Methods and apparatus for the generation of chemical libraries

What is claimed is:

1. A reaction block comprising:

a plurality of reaction chambers;

a first side wall and a second side wall, the first and second side walls having a first width and a first height;

a first end wall and a second end wall, the first and second end walls having a second width narrower than the first width, and the first height;

a first top surface bordered by the first and second side walls and the first and second end walls, the first top surface having:

a plurality of generally circular openings for removably receiving the reaction chambers, the reaction chambers each having a circular opening and a second top surface;

a gas port; and

a plurality of raised beads defining the edges of a plurality of chambers;

the raised beads having a third top surface which is in approximately the same plane as the second top surface of the reaction chambers;

a flexible septum which seals against the second and third respective top surfaces of the reaction chambers and the raised beads; and

a retainer plate which covers the septum.

2. A reaction block as in claim 1 wherein the retainer plate has a plurality of openings which spatially correspond to the openings of the reaction chambers.

3. A reaction block as in claim 2 wherein the retainer plate is held securely against the septum and the reaction block with a plurality of fasteners.

4. A reaction block as in claim 1 wherein the first top surface includes a first, a second, a third, and a fourth, separately controllable gas outlet ports, and wherein the raised beads define a first, a second, a third, and a fourth pressurization chambers. chambers.

5. A reaction block as in claim 4 wherein each pressurization chamber includes an equal number of the reaction chambers.

6. A reaction block as in claim 4 wherein each pressurization chamber includes 12 reaction chambers.

7. A reaction block as in claim 4 wherein the first, second, third, and fourth separately controllable gas outlet ports are in communication with the first, second, third, and fourth pressurization chambers, respectively.

8. A reaction block as in claim 5 wherein each of the pressurization chambers includes a waste basin which is recessed below the level of the second top surfaces of the reaction chambers.

9. A reaction block as in claim 8 wherein each of the four pressurization chambers has 12 of the reaction chambers.

10. A reaction block as in claim 1 wherein each of the generally circular openings on the first top surface have a keying notch, and wherein each of the reaction chambers have a keying protrusion.

11. A reaction block as in claim 1 wherein the block is made of aluminum.

12. A reaction block as in claim 11 wherein the block is made of 6061 aluminum.

13. A reaction block as in claim 11 wherein the aluminum has been anodized.

14. A reaction block as in claim 1 wherein the block is color coded.

15. A reaction block as in claim 1 wherein the block has an identification number.

16. A reaction block as in claim 1 wherein the septum is made from thermoplastic rubber.

17. A reaction block as in claim 16 wherein the septum is die cut with six registration holes and four access holes.

18. A reaction block as in claim 1 wherein the first and second side walls are fitted with one pin each to facilitate securing the block to a work station.

19. A reaction block as in claim 1 wherein the first and second end walls are fitted with two pins each to facilitate handling by a robotic gripper.

20. A reaction block as in claim 7 further including a bottom surface defined by the bottom edges of the first and second side walls and the first and second end walls, the bottom surface being parallel to the first top surface.

21. A reaction block as in claim 20 further including a first, second, third and fourth gas inlet ports in communication with the first, second, third, and fourth gas outlet ports of the first top surface, respectively.

22. A reaction block as in claim 21 wherein the first, second, third and fourth gas inlet ports are in the bottom surface.

23. A reaction block as in claim 22 further including an input port for receiving heating or cooling fluid.

24. A reaction block as in claim 23 wherein the input port for receiving heating or cooling fluid is in the bottom surface.

25. A reaction block as in claim 23 further including an output port for draining heating or cooling fluid.

26. A reaction block as in claim 25 wherein the output port for draining heating or cooling fluid is on the bottom surface.

27. A reaction block as in claim 26 further including a plurality of passages connecting the input port of receiving heating or cooling fluid to the output port for draining heating or cooling fluid.

28. A reaction block as in claim 20 wherein the bottom surface includes a magnet.

29. A reaction block as in claim 28 wherein the magnet is a bar magnet.

30. A reaction block as in claim 20 wherein each of the reaction chambers includes a drain tube.

31. A reaction block as in claim 30 further including a cavity extending upwardly from the bottom surface to a middle surface, the middle surface parallel to the first top surface and the bottom surface.

32. A reaction block as in claim 31 wherein the middle surface has openings for receiving the drain tubes.

33. A reaction block as in claim 32 wherein the cavity includes a machined step to facilitate mating with a microtiter plate.

34. A reaction block as in claim 33 wherein the cavity includes a second machined step to facilitate mating with a bottom seal.

35. A reaction block as in claim 34 wherein the bottom seal includes an o-ring which seals against the second machined step.

