Patent Publication Number: US-2002001541-A1

Title: Apparatus and methods for evaluating the quality of a combinatorial library of compounds

Description:
BACKGROUND OF THE INVENTION  
       [0001] This invention relates generally to the field of quality control, and in particular to the quality control of a combinatorial library of compounds. More specifically, the invention relates to apparatus and methods for evaluating the quality of a library of compounds, typically produced by encoded split-pool synthesis.  
       [0002] The split-pool approach for solid phase synthesis is often used to rapidly create large, diverse chemical libraries of potential therapeutic compounds. This approach yields single beads of solid phase support to which a few hundred picomolecules of product compound are tethered. After synthesis, a pool of beads often can contain several thousand compounds.  
       [0003] It is desirable to verify the success of the synthesis before screening the library against biological targets. Although the library may be tested for activity in a high throughput screen in the event the library synthesis does not proceed as expected, the probability of identifying active compounds in this manner is very low. Even worse, an active component may turn out to be an unexpected (and irreproducible) side product.  
       [0004] Split pool synthesis is a method of producing a large number of compounds utilizing a minimal number of reaction steps in multiple reaction vessels. Exemplary split pool synthesis processes are described in U.S. Pat. Nos. 5,846,839 and 5,503,805; and in Kenneth C. Lewis et al., “Characterization of a Split Pool Combinatorial Library,” LCGC, Vol. 16, No. 7, pp. 644-649, July 1998; and Z. Ni et al., “A Versatile Approach To Encoding Combinatorial Organic Synthesis Using Chemically Robust Secondary Amine Tags,” J. Med. Chem., 39:1601-1608 (1996). The complete disclosures of all of these references are herein incorporated by reference. Merely by way of example, one exemplary synthesis process proceeds by attaching a photo cleavable linking compound to 90% of the amines on the bead surface while protecting the remaining amino groups with N-(allyloxycarbonyl)(Alloc). The beads are split into 35 pools and the first building block is attached to the photo-linking compound. Dailkylamine tags for the first building block are attached to the 10% residual amines. The beads are pooled and resplit to attach the second building block. Dailkylamine tags for the second building block are attached, and the beads are pooled and resplit to attach the third building block set to the scaffold. The 35 samples are then stored as separate subpools of the library. In this way, each resulting bead contains a desired compound and a series of dailkylamine tags that serve as a record of the synthesis procedure.  
       [0005] The quality control of split pool libraries presents a variety of challenges to analytical chemists. First, such libraries provide a limited amount of compound. For example, the actual amount of compound can vary from less than 50 pmol to more than 1 nmol depending on the size and type of bead used as the solid support. Merely by way of example, some processes may utilize 130 μm d p  Tentagel beads (Nova Biochem, San Diego, Calif.), which can be loaded to approximately 400 pmol/bead. However, the actual loading on such beads is often less due to the variations in bead size, synthetic yield and efficiency of cleavage.  
       [0006] Another set of problems occurs after the sample is separated from the bead. Such problems can include those relating to contamination, adsorption, and the transfer of small volumes. Because the sampling of a split pool library is random, chemists must analyze enough beads to obtain meaningful statistics about the library. Therefore, the analysis method must handle limited quantities of sample in a high throughput manner.  
       [0007] Hence, it would be desirable to provide devices and methods to facilitate the evaluation of the quality of a library of combinatorial compounds. Such devices and methods should be able to handle limited quantities of sample in a high throughput manner. As such, the devices and methods should be compatible with existing analysis and fluid handling equipment. Further, the devices and methods should be efficient to reduce the time required to evaluate the quality of the library.  
       SUMMARY OF THE INVENTION  
       [0008] The invention provides exemplary devices and methods for evaluating the quality of a combinatorial library of compounds. One exemplary device comprises a holding plate having an array of apertures. A plurality of vials are removably held within the apertures, with each vial having an open top end. A seal member is disposed over the top ends of the vials, and a top plate is removably coupled to the holding plate to force the seal member against the top ends of the vials. Such a device is particularly advantageous in that it provides a way to keep the beads in a sealed environment during both ligand cleavage and tag cleavage. In this manner, the beads do not need to be transferred to separate vials during a quality control process. For example, following ligand cleavage, the tags are typically released for analysis by acid hydrolysis. This step typically requires the beads to be heated. By utilizing the device of the invention, the beads may remain within the same vials used for ligand cleavage, and simply placed in an oven during the hydrolysis process.  
