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Solutions for library encoding to create collections of discrete compounds Rhett L Affleck The ability to design, produce, analyze, and manage high quality combinatorial libraries depends on the encoding strategy applied. Several recent advances in encoding technology have extended the range of library design parameters. Enhancements of established techniques have made them more robust and versatile, while additional new and creative encoding concepts have been introduced. With better options now available, combinatorial chemists can more easily select an encoding technique to match resources, library design, and compound management criteria. Addresses Discovery Partners International, 9640 Towne Centre Drive, San Diego, CA 92121, USA; e-mail: [email protected] Current Opinion in Chemical Biology 2001, 5:257–263 1367-5931/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations HTS high-throughput screening LC liquid chromatography MS mass spectrometry
Introduction Combinatorial chemistry provides a wonderful approach for creatinging large libraries of compounds. The libraries can be used as an archive from which to isolate unique compounds of interest as determined by an appropriate assay. The advances in high-throughput screening (HTS) for drug discovery have provided the means to more efficiently pull the compound ‘needles’ out of the compound library ‘haystacks’, increasing the demand for more compounds. HTS advances in other fields are following those in the pharmaceutical field, and new developments are likewise generating a demand for compound libraries in areas such as polymers, catalysts, and dyes. Clearly, the field of combinatorial chemistry is growing, the design of libraries is getting ‘smarter’, and library encoding processes are improving. Mix-and-split synthesis and robotic liquid-handling have made the production of large libraries more manageable, but along with large libraries comes the need for practical encoding strategies such that individual library components can be identified after they are selected from a screen. Encoding and deconvolution strategies for combinatorial libraries have been previously reviewed [1,2]. This review covers recent advances in encoding technologies from mid-1999 through December 2000. In particular, processes that result in a final compound format of discrete compounds (that can be arrayed as single compounds for analysis) will be discussed. Single-compound screening is often thought to provide more easily interpretable and higher quality data [3–5,6•], whereas
efficiency gains from screening mixtures can be more than lost on the overall discovery process because of missing potentially active compounds or from following up on false leads. Nevertheless, a few mixture-based processes are included here because they result in the separation and analysis of individual library components. With the predominance of in-process tagging used for single-compound-per-bead libraries and the majority of solution-phase libraries prepared in arrays, it can be easy to mistakenly associate encoding methods with reactor types. For this reason, a brief overview of reactor types, defined as the largest discrete container of a single library element, is given. It should be realized that, at least theoretically, different reactor types can be used for each encoding method.
Reactor types When using mix-and-split synthesis on loose resin beads, using the definition given above, the reactor is a single resin bead. At the end of the synthesis, each bead contains a unique compound, and although a compound may have multiple copies, those copies are dispersed among all the other beads. Although functionalized resin is available in various inorganic and polymeric materials, chemists working with bead-based libraries tend to use well-characterized resin of the largest practical and available diameter, typically 150–250 µm polystyrene. The amount of compound on a bead is typically 0.1–1 nmol. With the miniaturization of HTS, this amount of compound is sometimes thought to be plenty. In most discovery organizations, however, compounds are not only needed for HTS but also for chemical characterization and follow-up assays. Losses due to liquid handling and sample splitting also need to be considered. Pharmaceutical companies usually target 5–50 µmol for lead discovery libraries. Because of this demand, research creating higher-loading ‘big beads’ using polystyrene beads [7], dendrimeric beads [8], crosslinked polymer disks [9], and grafted porous polymer disks [10] has recently been published. The last example blurs the boundaries between beads and macrosupports, defined below. Difficulties associated with big beads are mechanical stability and increased diffusion time for compounds to enter and leave the center of the beads. Containers or bags with mesh openings too small for resin to escape but big enough to allow for the exchange of reactants to the resin beads inside are often referred to as teabags. All beads within a teabag are exposed to the same synthesis steps, thus providing greater compound amounts per reactor than bead-based techniques. Macrosupports are defined as having all chemical linkages at their surface, while their interior volume is an unreactive
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Table 1 Advantages and disadvantages of various library-encoding strategies. Encoding method
Reactor type
Sensible library sizes
Mix-andsplit efficiency
Solid phase
Solution phase
Flexible Flexible reaction library conditions design
Known location of compounds
Comments
Bead
>100,000
X
X
Teabag
–
Macrosupport
>100,000
Reaction vessel
–
No advantage
Bead
–
No practical method
Teabag
1–100,000
X
X
X
X
X
Best overall method if solid phase is not limiting
Macrosupport
1–10,000
X
X
X
X
X
Fewer chemistries available than on resin in teabags
Reaction vessel
–
In-process tagged X
Best for very large libraries or if reagents are extremely expensive Less advantageous than pre-encoded
X
X
X
Macrosupports could offer more compound per ‘bead’
Pre-encoded
This is implemented as ‘spatially arrayed’ below, where the ‘labels’ are the array locations
Spatially arrayed Bead
This has been attempted in combination with microfluidics by Orchid
Teabag
–
No advantage
Macrosupport
1–8000
X
Reaction vessel
1–5000
X
X
support. The beads and ‘big beads’ mentioned above are more like sponges, with reactive groups throughout their volume. Examples of macrosupports are polystyrene-grafted polypropylene and fluoropolymer tubes [11] and covalently linked microarray supports [12]. Finally, reaction vessels are often used in spatially arrayed formats. Reaction vessels may contain solution-phase or solid-phase components, but in this case the contents of vessels are never mixed with the other vessels.
Encoding methodologies Several methods have been created to encode combinatorial libraries. No one method is best for all types of libraries. Choosing the best method for a particular library involves evaluating several parameters, including the designing chemist’s knowledge and resources. A practical choice will depend on the amount of compound desired, library size, flexibility of the chemistry, deadlines, and cost. In order to aid in the selection of encoding technologies, some comments regarding advantages and disadvantages of each
X
X
Chemical microarrays
X
As library size increases, complexity and difficulty increase
method are given. Table 1 outlines and generalizes the differences between methods, which are described below. Encoding methodologies can be categorized into two types: in-process tagged and pre-encoded. In-process tagging involves the addition of a set of tags to each library element at each diversity step to mark that element’s particular building block at that synthesis step (Figure 1). Tags can be added directly to the compounds or to their carriers. Following synthesis, a library element’s identity can be determined by reading its tag set. Pre-encoded library elements have their carriers or reactors labeled prior to synthesis. This label or barcode must remain associated with the reactor throughout the library synthesis. With this method, each barcode/reactor represents a library element and has a predetermined chemical destiny prior to synthesis. Conceptually, spatially arrayed libraries are a subset of pre-encoded libraries in which each library element is ‘labeled’ with its array position. Practically, spatially arrayed libraries are handled very differently than other pre-encoded libraries, so they are treated here as their own category.
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Figure 1 Comparison of mix-and-split processes between in-process tagged and pre-encoded libraries. A small 18-member (3 × 2 × 3) library is represented. A, B, C, M, N, X, Y, and Z are building blocks added during the first, second, or third step as shown in the figure.
