Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Utility Patent
1997-10-14
2001-01-02
Venkat, Jyothsna (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S006120, C435S007100, C436S501000, C436S518000, C548S559000, C554S103000, C554S213000, C562S400000, C562S426000, C562S433000, C562S456000, C562S490000, C562S493000, C568S663000
Utility Patent
active
06168913
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a process of coding and identifying individual members of a combinatorial chemical library synthesized on a plurality of solid supports. The process provides for attaching fluorine containing tags to solid supports that is later decoded by fluorine nuclear magnetic resonance spectroscopy.
BACKGROUND OF THE INVENTION
Mix and split combinatorial chemistry is a synthetic tool that provides libraries of numerous compounds that are structurally related. With this method, the libraries are constructed on a solid support by assembling sets of chemically reactive building blocks (hereinafter “units” or “monomers”) in many possible combinations.
To understand the mix and split method, one should first understand its predecessor, solid phase peptide synthesis. In solid phase peptide synthesis, one set of solid supports (e.g., beads) having reactive functionalities is reacted with an amino acid, followed by another amino acid, and so on. Once the desired polypeptide is formed, one can cleave the peptide from the bead. Thus, for example, if one reacts amino acids A, B and C in that sequence, one can form an ABC tripeptide. Further, one can also react amino acids A, B and C in five other sequences, ACB, BAC, BCA, CAB and CBA. If one were to allow duplication of each amino acid , for example AAA, one could generate up to 27 tripeptides by this method. One drawback to this method is that each tripeptide is individually synthesized, so that 27 syntheses are required to make all permutations of tripeptides made from amino acids A, B and C. On the other hand, at the end of each synthesis, one either has a pretty good idea of which tripeptide was made, or can easily cleave the product off of the bead and identify the compound by traditional analytical methods.
The mix and split method improves on its predecessor by simultaneously adding different monomers to a mixture of beads that already carry various units. Using the A, B and C amino acids as an example, three different pools of beads are reacted with A, B or C, respectively, and then mixed together. Thus, a third of this mixture are beads carrying A, a third are beads carrying B and a third are beads carrying C. This mixture is then split into three pools. Each pool is reacted with A, B or C, respectively. Thus, one pool will contain beads that carry one of AA, BA or CA, another will contain beads that carry one of AB, BB or CB, and the third will contain beads that carry one of AC, BC or CC. The three pools are then mixed together again and split again into three pools and reacted with A, B or C, respectively. Thus, one pool will now carry one of AAA, BAA, CAA, ABA, BBA, CBA, ACA, BCA or CCA, another will now carry one of AAB, BAB, CAB, ABB, BBB, CBB, ACB, BCB or CCB, and the third will now carry one of AAC, BAC, CAC, ABC, BBC, CBC, ACC, BCC or CCC. In nine reactions, the mix and split method generates all 27 tripeptide permutations from A, B and C.
Moreover, the mix and split method is no longer limited to peptide synthesis. Many different chemical units and many different types of reactions can now be used to form libraries of many different classes of compounds by mix and split combinatorial chemistry. Chemical units, both naturally-occurring and synthetic, can include compounds containing reactive functional groups such as nucleophiles, electrophiles, dienes, alkylating agents, acylating agents, diamines, nucleotides, amino acids, sugars, lipids or derivatives thereof, organic monomers, synthons, and combinations thereof. Alternatively, reactions can include alkylation, acylation, nitration, halogenation, oxidation, reduction, hydrolysis, substitution, elimination, addition, and the like. This method can produce non-oligomers, oligomers, or combinations thereof in extremely small amounts. Non-oligomers include a wide variety of organic molecules, e.g., heterocyclics, aromatics, alicyclics, aliphatics and combinations thereof, such as steroids, antibiotics, enzyme inhibitors, ligands, hormones, drugs, alkaloids, opioids, terpenes, porphyrins, toxins, catalysts, as well as combinations thereof. Oligomers include oligopeptides, oligonucleotides, oligosaccharides, polylipids, polyesters, polyamides, polyurethanes, polyethers, poly(phosphorus derivatives) e.g., phosphates, phosphonates, phosphoramides, phosphonamides, phosphites, phosphinamides, etc., poly (sulfur derivatives) e.g., sulfones, sulfonates, sulfites, sulfonamides, sulfenamides, etc., where for the phosphorous and sulfur derivatives the indicated heteroatom, for the most part, will be bonded to C, H, N, O and combinations thereof.
While the split and mix method can quickly generate a diverse number of compounds, this diversity also generates its greatest challenge—identifying individual compounds from the mixture. For example, if one picks up a bead from a mixture of beads carrying AAA, BAA, CAA, ABA, BBA, CBA, ACA, BCA or CCA, how does one determine which tripeptide is attached? Because each bead generally has only a small amount of product, the reaction history and composition of individual beads are hard to determine. In fact, the amounts of product on each bead are so small (depending on the size of the solid support, about 10 picomoles to 1 nanomole), and the structures on each bead are so similar (e.g., BAA vs. ABA), that traditional analysis such as proton or carbon nuclear magnetic resonance spectroscopy (NMR) and mass spectroscopy (MS) are generally insufficient for determining the compound structure on each bead.
Other attempts to analyze combinatorial constructs by tagging the solid support require that the tags be detached for analysis (See, e.g., International Patent Publication No. WO94/08051). However, detachment adds an extra reaction step to the overall construction, and the translation can become garbled during the process of detachment. Further, one still needs to have distinctive tags that are present on the bead in sufficient quantities for decoding.
Various synthetic techniques and strategies are important factors in determining the success of combinatorial chemistry and are well-known in the art. However, to optimize the power of the mix and split method as a synthetic tool, one must develop a method to readily identify the individual compounds attached to each bead of the generated compound libraries. In other words, one must be able to pick one bead from a library of many beads of many different compounds and easily identify the specific member of the library on that bead. U.S. patent application Ser. No. 08/717,710, filed on Sep. 13, 1996 by Hochlowski et al. (pending), discloses that IR or Raman readable tags can be used to code combinatorial libraries without detaching the tag or the library for analysis. However, the CombiChem industry continues to seek alternative coding schemes to expand the utility of the process.
BRIEF SUMMARY OF THE INVENTION
The present invention relates to coding Combinatorial Chemistry libraries synthesized on a plurality of solid supports by attaching “tags” that comprise fluorine containing compounds. The codes are created by varying singular tags, combining different tags and/or varying the ratio of different combinations of tags. By applying appropriate tags at particular stages of the synthesis of a combinatorial library, one can later determine which compound was made on a particular bead by fluorine nuclear magnetic resonance spectroscopy (FNMR).
Coding combinatorial libraries with fluorine tags has many advantages over the prior art. For the most part, fluorine is a robust moiety that is unaffected by the chemical reactions used to construct the library. Further, the fluorine tagged solid support can be read without detaching the tag from the solid support. In fact, this method does not require detachment of either the tag or the library member from the solid support to follow the reaction history of individual beads, or to determine the particular member of the library that is attached to the bead. In addition, the FNMR spectrum is distinctive e
Hochlowski Jill Edie
Norbeck Daniel W.
Sowin Thomas J.
Wade Warren S.
Whittern David N.
Abbott Laboratories
Collins Daniel W.
Ricigliano Joseph W.
Venkat Jyothsna
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