       [0009] Conveniently, the device may further include a bottom plate having a cavity for receiving bottom ends of the vials. Preferably, the bottom plate has a standard footprint to allow the device to be utilized with automated equipment, such as autosamplers. In a similar manner, the apertures in the holding plate are preferably spaced apart to correspond to a standard multi-well plate format; e.g., the 96-well format. In this manner, automated equipment may also be used to introduce and remove fluids from the vials. In this manner, the time to perform the quality control process is greatly reduced.  
       [0010] The top plate and the holding plate are preferably constructed of a rigid material that is resistant to acids. For example, the top plate and holding plate may be constructed of an anodized aluminum. Conveniently, a plurality of screws may be provided to removably couple the top plate to the bottom plate. In still another aspect, the seal member preferably comprises a resilient sheet having the same dimensions as the holding plate. The resilient sheet preferably includes a layer of PTFE, and the vials are preferably constructed of glass so that the sheet and the vials will be compatible with the ligand and tag cleavage processes.  
       [0011] In still yet another aspect, the top plate includes a plurality of through holes which are aligned with the vials when the top plate is coupled to the holding plate. In this way, the seal member may be pierced and fluids within the vials extracted through the through holes. Optionally, a resilient pad may be disposed adjacent the holding plate and has openings which correspond to the apertures of the holding plate. The resilient pad is useful in protecting the vials from breaking when the device is assembled.  
       [0012] The invention further provides an exemplary method for evaluating the quality of a combinatorial library of compounds. According to the method, a device is provided which comprises a holding plate and a plurality of vials that are held by the holding plate. At least one solid support is placed into at least some of the vials. Each solid support has a ligand and encoded tags. The solid supports are then sealed within the vials. Once sealed, the ligand is cleaved from the solid supports, and a ligand sample is removed from the vials. Following the ligand cleavage, the encoded tags are cleaved from the solid supports. To cleave the encoded tags, the vials are preferably heated while the solid supports are sealed within the vials. Samples of the cleaved tags are then removed from the vials. The ligand is then compared to a structure indicated by the tags for each solid support.  
       [0013] To seal the solid supports within the vials, a seal member is preferably placed over the vials and a top plate is coupled to the holding plate to force the seal member against the vials.  
       [0014] In one particular aspect, a photo-cleavage process is employed to cleave the ligands from the solid support. Conveniently, the vials may be turned upside-down and then placed under a lamp to photo-cleave the ligands. When the vials are turned upside-down, the solid supports remain within the bottom ends of the vials due to the surface tension of the fluids with the vials.  
       [0015] In another aspect, the top plate has through holes which are aligned with the vials. In this manner, the seal member is pierced to extract the ligand sample from the vials.  
       [0016] In yet another aspect, the encoded tags are cleaved by first removing the top plate and the seal member and introducing a cleaving solution, such as hydrochloric acid, into the vials. A fresh seal member is then placed over the vials and the top plate is replaced to seal the solid supports within the vials. Once properly sealed, the device is transferred to an oven where the vials are heated. Following heating, the top plate and the seal member are preferably removed to allow the cleaving solution to be dried. For example, the device may be placed in a vacuum oven to dry the hydrochloric acid.  
       [0017] Following drying of the cleaving solution, a solvent, such as acetonitrile is introduced into the vials. The top member and the seal member are then replaced. To remove the cleaved tags from the vials, the seal member is pierced and a sample is extracted. Preferably, the ligand sample and the samples of the cleaved tags are evaluated using mass spectrometry. These results are then compared to determine the quality of the combinatorial library of compounds. 
     
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
     [0018]FIG. 1 is an exploded perspective view of an exemplary device for evaluating the quality of a combinatorial library of compounds according to the invention.  
     [0019]FIGS. 2A and 2B illustrate an exemplary method for evaluating the quality of a combinatorial library of compounds utilizing the device of FIG. 1. 
    
    
     DETAILED DESCRIPTION OF THE SPECIFIC EMBODIMENTS  
     [0020] The invention provides exemplary devices and methods for evaluating the quality of a combinatorial library of compounds. In this way, the quality of a library may be determined by analyzing a population of single beads representative of the entire library prior to biological screening of the encoded library. The combinatorial library of compounds is preferably obtained using a split pool synthesis process, such as the processes previously described in the Background section of this application. However, it will be appreciated that the device and methods of the invention may be useful with any combinatorial library of compounds. The invention will finds its greatest use, however, with encoding technologies that utilize dailkylamine tags which are released by acid hydrolysis.  