36. A reaction block as in claim 34 wherein the bottom seal includes a one-way pressure valve.

37. A reaction block as in claim 22 wherein the first, second, third and fourth gas inlet ports include a quad ring seal.

38. A reaction block as in claim 21 wherein the first, second, third and fourth gas inlet ports include a valve.

39. A reaction block as in claim 38 wherein the valve is a schraeder valve.

40. A reaction block as in claim 38 wherein the valve is a bi-directional elastomeric valve.

41. A reaction block comprising:

a plurality of reaction chambers;

a first side wall and a second side wall, the first and second side walls having a first width and a first height;

a first end wall and a second end wall, the first and second end walls having a second width narrower than the first width, and the first height;

a first top surface bordered by the first and second side walls and the first and second end walls, the top surface having:

a plurality of generally circular openings for removably receiving the reaction chambers, the reaction chambers each having a circular opening and a second top surface;

a gas port; and

a plurality of raised beads defining the edges of a plurality of chambers;

the raised beads having a first top surface which is in approximately the same plane as the second top surface of the reaction chambers;

means for sealing against the second and third respective top surfaces of the reaction chambers and the raised beads; and

a retainer plate which covers the means for sealing.

42. A reaction block as in claim 41 wherein the retainer plate has a plurality of openings which spatially correspond to the openings of the reaction chambers.

43. A reaction block as in claim 42 further including means for fastening the retainer plate to the means for sealing the reaction block.

44. A reaction block as in claim 41 wherein the first top surface includes a first, a second, a third, and a fourth, separately controllable gas outlet ports, and wherein the raised beads define a first, a second, a third, and a fourth pressurization chambers.

45. A reaction block as in claim 44 wherein each of the four pressurization chambers includes an equal number of the reaction chambers.

46. A reaction block as in claim 45 wherein the first, second, third, and fourth separately controllable gas outlet ports are in communication with the first, second, third, and fourth pressurization chambers, respectively.

47. A reaction block as in claim 45 wherein each of the four pressurization chambers includes a waste basin which is recessed below the level of the top surfaces of the reaction chambers.

48. A reaction block as in claim 45 wherein each of the four chambers has 12 of the reaction chambers.

49. A reaction block as in claim 41 wherein each of the generally circular openings on the first top surface have a keying notch, and wherein each of the reaction chambers have a keying protrusion.

50. A reaction block as in claim 41 wherein the block is made of aluminum.

51. A reaction block as in claim 50 wherein the block is made of 6061 aluminum.

52. A reaction block as in claim 50 wherein the aluminum has been anodized.

53. A reaction block as in claim 41 wherein the block is color coded.

54. A reaction block as in claim 41 wherein the block has an identification number.

55. A reaction block as in claim 41 wherein the block has a bar code.

56. A reaction block as in claim 41 wherein the first and second side walls are fitted with one pin each to facilitate securing the block to a work station.

57. A reaction block as in claim 41 wherein the first and second end walls are fitted with two pins each to facilitate handling by a robotic gripper.

58. A reaction block as in claim 46 further including a bottom surface defined by the bottom edges of the first and second side walls and the first and second end walls, the bottom surface being parallel to the first top surface.

59. A reaction block as in claim 58 further including a first, second, third and fourth gas inlet ports in communication with the first, second, third, and fourth gas outlet ports of the first top surface, respectively.

60. A reaction block as in claim 59 wherein the first, second, third and fourth gas inlet ports are in the bottom surface.

61. A reaction block as in claim 60 further including an input port for receiving heating or cooling fluid.

62. A reaction block as in claim 61 wherein the input port for receiving heating or cooling fluid is in the bottom surface.

63. A reaction block as in claim 62 further including an output port for draining heating or cooling fluid.

64. A reaction block as in claim 63 wherein the output port for draining heating or cooling fluid is on the bottom surface.

65. A reaction block as in claim 64 further including a plurality of passages connecting the input port for receiving heating or cooling fluid to the output port for draining heating or cooling fluid.

66. A reaction block as in claim 58 wherein the bottom surface includes a magnet.

67. A reaction block as in claim 66 wherein the magnet is a bar magnet.

68. A reaction block as in claim 58 wherein each of the reaction chambers includes a drain tube.

69. A reaction block as in claim 68 further including a cavity extending upwardly from the bottom surface to a middle surface, the middle surface parallel to the first top surface and the bottom surface.

70. A reaction block as in claim 69 wherein the middle surface has openings for receiving the drain tubes.

71. A reaction block as in claim 70 wherein the cavity includes a machined step to facilitate mating with a microtiter plate.

