Partial alloc-deprotection approach for ladder synthesis of "one-bead one-compound" combinatorial libraries

The topologically segregated bilayer-bead concept has been applied to encoded "one-bead one-compound"(OBOC) combinatorial libraries to avoid the interference of coding tags with biological screening. In this paper, we report on the development of a novel partial Alloc-deprotection (PAD) approach and the use of this approach to establish a new ladder-synthesis method for OBOC combinatorial libraries to further exploit the concept. In the PAD approach, Alloc-protected beads are partially deprotected, sequentially layer by layer, starting from the outer layer toward the bead interior. The degree of deprotection (or thickness of each layer) is controlled by the time of exposure to the deprotecting agent, palladium. By repetitive use of the PAD approach, a small portion of Alloc-protected N termini in the bead interior is liberated in each synthetic cycle for generation of an additional ladder member such that each library bead will carry a full-length library compound on the bead surface and a series of truncated ladder members in the bead interior. For the libraries containing isobaric residues, a simple encoding strategy is introduced in the ladder-synthesis method so that the isobaric residues can be differentiated by the coding tags. One advantage of this encoding strategy is that the coding tags are confined together with the truncated ladder members in the bead interior, thus maintaining the arrangement that only the library compounds are displayed on the bead surface. The PAD approach of forming multiple concentric functional layers inside a bead is simple, reliable, and may have other applications in addition to OBOC combinatorial library bead encoding, such as the development of novel optically encoded beads for multiplex immunodiagnostics or even information recording.     

A novel peptide-based encoding system for "one-bead one-compound" peptidomimetic and small molecule combinatorial libraries

The "one-bead one-compound" (OBOC) combinatorial library method is highly efficient, especially when used with well-established on-bead binding or functional assays. Literally, millions of compounds can be screened concurrently within 1 to 2 days. However, structure determination of peptidomimetic and small molecule compounds on one single bead is not trivial. A novel, highly efficient, and robust peptide-based encoding system has been developed for OBOC peptidomimetic and small molecule combinatorial libraries. In this system, topologically segregated bifunctional beads, which are made by a simple biphasic solvent strategy, are employed for the preparation and screening of an OBOC combinatorial peptidomimetic and small molecule libraries. Testing molecules are on the outer layer, and the coding tags in the interior of the bead do not interfere with screening. The coding tag is a peptide containing a large number of unnatural alpha-amino acids derived from different building blocks used for generating the peptidomimetic or small molecule. By coupling common building blocks simultaneously to the scaffold of the testing compound and to the side chains of the alpha-amino acids on the coding peptide, extra synthetic steps are eliminated and the amount of undesirable side products is minimized. Positive bead decoding is easy and straightforward as there is no need for cleavage and retrieval of the coding tag, and positive beads can be sequenced directly with Edman degradation. To demonstrate the efficiency and simplicity of our encoding system, an encoded 158 400-member model peptidomimetic library has been generated and screened for ligands that bind to streptavidin. Potent and novel ligands with clear motifs have been identified.     

Global transformation of OBOC combinatorial peptide libraries into OBOC polyamine and small molecule libraries

In "one-bead-one-compound" (OBOC) combinatorial chemistry, a compound-bead library with hundreds of thousands to millions of diversities can be rapidly generated such that each bead displays only one chemical entity. The highly efficient "libraries-from-libraries" approach involves the global transformation of a peptide library into many small molecule solution-phase mixture libraries, but this approach has never been successfully applied to OBOC libraries. Here we report a novel approach that allows us to combine these two enabling technologies to efficiently generate OBOC encoded small molecule bead libraries. By using a topologically segregated bilayer bead and a "ladder-synthesis" method, we can prepare peptide libraries with the peptide on the bead surface and a series of peptide ladders in the bead interior. Various global transformation reactions can then be employed to transform the starting peptide library into a variety of peptidomimetic libraries. During the transformation reactions, the peptide ladders in the bead interior are also transformed in a predictable manner. As a result, individual compound bead can be decoded by analyzing the hydrogen fluoride-released encoding tags with matrix-assisted laser desorption ionization Fourier transform mass spectrometry. Using this novel approach, a random encoded dipeptide library was prepared and subsequently transformed into polyamine and poly- N-acetylamine sublibraries. Random beads isolated from these sublibraries were reliably decoded.     