     [0021] In a particularly preferable aspect, the quality control process involves comparing the ligand on each bead as analyzed by mass spectrometry to the structure indicated by the codes on the particular bead. Conveniently, the code on each bead may also be determined by utilizing mass spectrometry. One particular advantage of the invention is that both ligand cleavage and tag cleavage are accomplished while the beads remain within the same vials. Further, during acid hydrolysis where tag cleavage occurs, the invention provides for placing the vials in a heated environment to facilitate the hydrolysis process. By performing both ligand cleavage and tag cleavage while the beads are within the same vials, the time to complete the quality control process is greatly reduced since no transferring of beads is required. Another advantage of the invention is that the vials are preferably arranged according to standard, well accepted formats. For example, the vials may be placed in a standard 96-well format. In this way, automated equipment may be used to facilitate the quality control process, including robots, auto-samplers, and the like. In this manner, the time to complete the quality control process is further reduced.  
     [0022] Referring now to FIG. 1, an exemplary device  10  that may be utilized when evaluating the quality of a combinatorial library of compounds will be described. Device  10  is constructed of a holding plate  12  having a plurality of apertures  14  which extend through holding plate  12 . Apertures  14  are each configured to receive a vial  16 . Holding plate  12  preferably has a thickness that is small enough so that a bottom end  18  of vial  16  projects beyond a bottom surface of holding plate  12 . Vials  16  each include an open top end  20  into which beads and other fluids may be placed. When vials  16  are inserted into apertures  14 , top ends  20  extend above a top surface of holding plate  12 . Preferably, vials  16  are constructed of glass so that they will be compatible with the solvents and solutions used during both ligand cleavage and tag cleavage. Exemplary vials that may be used with the invention include tapered glass micro-vials, part no. C-4008-632C, commercially available from National Scientific.  
     [0023] Optionally, a resilient pad  22  may be placed on top of holding plate  12  to help prevent vials  16  from breaking when forced against holding plate  12  as described hereinafter. Pad  22  also includes a plurality of apertures which are aligned with apertures  14  of holding plate  12 . Exemplary materials that may be used to construct resilient pad  22  include rubber, soft plastics, and the like. Optionally, apertures  14  may be chamfered at the top surface of holding plate  12  so that resilient pad  22  is not needed.  
     [0024] Positioned above holding plate  12  is a top plate  24 . Top plate  24  includes a plurality of through holes  26  to allow access to vials  16  through top plate  24 . Positioned between top plate  24  and top ends  20  of vials  16  is a seal member  28 . When seal member  28  is forced against open top ends  20 , it forms a seal at each open top end  20 . In this way, the contents within vials  16  are sealed from the external environment. A variety of materials may be employed to construct seal member  28 , with a necessary requirement being that seal member  28  is sufficiently resilient to form a seal at open top ends  20 . One particularly preferable way to construct seal member  28  is by combining a silicon rubber sheet with a PTFE sheet. Merely by way of example, the silicon rubber sheet may be approximately 0.050 inches thick and the PTFE sheet approximately 0.005 inches thick. Seal member  28  is positioned such that the PTFE side is placed adjacent open top ends  20 .  
     [0025] Top plate  24  also includes a plurality of holes  30  which are aligned with threaded holes  32  in holding plate  12 . Corresponding holes are also included in seal member  28  and resilient pad  22 . In this way, screws  34  may be inserted through holes  30  and into holes  32  to secure top plate  24  to holding plate  12 . As screws  34  are tightened, top plate  24  forces seal member  28  against open top ends  20  of vials  16 . As previously described, pad  22  assists in preventing the glass vials from being crushed and broken. Top plate  24  and holding plate  12  are preferably constructed of a rigid material so that they will generally not flex as screws  34  are tightened. Exemplary materials that may be used to construct top plate  24  and holding plate  12  include anodized aluminum, stainless steel, ceramics, and the like. Further, top plate  24 , seal member  28 , pad  22 , and holding plate  12  are preferably all constructed of heat-resistant materials to allow device  10  to be placed in an oven during hydrolysis as described hereinafter.  
