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PubMed1996

Structurally homogeneous and heterogeneous synthetic combinatorial libraries.

We have designed and synthesized structurally homogeneous and heterogeneous nonpeptide libraries. Structurally homogeneous libraries are characterized by the presence of one common structural unit, a scaffold, in all library compounds (e.g. cyclopentane, cyclohexane, diketopiperazine, thiazolidine). In structurally heterogeneous libraries different organic reactions (acylation, etherification, reductive amination, nucleophilic displacement) were applied to connect bifunctional building blocks unrelated in structure (aromatic hydroxy acids, aromatic hydroxy aldehydes, amino alcohols, diamines, and amino acids). The focus of this communication is to document the use of bifunctional building blocks for the design and synthesis of structurally heterogeneous libraries of N-(alkoxy acyl)amino acids, N,N'-bis-(alkoxy acyl)diamino acids, N-acylamino ethers, N-(alkoxy acyl)amino alcohols, N-alkylamino ethers, and N-(alkoxy aryl)diamines.

Aldehydes↗
PubMed1996

Construction and screening of M13 phage libraries displaying long random peptides.

We have constructed two phage display libraries expressing N-terminal pIII fusions in M13 composed of 37 and 43 random amino acid domains, respectively. The D38 library expresses 37 random amino acids with a central alanine residue, and the DC43 library contains 43 random amino acids with a central cysteine flanked by two glycine residues, giving the displayed peptide the potential to form disulfide loops of various sizes. We demonstrate that the majority of random sequences in both libraries are compatible in pentavalent display with phage viability. The M13 phage display vector itself has been engineered to contain a factor Xa protease cleavage site to provide an alternative to acid elution during affinity selection. An in-frame amber mutation has been inserted between the pIII cloning sites to allow for efficient selection against nonrecombinant phage in the library. These libraries have been panned against mAb 7E11-C5, which recognizes the prostate-specific membrane antigen (PSM). Isolated phage display a consensus sequence that is homologous to a region in the PSM molecule.

Amino Acid Sequence↗
PubMed1996

Bifunctional scaffolds as templates for synthetic combinatorial libraries.

A small-molecule synthetic combinatorial library was designed and synthesized that features potential pharmacophores attached to a variety of small cyclic scaffolds. The synthesis of the library involved randomization of three types of building blocks: 20 amino acids, 10 aromatic hydroxy acids and 21 alcohols, totaling a library complexity of 4200 compounds. Mitsunobu polymer-supported etherification was used in the last randomization. The library compounds were attached to beads via an ester-bond linkage enabling both on-bead as well as in-solution screening. When the library was tested against a model target, streptavidin, specific binders were found. The structures of the most active compounds were determined from the fragmentation pattern in MS/MS experiments.

Alcohols↗
PubMed1996

The impact of polystyrene resins in solid-phase organic synthesis.

A major objective of the DIVERSOMER technology is to provide pure and characterized compounds for biological testing in order to prevent 'false negatives' in our libraries. On several occasions, analysis of the final products by 1H-NMR and MS, has revealed by-products from the polystyrene solid support. Subsequently, three alternative methods were studied to remove polystyrene by-products; (i) prewashing of the resin prior to execution of the synthesis; (ii) pretreatment of the resin with the cleavage conditions consistent with the solid-phase synthesis reaction scheme; and (iii) parallel purification.

Chemistry, Organic↗
PubMed1996

Synthetic library techniques: subjective (biased and generic) thoughts and views.

Various aspects of synthetic diversity generation and screening are discussed. Controversial issues are raised and different points of view are presented. We hope the article will stimulate thinking about the utilization of library techniques and start a discussion about questions concerning their application.

Bacterial Proteins↗
PubMed1996

Assessing the ability of chemical similarity measures to discriminate between active and inactive compounds.

A method for assessing the biological discriminating power of chemical similarity measures is presented. The main concern of this work was to develop an objective way of evaluating different similarity measures in terms of how well they distinguished between active and inactive compounds. In addition, we have explored the level of similarity required for optimal separation and commented on its implications for work in the field of chemical diversity studies. The results for one simple similarity measure showed that statistically significant separation could be achieved, and indicated a reasonable similarity value for future work.

Directed Molecular Evolution↗
PubMed1996

Structure and properties of TentaGel resin beads: implications for combinatorial library chemistry.

In view of the widespread use of TentaGel resin beads for the synthesis of combinatorial libraries, the properties of TentaGel resin have been examined using a combination of confocal laser microscopy and NMR spectroscopy. Evidence is presented that trypsin, a 23.5-kDa enzyme, can penetrate to the core of 90-microns TentaGel beads, and that the matrix of such beads permits molecular motion at a similar rate to that in solution. The beads act as a separate gel phase rather than as a porous solid. These conclusions have important implications for the bioassay of on-bead combinatorial chemical libraries.

Deuterium↗
PubMed1996

Novel alpha-glucosidase inhibitors identified using multiple cyclic peptide combinatorial libraries.

