino-1-deoxy-D-galactitol.

The monomer (0.3 g) and sorbitol (0.75 g) were dissolved in water and diluted to 5 ml in a glass test tube. Then TEMED (12 Al) and ammonium persulfate (150 Al of a 15 mg/ml water solution) were added. The solution was overlaid with diisopropylether and polymerized overnight at room temperature. A viscous polymer solution was obtained.

Example 10. Preparation of water soluble and insoluble copolymers from acrylamide and N-acrvlovl-N-hexyl-1-amino-l-deoxv-D-glucitoI (NHAGU).

Different amounts of monomers were dissolved in 50 mM Tris-HCl buffer pH 8.8 to give total monomer concentration 0.43, 0.7, 1.0, 1.29 and 1.57 M and ratios of acrylamide to the N-hexyl monomer of 7: 1,5 : 1 and 3: 1. The solutions (300 ILl) were polymerized in microtiter plates. Polymerization was started by adding to the 0.43 M

solution 4 ILl of TEMED and 5. 9 Al of ammonium persulfate (AP, 15 mg/ml), to the 0. 7 M solution 3. 9 ILl of TEMED and 5. 7 ILl of AP, to the 1 M solution 3. 8 ILl of TEMED and 5. 5 ILl of AP, to the 1. 29 M solution 3. 7 ILl of TEMED and 5. 4 ILl of AP and to the 1. 57 M solution 3. 6 ILl of TEMED and 5. 3 ul of AP. Some solutions 3remained transparent whereas in others a precipitate was formed. In figure 1, soluble polymers are shown as white, not dotted, circles, precipitated polymers as dotted circles, stronger dotted for more precipitated polymers. The solubility clearly depends on the total monomer concentration as well as the molar ratio of acrylamide to the amphiphatic monomer.

A 10 ml solution of acrylamide and N-acryloyl-N-hexy 1-1-ammo-l-deoxy-D-glucitol containing 4% (w/v) total monomer and 7: 1 molar ratio of acrylamide to N-hexyl monomer was polymerized with 10 ILl of TEMED and 120 Al of ammonium persulfate (15 mg/ml). There resulted a viscous solution which foamed after agitation.

Example 11. Preparation of water soluble and insoluble copolymers from N-

acryiovl-l-amino-l-deoxv-D-glucitol (1-NAGA) and N-acryloyl-N-hexvl-1-amino-1-deoxv-D-glucitoI (NHAGU).

Twelve solutions were prepared having different molar ratios and total concentrations of monomers as described in Example 10. After polymerization, the same TEMED and AP concentrations were used as in Example 10, in the microtiter wells the result shown in figure 2 was obtained. Most combinations gave polymers which did not form visible precipitates. This is in contrast with copolymers comprising acrylamide instead of N-acryloyl-1-amino-l-deoxy-D-glucitol, demonstrating that copolymers with a higher amount of hydrophobic residues remain water soluble by increasing the hydrophilicity of the hydrophilic monomer.

A 5 ml water solution of N-acryloyl-l-amino-1-deoxy-D-glucitol and N-acryloyl-N- hexyl-l-amino-1-deoxy-D-glucitol containing 0.75 g sorbitol and 6% (w/v) total monomer with 10: 1 molar ratio of the hydrophilic to amphiphatic monomer was polymerized by addition of 8 ILl TEMED and 100 ILl ammonium persulfate (15 mg/ml).

There resulted a viscous solution which foamed upon agitation.

When the total monomer concentration was increased to 9% (w/v) or above, it was not possible to obtain polymers which completely dissolved after dilution with water.

Example 12. Transparent and opaque gels from acrylamide. N-acryloyl-N-hexyl- l-amino-l-deoxv-D-glucitol (NHAGU) and N. N'-methvlene-bis-acrylamide.

Fifteen solutions were polymerized which contained from 0.43 to 1.57 M total monomer concentration and molar ratios of acrylamide to the N-hexyl monomer from 3: 1 to 7: 1. The same amount of TEMED and AP as decribed in Example 10 were added. Each solution contained the same percentage of the cross-linker (C= 1 %, w/w)

in relation to the total monomer. In figure 3 the transparent gels are represented by white circles without dots and opaque gels by circles with dots. The opacity of gels is dependent on the total monomer concentration as well as the ration of acrylamide and the amphiphatic monomer.

Example 13. Transparent and opaque gels from N-acrvlovl-1-amino-l-deoxv-D-glucitol

(1-NAGA). N-acrvlovl-N-hexvl-1-amino-l-deoxv-D-slucitol CNHAGU) and N. N'-methvlene-bisacrvlamide.