     [0026] In summary, top plate  24  may be secured to holding plate  12  to provide a seal over each of vials  16 . Access to vials  16  may be made simply by removing top plate  24  and seal member  28  or simply by piercing through seal member  28  through holes  26 . If the latter option is chosen, the pierced seal member may be replaced simply by removing top plate  24  and inserting a fresh seal member.  
     [0027] Holding plate  12  is preferably configured so that apertures  14  are arranged in a standard format. In this way, device  10  may be utilized with automated equipment, such as robots, autosamplers, and the like. As shown, holding plate  12  is configured in a standard 96-well format. However, it will be appreciated that other arrangements may be possible.  
     [0028] Device  10  further includes a bottom plate  36  which defines a cavity  38 . Bottom plate  36  also includes a plurality of holes  40  into which screws  34  are received when holding plate  12  is placed onto bottom plate  36 . When assembled, bottom ends  18  of vials  16  protrude into cavity  38 . Preferably, bottom plate  36  is constructed of a rigid material and thus provides a degree of protection to the glass vials. Bottom plate  36  preferably has a geometry which is patterned after standard plate formats to enable device  10  to be used with automated equipment as previously described. For example, many robots are configured to accommodate standard sized plates. By utilizing bottom plate  36 , device  10  is useful with such equipment without modification.  
     [0029] Referring now to FIGS. 2A and 2B, an exemplary method for evaluating the quality of a combinatorial library of compounds will be described. Conveniently, reference will also be made to device  10  of FIG. 1 when describing the method. Initially, a sample of beads is selected from the combinatorial library so that they may be analyzed to generate meaningful statistics that reflect the quality of the library. The number of beads sampled is preferably about three times the largest building block set. Merely by way of example, a thiazolidinone library may be constructed using three chemical steps. Thirty-five members are utilized with each of the three building block sets. In this way, the resulting library contains 42,875 members (35 3 ). For this example, the number of samples to be evaluated would be  105  beads/pool (3×35).  
     [0030] As shown in FIG. 2A, the method begins by placing vials  16  into holding plate  12  as shown in step  42 . A statistical sampling of beads is then placed into vials  16  as shown in step  44 . Preferably, each of vials  16  is filled with water prior to transferring the beads. Conveniently, the beads may be transferred into vials  16  using an automated bead-picker. For example, one such bead-picker that may be utilized with device  10  is described in U.S. Pat. No. 5,722,470, the complete disclosure of which is herein incorporated by reference. Conveniently, bottom plate  36  may include various holes in its bottom side to allow device  10  to be properly positioned on the bead-picking apparatus. Preferably, each vial  16  receives a single bead. However, in some cases, more than one bead may be placed into each vial. Once the beads are within vials  16 , the water is removed from vials  16  and the vials are dried. Conveniently, a fluid handling apparatus, such as a Hydra  96  device, commercially available from Robbins Scientific, may be employed to remove the water from vials  16 . Device  10  is then placed in a vacuum drier to dry any remaining water.  
     [0031] As shown in step  46 , ligand cleavage may be accomplished using an acid cleaving process or a photo cleaving process. For acid cleavage, an acid, such as TFA, is introduced into each of vials  16 . After a predetermined time has passed, such as approximately one-half hour, vials  16  are placed in a vacuum oven as shown in steps  48  and  50 . A solvent, such as acetonitrile, is then added to the vials (see step  51 ) to allow samples to be removed from the vials as described hereinafter in step  58 .  
     [0032] If the photo cleavage option is chosen, a solvent, such as methanol, is introduced into each vials  16 . Seal member  28  and top plate  24  are then attached to holding plate  12  to seal the methanol and beads within vials  16 , as shown in step  52 . Device  10  is then positioned by a light source to cleave the ligands from the beads. As one example, photo cleavage may occur by placing vials  16  beneath a 500 UV mercury/vapor discharge lamp. However, other light sources may also be used. Optionally, holding plate  12  may be turned upside-down so that bottom ends  18  of vials  16  are positioned vertically upward. Due to the tapered nature of vials  16  and the fluid tension of the methanol, the methanol and bead stays within bottom ends  16 . Typically, about 25 to about 30 microliters of methanol is inserted into vials  16  so that only a small amount of methanol will be at bottom ends  18 . The turning of holding plate  12  in an upside-down configuration is advantageous in that it conveniently exposes the beads within vials  16  to the lamp (it being appreciated that bottom plate  36  is not employed during photo cleavage). This is shown in step  54  of FIG. 2A. Preferably, vials  16  are held under the lamp for about two hours to cleave off the ligand.  
