WO2000050465A2

WO2000050465A2 - Styrene/divinylbenzene copolymers functionalized with a high degree of aminomethyl groups

Info

Publication number: WO2000050465A2
Application number: PCT/BR2000/000023
Authority: WO
Inventors: Clovis Ryuichi Nakaie; Eduardo Maffud Cilli; Guita Nicolaewski Jabilut; Regina Siqueira Haddad Carvalho
Original assignee: Conselho Nacional De Desenvolvimento Científico E Tecnólogico - Cnp9
Priority date: 1999-02-24
Filing date: 2000-02-23
Publication date: 2000-08-31
Also published as: BR9904682A; WO2000050465A3

Abstract

Resins containing apolar styrene-divinylbenzenc-type matrices may solvate in aqueous medium, depending on the amount of positively charged ammonium groups incorporated in its structure. Experiments with a resin containing 4.9 mmol/g aminomethyl groups and denominated aminomethyl-resin (hereafter referred as to EPM-1) showed that this resin presents a good solvation in polar organic solvents and even in water. Based on a special strategy already developed for estimation of pKa of ionizable groups of the resin, it was found a value around 5,5 for the EPM-1 amine group. As model of anionic solutes for chromatographic assays with EPM-1, differently negatively charged peptide fragments were synthesized and tested in chromatographic purification thus demonstrating that EPM-1 can be considered an alternative anion exchange resin also for other anionic solutes. Besides, for containing easily reactive primary amine groups in its structure, this resin can be also precursor for other resins with different chromatographic potentialities. In the same trend, chemical variation involving the divinylbenzene degree of crosslinking of this copolymer may affect its solvation characteristics, porosity, etc., hence modifying the use of this polymeric material.

Description

" COPOLYMER OF STYRENE AND DIVINYLBENZENE FUNCTIONALIZED WITH HIGH DEGREE OF AMINOMETHYL GROUPS (EPM-1) AND ITS USE AS NEW TYPE OF ANION EXCHANGE RESIN"

This invention refers to the use of a copolymer of styrene and 1% divinylbenzene functional ized with high degree of amino-methyl groups for use in column liquid chromatography, as a new anion exchange resin. The chemical synthesis approach of this resin intending to obtain a high amine group-substitution, its physical-chemistry characterization necessary for the correct use in liquid chromatography and finally the purification assay using peptides as model of negatively charged biological material are presented in this invention regarding the use of this polymeric material.