In-process tagged
Pre-encoded
Add building blocks and tags
A
B
C
Add building blocks
Mix Random split
Directed split
Add building blocks and tags
M
Add building blocks
N Mix
Random split
Directed split
Add building blocks and tags
Add building blocks X
Mixtures of beads grouped by last building block Locations of individual compounds unknown and in redundancy
In-process tagged libraries When very large libraries are desired, the most efficient means of producing one may be to create single-compoundper-bead libraries. Unfortunately, at the single-bead level, effective ways to pre-encode beads with rapidly readable labels are not yet available, thereby requiring the in-process tagging method to encode the library. This method takes full advantage of mix-and-split synthesis. Because the splitting steps are random for the library elements, however, only full matrices can be designed (e.g. a specific set of 10,000 10-mer peptides using all of 20 different amino acids at each step can not be made without making the entire 2010 [= 1013] member library). Also, redundant library copies must be made to ensure full library coverage. Furthermore, resources must exist to handle compound from single beads and to maintain high compound yields and tagging quality. Two of the most established and robust techniques of in-process tagging are the carbene insertion of cleavable electrophoretic molecular tags followed with decoding by electron capture detection/gas chromatography [13], and secondary amine coupling of cleavable dialkylamine tags followed with decoding by fluorescence detection LC/MS [14]. In a quality assessment of a library created with the first technique, Dolle et al. [15•] reported a statistical 84% compound confirmation for a 25,000-member library of statine amides. The latest advancement of the second technique employs a 50:50 mix of tag with and without an isotopic difference to give a characteristic 1:1 ratio doublet separated by the isotopic difference (2.012 units for 2 hydrogen versus 2 deuterium) in the mass spectrum [16•]. This new twist affords much greater sensitivity and selectivity of the tags. A technique using 19F-encoded libraries appeared in the literature recently [17], and Pirrung and Park [18] have prepared a library of 90 N-alkylglycines bearing substituted succinamides and decoded it using 19F NMR, taking
Y
Z Single copy of discrete compounds in known location
Current Opinion in Chemical Biology
advantage of the more readily accessible decode method and instrumentation. Two new techniques have appeared in the past year. The first uses trityl tags that can be cleaved with acid and detected by laser desorption/ionization time-of-flight (TOF) MS with or without matrix [19,20]. Tag cations may also be generated directly by laser irradiation and measured directly by TOF MS. Although this tagging method is inappropriate for libraries requiring strong acid conditions, it has the potential for much simpler and faster decoding. The second technique tags with fluorescent silica colloids [21,22•]. With the aid of polymer bridging to enhance the binding strength of the 2.5 µm silica particles to the polymer library beads, these tags are shown to be robustly attached during library synthesis. Tag fluorescence is also shown to survive harsh synthesis conditions. Each silica tag has multiple fluorophores, the combination of which represents a unique tag. As an example, with only six dyes, 64 unique tags could be made, increasing the potential size of encoded libraries. This technique uses overlaid fluorescent microscope images of a bead at appropriate wavelengths to assess the population of each tag type present. Although not inherent to the method, to date no in-process tagging technique is amenable to rapid tag-reading. The best techniques take several minutes. Thus, the most efficient overall process is to only identify compounds of interest — after screening identifies them as active compounds. Most compound locations remain unknown. Combined with the fact that single-bead libraries do not typically generate enough compound for follow-up analysis, re-synthesis of compounds becomes necessary, increasing the discovery process timeline and tying up chemistry resources, and doing so with only primary assay data to assess which compounds to pursue. The increased time and cost associated with assay
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Figure 2
The IRORI family of microreactors Resin capacity:
MacroKanTM ~ 300 mg
Compound generated:
~ 100 mg
RF Tag MicroKanTM ~ 30 mg
~ 10 mg
NanoKanTM ~ 8 mg
~ 2 mg
IRORI’s implementation of teabags. The MiniKanTM (intermediate in size between the MacroKan and the MicroKan) is not shown. The larger microreactors are constructed of polypropylene, and the NanoKans are made of ethylene tetrafluoroethylene (ETFE) and ceramic. Reproduced from [25••] with permission from the Journal of the American Chemical Society.
follow-up for single-bead libraries is sometimes overlooked because of the more obvious (but usually minor) savings in library production cost resulting from the small volume of the libraries. Nevertheless, this remains the only practical means to create libraries of greater than 100,000 members of discrete, encoded compounds.
Pre-encoded libraries Pre-encoded libraries take full advantage of mix-and-split synthesis. Library elements must be sorted between diversity synthesis steps, requiring the labeling method to be easily read. This method has been used almost exclusively with fairly large teabag or macrosupport reactors that can be easily handled and that can carry a label large enough to be read. The simplest system is a small library of teabags that can be visually and manually sorted. By combining visual tags and spatial arraying, Guiles et al. [23] extended the teabag technique to make a library of 1920 compounds. Radiofrequency labeling simplifies this method and, with automated sorting, libraries of up to 10,000 compounds are easily created [24]. In a significant development of 2000, IRORI’s new NanoKan System extends the pre-encoded method to libraries of up to 100,000 members [25••]. By using optically encoded ceramic caps instead of radiofrequency tags, the NanoKans are a smaller teabag package providing milligram quantities of each compound (Figure 2). Productivity of the system is also enhanced by automated workstations for NanoKan assembly, washing, arraying and cleaving, as well as sorting. In addition to the aforementioned optical tagging, the past year has also brought other novel techniques to the pre-encoded category. In one technique, ‘big beads’ are cut from a 2 mm
thick gel-type polymer in various shapes [26]. Using shape to discriminate library elements, 30 unique shapes were used to create a 150-member library. The monoliths had diffusion times of several hours, but did provide a few-thousand-fold increase in per-bead compound capacity compared with that of 200 µm beads. Another technique used solid-phase synthesis to create a four-member library of oligonucleotide 20-mers on fluorescently labeled 8.8 µm beads [27]. Library beads were sorted by fluorescence intensity on a flow cytometer. With flow sorter rates of 25,000 events and decreased bead size, this technique offers the potential of creating extremely large libraries. What remains to be determined is the number of independent bead sets that can be created whose labels can be optically distinguishable by flow cytometry. A tremendous advantage of pre-encoded libraries is that the precise location and identity of all library elements are known during and after synthesis. Presumably, the reactor size has been chosen to provide enough compound for follow-up assays. Thus, concerns about compound identity, turnaround time for follow-up assays, and compound re-synthesis are negated. Another advantage of pre-encoded libraries is the complete flexibility of library design. This includes the number of building blocks at each step, multiples of library element subsets, and sparse library matrices (Figure 3). Designing incomplete matrices may be desirable in order to eliminate specific building-block combinations or to react certain library subsets with additional steps. Adaptability to only solid-phase chemistries is a disadvantage of pre-encoded libraries.
Spatially arrayed libraries Applications of spatially arrayed libraries in the past few years have used arrays of test tubes, microtiter plates, or reactors. Larger libraries are usually in formats that can be accessed by liquid-handling robots. Libraries can be solution-phase or resin-based solid-phase, and combinations of solution and resin-based approaches are becoming more common [28]. Three interesting and very different approaches have recently been reported. Each of these new techniques takes advantage of macrosupports. The first approach uses a separate macrosupport unit for each library element. By keeping track of their spatial orientation while redistributing them for common reactions, this technique takes advantage of mix-and-split synthesis. Furka [29] theorizes on possible support units and redistribution patterns. Using a second approach, Scharn et al. [30] made an 8000-member library of bound and trifluoroacetic acid (TFA)-cleavable 1,3,5-triazines in a microarray, expanding the earlier peptide work of Frank [31]. Reactants are delivered by pipette in 1–2 µl droplets on 2 mm centers over either 18 cm × 26 cm cellulose or polypropylene membranes. Substitution rates were enhanced by microwave irradiation, and yields of 50–250 nmol were obtained. The third novel approach in this category uses a single thread as the macrosupport. Wrapping the thread multiple times around a cylinder and partitioning the cylinder lengthwise with
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Figure 3
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Representation of a sparse matrix library design, which is possible with pre-encoded and spatially arrayed libraries. (The same library scheme and building blocks of Figure 1 are used again.) Solid lines and bold letters represent reactions and compounds included in the library
design. Dashed lines and non-bold letters represent unwanted reactions and compounds of the full matrix, which are not in the library design (but which would nevertheless be made with other methods). Selected compounds may also be created in multiple copies, if desired.
boundaries of paraffin creates regions along the thread for common diversity synthesis steps. Repeating this process with cylinders of appropriately different diameters enables multiple diversity steps (Figure 4). Using this procedure, Schwabacher et al. [32••] created a small library of peptides on a cotton thread. Like the first approach mentioned above, this technique takes advantage of mix-and-split synthesis, and it does it in a simple and straightforward manner.