Twelve solutions were polymerized which contained from 0. 43 to 1. 29 M total monomer and from 3: 1 to 7: 1 molar ratio of the hydrophilic to the amphiphatic monomer. Each solution had the same percentage of the cross-linker (C= 1 %, w/w) in relation to the total monomer. Figure 4 shows that most combinations yielded transparent gels. That is different to the result obtained with acrylamide (Example 12), where most combinations yielded slightly to fully opaque gels.

Example 14. A transparent gel from N-acrvlovl-N-butvl-l-amino-l-deoxv-D-galactitol and 1. 2-dihvdroxvethvlene-bis-acrvlamide.

To a 5 ml water solution of the monomers (T=8%, C=3%) in a glass test tube TEMED (9 Al) and ammonium persulfate (100 Al, 15 mg/ml solution) were added.

After two hours a transparent gel was formed.

Example 15. Electrophoresis in the gel prepared from N-acrvloyl-N- (2- hvdroxvethvD-l-amino-l-deoxv-D-glucitol and N. N'-methvlene-bis-acrvlamide.

As described in Example 7, this monomer was obtained as a concentrated water solution. That solution was treated with a mixture of ion exchangers (containing a blue indicator, from Bio Rad) and then with activated charcoal. The solution was first filtered through a filter paper and then through a nitrocellulose membrane filter (0.45 jim). The concentration of the monomer solution was estimated by measuring the absorbance at 260nm of the appropriately diluted sample and comparing it to the standard curve obtained with crystalline N-acryloyl-N-methyl-l-amino-l-deoxy-D- galactitol. The concentrated solution was 40% (w/v). A part of it was diluted to give a 7% solution in 30 mM Tris-acetate buffer pH 8.4, containing 2 mM

ethylenediaminetetraacetic acid. In 20 ml of this solution N, N'-methylene-bis-acrylamide (42 mg) was dissolved to give C=3%. Then TEMED (23 Al) and ammonium persulfate (270 il, 15 mg/ml) were added and the gel polymerized in a plastic cassette (7 X 10 cm), having sample well formers about 5 mm apart from edge of the shorter side. The gel was approximately 3 mm thick and the sample wells were about 2 mm deep and 5 mm long. The gel was polymerized on a plastic support (PAGE GelBond, FMC). After 4 hours at room temperature, the cassette was opened and the gel placed in an electrophoretic apparatus for submerged gel electrophoresis. The apparatus (home made) was equipped with a pump for buffer circulation and the gel rested on a cooling plate. Three different DNA standard mixtures were applied to the gel. They included 1 kbp ladder (from BRL), 123 bp ladder (from BRL) and lambda/Hind ill fragments (from Biofinex). The gel was run at 20 C in 30 mM Tris-acetate buffer pH 8.4, containing 2 mM EDTA at 4 V/cm for 4 hours. Then it was stained with ethidium bromide (1 g/ml) and distained with water.

DNA bands were visualized under UV light. All DNA fragments migrated further than in the poly (NAT-Bis) gel (T=7%, C=3%) which was run as a control, demonstrating a larger effective pore size. In the 1 kbp ladder, 506 and 516 DNA bands were clearly resolved as were 3 and 4 kbp bands. In the 123 bp ladder, at least 12 bands well resolved. In the lambda/Hind m mixture, the 2.0 and 2.2 kbp fragments were well resolved.

Example 16. Electrophoresis in the gel prepared from N-acrvloyl-amino-N. N-bis (1deoxy-D-galactitol) and N. N'-methYlene-bis-acrYlamide.

The monomer (1. 6 g) and the cross-linker (48 mg) were dissolved in 10 ml of water and the solution treated with mixed ion exchanger and charcoal as described in Example 15. The filtered solution was diluted to 20 ml with 60 mM Tris-acetate pH 8.4, containing 4 mM EDTA. The gel was then polymerized, run and stained as described in Example 15. All DNA fragments migrated much further than in the corresponding poly (NAT-Bis) gel and somewhat further than the gel of Example 15.

The bands were slightly broader than in the gel of Example 15. In the 1 kbp ladder 506 and 516 DNA bands were resolved and in 123 bp ladder at least 10 bands were clearly visible.

Example 17. Electrophoresis in the se ! prepared from N-acrvlovl-N-ethvl-1-amino- 1-deoxv-D-ealactitol and N. N'-methvlene-bis-acrvlamide.

The gel (T=6%, C=3%) solution was prepared by dissolving the monomer and cross-linker in the running buffer. The solution was polymerized to a perfectly transparent gel, which was run for 3 h at 4 V/cm and stained as described in Example 15. The bromphenol blue migrated slightly ahead of 123 bp fragment. In the 1 kbp ladder, 506 and 516 bp bands were resolved and the distances between upper bands were slightly larger than in the corresponding N-acryloyl-N-methyl-1-amino-l-deoxy-D- galacitol gel run as a control at the same time. In the 123 bp ladder, at least 13 bands were distinguishable. In the lambda/Hind ill fragments, 2.0 and 2.2 kbp bands were resolved. After electrophoresis the gel was somewhat swollen (thicker) and swelling becaome more pronounced during staining and destaining. Such swelling was not noticed with other gels examined. Once the gel detached from the supporting plastic.

