Electrolysis: processes – compositions used therein – and methods – Electrolytic synthesis – Involving measuring – analyzing – or testing during synthesis
Reissue Patent
2000-05-09
2001-05-29
Bell, Bruce F. (Department: 1741)
Electrolysis: processes, compositions used therein, and methods
Electrolytic synthesis
Involving measuring, analyzing, or testing during synthesis
C422S051000, C422S051000, C422S051000, C422S051000, C422S063000, C422S082000, C422S119000, C422S129000, C422S138000, C422S186220, C422S198000, C422S198000, C422S232000, C422S233000, C422S236000, C436S052000, C436S055000, C436S047000, C436S048000, C436S049000, C436S165000, C436S169000, C206S305000, C206S459500
Reissue Patent
active
RE037194
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the field of combinatorial chemistry and, more particularly, to a technique for performing combinatorial chemistry using high throughput solid phase chemical synthesis within a plurality of thin elongate reaction vessels.
DESCRIPTION OF RELATED ART
Combinatorial chemistry involves the synthesis of a large variety of chemical compounds from a series of reactions or “chemical recipes”. Various combinatorial chemistry techniques have been used to create a large number or library of compounds and these large numbers of compounds can then be screened for various possible biological activities for pharmaceutical, agricultural or other purposes. Typically such a synthesis occurs in successive stages, each of which involves a chemical modification of the then existing molecules.
Geysen, in PCT Patent Appln. No., WO 90/09395 describes an approach to high-throughput synthesis of peptide oligomers by synthesizing these structures as they are attached to an array of “pins” that are dipped into different reaction mixtures. However, using this approach, quantities of synthesized compounds are limited. U.S. Pat No. 5,143,854 to Fodor et al. discloses a similar array synthesis method for peptides, oligonucleotides, and perhaps other oligomers by using lithographic techniques derived from the semiconductor industry, but applied to synthetic chemistry. Again, using these techniques, yields are limited, typically to subpicomoles, chemistries are also limited by the lithographic technique, and biological activities must be assessed on the lithographic array.
The concept of bead-based split synthesis to make large collections of oligomers was first disclosed by Furka et al. in the 14th Int'l Congress of Biochemistry, Prague, Czechoslovakia, Jul. 15, 1988. A mix and split methodology is used whereby solid phase reactive materials are randomly mixed and separated between reaction stages. The reactive materials are then exposed to reactive agents and conditions which are tracked. Dower et al. in PCT Patent Appln. No. WO 93/06121, also disclose a “split synthesis” methodology for synthesizing large numbers of different oligomers while they are attached to bead-based solid phase supports as disclosed by Merrifield in
Science,
vol. 236, Apr. 18, 1986, pgs 341-347. The oligomers on each bead can be identified directly or indirectly using an oligonucleotide “tag” that tracks each synthetic step. However, the quantity available on each bead is generally less than 1 nanomole and the process of identifying compounds involves PCR or some other tedious chemical method.
Still et al. in PCT Patent International Publ. No. WO 94/08051 have disclosed an improved bead-based split synthesis technique which includes a wide variety of chemical reactions not limited to oligomers. The beads disclosed by Still et al. are also “tagged,” but with a binary code utilizing independent chemical entities that are more readily detected and identified using Gas Chromatography techniques. Nonetheless, the quantities of synthetic compound yielded from this technique are still subnanomole and their identification is still based on chemical methods.
Houghten in European Patent Appln. No. 196174, describes yet another method applicable to the synthesis of peptides or oligonucleotides where solid support resin beads are partitioned using inert plastic mesh bags or “tea-bags” where they stay together during synthesis. Many “tea-bags” can be processed using split synthesis to generate sufficient quantities of an array of compounds. The methods as described by Houghten are specific to oligomeric reactions. However, others, like Cody et al., in U.S. Pat. No. 5,324,483, have generalized the basic “tea-bag” principle to include segregating beads during reactions in systems that are compatible with a wide range of chemical reactions. However, these systems are somewhat awkward to use, require that the beads be agitated, require many fluid-tight seals that must allow removal and insertion and may require physically large layout areas for a reasonable number of distinct compounds. In addition, detachment of compounds and delivery to bioassays would be cumbersome for large numbers of compounds.
Others such as Pavia et al., as disclosed in Bioorganic & Medicinal Chem. Letters. (1993), vol. 3, pgs. 387-396 have used robotic or automated systems for the synthesis of compounds in liquid or solid phase (e.g., on beads) with milligram quantities possible and some economies gained. All of these robotic systems allow reasonably flexible programming of parallel synthesis of an adequate quantity of compounds in separate reaction wells. However, these systems are essentially serial in nature and do not enjoy the enormous advantages of the split synthesis methods described above. Operation is generally limited to hundreds or thousands of compounds, and the automation can add extra constraints on the choices or efficiencies of the synthetic reaction steps used. Moreover, the solid phase bead-based systems require agitation as mentioned above.
Beattie et al., in U.S. Pat. No. 5,175,209 and Frank et al., in U.S. Pat. No. 4,689,405 describe a method of synthesizing oligomers in large quantities using mix and split techniques similar to those described above where solid support structures in the form of disks are sorted into reaction vessels and resorted after each step. The identity of the oligomer on each disk is defined by the reaction chamber in which such disk was located during each reaction step. Since the disks are large and can be marked, the sorting is deliberate and direct. Moreover, non-chemical (e.g., mechanical) methods may be used to mark and identify the disks as they go through each synthesis step and thereby identify the structures thereon. The quantities achievable using this method are greater than 1 mg. However, the methods described by Beattie et al. and Frank et al. are specific to oligomers, and the disk support structures do not provide a convenient interface to standard bioassays such as 96-well-plate-based tests. Compounds in solutions extracted from these disks are not easily loaded into standard 96-well plates. The sorting and loading methods for the disks may also be inadequate if the numbers of different structures desired reaches 100,000 or more. Moreover, the mesh-like solid supports described by Beattie et al. and Frank et al. may not be applicable to non-oligomeric chemistries.
Therefore, none of these methods is ideal for synthesizing greater than milligram quantities of non-oligomeric chemical compounds in combinatorial or otherwise large libraries in such a way that sampling such libraries into 96-well plates or other standard bio-assay formats is easily achieved.
It is therefore desirable to obtain a combinatorial chemical library synthesis system which is capable of a wide variety of synthetic operations creating medicinally relevant molecules as well as biopolymers.
It is also desirable to obtain a combinatorial chemical library synthesis system which provides a yield in excess of 1 milligram for each compound in the library with reasonable purity.
It is further desirable to obtain a system of executing combinatorial “mix and split” synthetic strategies for up to 100,000 distinct elements. Such a system should provide for random as well as deliberate mixing, which is important when one desires to reduce the library from the set of all combinations because of chemical or other constraints.
It is further desirable to obtain a combinatorial chemical library synthesis system which conveniently prepares samples for biological screening while allowing for quantitative partial extraction and compatibility with 96-well or high density screening formats.
It is also desirable to provide a combinatorial chemical library synthesis system which facilitates the deliberate sorting and selecting of library members.
SUMMARY OF THE INVENTION
The present invention provides all of the above desirable aspects, and a goal of the present invention is to provide a method of synthesizing a l
Grubbs Robert H.
Kirk Gregory L.
Bell Bruce F.
Goldstein Ronald B.
Heslin & Rothenberg, P.C.
Pharmacopeia Inc.
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