72. A reaction block as in claim 71 wherein the cavity includes a second machined step to facilitate mating with a bottom seal.

73. A reaction block as in claim 72 wherein the bottom seal includes an o-ring which seals against the second machined step.

74. A reaction block as in claim 72 wherein the bottom seal includes a one-way pressure valve.

75. A reaction block as in claim 59 wherein the first, second, third and fourth gas inlet ports include a quad ring seal.

76. A reaction block comprising:

a plurality of raised beads on a first top surface, the raised beads defining a plurality of chambers;

a plurality of reaction chambers;

a plurality of openings in the first top surface for receiving the reaction chambers; and

the first top surface having a gas port and each reaction chamber having a second top surface;

wherein the raised beads and the second top surfaces of the reaction chambers are in the same plane and sealed with means for sealing.

Description Submit all comments and votes

FIELD OF THE INVENTION

The present invention relates generally to combinatorial synthesis, and more particularly to methods and apparatus for the generation of chemical libraries of known composition.

BACKGROUND

The relationship between structure and function of molecules is a fundamental issue in the study of biological and other chemistry-based systems. Structure-function relationships are important in understanding, for example, the function of enzymes, cellular communication, and cellular control and feedback mechanisms. Certain macromolecules are known to interact and bind to other molecules having a specific three-dimensional spatial and electronic distribution. Any macromolecule having such specificity can be considered a receptor, whether the macromolecule is an enzyme, a protein, a glycoprotein, an antibody, an oligonucleotide sequence of DNA, RNA or the like. The various molecules to which receptors bind are known as ligands.

Pharmaceutical drug discovery is one type of research that relies on the study of structure-function relationships. Much contemporary drug discovery involves discovering novel ligands with desirable patterns of specificity for biologically important receptors. Thus, the time necessary to bring new drugs to market could be greatly reduced through the use of methods and apparatus which allow rapid generation and screening of large numbers of ligands.

A common way to generate such ligands is to synthesize libraries of ligands on solid phase resins. Techniques for solid phase synthesis of peptides are described, for example, in Atherton and Sheppard, Solid Phase Peptide Synthesis: A Practical Approach, IRL Press at Oxford University Press, Oxford, England, 1989. Techniques for solid phase synthesis of oligonucleotides are described in, for example, Gait, Oligonucleotide Synthesis: A Practical Approach, IRL Press at Oxford University Press, Oxford, England, 1984. Each of these references is incorporated herein by reference.

Techniques for solution and solid phase multiple component combinatorial array syntheses strategies include U.S. patent application Ser. No. 08/092,862 filed Jan. 13, 1994, which is assigned to the assignee of the present invention, and which is incorporated herein by reference. Other synthetic strategies that may be employed are described in, for example, Bunin and Ellman, "A General and Expedient Method for the Solid Phase Synthesis of 1,4-Benzodiazepine Derivatives," J. Amer. Chem. Soc. 114:10997-10998 (1992); Bunin et al., "The Combinatorial Synthesis and Chemical and Biological Evaluation of a 1,4-Benzodiazepine Library," Proc. Natl. Acad. Sci. 91:4708-4712 (1994); U.S. Pat. No. 5,288,514 entitled "Solid Phase and Combinatorial Synthesis of Benzodiazepine Compounds on a Solid Support," issued Feb. 22, 1994; and PCT Publication WI 94/08051, Apr. 14 (1994), each of which is incorporated herein by reference.

Since the introduction of solid phase synthesis methods for peptides, oligonucleotides and other polynucleotides, new methods employing solid phase strategies have been developed that are capable of generating thousands, and in some cases even millions, of individual peptide or nucleic acid polymers using automated or manual techniques. These synthesis strategies, which generate families or libraries of compounds, are generally referred to as "combinatorial chemistry" or "combinatorial synthesis" strategies.

Combinatorial chemistry strategies can be a powerful tool for rapidly finding novel ligands to receptors of interest. To date, three general strategies for generating combinatorial libraries have emerged: "spatially-addressable," "split-bead," and "recombinant" strategies These methods differ in one or more of the following aspects: reaction vessel design, polymer type and composition, control of physical variables such as time, temperature and atmosphere, isolation of products, solid-phase or solution-phase methods of assay (i.e., chemical analysis), simple or complex mixtures, and methods for finding or determining the structure of the individual library members.