Example 18. Electrophoresis in the gel prepared from N-acrvlovl-N-propvl-1- amino-1-deoxv-D-galactitol and N. N' -methylene-bis-acrylamide.

The gel was prepared and run essentially as described in Example 17. The gel was very slightly opaque, which was noticeable when looking through the long side of the gel.

The DNA bands were somewhat sharper and better resolved than in the gel of Example 17, although the migration distances were comparable. Bromphenol blue migrated in this gel approximately as the 246 bp fragment. The 506 and 516 bp bands were well resolved and at least 13 bands were distinguishable in the 123 bp ladder. The 2.0 and 2.2 kbp bands were also well resolved.

Example 19. Electrophoresis in the gel prepared from N-acrylovl-N-butyl-1-amino-l- deoxy-D-galactitol and N. N'-methylene-bis-acrylamide.

The gel was prepared and run essentially as described in Example 17. This gel was considerably more opaque than the gel of Example 18. The DNA fragments migrated a similar distance but the bands were generally broader. The 506 and 516 bp were distinguishable and the 2.0 and 2.2 kbp were resolved, although not so well as in other gels. Bromphenol blue concentrated as it entered the gel and it also changed the color (to paler blue). At the end of the run bromphenol blue migrated approximately the

same distance as the 1 kbp band from the 1 kbp ladder. This gel was mechanically weaker than the previous two gels and had a tendency to detach from the plastic support.

Example 20. Electrophoresis in the gels prepared from N-acrvloyl-N-hexyl-1- amino-1-deoxv-D-g : lucitol (NHAGU). acrylamide and N. N'-methylene-bis-acrylamide in the presence of 0. 02% SDS.

The gels (T= 12% C= 1%) contained various molar ratios of acrylamide and the N-hexyl monomer. The gel was polymerized in 0.375 M Tris-HCl pH 8.8 containing 0.02% SDS in glass tubes (inner diameter 4 mm, length 7 cm). A stacking gel (T=4%, C= 1%) was polymerized in 0.125 M Tris-HCl pH 6.8. The running buffer was 50 mM Tris-0.384 M glycine buffer pH 8.3. Bovine serum albumin was heated in the stacking gel buffer containing 1% SDS and 3% mercaptoethanol and applied to the gel.

The gels were run until bromphenol blue in the control gel reached the bottom of the gel. Proteins were detected by Coomassie Brilliant Blue R-250 staining. As shown in figure 5, the migration of BSA was dependent on the ratio of acrylamide and the N-hexyl monomer, slightly decreasing in the gels with higher amounts of the amphiphatic monomer. The first gel was polymerized without N-acryloyl-N-hexyl-l-amino-l-deoxy-D-glucitol (NHAGU), the last one with N-acryloyl-l-amino-l-deoxy-D-glucitol (1-NAGA, 20: 1 molar ratio).

Example 21. Electrophoresis in the gels prepared from N-acrYloYl-N-hexyl-l- amino-l-deoxy-D-glucitol (NHAGU). acrylamide and N. N'-methylene-bis-acrylamide in the presence of 0.05% SDS.

The gels were prepared and run as described in Example 20. At this higher concentration of SDS the protein was retarded even in the gel with 160: 1 molar ratio of acrylamide to the N-hexyl monomer, as shown in figure 6. BSA migrated only very little in the gel with the 20: 1 ratio.

Example 22. Electrophoresis in the gels prepared from N-acrvlovl-N-hexyI-1- amino-l-deoxy-D-glucitol (NHAGU). acrvlamide and N. N'-methylene-bis-acrvlamide in the presence of 0.1 % SDS.

The gels were prepared and run as described in Example 20. As can be seen from figure 7, in the 90: 1 gel BSA migrated approximately half the distance it migrated in the gel without NHAGU (first gel) and it hardly entered the 20: 1 gel.

Example 23. Electrophoresis in the gels prepared from N-acrvlovl-N-hexvl-l- amino-1-deoxv-D-glucitoI (NHAGU). acrylamide and N. N'-methylene-bis-acrylamide in the presence of 0.2% SDS.

The gels were prepared and run as described in Example 20. Figure 8 shows that in the 160: 1 gel BSA migrated less than half the distance it migrated in the gel without NHAGU (first gel). The protein remained on the top of the 40: 1 and 20: 1 gels.