A novel and rapid encoding method based on mass spectrometry for "one-bead-one-compound" small molecule combinatorial libraries

A novel and efficient encoding method based on mass spectrometry for "one-bead-one-compound" small molecule combinatorial libraries has been developed. The topologically segregated bifunctional resin beads with orthogonal protecting groups in the outer and inner regions are first prepared according to our previously published procedure. Prior to library synthesis, the inner core of each bead is derivatized with 3-4 different coding blocks on a cleavable linker. Each functional group on the scaffold is encoded by an individual coding block containing a functional group with the same chemical reactivity. During the library synthesis, the same chemical reactions take place on the scaffold (outer layer of the bead) and coding blocks (inner core of the bead) concurrently. After screening, the coding tags in the positive beads are released, followed by molecular mass determination using matrix-assisted laser desorption ionization Fourier transform mass spectrometry. The chemical structure of library compounds can be readily identified according to the molecular masses of the coding tags. The feasibility and efficiency of this approach were demonstrated by the synthesis and screening of a model small molecule library containing 84 672 member compounds, with a model receptor, streptavidin. Streptavidin binding ligands with structural similarity (17) were identified. The decoding results were clear and unambiguous.     

Rainbow beads: a color coding method to facilitate high-throughput screening and optimization of one-bead one-compound combinatorial libraries

We have developed a new color-encoding method that facilitates high-throughput screening of one-bead one-compound (OBOC) combinatorial libraries. Polymer beads displaying chemical compounds or families of compounds are stained with oil-based organic dyes that are used as coding tags. The color dyes do not affect cell binding to the compounds displayed on the surface of the beads. We have applied such rainbow beads in a multiplex manner to discover and profile ligands against cell surface receptors. In the first application, a series of OBOC libraries with different scaffolds or motifs are each color-coded; small samples of each library are then combined and screened concurrently against live cells for cell attachment. Preferred libraries can be rapidly identified and selected for subsequent large-scale screenings for cell surface binding ligands. In a second application, beads with a series of peptide analogues (e.g., alanine scan) are color-coded, combined, and tested for binding against a specific cell line in a single-tissue culture well; the critical residues required for binding can be easily determined. In a third application, ligands reacting against a series of integrins are color-coded and used as a readily applied research tool to determine the integrin profile of any cell type. One major advantage of this straightforward and yet powerful method is that only an ordinary inverted microscope is needed for the analysis, instead of sophisticated (and expensive) fluorescent microscopes or flow cytometers.     

Synthesis, screening, and sequencing of cysteine-rich one-bead one-compound peptide libraries

Cysteine-rich peptides are valued as tags for biarsenical fluorophores and as environmentally important reagents for binding toxic heavy metals. Due to the inherent difficulties created by cysteine, the power of one-bead one-compound (OBOC) libraries has never been applied to the discovery of short cysteine-rich peptides. We have developed the first method for the synthesis, screening, and sequencing of cysteine-rich OBOC peptide libraries. First, we synthesized a heavily biased cysteine-rich OBOC library, incorporating 50% cysteine at each position (Ac-X8-KM-TentaGel). Then, we developed conditions for cysteine alkylation, cyanogen bromide cleavage, and direct MS/MS sequencing of that library at the single bead level. The sequencing efficiency of this library was comparable to a traditional cysteine-free library. To validate screening of cysteine-rich OBOC libraries, we reacted a library with the biarsenical FlAsH and identified beads bearing the known biarsenical-binding motif (CCXXCC). These results enable OBOC libraries to be used in high-throughput discovery of cysteine-rich peptides for protein tagging, environmental remediation of metal contaminants, or cysteine-rich pharmaceuticals.     