The methodology of the chemical peptides synthesis, denominated of solid phase, (Gross, E. and Meinhofer, J., eds. Acad. Press, N.Y. [1980], The Peptides, vol. 2, 1), and thoroughly disseminated in practically all the areas of the biological field, is based upon the step-wise lengthening of the peptide chain starting from the carboxy-terminal amino acid of the sequence bound to a specific functional group present in the granular resin structure. After the peptide synthesis assembly, the peptide chains are cleaved from the resin for further purification of this biological material. The resin serves therefore as solid support for obtaining the desired peptide sequence. Among the several polymers synthesized, one may mention the chloromethyl-resin (Gross, E. and Meinhofer, J., eds. Acad. Press, N.Y. [1980], The Peptides, vol. 2, 1), that contains choromethyl sites for the binding of the carboxy-terminal amino acid (forming an ester bound) or the benzidrylamino-resin (BAR) (J. Org. Chem. 39, 44 [1974]) and the methyl-benzidrylamino-resin (MBAR), Peptides 2, 45 (1981), containing instead, phenylmethylamine or toluylmethylamine-groups spread throughout the styrene-1 % divinylbenzene copolymer matrix, respectively. Independently of the functional group linked to the polymeric matrix, these resins show a better solvation in apolar solvent as the chloroform or dichloromethane (DCM) and as a consequence, the peptide synthesis is carried out usually with this type of organic solvent. This solvation property of the resin beads can however, be strongly affected if a great amount of amine groups in protonated forms are inserted in its structure. The large amount of positively charged groups induces the resin to start solvating more intensively in polar organic solvents such as dimethylformamide (DMF) or dimethylsulfoxide (DMSO) but also in water. This observation was made previously by the same inventors here, v.g. J. Braz. Chem. Soc. 3, 30 (1992), when investigating the BAR. Based on this solvation properties in aqueous solution, it was proven that this resin could be considered as an alternative resin for anion exchange liquid chromatography, v.g. Hodges, R.S. and Smith, J. A., eds., in Peptides: Chemistry, Structure and Biology, Escom, Leiden, 252, (1994). This resin was therefore the first developed exclusively for use in peptide synthesis but applied alternatively as solid support for column chromatography. Following this same strategy, the synthesis, physical-chemical characterization and the potentiality of its use as anion exchange resin of an other polymer also employed for peptide synthesis will be detailed. It is the aminomethyl-resin (AMR) and is constituted of aminomethyl-groups attached to a styrene- 1% of divinylbenzene resin, v.g. J. Org. Chem. 43, 2845 (1978). It will be demonstrated that, when synthesizing in more forceful conditions, highly amine-substitution is achieved (up to 7 mmol/g of the resin). This extremely high degree of amine group substitution of the resin (well above values about 0,5 mmol/g used commonly for the peptide synthesis) if in the protonated form, induces the resin beads to start swelling in aqueous solution, making possible its use as anion exchange resin in conventional liquid chromatography. As already emphasized, the tested aminomethyl-resin batch that contains 4,9 mmol/g of protonated amine groups will be hereafter referred as to EPM-1 in this text and no more AMR, because besides its conventional use in peptide synthesis, it will have demonstrated its potentiality as anion exchange resin. Although taking into account the 4,9 mmol/g batch as an example, the invention herein applied embraces any lots of AMR presenting high amino group loading and that, in reason of its high solvation in aqueous media, present this chromatographic character. Table I details as follows, for exemplificatory purposes, the more relevant characteristics of the most common commercial anion exchange resins. As a general rule, the majority are of the polar type as dextran or agarose. However, some of them are of the styrene- divinylbenzene-type, whose basic structure is represented in the Figure 1 and are as well comprised herein as in the scope of this invention but containing secondary, tertiary or quaternary amino groups in their structure. The denoted EPM-1, whose feasibility for employment as an anion exchanger support will be here demonstrated, is unique if compared with most commercial resins known so far because it contains reactive primary amino groups in its structure. The steps developed in the process subject of the invention begin with the resin synthesis in peculiar conditions, aiming to obtain high degree of aminomethyl-group incorporation. Next, relevant solvation tests of the resin properties in different solvents (organic or not) are carried out to envisage its chromatographic potential which is strongly dependent upon the swelling degree of resin in each solvation medium. One method recently introduced by the inventors is applied, where a new solvent polarity parameter is proposed based on the swelling of model peptidyl -resins, (J. Org. Chem. 61, 8992 [1996]) therefore diameters (dry and swollen) of resin beads will be measured in several solvents with a microscope. Besides the influence of the solvent, it will also be investigated the effect of the ionic strength of the media on EPM-1 swelling property because salt gradient is usually applied for the elution of ionic solutes in chromatography. These tests will always be carried out comparatively with some of those commercial resins already mentioned.

The previous knowledge of the value of the pKa of the amino group of EPM-1 is also an important pre-requisite for its appropriate use as anion exchange support. In order to obtain this EPM-1 value, we will apply the method developed recently by the inventors, (described in Chromatographia 43, 82 [1996]) as for to the BAR where swelling degree of EPM-1 will be measured in several pHs. Next, these swelling values are correlated with the pH of the media in a figure. As the swelling degree of the resin decreases as the deprotonation of the amine groups increases during the pH elevation, a decreasing sigmodal-type curve is obtained and the middle of the inflection of this curve corresponds to the resin pKa amino group. In the case of the BAR, we have found a pKa around 7 (v.g Chromatographia 43, 82 [1996]). Among the several negatively charged solutes to be tested with EPM-1, peptide sequences containing -1 and -3 net charges in neutral pH are chosen and salt and pH gradient will be comparatively tested during the purification. a) EPM-1 synthesis. The synthesis of the copolymer of styrene with 1% of divinylbenzene functionalized with aminomethyl-groups followed the protocol described previously, in J. Org. Chem. 43, 2845 (1978). This process includes two steps as shown in the Figure 2 and the following steps detail the protocol necessary for the EPM-1 synthesis containing 4,9 mmol of amino groups for gram of the resin in the chloride form. a.1. Copolymer N-(hydroxymethyP-phtalimidomethylation.

In this first step, N-(hydroxymethyl)phthalimide (14 g, 79 mmol) were added to 12 g of copoly(styrene-l% divinylbenzene) resin in 50% TFA/DCM as solvent (180 ml). Next, 50 g of trifluoromethanesulfonic acid (catalyst) were added slowly and under stirring to the suspension. Stirring was continued for 5 hours at room temperature. After the reaction, the resin was filtered and washed 6 times with 50% TFA/DCM, DCM, EtOH and DCM in a sintered glass funnel. The resin was dried under vacuum until constant weight (25,12 g). a.2. Hydrolysis.

