Drug – bio-affecting and body treating compositions – Solid synthetic organic polymer as designated organic active... – Ion exchange resin
Reexamination Certificate
2000-02-17
2002-09-10
Page, Thurman K. (Department: 1615)
Drug, bio-affecting and body treating compositions
Solid synthetic organic polymer as designated organic active...
Ion exchange resin
C424S124000
Reexamination Certificate
active
06447764
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method for isolating organic compounds with anionic character from aqueous systems using cationic polymer nanoparticles.
BACKGROUND OF THE INVENTION
The isolation and characterization of anionic organic compounds represents a big challenge particularly in environmental analysis and in the fields of biotechnology. Frequently, compounds which are to be isolated are present in aqueous systems at high dilution and/or in combination with other classes of substances. Therefore, a suitable extraction method must fulfill the following requirements:
selectivity that is as high as possible
no necessity of a previous derivatization of the substances to be isolated
effective separability of the exracts from the aqueous system
simple concentration and release of the isolated substances in high purity.
The quantitative detection of environmentally relevant and deleterious substances in the ground and water is becoming more important. Hence, it is of great interest to not only go below the limit of detection of the previous customary methods but to also obtain information on the exact composition of the substance mixtures for several classes of substances such as, for example, nitrophenols, chlorophenols and phenoxycarboxylic acids that can be present in drinking water or ground water as impurities.
Up to now, phenols and phenol derivatives have been mainly determined as a cumulative parameter according to German standard specification (DIN) 38409 part 16 (H16). This determination of the phenol index results from a color reaction with 4-aminoantipyrin and does not give any information on the composition of the sample but only on its total phenol content. Solid phase extraction methods with PR C-18 phases are also applied, among others, for isolating the substance classes of nitrophenols and chlorophenols. However, for this it is necessary to derivatize the samples before the actual extraction, for example, by the reaction with acetic anhydride. With different reactivity of the individual phenol derivatives, this additional reaction step can lead to a falsification of the results of the analysis. Similar problems arise with the extraction of phenoxycarboxylic acids that also have to be derivatized before the solid phase extraction.
The isolation of anionic organic systems in the form of peptides, nucleic acids and nucleic acid derivatives also plays an important role in the field of biological, pharmaceutical and medicinal applications. For example, synthetic oligonucleotides with their polyanionic character represent a new class of therapeutic substances that are employed, for example, in the antisense and antigen strategy for controlling gene expression. Usually, modified nucleic acids are used for this which are only composed of a small number of nucleotides of up to approximately 25 nucleotide units. In order to obtain information on the metabolism and the pharmakinetics of these active ingredients within the course of pre-clinical and clinical studies, it is necessary to isolate the employed nucleic acids and their degradation products from biological media such as blood, urine or cell extracts. In order to make them accessible for characterization by means of sensitive analytical methods, the substances should be present in high yield and purity after separation and, in particular, should not have any impurities due to foreign electrolytes.
Up to now, oligonucleotides and their derivatives have mainly been extracted from biological media via chromatography using ion exchange resins. The elution of the nucleic acids ensues after the separation of the serum components with buffers of high ionic strength. Consequently, the high salt content of the samples necessitates additional purification steps for de-salting in order to permit characterization of the material. The use of a two-stage solid phase extraction for isolating oligonucleotides from human blood plasma using a combination of ion exchange chromatography and reversed phase chromatography is described in J. M. Leeds, M. J. Graham, L. Truong, L. L. Cummins, Anal. Biochem. 235, 36-43 (1996). This method includes a membrane dialysis for removing salt residues as a further processing step after the extraction. The samples can only then be employed for capillary electrophoretic analysis. The low rate of retrieval of approximately 40% and the fact that it is limited to the isolation of oligonucleotides with more than 15 nucleotide units are disadvantageous with this method.
For some applications with larger DNA fragments it is necessary to extract these from complex buffer systems and solutions containing enzymes. Thus, for example, aside from polymerases and buffer salts, a PCR reaction often contains certain detergents that are required for stabilizing the enzyme. Classically, the separation of nucleic acids from aqueous solutions occurs by means of phenol extraction or by precipitation with ethanol. The use of inorganic carrier materials based on silica gel for extracting nucleic acids with a length of 40 base pairs to 50 kilobases is described, for example, in WO 9521177-A1. The adsorptive binding of nucleic acids to the glass surface occurs at a pH value smaller than 7.5 in the presence of high salt concentrations with addition of a chaotropic salt. The release of the bound nucleic acids is achieved by decreasing the ionic strength. The isolation of a plasmid from cell lysates using extraction columns that contain the two adsorbents—an ion exchange resin and silica gel—in two different segments is described in DE 4139664-A1. In this case, the nucleic acids to be isolated are first applied to the anion exchanger segment with a buffer of low ionic strength, washed and subsequently eluted onto the silica gel segment with a buffer of high ionic strength. After a further wash step, the nucleic acids are eluted with buffers of lower ionic strength.
The use of magnetic microparticles for separating polynucleotides is described in EP 281390-A2, for example. In this case, binding occurs over an amino group of the carrier material in phosphate buffer containing detergent. After magnetic separation of the charged particles, the nucleic acids are released by addition of phosphate buffer containing 50% formamide.
DESCRIPTION OF THE INVENTION
An object of the present invention was to develop a simple and inexpensive method for isolating anionic organic substances from aqueous systems. The term aqueous system here means a system in which water is a component, usually the main component, for example diluted solutions, biological media and complex buffer systems. The method is suitable for extracting low molecular compounds with anionic character such as, for example, phenol derivatives, as well as for isolating high molecular, polyanionic substances such as, for example, peptides, nucleic acids and their derivatives.
The fundamental principle of the method is based on the use of cationic polymer nanoparticles with covalently bound pH sensitive groups, especially terminal groups. The selective binding of the substances to be isolated to the particles is ensured by the cooperation of two interaction forces that are independent of each other, electrostatic and hydrophobic interactions. The affinity of the present substances for the particle surface is dependent on its anionic character as well as on its hydrophobicity. This combination permits a selective separation of the substances to be isolated from complex mixtures and highly diluted solutions by suitable selection of the extraction and wash conditions. Basic groups present on the particle surface bring about the electrostatic interactions. This part of the interaction forces can be influenced by alterations in the pH value of the medium. Thereby, the substances bound to the particle can be selectively released after their isolation by abolishing the electrostatic interactions. This allows the provision of a method for isolating anionic organic substances from aqueous systems, characterized in that these substances are reversibly bound to cation
Bayer Ernst
Fritz Hans
Gerster Michael
Maier Martin
Schewitz Jens
Degussa - AG
Fubara Blessing
Juneau Todd L.
Nath Gary M.
Nath & Associates PLLC
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