Although this review is not focused on these techniques, there are a few recent papers that deserve mention.
With the exception of the three new macrosupport techniques mentioned above, the major advantage of spatially arrayed libraries is that they can be used for both solutionphase and solid-phase chemistries. The major disadvantage of all spatially arrayed techniques is that to enable larger numbers of compounds to be synthesized in parallel, sacrifices in reaction flexibility must be made. Although arrayed reactors may enable solution-phase chemistry, conditions such as high or low temperature, high pressure, addition of solid-phase reactants, and removal of solid-phase products may not all be conveniently utilized without extreme expense.
Mixtures When library production methods result in library members as mixtures in one or multiple fractions, analysis and assay techniques must then determine which individual library components are responsible for the resulting positive assay indications from these mixtures of compounds. Mixtures can be in solution phase or in solid phase (usually on resin beads) format. Library generation is fairly straightforward, taking advantage of mix-and-split synthesis without encoding. However, these libraries usually need deconvolution strategies to identify the active compounds from the data.
Deconvolution schemes involve assays of mixtures of compounds wherein any individual compound is assayed multiple times in different mixture groups. With a valid scheme, data from active mixture groups should produce definitive active compounds. Deconvolution strategies will depend on the library size and structure. Topiol et al. [33] have extensively examined mix-and-split schemes for optimal efficiency, considering the parameters of library size, number of diversity steps, number of reaction vessels, and number of compounds per final pool. Two of the main criticisms of mixtures are the amount of compound necessary for the redundant assays and the loss of sensitivity of an assay towards any particular compound. This second point is clearly shown in experiments by Boger et al. [6•], who compare 10-compound mixtures with 100-compound mixtures and observe that with the larger mixtures comes a loss of the less-distinguishable activities and subtle information. From a large mixture of compounds, individual components of interest can be culled out firstly if there is a very highly selective screen that can not only select but also separate the active components from the mixture and secondly if analytical techniques exist to identify these components. DNA and peptide libraries make the second of these criteria obtainable, and Alam et al. [34] take advantage of this by preparing and screening a 3,200,000-member library of pentapeptides to identify DNA-binding peptide ligands. Target DNA on magnetic beads was used to pull the active ligands from the bead-based combinatorial library. In a more general approach
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Figure 4
(a)
Combinatorial chemistry on a thread. (a) A thread is wrapped around a cylinder. The cylinder is then partitioned lengthwise into three regions. (b) A different reactant or building block (red, blue and green) is added to each region. (c) The thread now has repeating domains. (d) The process is repeated around an appropriately sized cylinder for four building blocks. (e) Four new building blocks (black, yellow, cyan and magenta) are added. (f) All combinations are represented. Reproduced from [32••] with permission from the Journal of the American Chemical Society.
(b)
(c)
(d)
(e)
(f)
for small-molecule libraries, Eliseev [35] added target macromolecules directly to a solution-phase library to select for high-affinity ligands. Size exclusion chromatography separates the receptor with bound ligands from the mixture, and desalting and LC–MS followed to separate and identify highaffinity ligands. Although MS does not distinguish between isomeric compounds, tagging the library components with mass tags can be helpful. Furthermore, a new regiochemical tagging tool for identification of isomeric library components has been described [36]. Although the above-mentioned techniques may be limited in both the applicable assay types and in the library design, the incredibly efficient screening obtained with testing of mixtures makes it very attractive in certain circumstances. If solution-phase mixtures can be separated and identified during or after synthesis, they begin to look like their encoded solid-phase library cousins. Curran and Zhiyong [37] describe techniques to both tag solution-phase compounds with fluorous tags and to purify them by fluorous reverse-phase silica gels. In a more recent paper [38•], more tags with differing retention times are presented. These techniques offer the basis for solution-phase mixture synthesis along with the ability to provide pure compounds as the final format.
beads (in-process tagged) are becoming larger and teabags (pre-encoded) are becoming smaller, thereby expanding the limits of both techniques. Likewise, encoding options have evolved, and potential library size is constantly increasing. The selection of a practical and efficient encoding method depends on several parameters, involving primarily the size of the library and the amounts of compounds desired. These criteria may narrow the remaining choices significantly. Furthermore, the ultimate decision must, of course, take into consideration solid-phase versus solution-phase experience, chemistry flexibility, imposed deadlines, and the myriad costs of capital equipment, manpower, facilities, and reagents. Thanks to the ongoing quest to improve library methodologies, more and better tools are continually becoming available to the combinatorial chemist.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Czarnik AW: Encoding methods for combinatorial chemistry. Curr Opin Chem Biol 1997, 1:60-66.
2.
Barnes C, Balasubramanian S: Recent developments in the encoding and deconvolution of combinatorial libraries. Curr Opin Chem Biol 2000, 4:346-350.
3.
Tiebes D: Combinatorial chemistry. In Combinatorial Chemistry. Edited by Jung G. Weinheim: Wiley-VCH; 1999:1-34.
4.
Austel V: Solution-phase combinatorial chemistry. In Combinatorial Chemistry. Edited by Jung G. Weinheim: Wiley-VCH; 1999:77-123.
5.
Turner R, Sterrer S, Wiesmuller KH, Meyer-Almes FJ: How to scaleup while scaling down: EVOscreenTM, a miniaturized ultra-highthroughput screening system. In Combinatorial Chemistry. Edited by Jung G. Weinheim: Wiley-VCH; 1999:441-461.
Summary and conclusions There are several viable options for encoding combinatorial libraries such that arrays of discrete compounds may be produced to enable ‘single compound’ assays. While established techniques continue to become more robust and flexible, several newly invented techniques have also been successful. Overall, the industry continues to advance with respect to many facets of the design and outcome of compound libraries:
Solutions for library encoding to create collections of discrete compounds Affleck
6. •
Boger DL, Dechantsreiter MA, Ishii T, Fink BE, Hedrick MP: Assessment of solution-phase positional scanning libraries based on distamycin A for the discovery of new DNA binding agents. Bioorg Med Chem 2000, 8:2049-2057. A good paper that contrasts the efficiency of larger mixtures with the disadvantage of the lower quality screening data associated with those larger mixtures. 7.
Lee D, Sello JK, Schreiber SL: Pairwise use of complexitygenerating reactions in diversity-oriented organic synthesis. Org Lett 2000, 2:709-712.
8.
Fromont C, Bradley M: High-Loading resin beads for solid phase synthesis using triple branching symmetrical dendrimers. Chem Commun 2000, 4:283-284.
9.
Hird N, Hughes I, Hunter D, Morrison MGJT, Sherrington DC, Stevenson L: Polymer discs — an alternative support format for solid phase synthesis. Tetrahedron 1999, 55:9575-9584.
10. Tripp JA, Stein JA, Svec F, Fréchet JMJ: ‘Reactive filtration’: use of functionalized porous polymer monoliths as scavengers in solution-phase synthesis. Org Lett 2000, 2:195-198. 11. Li W, Czarnik AW, Lillig J, Xiao XY: Kinetic study of organic reactions on polystyrene grafted microtubes. J Comb Chem 2000, 2:224-227.
