Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is organic
Reexamination Certificate
2001-07-03
2003-11-25
Ponnaluri, Padmashri (Department: 1639)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is organic
C435S004000, C436S518000, C436S545000, C436S120000, C546S207000, C546S208000
Reexamination Certificate
active
06653153
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to combinatorial chemistry, and more specifically to combinatorial libraries comprising solid supports in the form of support units (particles, beads and the like), each of which is labeled with one or more metals that provide a code for identifying the compounds that are or were attached to the bead.
BACKGROUND OF THE INVENTION
In the field of combinatorial chemistry, libraries of chemical compounds are made for screening to determine which chemical compounds are active for a particular use, such as agonism or antagonism of a receptor. Usually this screening is carried out by performing assays on each member of the library or groups of members of the library. The compounds that have the desired activity as determined by the assay method are then made on a larger scale for more thorough testing.
Numerous strategies have been designed for testing and tracking the compounds being tested in these mass screenings so that the compounds that have activity in the assays can be readily identified after a positive assay. One of these strategies involves the synthesis of compounds (often referred to as ligands) on the solid support units such that each support unit carries a single compound. The compounds can then be assayed individually, either while they are still attached to the support unit, or more typically, after being cleaved from the support unit. Identification of the compound that is or was on the support unit after a positive assay result is still an ongoing source of difficulty. Since large numbers of support units are used (typically in the hundreds or thousands), the individual support units are not handled and tracked separately. For example, in a split pool synthesis, the support units are synthesized and manipulated in groups for each synthetic step and for assays. Even though each support unit may have only one kind of ligand bound to it, the individual support units are mixed with a large number of other support units, each having a different ligand bound to it. This kind of mass screening makes it impractical or impossible to keep track of the individual ligands as they are synthesized and assayed. As a result, after the assays have been completed, the ligands that are present on the beads that have the desired activity must still be identified. Either the ligand can be analyzed, as by mass spectrometry, or the ligand can be identified based on information contained in the support unit to which it is or was previously attached. To make analysis of the compound (the ligand) being assayed easier, schemes have been developed for encoding the support units by placing chemical markers or “tags” on the support units and then using those tags to identify the chemical compound (ligand) that was originally synthesized on the support unit.
These chemical markers have taken at least two forms. In one, a unique sequencable oligomer, such as a polynucleotide or polypeptide oligomer, is synthesized in parallel with the compound that is being tested on the support unit. The nucleotide or peptide sequence is then determined for the units that have positive assays to determine the compound that has the desired activity in the assay. See for example, WO 93/06121; Brenner, et al., Proc. Natl. Acad. Sci. USA (1992), 89, 5381; Kerr, et al., J. Am. Chem. Soc (1993), 115, 2529; Lebl, Pept. Res. (1993), 6 (3), 161; and Lebl, Proc Natl. Acad. Sci. USA, (1996), 93, 8194. This approach to chemical coding requires the synthesis of a complete second, parallel library of oligomers that serve as chemical markers. This method can be very cumbersome and has the limitation that the syntheses of the oligomer/chemical marker and the molecules being tested must be compatible with one another.
A second approach to marking the support units involves attaching combinations of chemical markers to the support unit. In this approach, the information that identifies the support unit is carried in the combination of what markers are present and what markers are not present, and does not rely on the sequence of the markers. The chemical markers can each be attached directly to the support unit in some way or can be attached to each other and then attached as a group to the support unit. The information needed to identify the chemical compound that was synthesized on the support unit for testing is not retrieved by sequencing the markers, but rather is obtained by determining which markers are present and which markers are absent. This approach is inherently easier, since making and then later analyzing a molecular sequence is much more time consuming and difficult than just creating a code by attaching individual markers to a support and then later determining what markers are present without having to determine the order in which they are attached. Furthermore, only a few kinds of sequences can be determined using automated technology, such as polypeptides and polynucleotides.
The chemical markers can be used to provide a code, based on which chemical markers are present and which markers are absent. One very convenient and efficient kind of code is a binary code, where each chemical marker is represented as a digit in a binary number, with its presence or absence representing the two choices (i.e. “1” or “0”) for the binary digit. Examples of organic chemical markers that have been used in this approach include aryl ether carbenes, which are attached to the support unit at low levels compared with the molecule being synthesized during each step of a split pool synthesis, and are then decoded by cleavage of the aryl carbene residues from the support unit followed by gas chromatographic analysis. See for example, Still, et al. U.S. Pat. No. 5,563,324; Still et al., WO 94/08051; Still et al., WO 95/26640; Still et al., Proc. Natl. Acad. Sci. USA (1993), 90, 10922. Another example of a binary encoding scheme using organic markers is based on secondary amines assembled as N-amidomethyl polyglycines, Ni, et al, J. Med. Chem. (1996), 1601; Gallop, et al., U.S. Pat. No. 5,846,839.
Other methods for encoding support units use physical encoding, such as bar codes, as for example WO 97/15390. Radio frequency has also been utilized, as for example by IRORI, in Ang. Chem. Int. Ed. Engl. (1995), 34 (20), 2289; Ontogene, in J. Am. Chem. Soc. (1995), 117, 10787; and Mandecki, in WO 97/19958. Other marking methods include fluoroescence encoding, as for example in WO 95/32425 and Egner et al., Chem. Comm (1997), 735; and isotope ratio encoding, as for example in Geysen, et al., Chem. Biology (1997), 3 (8), 679; Geysen et al., WO 97/37953; Wagner et al, Combinatorial Chem. High Throughput Screening (1998), 1, 143; and Weinstock et al., WO 97/29371. These methods all have limited utility for general combinatorial libraries.
A variation on chemical encoding involves the use of metal ions rather than organic chemical residues to encode combinatorial libraries on solid supports. See Rink, et al., WO 96/30392, which reference is incorporated by reference into this application in its entirety. In this method, soluble salts (e.g. nitrates) of the lanthanide metal series are absorbed into the support units at each step of the split pool synthesis of the organic compounds (ligands) that are being attached to the support units. The metal salts in solution are added to the beads, which are suspended in the solvent.
A different metal salt or salts may be added to the support after each new step in the split pool synthesis. Analysis of the metal content of the support units and comparison with a key of what the various metals represent enables identification of the compounds.
Although the metal salts are not covalently attached to the support units, they are reported to remain in the beads throughout the synthesis in the form of soluble salts. The inventors report that sufficient quantities of the soluble metal salts “surprisingly” remain in the beads through the subsequent reaction steps so that the presence or absence of the metal salts can be determined at the end of the process by methods t
Chapman Kevin
Wang Tiebang
Xiong Yusheng
McGinnis James L.
Merck & Co. , Inc.
Ponnaluri Padmashri
Tran My Chau T.
Winokur Melvin
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