(19)F-encoded combinatorial libraries: discovery of selective metal binding and catalytic peptoids

A (19)F NMR method for encoding of combinatorial libraries has been developed. Aryl fluorides whose chemical shifts are modified by aromatic substituents were prepared and attached to resin support beads that were used in the split-pool synthesis of peptoids. The detection of the (19)F NMR signal of tags derived from a single "big bead" was demonstrated. The library diversity arises from peptoid amines and the cyclic anhydrides used in their acylation. The resulting 90-compound library was examined for metal ion binding, and novel ligands for iron and copper were discovered. Their binding constants were determined to be in the low micromolar range using conventional methods. The library was also examined for autocatalysis of acylation, and a molecule possessing the catalytic triad of serine proteases was deduced.     

Image subtraction approach to screening one-bead-one-compound combinatorial libraries with complex protein mixtures

To screen one-bead-one-compound (OBOC) combinatorial bead libraries,(1) one generally uses tagged purified protein as the screening probe. Compound beads that interact with the purified protein are then identified, for example, via an enzyme-linked colorimetric assay, and isolated for structure determination. In this report, we demonstrate a rapid and efficient method to screen OBOC combinatorial libraries utilizing two protein mixtures as screening probes, and by comparing optical images of the beads stained by one protein mixture but not the other, ligand beads unique to one of the two protein mixtures can be identified. The significance of this method is that it allows for rapid selection of ligands directed against proteins unique to one mixture while screening out positive beads resulting from proteins common to both mixtures as well as beads that are positive as a result of interactions with chemical and protein components found in the assay itself. The method is fast, efficient, and uses off-the-shelf equipment.     

OBOC small-molecule combinatorial library encoded by halogenated mass-tags

[reaction: see text] A bromine-/chlorine-containing mass-tag encoding strategy for a small-molecule OBOC combinatorial library is reported. The resulting MALDI FTMS isotope pattern of each tag clearly defines the component building blocks of each "hit" bead in an 1890-member demonstration library screened on-bead for binding against streptavidin via both enzyme-linked colorimetric and Quantum Dot/COPAS assays.     

Imaging combinatorial libraries by mass spectrometry: from peptide to organic-supported syntheses

Supported peptide and drug-like organic molecule libraries were profiled in single nondestructive imaging static secondary ion mass spectrometric experiments. The selective rupture of the bond linking the compound and the insoluble polymeric support (resin) produced ions that were characteristic of the anchored molecules, thus allowing unambiguous resin bead assignment. Very high sensitivity and specificity were obtained with such a direct analytical method, which avoids the chemical release of the molecules from the support. Libraries issued from either mix-and-split or parallel solid-phase organic syntheses were profiled, demonstrating the usefulness of such a technique for characterization and optimization during combinatorial library development. Moreover, the fact that the control was effected at the bead level whatever the structure and quantity of the anchored molecules allows the sole identification of active beads selected from on-bead screening. Under such circumstances, the time-consuming whole-library characterization could thus be suppressed, enhancing the throughput of the analytical process.     

High-throughput sequence determination of cyclic peptide library members by partial Edman degradation/mass spectrometry

Cyclic peptides provide attractive lead compounds for drug discovery and excellent molecular probes in biomedical research. Large combinatorial libraries of cyclic peptides can now be routinely synthesized by the split-and-pool method and screened against biological targets. However, post-screening sequence determination of hit peptides has been problematic. In this report, a high-throughput method for the sequence determination of cyclic peptide library members has been developed. TentaGel microbeads (90 mum) were spatially segregated into outer and inner layers; cyclic peptides were displayed on the bead surface, whereas the inner core of each bead contained the corresponding linear peptide as the encoding sequence. After screening of the cyclic peptide library against a macromolecular target, the identity of hit peptides was determined by sequencing the linear encoding peptides inside the bead using a partial Edman degradation/mass spectrometry method. On-bead screening of an octapeptide library (theoretical diversity of 160 000) identified cyclic peptides that bind to streptavidin. A 400-member library of tyrocidine A analogues was synthesized on TentaGel macrobeads and solution-phase screening of the library directly against bacterial cells identified a tyrocidine analogue of improved antibacterial activity. Our results demonstrate that the new method for cyclic peptide sequence determination is reliable, operationally simple, rapid, and inexpensive and should greatly expand the utility of cyclic peptides in biomedical research.     

