Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-11-23
2003-04-01
Horlick, Kenneth R. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007500, C536S023100, C536S024300, C536S024330, C436S518000
Reexamination Certificate
active
06541203
ABSTRACT:
BACKGROUND OF THE INVENTION
The goal of pharmaceutical chemistry has always been to identify chemical compounds that have the ability to affect specific biological pathways or reactions so that they are useful as therapeutic agents or as probes of biological activity. Recent developments in synthetic chemistry have greatly expanded scientists'ability to rapidly produce potentially interesting chemical compounds, and to assay their biological and chemical activities. Most notably, the burgeoning field of combinatorial chemistry provides techniques for the rapid and facile generation of large numbers of compounds (see, e.g., “Combinatorial Chemistry”,
Chem. and Eng. News
, Feb. 24, 1997, p. 43; Thompson, L. A., Ellman, J. A.,
Chem. Rev
. 1996, 96, 555).
Combinatorial methods are available for use in the solution phase, the solid phase, or combinations thereof. One particularly powerful solid-phase technique, known as “split and pool” synthesis, allows large numbers of compounds to be produced, each of which is separately attached to its own solid support (Furka, A. et al., Int.
J. Pept. Protein Res
. 1991, 37, 487-493). However, one complication of this method is that, because the solid supports are recovered as mixed pools, either deconvoluting or encoding strategies are required to determine the chemical structure of compounds with desired activities.
A large variety of different deconvolution and encoding techniques have been developed to facilitate the analysis of chemical compounds produced by split-and-pool techniques (See, e.g., Czarnik, A. W., Curr. Op.
Chem. Biol
., 1997, 1, 60). One of the earliest encoding methods employed oligonucleotide tags for identification of libraries of random oligomers (WO 93/06121). Although an advantage of this method is that tag analysis can be readily accomplished through polymerase chain reaction amplification (PCR), the tags are not sufficiently stable to survive the synthetic conditions required for production of small molecule libraries.
Other available encoding schemes include the use of fluorophenyl ether tags (Ohlmeyer et al.,
Proc. Natl. Acad. Sci. USA
1993, 90, 10922; Nestler et al.,
J. Org. Chem
. 1994, 59, 4723). These tags have the advantage that they are resistant to most of the reaction conditions used for organic synthesis. However, the tags are analyzed by gas chromatography and electron capture, and therefore cannot be assayed without first being cleaved from the solid support. Difficulties are often encountered in attempts to analyze the small quantities of tag that are released in these methods.
There has been one report of a tag compound, 3, 5-dimethoxy benzoic acid labeled with
13
C, being analyzed directly on a solid support (a Wang resin), without first being released by cleavage (
J. Am. Chem. Soc
. 1996, 118, 2305; WO 97/14814). However, the method described lacked sensitivity, and required time-intensive
13
C nuclear magnetic resonance (NMR) spectroscopy techniques. Unfortunately, the technique therefore cannot be practically applied to the rapid analysis of large numbers of compounds.
Therefore, there remains a need to develop an improved system for encoding reaction history or direct structural information about chemical compounds synthesized on the solid phase. Preferably, the system should allow on-bead analysis, and should employ chemically robust detectable tags.
SUMMARY OF THE INVENTION
The present invention provides an improved system for encoding the reaction history and/or molecular structure of chemical compounds attached to a solid support. In general, the invention provides an identification unit comprising a binding pair in which a first ligand, component one, is associated with the solid phase, and a second ligand, component two, is employed to specifically recognize the first ligand. Component one need not be separable from the solid support. Component one is sometimes referred to herein as the “tag”.
In certain preferred embodiments of the invention, component one is attached to the solid phase during the synthesis of the chemical compound. In such circumstances, component one preferably designates a particular reaction step so that the synthetic history of the chemical compound is recorded in a series of component ones attached to the solid support. Each component one is then preferably detected by means of its component two binding partner, after the synthesis is complete.
Component two may be any ligand capable, of specific interaction with a component one. Component two need not be chemically robust, as it is not present during the synthesis reactions. However, component two must be selected so that its interaction with component one is detectable. Any method of detection is sufficient. For example, component two may include (i.e., may be covalently linked to) or be otherwise associated with (i.e., by other than covalent linkage) a fluorescent, luminescent, or radioactive moiety. In certain preferred embodiments, component two is associated with a detectable moiety such as a nucleic acid molecule (having a selected nucleotide sequence defining a particular component one-component two interaction) whose signal is subject to amplification (e.g., by PCR), so that the detectable moiety can be identified even when present at very low levels. The steps involved in detecting this binding interaction preferably include, (1) providing a solid phase, a chemical compound and a tag, (2) contacting the tag with the binding partner, and (3) detecting a detectable moiety, which detectable moiety may be any moiety capable of being identified.
The present invention also provides methods of identifying chemical compounds attached to a support by (1) providing a solid support to which a chemical compound whose structure is to be determined is attached, along with a tag selected to represent a structural or synthetic feature of the chemical compound; (2) contacting the solid support with a binding partner that binds specifically and detectably to the tag; and (3) detecting binding of the binding partner to the tag, the existence of such binding being indicative of the presence of the tag on the solid support, which presence is in turn indicative of the existence of the structural or synthetic feature of the chemical compound. Preferably, (1) the step of providing involves providing a solid support comprising a plurality of attached tags, each of which is selected to represent a particular structural or synthetic feature of the chemical compound; (2) the step of contacting comprises (a) providing a plurality of binding partners, each of which specifically and detectably binds to one tag; and (b) contacting the solid support with each of the binding partners (simultaneously or sequentially); and (c) the step of detecting comprises detecting each binding partner/tag binding interaction, and thereby determining the existence of the structural or synthetic features of the compound.
Finally, the present invention provides methods for analyzing the tags off the bead. Specifically, the present invention provides techniques for creating spatially encoded split and pool libraries using the tags and binding partners described herein. According to this aspect of the present invention, (1) the beads, with attached compounds and tags, are distributed into microtiter plates at one bead per well; (2) the tags are detached from the beads by a specific releasing chemistry; (3) the tags are arrayed onto a slide; and (4) the compound represented by the tags are identified by the steps comprising (a) providing a plurality of binding partners; (b) contacting the tags arrayed on each slide with each of the binding partners; and (c) detecting each binding partner/tag binding interaction.
In another aspect, the present invention provides a kit comprising (1) a collection of chemically robust tag components capable of attachment to a solid support, and (2) a corresponding collection of detectable binding partners, each of which interacts selectively with one tag component.
Definitions
“Encoded combinatorial library” An enc
Choate Hall & Stewart
Herschbach Jarrell Brenda
Horlick Kenneth R.
President and Fellows of Harvard College
Wilder Cynthia
LandOfFree
Detecting structural or synthetic information about chemical... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Detecting structural or synthetic information about chemical..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Detecting structural or synthetic information about chemical... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3007117