23. Guiles JW, Lanter CL, Rivero RA: A visual tagging process for mix and sort combinatorial chemistry. Angew Chem Int Ed Engl 1998, 37:926-928. 24. Herpin TF, Van Kirk KG, Salvino JM, Yu ST, Labaudinière RF: Synthesis of a 10000 member 1,5-benzodiazepine-2-one library by the directed sorting method. J Comb Chem 2000, 2:513-521. 25. Nicolaou KC, Pfefferkorn JA, Mitchell HJ, Roecker AJ, Barluenga S, •• Cao GQ, Affleck RL, Lillig JE: Natural product-like combinatorial libraries based on privileged structures. 2. Construction of a 10000-membered benzopyran library by directed split-and-pool chemistry using NanoKans and optical encoding. J Am Chem Soc 2000, 122:9954-9967. An excellent paper demonstrating the ease and utility of IRORI’s NanoKan System. In a major milestone (and by the only practical means available), they produced 1–2 mg of discrete compounds for their 10,000-member sparsematrix library. Quality controls were run prior to, during, and following the library production to stepwise monitor and evaluate the success of the library. The entire library was synthesized, cleaved, and automatically formatted into microtiter plates in a remarkable eight days. 26. Vaino AR, Janda KD: Euclidean shape-encoded combinatorial chemical libraries. Proc Natl Acad Sci USA 2000, 97:7692-7696. 27.
12. Fodor SPA, Read JL, Pirrung MC, Stryer L, Lu AT, Solas D: Lightdirected, spatially addressable parallel chemical synthesis. Science 1991, 251:767-773. 13. Ohlmeyer MHJ, Swanson RN, Dillard LW, Reader JC, Asouline G, Kobayashi R, Wigler M, Still WC: Complex synthetic chemical libraries indexed with molecular tags. Proc Natl Acad Sci USA 1993 90:10922-10926. 14. Fitch WL, Baer TA, Chen W, Holden F, Holmes CP, Maclean D, Shah N, Sullivan E, Tang M, Waybourn P: Improved methods for encoding and decoding dialkylamine-encoded combinatorial libraries. J Comb Chem 1999, 1:188-194. 15. Dolle RE, Guo J, O’Brien L, Jin Y, Piznik M, Bowman KJ, Li W, • Egan WJ, Cavallaro CL, Roughton AL et al.: A statistical-based approach to assessing the fidelity of combinatorial libraries encoded with electrophoric molecular tags. Development and application of tag decode-assisted single bead LC/MS analysis. J Comb Chem 2000, 2:716-731. With mix-and-split synthesis on single-bead-per-compound libraries, there is often just enough compound from a bead to run the biological screen. Although much chemistry optimization and quality control is performed in advance of library production, it is desirable to have quality assurance (QA) of the actual produced library. This paper details such a statistical QA procedure. 16. Lane SJ, Pipe A: Single bead and hard tag decoding using • accurate isotopic difference target analysis-encoded combinatorial libraries. Rapid Commun Mass Spectrom 2000, 14:782-793. The secondary amine methodology has been further refined for higher sensitivity and selectivity of the dialkylamine tags. This is accomplished by using 50:50 mixes of tags with 2H:2D or 12C:13C, giving doublet pairs in the mass spectrum separated by 2.012 u or 2.0068 u, respectively. 17.
Hochlowski JE, Whittern DN, Sowin TJ: Encoding of combinatorial chemistry libraries by fluorine-19 NMR. J Comb Chem 1999, 1:291-293.
18. Pirrung MC, Park K: Discovery of selective metal-binding peptoids using 19F encoded combinatorial libraries. Bioorg Med Chem Lett 2000, 10:2115-2118. 19. Shchepinov MS, Chalk R, Southern EM: Trityl mass-tags for encoding in combinatorial oligonucleotide synthesis. Nuc Acid Sym Ser 1999 42:107-108. 20. Shchepinov MS, Chalk R, Southern EM: Trityl tags for encoding in combinatorial synthesis. Tetrahedron 2000, 56:2713-2724. 21. Battersby BJ, Bryant D, Meutermans W, Matthews D, Smythe ML, Trau M: Toward larger chemical libraries: encoding with fluorescent colloids in combinatorial chemistry. J Am Chem Soc 2000, 122:2138-2139. 22. Grøndahl L, Battersby BJ, Bryant D, Trau M: Encoding combinatorial • libraries: a novel application of fluorescent silica colloids. Langmuir 2000, 16:9709-9715. These two papers (see also [21]) on encoding with fluorescent colloids are interesting for a couple of reasons. First, the colloids are analyzed on the beads in a non-destructive manner. Second, by varying the ratios of different colored tags, many tag sets can be created with the potential to encode very large libraries.
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Nanthakumar A, Pon RT, Mazumder A, Yu S, Watson A: Solid-phase oligonucleotide synthesis and flow cytometric analysis with microspheres encoded with covalently attached fluorophores. Bioconjugate Chem 2000, 11:282-288.
28. South MS, Dice TA, Parlow JJ: Polymer-assisted solution-phase (PASP) library synthesis of α-ketoamides. Biotech Bioeng 2000, 71:51-57. 29. Furka A: Redistribution in combinatorial synthesis. A theoretical approach. Comb Chem High Throughput Screen 2000, 3:197-209. 30. Scharn D, Wenschuh H, Reineke U, Schneider-Mergener J, Germeroth L: Spatially addressed synthesis of amino- and aminooxy-substituted 1,3,5-triazine arrays on polymeric membranes. J Comb Chem 2000, 2:361-369. 31. Frank R: Spot-synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support. Tetrahedron 1992, 48:9217-9232. 32. Schwabacher AW, Shen Y, Johnson CW: Fourier transform •• combinatorial chemistry. J Am Chem Soc 1999, 121:8669-8670. A unique and creative addition to encoding processes for combinatorial chemistry. Spatially encoded compounds are synthesized in parallel along a single thread. Parallel synthesis of building blocks is achieved by wrapping the thread around a cylinder and exposing a radial section of the cylinder to a single building block for that step. 33. Topiol S, Davies J, Vijayakuma S, Wareing JR: Computer aided analysis of split and mix combinatorial libraries. J Comb Chem 2001, 3:20-27. 34. Alam MR, Maeda M, Sasaki S: DNA-binding peptides searched from the solid-phase combinatorial library with the use of magnetic beads attaching the target duplex DNA. Bioorg Med Chem 2000, 8:465-473. 35. Eliseev AV: Emerging approaches to target-assisted screening of combinatorial mixtures. Drug Dev Discov 1998, 1:106-115. 36. Nazarpack-Kandlousy N, Chernushevich IV, Meng LJ, Yang Y, Eliseev AV: Regiochemical tagging: a new tool for structural characterization of isomeric components in combinatorial mixtures. J Am Chem Soc 2000, 122:3358-3366. 37.
Curran DP, Zhiyong L: Fluorous synthesis with fewer fluorines (light fluorous synthesis): separation of tagged from untagged products by solid-phase extraction with fluorous reverse-phase silica gel. J Am Chem Soc 1999, 121:9069-9072.
38. Zhang Q, Luo Z, Curran DP: Separation of ‘light fluorous’ reagents • and catalysts by fluorous solid-phase extraction: synthesis and study of a family of triarylphosphines bearing linear and branched fluorous tags. J Org Chem 2000, 65:8866-8873. These two papers (see also [37]) on ‘light fluorous’ synthesis deal with tagging and purifying solution-phase compounds on the basis of their fluorous content. Although not specifically dealt with in these articles, an implication of this technology would be the ability to create fluorous blocking groups that could lead to ‘sorting’ of solution-phase compounds with a separation technique that is orthogonal to the properties of the compounds. This would allow solution-phase mix-and-split synthesis.