A Cleavable Scaffold Strategy for the Synthesis of One-Bead One-Compound Cyclic Peptoid Libraries That Can Be Sequenced By Tandem Mass Spectrometry

Many macrocyclic depsipeptides or related compounds have interesting medicinal properties and often display more favorable pharmacokinetic properties than linear analogues. Therefore, there is considerable interest in the development of large combinatorial libraries of macrocyclic peptidomimetic compounds. However, such molecules cannot be easily sequenced by tandem mass spectrometry, making it difficult to identify hits isolated from library screens using one bead one compound libraries. Here we report a strategy to solve this problem by placing a methionine in both the linker connecting the cyclic molecule to the bead as well as within the cycle itself. Treatment with CNBr both linearizes the molecule at a specific position and releases the molecule from the bead, making its characterization by tandem MALDI mass spectrometry straightforward.     

Automated mass spectrometric sequence determination of cyclic peptide library members

Cyclic peptides have come under scrutiny as potential antimicrobial therapeutic agents. Combinatorial split-and-pool synthesis of cyclic peptides can afford single compound per well libraries for antimicrobial screening, new lead identification, and construction of quantitative structure-activity relationships (QSAR). Here, we report a new sequencing protocol for rapid identification of the members of a cyclic peptide library based on automated computer analysis of mass spectra, obviating the need for library encoding/decoding strategies. Furthermore, the software readily integrates with common spreadsheet and database packages to facilitate data visualization and archiving. The utility of the new MS-sequencing approach is demonstrated using sonic spray ionization ion trap MS and MS/MS spectrometry on a single compound per bead cyclic peptide library and validated with individually synthesized pure cyclic D,L-alpha-peptides.     

Directed-sorting method for synthesis of bead-based combinatorial libraries of heterogeneous catalysts

The synthesis and analysis of inorganic material combinatorial libraries by a directed-sorting, split-pool bead method was demonstrated. Directed-sorting, split-pool, metal-loaded libraries were synthesized by adsorbing metal salts (H2PtCl6, SnCl2, CuCl2, and NiCl2) and metal standards (Pt, Cu, Ni in HCl) onto 2-mg porous gamma-alumina beads in 96- or 384-well plates. A matrix algorithm for the synthesis of bead libraries treated each bead as a member of a row or column of a given matrix. Computer simulations and manual tracking of the sorting process were used to assess library diversity. The bead compositions were analyzed by energy-dispersive X-ray spectroscopy, X-ray fluorescence spectroscopy, electron probe microanalysis, inductively coupled plasma atomic emission spectroscopy, and inductively coupled plasma mass spectroscopy. The metal-loaded beads were analyzed by laser-activated membrane introduction mass spectroscopy (LAMIMS) for catalytic activity using methylcyclohexane dehydrogenation to toluene as a probe reaction. The catalytic activity of individual beads that showed minimal (approximately 20% of that of Pt on alumina) to high conversion could be determined semiquantitatively by LAMIMS. This method, therefore, provides an alternative to screening using microreactors for reactors that employ catalysts in the form of beads. The directed-sorting method offers the potential for synthesis of focused libraries of inorganic materials through relatively simple benchtop split-pool chemistry.     

Functional Peptides from One-bead One-compound High-throughput Screening Technique

Combinatorial chemistry provides a cost-effective method for the rapid discovery of new functional peptides. One-bead one-compound(OBOC) high-throughput screening technique offers a lot of structurally diverse peptides to be rapidly synthesized and screened for binding to a target of interest. The OBOC peptide library screening involves three main steps: library construction, positive beads separation, and peptide sequencing. This review mainly summarizes some special technique tips during functional peptide screening and potential future directions of the OBOC high-throughput screening technique.     

