Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
1997-04-04
2001-07-17
Gerstl, Robert (Department: 1626)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
active
06262257
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the fields of separation technology, environmental remediation, and biomedical applications such as dialysis and drug delivery. More particularly, it concerns ion- and neutral molecule-binding, ion- and neutral molecule-separation or ion- and neutral molecule-detection, using calix[n]pyrrole, calix[m]pyridino[n]pyrrole, or calix[m]pyridine macrocycles. A new type of liquid chromatography is provided herein, that of Hydrogen Bonding Liquid Chromatography, that is based on noncovalent interactions and provides efficient and effective separation of heretofore difficult-to-separate anions and molecules.
BACKGROUND OF THE INVENTION
Anions play essential roles in biological processes; indeed, it is believed that they participate in 70% of all enzymatic reactions. There is, therefore, intense effort being devoted to the problem of anion complexation and recognition. In the molecular recognition arena, a number of research groups have followed Nature's lead and have designed and synthesized receptors that use hydrogen bonds alone, or in concert with electrostatic interactions, to coordinate to anions. Nonetheless, there remains at present a critical need for additional anion complexing agents that are either easy to make or inherently selective in their substrate binding properties.
Additionally, the separation of anionic mixtures using chromatographic techniques involving anion binding interactions is a little studied area of chemistry. Current techniques for purification of anions such as oligonucleotide fragments, polyphosphate-containing molecules such as ATP, ADP and AMP, carboxylates, or N-protected amino acids involve derivatization prior to separation, leading to decreased yields and cumbersome methodology, or involve a salt-separation step following chromatography. This is a very important area to both scientists and clinicians as both mono- and di-nucleotides, natural and synthetic oligonucleotides play critical roles in modern biotechnology as well as medicine. Oligonucleotides are used, for instance, as hybridization probes in blot analyses, primers for PCR amplification, and for site-specific mutagenesis. Furthermore, in some areas, oligonucleotide-derived products are currently being used as probes for the detection of genetic diseases and for proviral HIV, the causative agent of Acquired Immunodeficiency Syndrome (AIDS). Oligonucleotides are also being considered as potential chemotheraputic agents, both directly, i.e., in gene therapy, and in an antisense fashion.
The above-mentioned applications require oligonucleotide materials of impeccable purity (often greater that >99.99%). Such purity, however, is not readily obtained using existing technology. Presently, gene products and other oligonucleotide-type materials are purified using polyacrylamide gel electrophoresis (PAGE). This approach suffers from the requirement of using toxic materials and is painfully manpower-demanding and low-yielding.
Liquid chromatographic techniques, in particular, high performance liquid chromatography (HPLC) and High Performance Affinity Chromatography (HPAC) employing speciality silica gels are currently used to separate biological molecules. Indeed, silica gel phases with bonded groups, such as linear hydrocarbons, amino groups, cyano-groups, carboxylic amides and amino acids, are all known. Unfortunately, few, if any, of these phases are efficacious for the efficient, high-yielding separation of nucleotides and oligonucleotides. Those that work best for this purpose are ion exchange columns which, on a limited basis, can sometimes separate oligonucleotides containing 40 or fewer residues. However, this technique still suffers from several limitations, including the requirement for severe conditions, such as elution at pH 2.7, for routine operation. The use of high concentration buffers (greater than 1 M) and gradients that often include formic acid or formamide, also limits the half-lives of ion exchange columns. Reverse phase columns use column media, such as silica gel with appended groups such as alkyl chains, that separate species on the basis of hydrophobic effects. Reverse phase columns may be used at pressures up to 5000 psi. However, in order to separate species such as oligonucleotides on this type of column, protecting groups must first be appended to the oligonucleotide, and ion-pairing reagents must be used, requiring an additional purification step after the chromatographic separation.
A type of stationary phase has been previously described in which purine and pyrimidine bases are bonded to silica gel. With this support, base-pairing interactions between the nucleic acid base pairs and the modified silica gel were expected to improve the resolution obtained in nucleic acid separation procedures. Although such stationary phases were used to separate nucleic acid-free bases, purine alkaloids, nucleosides, and mono- and oligonucleotides, this approach unfortunately has demonstrated the least success in the case of oligonucleotide separation, which is the most important area. Thus, prior to the present invention, there remained a critical need for improved solid supports that would effectively separate nucleotides and oligonucleotides.
Additionally, sapphyrin (an expanded porphyrin known to bind anions) has been attached to a functionalized silica gel. The separatory properties of this material were not sufficient to separate oligonucleotides successfully due to broad peaks observed for (3- to 9-mer) oligonucleotide mixtures. Furthermore, synthetic challenges are associated with preparing the requisite functionalized sapphyrins since they require over 20 synthetic steps to produce.
Current technology for dialysis in medical applications relies on membranes, such as microfiltering cellophane, to filter anions such as chloride anion or phosphate-containing anions from the blood stream. Aluminum hydroxide or calcium carbonate cocktails must be consumed by the dialysis patient in order to bind the anionic species. A major drawback of this technology is that aluminum builds up in cellular membranes to toxic levels over time causing ailments including dementia and death. Calcium carbonate offers a less toxic substitute, however, it is less efficient and is associated with hypercalcemia.
Water-soluble anion binding agents are desired as drug delivery agents. For example, many anti-viral drugs only show activity when phosphorylated. However, many phosphorylated drug derivatives are too polar to pass through cell wall membranes. A water-soluble anion binding agent may be able to encapsulate the negative charge and so allow the drug to pass though cell walls.
The synthesis of new molecular devices designed to sense and report the presence of a particular substrate is an area of analytical chemistry that is attracting attention. The detection of anionic species is a particular challenge, as anions are difficult to bind and are generally larger than cations leading to a smaller charge-to-radius ratio.
Molecular recognition of neutral compounds presents a challenge in the area of supramolecular chemistry. Binding of substrates, such as short-chain alcohols and simple monoamides, is particularly difficult because these molecules have few functionalized sites available for hydrogen bonding, and they lack the large hydrocarbon surfaces necessary to participate in efficient hydrophobic or &pgr;—&pgr; stacking interactions. Association constants for neutral substrate-synthetic receptor complexes are thus generally modest, even though the architectural complexity of the receptors is often high.
Cation binding agents may be useful as sensors for particular cations or as sequestering agents. Additionally, particular cation-complexes may be useful in medicine as imaging agents or in the treatment of disease.
One aspect of the present invention involves calixpyrroles. Calixpyrroles represent a subset of a class of macrocycles that was previously termed porphyrinogens. Porphyrinogens
Allen William E.
Andrievsky Andrei
Brown Christopher T.
Gale Philip A.
Gebauer Andreas
Board of Regents University of Texas System
Gerstl Robert
Thorpe North & Western, llp
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