Solutions for library encoding to create collections of discrete compounds Rhett L Affleck The ability to design, produce, analyze, and manage high quality combinatorial libraries depends on the encoding strategy applied. Several recent advances in encoding technology have extended the range of library design parameters. Enhancements of established techniques have made them more robust and versatile, while additional new and creative encoding concepts have been introduced. With better options now available, combinatorial chemists can more easily select an encoding technique to match resources, library design, and compound management criteria. Addresses Discovery Partners International, 9640 Towne Centre Drive, San Diego, CA 92121, USA; e-mail: [email protected] Current Opinion in Chemical Biology 2001, 5:257–263 1367-5931/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations HTS high-throughput screening LC liquid chromatography MS mass spectrometry
Introduction Combinatorial chemistry provides a wonderful approach for creatinging large libraries of compounds. The libraries can be used as an archive from which to isolate unique compounds of interest as determined by an appropriate assay. The advances in high-throughput screening (HTS) for drug discovery have provided the means to more efficiently pull the compound ‘needles’ out of the compound library ‘haystacks’, increasing the demand for more compounds. HTS advances in other fields are following those in the pharmaceutical field, and new developments are likewise generating a demand for compound libraries in areas such as polymers, catalysts, and dyes. Clearly, the field of combinatorial chemistry is growing, the design of libraries is getting ‘smarter’, and library encoding processes are improving. Mix-and-split synthesis and robotic liquid-handling have made the production of large libraries more manageable, but along with large libraries comes the need for practical encoding strategies such that individual library components can be identified after they are selected from a screen. Encoding and deconvolution strategies for combinatorial libraries have been previously reviewed [1,2]. This review covers recent advances in encoding technologies from mid-1999 through December 2000. In particular, processes that result in a final compound format of discrete compounds (that can be arrayed as single compounds for analysis) will be discussed. Single-compound screening is often thought to provide more easily interpretable and higher quality data [3–5,6•], whereas
efficiency gains from screening mixtures can be more than lost on the overall discovery process because of missing potentially active compounds or from following up on false leads. Nevertheless, a few mixture-based processes are included here because they result in the separation and analysis of individual library components. With the predominance of in-process tagging used for single-compound-per-bead libraries and the majority of solution-phase libraries prepared in arrays, it can be easy to mistakenly associate encoding methods with reactor types. For this reason, a brief overview of reactor types, defined as the largest discrete container of a single library element, is given. It should be realized that, at least theoretically, different reactor types can be used for each encoding method.
Reactor types When using mix-and-split synthesis on loose resin beads, using the definition given above, the reactor is a single resin bead. At the end of the synthesis, each bead contains a unique compound, and although a compound may have multiple copies, those copies are dispersed among all the other beads. Although functionalized resin is available in various inorganic and polymeric materials, chemists working with bead-based libraries tend to use well-characterized resin of the largest practical and available diameter, typically 150–250 µm polystyrene. The amount of compound on a bead is typically 0.1–1 nmol. With the miniaturization of HTS, this amount of compound is sometimes thought to be plenty. In most discovery organizations, however, compounds are not only needed for HTS but also for chemical characterization and follow-up assays. Losses due to liquid handling and sample splitting also need to be considered. Pharmaceutical companies usually target 5–50 µmol for lead discovery libraries. Because of this demand, research creating higher-loading ‘big beads’ using polystyrene beads [7], dendrimeric beads [8], crosslinked polymer disks [9], and grafted porous polymer disks [10] has recently been published. The last example blurs the boundaries between beads and macrosupports, defined below. Difficulties associated with big beads are mechanical stability and increased diffusion time for compounds to enter and leave the center of the beads. Containers or bags with mesh openings too small for resin to escape but big enough to allow for the exchange of reactants to the resin beads inside are often referred to as teabags. All beads within a teabag are exposed to the same synthesis steps, thus providing greater compound amounts per reactor than bead-based techniques. Macrosupports are defined as having all chemical linkages at their surface, while their interior volume is an unreactive
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Table 1 Advantages and disadvantages of various library-encoding strategies. Encoding method
Reactor type
Sensible library sizes
Mix-andsplit efficiency
Solid phase
Solution phase
Flexible Flexible reaction library conditions design
Known location of compounds
Comments
Bead
>100,000
X
X
Teabag
–
Macrosupport
>100,000
Reaction vessel
–
No advantage
Bead
–
No practical method
Teabag
1–100,000
X
X
X
X
X
Best overall method if solid phase is not limiting
Macrosupport
1–10,000
X
X
X
X
X
Fewer chemistries available than on resin in teabags
Reaction vessel
–
In-process tagged X
Best for very large libraries or if reagents are extremely expensive Less advantageous than pre-encoded
X
X
X
Macrosupports could offer more compound per ‘bead’
Pre-encoded
This is implemented as ‘spatially arrayed’ below, where the ‘labels’ are the array locations
Spatially arrayed Bead
This has been attempted in combination with microfluidics by Orchid
Teabag
–
No advantage
Macrosupport
1–8000
X
Reaction vessel
1–5000
X
X
support. The beads and ‘big beads’ mentioned above are more like sponges, with reactive groups throughout their volume. Examples of macrosupports are polystyrene-grafted polypropylene and fluoropolymer tubes [11] and covalently linked microarray supports [12]. Finally, reaction vessels are often used in spatially arrayed formats. Reaction vessels may contain solution-phase or solid-phase components, but in this case the contents of vessels are never mixed with the other vessels.
Encoding methodologies Several methods have been created to encode combinatorial libraries. No one method is best for all types of libraries. Choosing the best method for a particular library involves evaluating several parameters, including the designing chemist’s knowledge and resources. A practical choice will depend on the amount of compound desired, library size, flexibility of the chemistry, deadlines, and cost. In order to aid in the selection of encoding technologies, some comments regarding advantages and disadvantages of each
X
X
Chemical microarrays
X
As library size increases, complexity and difficulty increase
method are given. Table 1 outlines and generalizes the differences between methods, which are described below. Encoding methodologies can be categorized into two types: in-process tagged and pre-encoded. In-process tagging involves the addition of a set of tags to each library element at each diversity step to mark that element’s particular building block at that synthesis step (Figure 1). Tags can be added directly to the compounds or to their carriers. Following synthesis, a library element’s identity can be determined by reading its tag set. Pre-encoded library elements have their carriers or reactors labeled prior to synthesis. This label or barcode must remain associated with the reactor throughout the library synthesis. With this method, each barcode/reactor represents a library element and has a predetermined chemical destiny prior to synthesis. Conceptually, spatially arrayed libraries are a subset of pre-encoded libraries in which each library element is ‘labeled’ with its array position. Practically, spatially arrayed libraries are handled very differently than other pre-encoded libraries, so they are treated here as their own category.
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Figure 1 Comparison of mix-and-split processes between in-process tagged and pre-encoded libraries. A small 18-member (3 × 2 × 3) library is represented. A, B, C, M, N, X, Y, and Z are building blocks added during the first, second, or third step as shown in the figure.