Of these general strategies, several sub-strategies have been developed. One spatially-addressable strategy that has emerged involves the generation of peptide libraries on immobilized pins that fit the dimensions of standard, 96 well microtiter plates. See PCT patent publication Nos. 91/17271 and 91/19818, each of which is incorporated herein by reference. This method has been used to identify peptides which mimic discontinuous epitopes, Geysen et al., "Screening Chemically Synthesized Peptide Libraries for Biologically Relevant Molecules," Bioorg Med Chem. Lett. 3: 397-404 (1993), and to generate benzodiazepine libraries, U.S. Pat. No. 5,288,514 entitled "Solid Phase and Combinatorial Synthesis of Benzodiazepine Compounds on a Solid Support," issued Feb. 22, 1994 and Bunin et al., "The Combinatorial Synthesis and Chemical and Biological Evaluation of a 1,4-Benzodiazepine Library," Proc. Natl. Acad Sci. 91:4708-4712 (1994). The structures of the individual library members can be determined by analyzing the pin location (in the microtiter plate) in conjunction with the sequence of reaction steps (called a "synthesis histogram") performed during the synthesis.

A second, related spatially-addressable strategy that has emerged involves solid-phase synthesis of polymers in individual reaction vessels, where the individual vessels are arranged into a single reaction unit. An illustrative example of such a reaction unit is a standard 96-well microtiter plate; the entire plate comprises the reaction unit and each well corresponds to a single reaction vessel. This approach is an extrapolation of traditional single-column solid-phase synthesis.

As is exemplified by the 96-well plate reaction unit, each reaction vessel is spatially defined by a two-dimensional matrix. Thus, the structures of individual library members can be determined by analyzing the sequence of reactions to which each well was subjected.

Another spatially-addressable strategy employs "teabags" (i.e., small, porous sacks) to hold synthesis resin. The reaction sequence to which each teabag is subject is recorded. This recorded reaction sequence determines the structure of the oligomer synthesized in each teabag. See for example, Lam et al., "A New Type of Synthetic Peptide Library for Identifying Ligand-Binding Activity," Nature 354:82-84 (1991), Houghton et al., "Generation and Use of Synthetic Peptide Combinatorial Libraries for Basic Research and Drug Discovery," Nature 354:84-86 (1991), and Jung et al., "Multiple Peptide Synthesis Methods and Their Applications," Agnew. Chem. Int. Ed. Engl. 31:367-383 (1992), each of which is incorporated herein by reference.

In another recent development, the techniques of photolithography, chemistry and biology have been combined to create large collections of oligomers and other compounds on the surface of a substrate. See U.S. Pat. No. 5,143,854 and PCT patent publication Nos. 90/15070 and 92/10092, each of which is incorporated herein by reference.

Recombinant methods for preparing collections of oligomers have also been developed. See PCT patent publication nos. 91/17271 and 91/19818, each of which is incorporated herein by reference. Using these methods, one can identify each oligomer in the library by determining the DNA coding sequences in a recombinant organism or phage. However, since the library members are generated in vivo (i.e., within the organism or phage), recombinant methods are limited to polymers whose synthesis can occur in the cell. Thus, these methods typically have been restricted to constructing peptide libraries.

A third general strategy that has emerged involves the use of "split-bead" combinatorial synthesis strategies. See Furka et al., "General Methods for Rapid Synthesis of Multicomponent Peptide Mixtures," Int. J. Pept. Protein Res. 37: 487-493, (1991) which is incorporated herein by reference. In this method, beads are apportioned into smaller groups. These smaller groups (called "aliquots") each contain a number of beads that is evenly divisible into the total number of beads. Each aliquot exposed to a monomer, and the beads are pooled together again. The beads are mixed, reapportioned into aliquots, and then exposed to a second monomer. The process is repeated until the desired library is generated.

A technique for synthesizing labelled combinatorial chemistry libraries is described in co-pending application Ser. No. 08/303,766, filed Feb. 2, 1995, entitled "Methods and Apparatus for Synthesizing Labeled Combinatorial Chemical Libraries," filed Feb. 2, 1995, assigned to the assignee of the present invention, and incorporated herein by reference. In a preferred embodiment of that invention, each synthesized compound is associated with a unique identifier tag. The identifier tag relates a signal to a detector upon excitation with electromagnetic radiation.

To aid in the generation of combinatorial chemical libraries, scientific instruments have been produced which automatically perform many or all of the steps required to generate such libraries. An example of an automated combinatorial chemical library synthesizer is the Model 396 MPS fully automated multiple peptide synthesizer, manufactured by Advanced ChemTech, Inc. ("ACT") of Louisville, Ky.

The Model 396 MPS is capable of generating up to 96 different peptides or other small molecules in a single run. The syntheses occur simultaneously, with one amino acid being added to each growing polypeptide chain before addition of the next successive amino acid to any polypeptide chain. Thus, each growing polypeptide chain contains the same number of amino acid residues at the end of each synthesis cycle.