REFERENCES 1. Kozulic, B. European Patent Application, 88810717.4 (EP 0367886) 2. Kozulic, M. , Kozulic, B. , and Mosbach, K (1987) Anal. Biochem. 163,506-512 3. Kozulic, B. , Mosbach, K. , and Pietrzak. , M. (1988) Anal. Biochem. 170,478-484 4. Kozulic, B. , and Mosbach, K. (1988) Patent Application, PCT/EP88/00515 (EP 0318551) 5. Park, T. G. , and Hoffman, A. S. (1990) Journal of Biomedical Materials Research 24,21-38 6. von Morze, Herwig, European Patent Application 83303047.1 (EP 0095901) 7. European Patent Application 79102502.6 (EP 0007116) 8. European Patent Application 80103828.6 (EP 0022529)

9. Hodge, J. E., and Moy, B. F. (1963) J. Org. Chem. 28, 2784-2789 10. Morgan. S. E., and McCormick, C. L. (1990) Prog. Polym. Sci. 15, 103-145 11. Goubran R. , and Garti, N. (1988) J. Dispersion Science And Technology 9,131- 148 12. Laemmli, U. K. (1970) Nature 277, 680-685 13. Terabe, S. , Otsuka, K. , Ichikawa, K. , Tsuchiya, A. , and Ando, T. (1984) Anal.

Biochem. 56,111-113

Claims

Claims 1. A method of separating molecules on the basis of their hydrophobicity, comprising

subjecting the molecules to an electric field in an electrophoretic gel comprising L immobilised amphiphatic molecules.
2. A method of separating molecules, comprising contacting a mixture of said molecules with a separation medium while being subjected to an electric field, wherein said separation medium comprises a polymer containing hydrophilic and hydrophobic residues, said hydrophobic residues being incorporated into the polymer by a freeradical polymerisation reaction including a monomer having the hydrophobic residue, wherein separation is effected by hydrophobic interactions between said molecules and said hydrophobic residues of the polymer.
3. A method for gel-electrophoretic separation of molecules characterised by electrophoretic mobilities dependent on the hydrophobicity of the gel and the separating molecules using a gel with incorporated immobilized amphiphatic molecules.
4. A method according to claim 1,2 or 3, wherein the electrophoretic gel or separation medium comprises a polymer including an acrylic monomer of the formula:

where Rl is H or (CHOHCHOH, m being 0,1 or 2; Rz is monohydroxyalkyl, polyhydroxyalkyl or hydrocarbon radical having from 2 to 30 carbon atoms, and R is hydrophobic; R3 is H or CH3 ; and n is an integer in the range 1-4.
5. A method according to claim 4, wherein the monomer comprises

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N-acryloyl-N-ethyl-l-amino-l-deoxy-D-galactitol.
6. A method according to claim 4, wherein the monomer comprises N-acryloyl-Npropyl-1-amino-1-deoxy-D-galactitol.
7. A method according to claim 4, wherein the monomer comprises N-acryloyl-N- butyl-1-amino-1-deoxy-D-galactitol.
8. A method according to claim 4, wherein the monomer comprises N-acryloyl-N-

hexyl-1-amino-l-deoxy-D-glucitol.
9. A method according to claim 4, wherein the monomer comprises N-acryloyl-N- octyl-1-amino-1-deoxy-D-glucitol.
10. A method according to claim 4, wherein the monomer comprises N-acryloyl-Nphenylethyl-1-amino-l-deoxy-D-galactitol.
11. A method according to claim 4, wherein the monomer comprises N-acryloyl-N- (2hydroxyethyl)-1-amino-1-deoxy-D-glucitol.
12. A method according to claim 4, wherein the monomer comprises N-acryloyl-N- (2- hydroxyethyl)-l-ammo-l-deoxy-D-gaIactitol.
13. A method according to claim 4, wherein the monomer comprises N-acryloyl-amino- N, N-bis (l-deoxy-D-galactitol).
14. A method according to claim 4, wherein the monomer comprises N-acryloyl-aminoN, N-bis (l-deoxy-D-glucitol).
15. A method according to claim 4, wherein the monomer is substantially as herein described.

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16. A method according to claim 4, wherein the polymer comprises a monomer according to any one of claims 4 to 15.
17. A method according to claim 4, wherein the polymer contains repeating units from only one of the monomers of any one of claims 4 to 15.
18. A method according to claim 4, wherein the polymer contains repeating units from at least two of the monomers of any one of claims 4 to 15.
19. A method according to claim 4, wherein the polymer contains repeating units from a monomer of any one of claims 4 to 15 and repeating units from at least one other monomer having an ethylenic double bond.
20. A method according to claim 4, wherein the polymer is cross-linked by a crosslinker having at least two ethylenic double bonds.
21. A method according to claim 1, substantially as herein described.