In-process tagged
Pre-encoded
Add building blocks and tags
A
B
C
Add building blocks
Mix Random split
Directed split
Add building blocks and tags
M
Add building blocks
N Mix
Random split
Directed split
Add building blocks and tags
Add building blocks X
Mixtures of beads grouped by last building block Locations of individual compounds unknown and in redundancy
In-process tagged libraries When very large libraries are desired, the most efficient means of producing one may be to create single-compoundper-bead libraries. Unfortunately, at the single-bead level, effective ways to pre-encode beads with rapidly readable labels are not yet available, thereby requiring the in-process tagging method to encode the library. This method takes full advantage of mix-and-split synthesis. Because the splitting steps are random for the library elements, however, only full matrices can be designed (e.g. a specific set of 10,000 10-mer peptides using all of 20 different amino acids at each step can not be made without making the entire 2010 [= 1013] member library). Also, redundant library copies must be made to ensure full library coverage. Furthermore, resources must exist to handle compound from single beads and to maintain high compound yields and tagging quality. Two of the most established and robust techniques of in-process tagging are the carbene insertion of cleavable electrophoretic molecular tags followed with decoding by electron capture detection/gas chromatography [13], and secondary amine coupling of cleavable dialkylamine tags followed with decoding by fluorescence detection LC/MS [14]. In a quality assessment of a library created with the first technique, Dolle et al. [15•] reported a statistical 84% compound confirmation for a 25,000-member library of statine amides. The latest advancement of the second technique employs a 50:50 mix of tag with and without an isotopic difference to give a characteristic 1:1 ratio doublet separated by the isotopic difference (2.012 units for 2 hydrogen versus 2 deuterium) in the mass spectrum [16•]. This new twist affords much greater sensitivity and selectivity of the tags. A technique using 19F-encoded libraries appeared in the literature recently [17], and Pirrung and Park [18] have prepared a library of 90 N-alkylglycines bearing substituted succinamides and decoded it using 19F NMR, taking
Y
Z Single copy of discrete compounds in known location
Current Opinion in Chemical Biology
advantage of the more readily accessible decode method and instrumentation. Two new techniques have appeared in the past year. The first uses trityl tags that can be cleaved with acid and detected by laser desorption/ionization time-of-flight (TOF) MS with or without matrix [19,20]. Tag cations may also be generated directly by laser irradiation and measured directly by TOF MS. Although this tagging method is inappropriate for libraries requiring strong acid conditions, it has the potential for much simpler and faster decoding. The second technique tags with fluorescent silica colloids [21,22•]. With the aid of polymer bridging to enhance the binding strength of the 2.5 µm silica particles to the polymer library beads, these tags are shown to be robustly attached during library synthesis. Tag fluorescence is also shown to survive harsh synthesis conditions. Each silica tag has multiple fluorophores, the combination of which represents a unique tag. As an example, with only six dyes, 64 unique tags could be made, increasing the potential size of encoded libraries. This technique uses overlaid fluorescent microscope images of a bead at appropriate wavelengths to assess the population of each tag type present. Although not inherent to the method, to date no in-process tagging technique is amenable to rapid tag-reading. The best techniques take several minutes. Thus, the most efficient overall process is to only identify compounds of interest — after screening identifies them as active compounds. Most compound locations remain unknown. Combined with the fact that single-bead libraries do not typically generate enough compound for follow-up analysis, re-synthesis of compounds becomes necessary, increasing the discovery process timeline and tying up chemistry resources, and doing so with only primary assay data to assess which compounds to pursue. The increased time and cost associated with assay
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Figure 2
The IRORI family of microreactors Resin capacity:
MacroKanTM ~ 300 mg
Compound generated:
~ 100 mg
RF Tag MicroKanTM ~ 30 mg
~ 10 mg
NanoKanTM ~ 8 mg
~ 2 mg
IRORI’s implementation of teabags. The MiniKanTM (intermediate in size between the MacroKan and the MicroKan) is not shown. The larger microreactors are constructed of polypropylene, and the NanoKans are made of ethylene tetrafluoroethylene (ETFE) and ceramic. Reproduced from [25••] with permission from the Journal of the American Chemical Society.
follow-up for single-bead libraries is sometimes overlooked because of the more obvious (but usually minor) savings in library production cost resulting from the small volume of the libraries. Nevertheless, this remains the only practical means to create libraries of greater than 100,000 members of discrete, encoded compounds.
Pre-encoded libraries Pre-encoded libraries take full advantage of mix-and-split synthesis. Library elements must be sorted between diversity synthesis steps, requiring the labeling method to be easily read. This method has been used almost exclusively with fairly large teabag or macrosupport reactors that can be easily handled and that can carry a label large enough to be read. The simplest system is a small library of teabags that can be visually and manually sorted. By combining visual tags and spatial arraying, Guiles et al. [23] extended the teabag technique to make a library of 1920 compounds. Radiofrequency labeling simplifies this method and, with automated sorting, libraries of up to 10,000 compounds are easily created [24]. In a significant development of 2000, IRORI’s new NanoKan System extends the pre-encoded method to libraries of up to 100,000 members [25••]. By using optically encoded ceramic caps instead of radiofrequency tags, the NanoKans are a smaller teabag package providing milligram quantities of each compound (Figure 2). Productivity of the system is also enhanced by automated workstations for NanoKan assembly, washing, arraying and cleaving, as well as sorting. In addition to the aforementioned optical tagging, the past year has also brought other novel techniques to the pre-encoded category. In one technique, ‘big beads’ are cut from a 2 mm
thick gel-type polymer in various shapes [26]. Using shape to discriminate library elements, 30 unique shapes were used to create a 150-member library. The monoliths had diffusion times of several hours, but did provide a few-thousand-fold increase in per-bead compound capacity compared with that of 200 µm beads. Another technique used solid-phase synthesis to create a four-member library of oligonucleotide 20-mers on fluorescently labeled 8.8 µm beads [27]. Library beads were sorted by fluorescence intensity on a flow cytometer. With flow sorter rates of 25,000 events and decreased bead size, this technique offers the potential of creating extremely large libraries. What remains to be determined is the number of independent bead sets that can be created whose labels can be optically distinguishable by flow cytometry. A tremendous advantage of pre-encoded libraries is that the precise location and identity of all library elements are known during and after synthesis. Presumably, the reactor size has been chosen to provide enough compound for follow-up assays. Thus, concerns about compound identity, turnaround time for follow-up assays, and compound re-synthesis are negated. Another advantage of pre-encoded libraries is the complete flexibility of library design. This includes the number of building blocks at each step, multiples of library element subsets, and sparse library matrices (Figure 3). Designing incomplete matrices may be desirable in order to eliminate specific building-block combinations or to react certain library subsets with additional steps. Adaptability to only solid-phase chemistries is a disadvantage of pre-encoded libraries.
Spatially arrayed libraries Applications of spatially arrayed libraries in the past few years have used arrays of test tubes, microtiter plates, or reactors. Larger libraries are usually in formats that can be accessed by liquid-handling robots. Libraries can be solution-phase or resin-based solid-phase, and combinations of solution and resin-based approaches are becoming more common [28]. Three interesting and very different approaches have recently been reported. Each of these new techniques takes advantage of macrosupports. The first approach uses a separate macrosupport unit for each library element. By keeping track of their spatial orientation while redistributing them for common reactions, this technique takes advantage of mix-and-split synthesis. Furka [29] theorizes on possible support units and redistribution patterns. Using a second approach, Scharn et al. [30] made an 8000-member library of bound and trifluoroacetic acid (TFA)-cleavable 1,3,5-triazines in a microarray, expanding the earlier peptide work of Frank [31]. Reactants are delivered by pipette in 1–2 µl droplets on 2 mm centers over either 18 cm × 26 cm cellulose or polypropylene membranes. Substitution rates were enhanced by microwave irradiation, and yields of 50–250 nmol were obtained. The third novel approach in this category uses a single thread as the macrosupport. Wrapping the thread multiple times around a cylinder and partitioning the cylinder lengthwise with
Solutions for library encoding to create collections of discrete compounds Affleck
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Figure 3
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AMY AMY AMY
AMZ
ANX
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ANZ
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BMX
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Representation of a sparse matrix library design, which is possible with pre-encoded and spatially arrayed libraries. (The same library scheme and building blocks of Figure 1 are used again.) Solid lines and bold letters represent reactions and compounds included in the library
design. Dashed lines and non-bold letters represent unwanted reactions and compounds of the full matrix, which are not in the library design (but which would nevertheless be made with other methods). Selected compounds may also be created in multiple copies, if desired.
boundaries of paraffin creates regions along the thread for common diversity synthesis steps. Repeating this process with cylinders of appropriately different diameters enables multiple diversity steps (Figure 4). Using this procedure, Schwabacher et al. [32••] created a small library of peptides on a cotton thread. Like the first approach mentioned above, this technique takes advantage of mix-and-split synthesis, and it does it in a simple and straightforward manner.