The syntheses occur in an ACT proprietary plastic reaction block having 96 reaction chambers. While the ACT reaction blocks work for their intended purpose, they possess several shortcomings.

First, ACT reaction blocks are machined from a single piece of plastic. Thus, they require extremely intricate machining, and are quite expensive to manufacture. Furthermore, since ACT reaction blocks are in the form of a single unit, should a portion of a block become damaged or contaminated in some way, the entire reaction block would have to be discarded; there is no way to replace individual portions of an ACT block.

An additional drawback of the plastic ACT reaction blocks is that they cannot be efficiently heated or cooled to aid in chemical reactions that may require such heating or cooling.

Certain processes and chemistries require that the chemical reagents (which may be reactants, solvents, or reactants dissolved in solvents) be kept under an inert or anhydrous atmosphere to prevent reactive groups from reacting with molecular oxygen, water vapor, or other agents commonly found in air. Examples of atmosphere or moisture sensitive chemistries include peptide chemistry, nucleic acid chemistry, organometallic, heterocyclic, and chemistries commonly used to construct combinatorial chemistry libraries. Accordingly, such reagents must be stored and used under an anhydrous or inert atmosphere, such as one of argon, nitrogen, or other gases or mixtures of gases. Typically, containers of such reagents (and containers in which reactions using these reagents take place) are sealed from outside air by a gas impermeable septum. Reagents may be removed from or introduced into a septum sealed container via a non-coring pipetting needle that pierces the septum.

The composition of the septum depends on the chemistry involved, but common materials include thermoplastic rubber (TPR), natural rubber, teflon (typically used as a lining), and EPDM.

While the ACT reaction block can maintain an inert atmosphere when locked in place on the work station of the Model 396 MPS, there is no way to maintain an inert atmosphere once an ACT reaction block is removed from the work station. Thus, the reaction block must remain docked at the work station during the entire synthesis cycle. Since many reactants require several hours to react, this represents significant down time for the Model 396 MPS pipetting station, as it remains idle during the reaction period.

Additionally, creating an effective seal that maintains an inert atmosphere within the ACT reaction block is difficult due to the design of the block. To create such a seal, a top plate fitted with a rubber gasket is clamped onto the reaction chamber using six set screws. The screws are hand tightened to create the seal. The top of the block is machined such that a raised rim or bead separates the 96 reaction chambers into four sections of 24 reaction chambers each. Thus, individual chambers within a group of 24 are not sealed with a raised bead but rather sealed with a flat junction between the septa and the flat top of the machined polymeric reaction block. This design provides an inferior seal and allows solvent from the reaction chambers to cross contaminate reaction chambers within each group of 24 by creeping along the underside of the septa material or alternatively, by creeping along the gas passages machined into the top of the reaction block. Proper adjustment of the screws to distribute pressure evenly across each of the four sections (to create an effective seal) requires careful manipulation and cannot always be accomplished successfully.

A poorly formed seal can also create a problem with reagent cross-contamination. If the gasket does not seal evenly around each reaction chamber, reagents may seep from one reaction vessel into another.

While the ACT reaction block includes 96 reaction chambers, the compounds generated in the ACT reaction block cannot be directly transferred into a standard 96-well microtiter plate because the distance between the outlets of the reaction chambers is too great. For each reaction chamber to have the volume needed to perform reactions, the 96-reaction chamber ACT reaction block must necessarily be too large to mate with a standard 96-well microtiter plate. When reactions are complete, the user must transfer the contents of the reaction chambers into an array of 96 flat bottom glass vials supported in a plastic frame. The user must then manually pipette fluid from the glass vials into a microtiter plate for further analysis. This arrangement presents several disadvantages. First, the glass vials must be cleaned between uses, which increases the chance for contamination. Furthermore, the labor intensive nature of be the transfer increases a chance for error. Finally, this process cannot easily be automated.

The reagent delivery system of the Model 396 MPS also suffers limitations. While the septum-sealed reagent containers from which the reactants are drawn can be sealed under an inert or anhydrous atmosphere, the volume of reagent removed is not replaced with an equivalent or greater volume of inert gas. As reagents are withdrawn from the reagent containers, a partial vacuum is generated within the containers. If the pressure difference between the inside of the container and the external atmosphere is great enough, outside air may seep into the container through needle holes previously made in the septum.

The Model 396 MPS also employs a capacitance detector that can determine the fluid surface level in a reagent bottle. During operation of the Model 396 MPS, fluid is removed from reagent bottles by inserting a pipetting needle just below the fluid surface level such that reagent directly at the reagent-atmosphere interface is withdrawn. While this operation permits only the very tip of a pipetting needle to be contaminated, this operation