20 g of the resin obtained above was stirred under reflux for 24 hours in 5% hydrazine in ethanol (500 ml). After this treatment the resin was quickly filtered (hot) and washed in boiling ethanol and methanol, followed by water, ethanol and DCM washing. The product was dried under vacuum and the amine group degrees (5,9 mmol/g in unprotonated or 4,9 in protonated form, chloridrate) were determined with the picric acid method, v.g. Anal. Chim. Acta 58, 248, (1972), elemental analysis and amino acid analysis (after previous derivation of the resin with alanine residue). The synthesized resin was submitted to a special fractionation treatment in order to obtain a more homogeneously sized population of beads. For this, the synthesized batch was treated with suspension/ precipitation processes in EtOH and in DCM and after drying, it was filtrated in different metal-pore sieves. After this treatment, a 4-5% standard deviation of the bead sizes was achieved. A total of 19 g of EPM-1 was obtained after these treatments. b) Evaluation of the solvation properties of EPM-1. We had already mentioned that although it contains originally an apolar character (styrene with divinylbenzene), the introduction of great amounts of positive charges in its structure, given by protonated amino groups can alter significantly its physical-chemistry property increasing its solvation capacity in polar solvents including water. Table II shows therefore the EPM-1 swelling data in protonated form (Cl^") either in an apolar (DCM) and in some polar solvents (MeOH, DMF, DMSO and water), as comparison. The swelling parameters that we use most (J. Org. Chem. 61, 8992, [1996]) are either the volume of solvent inside the bead (in absolute value) or this percentage value in relation to the total volume of the swollen bead, both calculated starting from the diameters of the dry and swollen data and applying the equation of volume of a sphere. It is confirmed by the data displayed in Table II that higher the ammonium group degree of the resin, the better the swelling in polar solvents. The good swelling measured in water seems to credit this resin to present also an anion exchange function.

The study of the solvation properties of EPM-1 was also extended for solutions where their ionic strength were varied, because liquid chromatography is usually carried out applying the ionic strength or pH gradients. Thus, Table III shows the average swelling of EPM-1 beads in water changing the ionic strength (ammonium acetate) of the solution. Only a small decrease in the degree of swelling is observed for the EPM-1 as the ionic strength ranged from 0.02 to 1.0. c). Estimation of the EPM-1 amine group pKa. As already referred to in the introductory section here, the protocol developed to estimate the pKa of functional groups of resin, v.g. Chromatographia 43, 82, (1996), is based upon the swelling measurement of beads in all pH range of the aqueous solution. By correlating the swelling degree with the pH of the media, a decreasing sigmoidal curve is obtained as the pH increases and the inflection point of this curve corresponds to the pKa of the amino group of the resin. The reduction of the resin bead solvation during the pH increase is due to the disappearance of positive sites in the resin provoked by the ammonium group deprotonation of the resin. Table IV shows the swelling data obtained with a microscope in 2-11 pH range and the mentioned sigmoidal curve is represented in the Figure 3. By considering the inflection point of the curve one may estimate that the amine group pKa of EPM-1 is located around 5.5. The swelling values measured in this study were obtained in the following buffers systems (after previous equilibrium and pre-washing in MeOH to facilitate the further solvation in aqueous media): pH 2 to 2.9: phtalate / HC1; pH 3.3 to 5.5: HC1 / NaAc; pH 5.6 to 7.9: NaH₂PO₄ / Na₂HPO₄; pH 8.0 to 8.9: Tris / HC1; pH 9.0 to 10.3: Na₂CO₃ / NaHCO₃ and pH 10.4 to 12.3: Na₂CO₃ / NaHCO₃ / NaOH. In all cases, the ionic strength of the solution was maintained at 0.05. d). Peptide Synthesis. The present invention is particularly related to the separation of ionic solutes. As na example of these compounds and considering the biological relevance of peptides, this class of macromolecules was selected for the final test of the use of EPM-1 as a novel anion exchange resin. The ionic separation of peptides is mainly based upon the charges of the amine and carboxyl-groups at the peptide extremities but also the ionizable groups present at side chains of some amino acid residues (ex: aspartic acid, glutamic acid, lysine, arginine and histidine). The binding between two or more amino acids forms a peptide that can be electrically neutral, positive or negative, depending on the pKa of each group and of the pH of the medium. With these considerations, we decided to choose the following examples of peptide sequences for testing the EPM-1 chromatographic property:

PI = Asp-Arg-Val-Tyr-Ile-His-Pro-CONH2 (+2) P2 = Asp-Glu-Val-Tyr-Ile-His-for-Phe-COO ^" (-1) P3 = Asp-Glu-Val-Tyr-Ile-Glu-for-Phe-COO ^_ (-3) The peptide PI was used for this study due to its positive charge in neutral pH thus serving as a control for occurrence of unspecific interactions in the EPM-1 matrix (it should elute in the void volume of the column of the resin). The peptides P2 and P3 seems to be valuable models for the present assay as they have equivalent sizes but differing in the net charge at neutral pH. It is expected that this difference in the electric charge between both peptides can be sufficient for their separation with the EPM-1 solid support. All the peptides were synthesized in laboratory by the already solid phase methodology. e) Anion exchange chromatography of peptides with the EPM-1

The chromatographic purifications herein describe were based on the on the following experimental protocol: a mixture of peptides PI, P2 and P3 (about 10 mg each) is applied in a column contening 0,65 g (3,2 mmol of ammonium groups per column) of the EPM-1 equilibrated in a 0.02 M, pH 5.0 ammonium acetate buffer. The swollen volume of resin in this condition was 5.6 mL. e.l). pH gradient. After the injection of the sample, the pH gradient is applied starting from the initial solvent system to AcOH 10% solution (120 mL each). After the gradient, the resin is washed sequentially with 5% AcOH (50 ml) and 50% AcOH (250 mL). The collected solvent volume per tube is 5 mL using a 15 mL/h flow rate and the elution of the peptides was monitored at 275 nm. The Figure 4 depicted the elution profile obtained with this experimental protocol. Worth of note, the positively charged peptide PI was not retained in the column, eluting in the void volume of the column. The negatively charged (-1 and -3) peptides were retained in the cationic resin and eluted separated but with significant retention of the latter which was only eluted from the column when 20% AcOH solution, pH 2 was added to the resin. e.2). salt gradient The ionic strength (or salt) gradient was carried out in the same column above mentioned with a 0.02 to 0.5 M ammonium acetate solutions (pH 5). Figure 5 shows the fractionation profile obtained in these experimental conditions. A very poor resolution of peaks was observed with a significant spreading of the P2 component and until the end of the gradient (0.25) there was no elution of the P3 peptide. At last, if we compare the chromatographic assays, it is possible to conclude that EPM-1 really works as anion exchange resin but with better results when pH instead of salt gradient is used. Certainly, specific alterations in the purification conditions should be tested for each type of negatively charged solute during the purification with this alternative anion exchange resin. In summary, the EPM-1 or any other batch of aminomethylated-resin but containing large amount of positively charged ammonium groups (as a general rule, 1 mmol/g) which present acceptable solvation in aqueous medium can be useful for the anion exchange fractionation of different types of solutes. The present invention comprises therefore all and any use of this resin as anion exchange support, independently of the solutes to be purified. Moreover differing from other commercial anion exchange resins, EPM-1 type polymer contains more reactive primary amino functions thus this patent also claims for rights on any possible EPM-1 derivatives modified at its amino groups and that turn it useful for any other chromatography purpose. As an example, its amine group succinylation will introduce negative sites in the resin thus transforming it potentially in a cation exchange resin. At last the claims made to this invention also involve other EPM-1 derivatives as a consequence of modification in its polymeric matrix (bead size, porosity, density, etc) which may turns them useful also for some type of liquid chromatography. As a representative example, one can synthesize the same EPM-1 but varying the percentage of divinylbenzene- crosslinkage of the resin, and it will fall the same under the scope of this invention as this structural modification certainly affects the physical-chemical properties of the original polymer altering significantly its potentiality.

Claims

1) " COPOLYMER OF STYRENE AND DIVINYLBENZENE FUNCTIONALIZED WITH HIGH DEGREE OF AMINOMETHYL GROUPS (EPM-1) AND ITS USE AS NEW TYPE OF ANION EXCHANGE RESIN ", characterized by the use of a copolymer (styrene- 1% divinylbenzene) functionalized with high degree of aminomethyl groups, as a new ion exchange resin of several types of anionic compounds in liquid chromatography.

2) " COPOLYMER OF STYRENE AND DIVINYLBENZENE FUNCTIONALIZED WITH HIGH DEGREE OF AMINOMETHYL GROUPS (EPM-1) AND ITS USE AS NEW TYPE OF ANION EXCHANGE RESIN ", characterized by having a good solvation property in polar media including aqueous solution due to highly amount of protonated ammonium groups in its polymeric structure.