Although this review is not focused on these techniques, there are a few recent papers that deserve mention.
With the exception of the three new macrosupport techniques mentioned above, the major advantage of spatially arrayed libraries is that they can be used for both solutionphase and solid-phase chemistries. The major disadvantage of all spatially arrayed techniques is that to enable larger numbers of compounds to be synthesized in parallel, sacrifices in reaction flexibility must be made. Although arrayed reactors may enable solution-phase chemistry, conditions such as high or low temperature, high pressure, addition of solid-phase reactants, and removal of solid-phase products may not all be conveniently utilized without extreme expense.
Mixtures When library production methods result in library members as mixtures in one or multiple fractions, analysis and assay techniques must then determine which individual library components are responsible for the resulting positive assay indications from these mixtures of compounds. Mixtures can be in solution phase or in solid phase (usually on resin beads) format. Library generation is fairly straightforward, taking advantage of mix-and-split synthesis without encoding. However, these libraries usually need deconvolution strategies to identify the active compounds from the data.
Deconvolution schemes involve assays of mixtures of compounds wherein any individual compound is assayed multiple times in different mixture groups. With a valid scheme, data from active mixture groups should produce definitive active compounds. Deconvolution strategies will depend on the library size and structure. Topiol et al. [33] have extensively examined mix-and-split schemes for optimal efficiency, considering the parameters of library size, number of diversity steps, number of reaction vessels, and number of compounds per final pool. Two of the main criticisms of mixtures are the amount of compound necessary for the redundant assays and the loss of sensitivity of an assay towards any particular compound. This second point is clearly shown in experiments by Boger et al. [6•], who compare 10-compound mixtures with 100-compound mixtures and observe that with the larger mixtures comes a loss of the less-distinguishable activities and subtle information. From a large mixture of compounds, individual components of interest can be culled out firstly if there is a very highly selective screen that can not only select but also separate the active components from the mixture and secondly if analytical techniques exist to identify these components. DNA and peptide libraries make the second of these criteria obtainable, and Alam et al. [34] take advantage of this by preparing and screening a 3,200,000-member library of pentapeptides to identify DNA-binding peptide ligands. Target DNA on magnetic beads was used to pull the active ligands from the bead-based combinatorial library. In a more general approach
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Figure 4
(a)
Combinatorial chemistry on a thread. (a) A thread is wrapped around a cylinder. The cylinder is then partitioned lengthwise into three regions. (b) A different reactant or building block (red, blue and green) is added to each region. (c) The thread now has repeating domains. (d) The process is repeated around an appropriately sized cylinder for four building blocks. (e) Four new building blocks (black, yellow, cyan and magenta) are added. (f) All combinations are represented. Reproduced from [32••] with permission from the Journal of the American Chemical Society.
(b)
(c)
(d)
(e)
(f)
for small-molecule libraries, Eliseev [35] added target macromolecules directly to a solution-phase library to select for high-affinity ligands. Size exclusion chromatography separates the receptor with bound ligands from the mixture, and desalting and LC–MS followed to separate and identify highaffinity ligands. Although MS does not distinguish between isomeric compounds, tagging the library components with mass tags can be helpful. Furthermore, a new regiochemical tagging tool for identification of isomeric library components has been described [36]. Although the above-mentioned techniques may be limited in both the applicable assay types and in the library design, the incredibly efficient screening obtained with testing of mixtures makes it very attractive in certain circumstances. If solution-phase mixtures can be separated and identified during or after synthesis, they begin to look like their encoded solid-phase library cousins. Curran and Zhiyong [37] describe techniques to both tag solution-phase compounds with fluorous tags and to purify them by fluorous reverse-phase silica gels. In a more recent paper [38•], more tags with differing retention times are presented. These techniques offer the basis for solution-phase mixture synthesis along with the ability to provide pure compounds as the final format.
beads (in-process tagged) are becoming larger and teabags (pre-encoded) are becoming smaller, thereby expanding the limits of both techniques. Likewise, encoding options have evolved, and potential library size is constantly increasing. The selection of a practical and efficient encoding method depends on several parameters, involving primarily the size of the library and the amounts of compounds desired. These criteria may narrow the remaining choices significantly. Furthermore, the ultimate decision must, of course, take into consideration solid-phase versus solution-phase experience, chemistry flexibility, imposed deadlines, and the myriad costs of capital equipment, manpower, facilities, and reagents. Thanks to the ongoing quest to improve library methodologies, more and better tools are continually becoming available to the combinatorial chemist.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Czarnik AW: Encoding methods for combinatorial chemistry. Curr Opin Chem Biol 1997, 1:60-66.
2.
Barnes C, Balasubramanian S: Recent developments in the encoding and deconvolution of combinatorial libraries. Curr Opin Chem Biol 2000, 4:346-350.
3.
Tiebes D: Combinatorial chemistry. In Combinatorial Chemistry. Edited by Jung G. Weinheim: Wiley-VCH; 1999:1-34.
4.
Austel V: Solution-phase combinatorial chemistry. In Combinatorial Chemistry. Edited by Jung G. Weinheim: Wiley-VCH; 1999:77-123.
5.
Turner R, Sterrer S, Wiesmuller KH, Meyer-Almes FJ: How to scaleup while scaling down: EVOscreenTM, a miniaturized ultra-highthroughput screening system. In Combinatorial Chemistry. Edited by Jung G. Weinheim: Wiley-VCH; 1999:441-461.
Summary and conclusions There are several viable options for encoding combinatorial libraries such that arrays of discrete compounds may be produced to enable ‘single compound’ assays. While established techniques continue to become more robust and flexible, several newly invented techniques have also been successful. Overall, the industry continues to advance with respect to many facets of the design and outcome of compound libraries:
Solutions for library encoding to create collections of discrete compounds Affleck
6. •
Boger DL, Dechantsreiter MA, Ishii T, Fink BE, Hedrick MP: Assessment of solution-phase positional scanning libraries based on distamycin A for the discovery of new DNA binding agents. Bioorg Med Chem 2000, 8:2049-2057. A good paper that contrasts the efficiency of larger mixtures with the disadvantage of the lower quality screening data associated with those larger mixtures. 7.
Lee D, Sello JK, Schreiber SL: Pairwise use of complexitygenerating reactions in diversity-oriented organic synthesis. Org Lett 2000, 2:709-712.
8.
Fromont C, Bradley M: High-Loading resin beads for solid phase synthesis using triple branching symmetrical dendrimers. Chem Commun 2000, 4:283-284.
9.
Hird N, Hughes I, Hunter D, Morrison MGJT, Sherrington DC, Stevenson L: Polymer discs — an alternative support format for solid phase synthesis. Tetrahedron 1999, 55:9575-9584.
10. Tripp JA, Stein JA, Svec F, Fréchet JMJ: ‘Reactive filtration’: use of functionalized porous polymer monoliths as scavengers in solution-phase synthesis. Org Lett 2000, 2:195-198. 11. Li W, Czarnik AW, Lillig J, Xiao XY: Kinetic study of organic reactions on polystyrene grafted microtubes. J Comb Chem 2000, 2:224-227.
