Methods for detection of chloral hydrate in dichloroacetic acid

Details Methods for detection of chloral hydrate in dichloroacetic acid Methods for detection of chloral hydrate in dichloroacetic acid

2002-01-29

2003-11-11

Riley, Jezia (Department: 1850)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S006120, C536S023100, C536S025300

Reexamination Certificate

active

06645716

ABSTRACT:

FIELD OF THE INVENTION
This invention relates generally to novel methods for detecting chloral hydrate in dichoroacetic acid.
BACKGROUND OF THE INVENTION
It is well known that most of the bodily states in multicellular organisms, including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic or other functions, contribute in major proportion to many diseases and regulatory functions in animals and man. For disease states, classical therapeutics has generally focused upon interactions with such proteins in efforts to moderate their disease-causing or disease-potentiating functions. In newer therapeutic approaches, modulation of the actual production of such proteins is desired. By interfering with the production of proteins, the maximum therapeutic effect may be obtained with minimal side effects. It is therefore a general object of such therapeutic approaches to interfere with or otherwise modulate gene expression, which would lead to undesired protein formation.
One method for inhibiting specific gene expression is with the use of oligonucleotides, especially oligonucleotides which are complementary to a specific target messenger RNA (mRNA) sequence. Several oligonucleotides are undergoing clinic trials for such uses. Oligonucleotides can also serve as competitive inhibitors of transcription factors, which interact with double-stranded DNA during regulation of transcription, to modulate their action. Several recent reports describe such interactions (see, for example, Bielinska, A., et. al.,
Science,
250 (1990), 997-1000; and Wu, H., et. al.,
Gene,
89, (1990), 203-209).
In addition to such use as both indirect and direct regulators of proteins, oligonucleotides and their analogs also have found use in diagnostic tests. Such diagnostic tests can be performed using biological fluids, tissues, intact cells or isolated cellular components. As with gene expression inhibition, diagnostic applications utilize the ability of oligonucleotides and their analogs to hybridize with a complementary strand of nucleic acid.
Oligonucleotides and their analogs are also widely used as research reagents. They are useful for understanding the function of many other biological molecules as well as in the preparation of other biological molecules. For example, the use of oligonucleotides and their analogs as primers in PCR reactions has given rise to an expanding commercial industry. PCR has become a mainstay of commercial and research laboratories, and applications of PCR have multiplied. For example, PCR technology now finds use in the fields of forensics, paleontology, evolutionary studies and genetic counseling. Commercialization has led to the development of kits which assist non-molecular biology-trained personnel in applying PCR. Oligonucleotides and their analogs, both natural and synthetic, are employed as primers in such PCR technology.
Oligonucleotides and their analogs are also used in other laboratory procedures. Several of these uses are described in common laboratory manuals such as
Molecular Cloning, A Laboratory Manual,
Second Ed., J. Sambrook, et al., Eds., Cold Spring Harbor Laboratory Press, 1989; and
Current Protocols In Molecular Biology,
F. M. Ausubel, et al., Eds., Current Publications, 1993. Such uses include as synthetic oligonucleotide probes, in screening expression libraries with antibodies and oligomeric compounds, DNA sequencing, in vitro amplification of DNA by the polymerase chain reaction, and in site-directed mutagenesis of cloned DNA. See Book 2 of
Molecular Cloning, A Laboratory Manual,
supra. See also “DNA-protein interactions and The Polymerase Chain Reaction” in Vol. 2 of
Current Protocols In Molecular Biology,
supra. Oligonucleotides and their analogs have also been developed and used in molecular biology in a variety of procedures as probes, primers, linkers, adapters, and gene fragments.
The widespread use of such oligonucleotides has increased the demand for rapid, inexpensive and efficient procedures for their modification and synthesis. Early synthetic approaches to oligonucleotide synthesis included phosphodiester and phosphotriester chemistries. Khorana et al.,
J. Molec. Biol.
72 (1972), 209; Reese,
Tetrahedron Lett.
34 (1978), 3143-3179. These approaches eventually gave way to more efficient modem methods, such as the use of the popular phosphoramidite technique (see, e.g.,
Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach,
Beaucage, S. L.; Iyer, R. P.,
Tetrahedron,
48 (1992) 2223-2311 and references cited therein), wherein a nucleoside or oligonucleotide having a free hydroxyl group is reacted with a protected cyanoethyl phosphoramidite monomer in the presence of a weak acid to form a phosphite-linked structure. Oxidation of the phosphite linkage followed by hydrolysis of the cyanoethyl group yields the desired phosphodiester or phosphorothioate linkage.
Solid phase techniques continue to play a large role in oligonucleotide synthetic approaches. Typically, the 3′-most nucleoside is anchored to a solid support which is functionalized with hydroxyl or amino residues. The additional nucleosides are subsequently added in a step-wise fashion to form the desired linkages between the 3′-functional group of the incoming nucleoside and the 5′-hydroxyl group of the support bound nucleoside. Implicit to this step-wise assembly is use of a protecting group to render unreactive the 5′-hydroxy group of the incoming nucleoside. Following coupling, the 5′-hydroxy group is removed through the judicious choice of a deprotecting reagent.
Dichloroacetic acid (DCA) is a commonly used reagent for deblocking nucleotides during oligonucleotide synthesis. Because the addition of new nucleosides involves the repeated use of dichloroacetic acid for deprotecting the 5′-hydroxy group, it is important that this reagent be as free as possible of contaminants which may propagate impurities and produce improper sequences of the target oligonucleotide. Accordingly, methods are needed for detecting such impurities in dichloroacetic acid. The present invention is directed to these, as well as other, important ends.
SUMMARY OF THE INVENTION
It has been discovered that chloral hydrate is a common contaminant in commercially prepared dichloroacetic acid. It has been further discovered that chloral hydrate reacts with the 5′-hydroxy group of nucleosides during the course of oligonucleotide synthesis to form undesired side-products that are removed, if at all, only with great difficulty. It has been further discovered that chloral hydrate in dichloroacetic acid can be detected and its concentration accurately measured, by comparing the integral of a nuclear magnetic resonance peak of the CH proton of chloral hydrate with a known amount of internal standard.
Accordingly, it is an object of the present invention to provide methods for detecting chloral hydrate in dichloroacetic acid.
It is a further object of the present invention to provide methods for measuring the concentration of chloral hydrate in dichloroacetic acid, particularly, for detecting chloral hydrate and measuring its concentration in dichloroacetic acid which is to be used as a deprotecting reagent in oligonucleotide synthesis.
It is a further object of the present invention to provide methods for preparing oligonucleotides that are free of the impurity which is caused by chloral hydrate present in dichloroacetic acid.
These, as well as other important objects, will be become apparent during the following detailed description.
DETAILED DESCRIPTION OF THE INVENTION
In a first embodiment, the present invention provides an analytical method comprising determining whether or not a nuclear magnetic resonance spectrum taken from a sample of dichloroacetic acid includes a nuclear magnetic resonance peak associated with a CH proton of chloral hydrate. In certain preferred embodiments, the method further comprises comparing an integral of said nuclear magnetic resonance peak associated

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