     [0033] As shown in step  56 , bottom plate  36  is then coupled to holding plate  12  and device  10  is placed in an autosampler to extract samples of the ligand from each of the vials as shown in step  58 . An exemplary auto-sampler that may be employed is a Leap Technologies HTS PAL Auto-sampler, commercially available from Leap Technologies. The auto-sampler has a needle which is separately inserted through each through hole  26 , through seal member  28  and into vials  16  where the sample is extracted. Alternatively, an auto-sampler having an array of needles may be employed. As shown in step  60 , the autosampler then transfers the sample to a mass spectrometer which analyzes the sample. Attachment of bottom plate  36  to holding plate  12  is advantageous in that it properly positions device  10  within the auto-sampler. In this way, the needles will be properly positioned so that they may be inserted through the through holes. An exemplary mass spectrometer which may be utilized with the invention is a LC-MSD, Series 1100 spectrometer, commercially available from Hewlett-Packard.  
     [0034] Following removal of the ligand sample, top plate  24  and seal member  28  are removed from holding plate  12  as illustrated in step  62 . A cleaving solution, such as hydrochloric acid, is then introduced into each of vials  16  to cleave the tags from the beads, as shown in step  64 . Because the seal member was previously pierced, it is replaced with a new seal member, and top plate  24  is again secured to holding plate  12  to seal the hydrochloric acid and the beads within vials  16 , as shown in step  66 . Bottom plate  36  is also attached and device  10  is then placed into an oven to begin the hydrolysis process as shown in step  68 . Preferably, vials  16  are heated to a temperature of about 135° C. for approximately 12 hours. However, it will be appreciated that this temperature and time period may be varied. Conveniently, a Thermolyne 62700 oven may be used. As previously described, the components of device  10  are constructed so that they are compatible with such an elevated temperature.  
     [0035] Following hydrolysis, top plate  24  and seal member  28  are removed as shown in step  70  of FIG. 2B. The hydrochloric acid is then dried, preferably by placing holding plate  12  within a vacuum oven. As shown in step  72 , a solvent is then added to each of the vials to allow the cleaved tags to dissolve within the solvent. In this manner, a convenient way is provided to remove samples of the targets from each of the vials. Preferably, acetonitrile is placed into each of the vials. Top plate  24  and seal member  28  are then adhered to holding plate  12  as shown in step  74 .  
     [0036] Once the tags have dissolved into the solvent, device  10  is placed back on the auto-sampler which pierces through seal member  28  and extracts a sample of the cleaved tags as shown in step  76 . These samples are then placed in the mass spectrometer as shown in step  78 . An exemplary mass spectrometer that may be utilized is a LC-MSD, Series 1100 spectrometer, commercially available from Hewlett-Packard.  
     [0037] As an alternative, following step  70 , the tags may be converted to dansyl derivatives as described in Kenneth C. Lewis et al., “Characterization of a Split Pool Combinatorial Library,” LCGC, Vol. 16, No. 7, pp. 644-649, July 1998; and Z. Ni et al., “A Versatile Approach To Encoding Combinatorial Organic Synthesis Using Chemically Robust Secondary Amine Tags,” J. Med. Chem., 39:1601-1608 (1996), previously incorporated by reference. The method then proceeds with steps  74  and  76 . The extracted sample is then placed in LC fluorescence, and step  80  is performed to complete the comparison.  
     [0038] Once the sample containing the tags is placed in the mass spectrometer, the ligand on each bead as analyzed by mass spectrometry is compared to the structure indicated by the codes on the beads as illustrated in step  80 . Hence, by utilizing device  10 , optimized methods are provided for analyzing dailkylamine tags in encoded, combinatorial chemistry. Because device  10  may be placed within a heated environment, both ligand cleavage and tag cleavage may occur while the beads remain within the same vials. Moreover, by providing device  10  with a standard footprint, it may be utilized with automated processing and handling equipment to further reduce the amount of time required to complete the quality control process.  
     [0039] The invention has now been described in detail for purposes of clarity of understanding. However, it will be appreciated that certain changes and modifications may be practiced within the scope of the appended claims.