23. Guiles JW, Lanter CL, Rivero RA: A visual tagging process for mix and sort combinatorial chemistry. Angew Chem Int Ed Engl 1998, 37:926-928. 24. Herpin TF, Van Kirk KG, Salvino JM, Yu ST, Labaudinière RF: Synthesis of a 10000 member 1,5-benzodiazepine-2-one library by the directed sorting method. J Comb Chem 2000, 2:513-521. 25. Nicolaou KC, Pfefferkorn JA, Mitchell HJ, Roecker AJ, Barluenga S, •• Cao GQ, Affleck RL, Lillig JE: Natural product-like combinatorial libraries based on privileged structures. 2. Construction of a 10000-membered benzopyran library by directed split-and-pool chemistry using NanoKans and optical encoding. J Am Chem Soc 2000, 122:9954-9967. An excellent paper demonstrating the ease and utility of IRORI’s NanoKan System. In a major milestone (and by the only practical means available), they produced 1–2 mg of discrete compounds for their 10,000-member sparsematrix library. Quality controls were run prior to, during, and following the library production to stepwise monitor and evaluate the success of the library. The entire library was synthesized, cleaved, and automatically formatted into microtiter plates in a remarkable eight days. 26. Vaino AR, Janda KD: Euclidean shape-encoded combinatorial chemical libraries. Proc Natl Acad Sci USA 2000, 97:7692-7696. 27.
12. Fodor SPA, Read JL, Pirrung MC, Stryer L, Lu AT, Solas D: Lightdirected, spatially addressable parallel chemical synthesis. Science 1991, 251:767-773. 13. Ohlmeyer MHJ, Swanson RN, Dillard LW, Reader JC, Asouline G, Kobayashi R, Wigler M, Still WC: Complex synthetic chemical libraries indexed with molecular tags. Proc Natl Acad Sci USA 1993 90:10922-10926. 14. Fitch WL, Baer TA, Chen W, Holden F, Holmes CP, Maclean D, Shah N, Sullivan E, Tang M, Waybourn P: Improved methods for encoding and decoding dialkylamine-encoded combinatorial libraries. J Comb Chem 1999, 1:188-194. 15. Dolle RE, Guo J, O’Brien L, Jin Y, Piznik M, Bowman KJ, Li W, • Egan WJ, Cavallaro CL, Roughton AL et al.: A statistical-based approach to assessing the fidelity of combinatorial libraries encoded with electrophoric molecular tags. Development and application of tag decode-assisted single bead LC/MS analysis. J Comb Chem 2000, 2:716-731. With mix-and-split synthesis on single-bead-per-compound libraries, there is often just enough compound from a bead to run the biological screen. Although much chemistry optimization and quality control is performed in advance of library production, it is desirable to have quality assurance (QA) of the actual produced library. This paper details such a statistical QA procedure. 16. Lane SJ, Pipe A: Single bead and hard tag decoding using • accurate isotopic difference target analysis-encoded combinatorial libraries. Rapid Commun Mass Spectrom 2000, 14:782-793. The secondary amine methodology has been further refined for higher sensitivity and selectivity of the dialkylamine tags. This is accomplished by using 50:50 mixes of tags with 2H:2D or 12C:13C, giving doublet pairs in the mass spectrum separated by 2.012 u or 2.0068 u, respectively. 17.
Hochlowski JE, Whittern DN, Sowin TJ: Encoding of combinatorial chemistry libraries by fluorine-19 NMR. J Comb Chem 1999, 1:291-293.
18. Pirrung MC, Park K: Discovery of selective metal-binding peptoids using 19F encoded combinatorial libraries. Bioorg Med Chem Lett 2000, 10:2115-2118. 19. Shchepinov MS, Chalk R, Southern EM: Trityl mass-tags for encoding in combinatorial oligonucleotide synthesis. Nuc Acid Sym Ser 1999 42:107-108. 20. Shchepinov MS, Chalk R, Southern EM: Trityl tags for encoding in combinatorial synthesis. Tetrahedron 2000, 56:2713-2724. 21. Battersby BJ, Bryant D, Meutermans W, Matthews D, Smythe ML, Trau M: Toward larger chemical libraries: encoding with fluorescent colloids in combinatorial chemistry. J Am Chem Soc 2000, 122:2138-2139. 22. Grøndahl L, Battersby BJ, Bryant D, Trau M: Encoding combinatorial • libraries: a novel application of fluorescent silica colloids. Langmuir 2000, 16:9709-9715. These two papers (see also [21]) on encoding with fluorescent colloids are interesting for a couple of reasons. First, the colloids are analyzed on the beads in a non-destructive manner. Second, by varying the ratios of different colored tags, many tag sets can be created with the potential to encode very large libraries.
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Nanthakumar A, Pon RT, Mazumder A, Yu S, Watson A: Solid-phase oligonucleotide synthesis and flow cytometric analysis with microspheres encoded with covalently attached fluorophores. Bioconjugate Chem 2000, 11:282-288.
28. South MS, Dice TA, Parlow JJ: Polymer-assisted solution-phase (PASP) library synthesis of α-ketoamides. Biotech Bioeng 2000, 71:51-57. 29. Furka A: Redistribution in combinatorial synthesis. A theoretical approach. Comb Chem High Throughput Screen 2000, 3:197-209. 30. Scharn D, Wenschuh H, Reineke U, Schneider-Mergener J, Germeroth L: Spatially addressed synthesis of amino- and aminooxy-substituted 1,3,5-triazine arrays on polymeric membranes. J Comb Chem 2000, 2:361-369. 31. Frank R: Spot-synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support. Tetrahedron 1992, 48:9217-9232. 32. Schwabacher AW, Shen Y, Johnson CW: Fourier transform •• combinatorial chemistry. J Am Chem Soc 1999, 121:8669-8670. A unique and creative addition to encoding processes for combinatorial chemistry. Spatially encoded compounds are synthesized in parallel along a single thread. Parallel synthesis of building blocks is achieved by wrapping the thread around a cylinder and exposing a radial section of the cylinder to a single building block for that step. 33. Topiol S, Davies J, Vijayakuma S, Wareing JR: Computer aided analysis of split and mix combinatorial libraries. J Comb Chem 2001, 3:20-27. 34. Alam MR, Maeda M, Sasaki S: DNA-binding peptides searched from the solid-phase combinatorial library with the use of magnetic beads attaching the target duplex DNA. Bioorg Med Chem 2000, 8:465-473. 35. Eliseev AV: Emerging approaches to target-assisted screening of combinatorial mixtures. Drug Dev Discov 1998, 1:106-115. 36. Nazarpack-Kandlousy N, Chernushevich IV, Meng LJ, Yang Y, Eliseev AV: Regiochemical tagging: a new tool for structural characterization of isomeric components in combinatorial mixtures. J Am Chem Soc 2000, 122:3358-3366. 37.
Curran DP, Zhiyong L: Fluorous synthesis with fewer fluorines (light fluorous synthesis): separation of tagged from untagged products by solid-phase extraction with fluorous reverse-phase silica gel. J Am Chem Soc 1999, 121:9069-9072.
38. Zhang Q, Luo Z, Curran DP: Separation of ‘light fluorous’ reagents • and catalysts by fluorous solid-phase extraction: synthesis and study of a family of triarylphosphines bearing linear and branched fluorous tags. J Org Chem 2000, 65:8866-8873. These two papers (see also [37]) on ‘light fluorous’ synthesis deal with tagging and purifying solution-phase compounds on the basis of their fluorous content. Although not specifically dealt with in these articles, an implication of this technology would be the ability to create fluorous blocking groups that could lead to ‘sorting’ of solution-phase compounds with a separation technique that is orthogonal to the properties of the compounds. This would allow solution-phase mix-and-split synthesis.