1,3,5-triazine-based mass spectral tagging of one-bead one-compound libraries

A triazine-based mass encoding strategy that accommodates cleavable linker, isotopic labeling, and diversity receptor moieties is reported. The resulting triazine-based tags, which are coupled to bifunctionalized TentaGel resin in a one-pot transformation, enable the construction of a 1-oxa-2,8-diazaspiro[4.5]dec-2-ene-7-carboxamide library and facilitate decoding by equalizing the ionization potential of the liberated tags in single bead MALDI-TOF experiments as well as balancing the reactivity of the starting tags in the resin coupling step. [reaction: see text].     

Software for automating analysis of encoded combinatorial libraries

This paper describes the applications which are used to automate the analysis of encoded combinatorial libraries. Commercial packages from MDL, Oracle and Agilent are linked with application software written in C/C++, in Microsoft Access and in ChemStation macro language. Encoding correspondence lists for each of up to three synthetic steps are conveniently associated with building block lists using the first application, CodeGen. The second application Decode allows the user to identify the individual beads picked onto a 96-well plate and the pool number for each bead. The decoding chromatography data for each well is then loaded into the program. The chromatography data is used to identify the tags used in the synthesis. Along with the building block information from ISIS/Host, the building block used in each step of the synthesis can be identified. A third routine, Code-to-Structure, takes the coded library building blocks and creates the connection table in ISIS for each structure found by the decode program. For quality control of encoded library synthesis, the decoded structures on a set of beads is compared to the LC/UV/MS data for the ligand cleaved from the same bead. CAPTURE, a GlaxoSmithKline proprietary application, is used to display and analyze the decoded structures and associated mass spectral data. This application uses simple isotopic composition and electrospray ionization rule sets to predict mass spectra and judge the concordance of a structure- mass spectrum data set. An ancillary program, EIC, is used to extract predicted single ion chromatograms from the full scan LC/MS data.     

Erythropoietin mimetics derived from solution phase combinatorial libraries.

The erythropoietin receptor (EPOr) is activated by ligand-induced homodimerization, which leads to the proliferation and differentiation of erythroid progenitors. Through the screening of combinatorial libraries of dimeric iminodiacetic acid diamides, novel small molecule binders of EPOr were identified in a protein binding assay. Evaluation of a series of analogues led to optimization of binding subunits, and these were utilized in the synthesis of higher order dimer, trimer, and tetramer libraries. Several of the most active EPOr binders were found to be partial agonists and induced concentration-dependent proliferation of an EPO-dependent cell line (UT-7/EPO) while having no effect on a cell line lacking the EPOr (FDC-P1). An additional compound library, based on a symmetrical isoindoline-5,6-dicarboxylic acid template and including the optimized binding subunits, was synthesized and screened leading to the identification of additional EPO mimetics.     

Identification of small molecule inhibitors that distinguish between non-transferrin bound iron uptake and transferrin-mediated iron transport

Chemical genetics is an emerging field that takes advantage of combinatorial chemical and small molecule libraries to dissect complex biological processes. Here we establish a fluorescence-based assay to screen for inhibitors of iron uptake by mammalian cells. Using this approach, we screened the National Cancer Institute's Diversity Set library for inhibitors of non-transferrin bound iron uptake. This screen identified 10 novel small molecule inhibitors of iron transport with IC(50) values that ranged from 5 to 30 microM. Of these ten compounds, only two blocked uptake of iron mediated by transferrin. Thus, this study characterizes the first small molecule inhibitors that distinguish between different pathways of iron transport.     

Optical barcoding of colloidal suspensions: applications in genomics,proteomics and drug discovery

The enormous amount of information generated through sequencing of the human genome has increased demands for more economical and flexible alternatives in genomics, proteomics and drug discovery. Many companies and institutions have recognised the potential of increasing the size and complexity of chemical libraries by producing large chemical libraries on colloidal support beads. Since colloid-based compounds in a suspension are randomly located, an encoding system such as optical barcoding is required to permit rapid elucidation of the compound structures. We describe in this article innovative methods for optical barcoding of colloids for use as support beads in both combinatorial and non-combinatorial libraries. We focus in particular on the difficult problem of barcoding extremely large libraries, which if solved, will transform the manner in which genomics, proteomics and drug discovery research is currently performed.