3) " COPOLYMER OF STYRENE AND DIVINYLBENZENE FUNCTIONALIZED WITH HIGH DEGREE OF AMINOMETHYL GROUPS (EPM-1) AND ITS USE AS NEW TYPE OF ANION EXCHANGE RESIN ", characterized by the use of the above mentioned polymer according to claim 1, synthesized in two steps where the former include the addition of N-(hydroxymethyl)phtalimide (14 g, 79 mmol) in 50% TFA/DCM as solvent (180 ml) upon 12 g of the copolymer(styrene-l% of divinylbenzene) and next, a slowly addition of 50 g (333 mmol) of the trifluoromethanesulfonic acid (catalyst) under stirring; once the addition is completed, the reaction mixture is stirred at room temperature and after the end of the reaction, the resin is collected in a sintered glass funnel and washed 6 times by filtration with 50% TFA/DCM, DCM, EtOH and DCM, being the resin at last, dried under vacuum until constant weight (25,12 g).

4) " COPOLYMER OF STYRENE AND DIVINYLBENZENE FUNCTIONALIZED WITH HIGH DEGREE OF AMINOMETHYL GROUPS (EPM-1) AND ITS USE AS

NEW TYPE OF ANION EXCHANGE RESIN ", characterized by the use of the above mentioned polymer according to the claim 1, obtained by two step synthesis where the second stated with the reaction of the N-(hydroxymethyl)-phatalimidomethylated resin (20 g) with 5% hydrazine/ethanol (500 ml) under reflux for 24 hours; after this period, the resin is quickly filtered (hot) and washed with ethanol and methanol in ebullition, followed by H₂O, ethanol and DCM washings and the obtained dry resin is submitted to a special purification treatment in order to obtain a more narrowly sized population of beads; for this, the resin batch was submitted to suspension/precipitation processes in EtOH and DCM and after drying, the beads were filtrated metal pore-sieves until the lowering of the standard deviation to about 4 to 5%.

5) " COPOLYMER OF STYRENE AND DIVINYLBENZENE FUNCTIONALIZED WITH HIGH DEGREE OF AMINOMETHYL GROUPS (EPM-1) AND ITS USE AS NEW TYPE OF ANION EXCHANGE RESIN ", characterized by presenting its amine group pKa around 5,5. 6) " COPOLYMER OF STYRENE AND DIVINYLBENZENE FUNCTIONALIZED WITH HIGH DEGREE OF AMINOMETHYL GROUPS (EPM-1) AND ITS USE AS NEW TYPE OF ANION EXCHANGE RESIN ", characterized by not showing significant shrinking of beads as the ionic strength of the medium increases. 7) " COPOLYMER OF STYRENE AND DIVINYLBENZENE FUNCTIONALIZED WITH HIGH DEGREE OF AMINOMETHYL GROUPS (EPM-1) AND ITS USE AS NEW TYPE OF ANION EXCHANGE RESIN ", characterized by resin derivatives according to claims 3 and 4 and comprising those synthesized from reaction with dicarboxylic acids which may generate cation exchange resins.

8) " COPOLYMER OF STYRENE AND DIVINYLBENZENE FUNCTIONALIZED WITH HIGH DEGREE OF AMINOMETHYL GROUPS (EPM-1) AND ITS USE AS NEW TYPE OF ANION EXCHANGE RESIN ", characterized by resin derivatives according to claims 3 and 4 and comprising those synthesized from reactions with different amine group acylation agents which may introduce aliphatic or aromatic moieties in the original AMR affecting its physico-chemical properties such as solvation and porosity.

9) " COPOLYMER OF STYRENE AND DIVINYLBENZENE FUNCTIONALIZED WITH HIGH DEGREE OF AMINOMETHYL GROUPS (EPM-1) AND ITS USE AS

NEW TYPE OF ANION EXCHANGE RESIN ", characterized by resin derivatives according to claims 3 and 4 and comprising those containing percentage of the crosslinking divinylbenzene different from the unity.

10) " COPOLYMER OF STYRENE AND DIVINYLBENZENE FUNCTIONALIZED WITH HIGH DEGREE OF AMINOMETHYL GROUPS (EPM-1) AND ITS USE AS

NEW TYPE OF ANION EXCHANGE RESIN ", characterized by resin derivatives according to claims 3 and 4 and comprising those synthesized through different protocols which allow polymers with different surface area, bead size, etc for application as novel solid support for chromatography.