Twenty-six cyclic synthetic peptide combinatorial libraries (disulfides and lactams) of varying size and composition, representing 6.8 x 10(3) to 4.7 x 10(7) individual peptides, were synthesized along with their respective linear analogs. One of the hexapeptide lactam libraries (cyclo[xXxXxN]) was found to have significant alpha-glucosidase inhibitory activity. This library was carried through an iterative process of synthesis and screening, during which all of the five mixture positions (x and X) were successively defined. As the result of this process, potent and selective alpha-glucosidase inhibitors were identified.

Amino Acid Sequence↗
PubMed1996

Analysis of novel streptavidin-binding peptides, identified using a phage display library, shows that amino acids external to a perfectly conserved consensus sequence and to the presented peptides contribute to binding.

Streptavidin-binding peptides containing the consensus amino acid sequence motif EPDW were identified using a phage display library. Phage presenting peptides containing these sequences bound streptavidin in a biotin-sensitive fashion and could be eluted with biotin. The previously identified 'streptag' peptide sequence (AWRHPQGG) competed with phage presenting the EPDW consensus sequence for streptavidin binding. Furthermore, the EPDW sequence has two amino acids in common with yet another previously identified streptavidin-binding sequence, GDWVFI, which has similar biochemical properties. Binding inhibition studies revealed that residues flanking EPDW, as well as residues of the modified phage pIII product to which displayed peptides are fused, contributed to streptavidin binding. The derivation of small molecules based on the structure of peptides selected using display methods is a potentially important application of phage display technology. The relevance of the observations made here for that application are discussed. Finally, a group of 'nuisance' peptides of the consensus sequence WHWWXW, whose binding specificity has not been fully elucidated, but which have been isolated in a number of biopanning experiments, including those that do not utilize streptavidin, are also described.

Amino Acid Sequence↗
PubMed1996

Quantitation of combinatorial libraries of small organic molecules by normal-phase HPLC with evaporative light-scattering detection.

The advantages of evaporative light-scattering detection over UV detection for the quantitation of combinatorial libraries composed of small organic compounds by HPLC are described. The detector's response is independent of the sample chromophore, which makes it well-suited to chromatographic analyses of mixtures of dissimilar solutes. Thus, HPLC with evaporative light-scattering detection offers to potential for reducing false positive or false negative results in screening assays, because of its ability to detect the presence of impurities that absorb poorly in the UV (e.g., those impurities originating from the polymeric support). Furthermore, the evaporative light-scattering detector exhibits a nearly equivalent response to compounds of similar structural class. Hence, rapid quantitation of compound libraries may be carried out with the use of a single external standard. For example, the quantitation errors, based on a single external standard, for a series of steroids, hydantoins, and BOC- and Fmoc-protected amino acids by normal-phase HPLC with evaporative light-scattering detection average approximately +/-10%. The application of the evaporative light-scattering detector to the quantitation of low-level sample impurities and the detector's compatibility with gradient elution are also described.

Amino Acids↗
PubMed1996

Identification of cyclized calmodulin antagonists from a phage display random peptide library.

To isolate peptide ligands that bound calmodulin (CaM) specifically, we screened an M13 phage library displaying cyclized octamer random peptides with immobilized bovine CaM. Isolates were recovered, sequenced, and deduced to express nine independent peptides, five of which contained the sequence Trp-Gly-Lys (WGK). Four of the nine peptide sequences were synthesized in cyclized, biotinylated form. All of the peptides required Ca2+ to bind CaM. The cyclized, disulfide-bonded form of one such peptide, SCLRWGKWSNCGS, bound CaM better than its reduced form or an analogue in which the cysteine residues were replaced by serine. The cyclized peptide also exhibited the ability to inhibit CaM-dependent kinase activity. Systematic alanine substitution of residues in this peptide sequence implicate the tryptophan residue as being critical for binding, with other residues contributing to binding to varying degrees. Cloning of ligand targets (COLT) confirmed the specificity of one of the cyclized peptides, yielding full-length and C-terminal CaM clones, in addition to a full-length clone of troponin C, a CaM-related protein. This study has demonstrated that conformationally constrained peptides isolated from a phage library acted as specific, Ca(2+)-dependent CaM ligands.

Amino Acid Sequence↗
PubMed1996

Discovery of a herbicidal lead using polymer-bound activated esters in generating a combinatorial library of amides and esters.

A combinatorial library containing mixtures of amides and esters was prepared through solid-phase chemistry. The advantages of using solid-phase chemistry over solution-phase chemistry to prepare this library are discussed. The library was screened through a high-throughput whole organism herbicidal assay upon which a mixture containing amides was found to have herbicidal activity. Deconvolution of the mixture provided N-(3-benzoylphenyl)-3-(1,1-dimethylethyl)-1-methyl)-1 H-pyrazole-5-carboxamide as a herbicidal lead with broadleaf and narrowleaf pre-emergence herbicidal activity as low as 100 g/ha on some weed species. This study represents the first report of an agrochemical discovered using a combinatorial approach.

Amides↗
PubMed1996

Automating combinatorial chemistry: a primer on benchtop robotic systems.

Benchtop robotic systems are inexpensive, flexible automation tools with potential applications in a wide array of disciplines such as combinatorial chemistry, high-throughput screening, and genomics. We explain the basic components of a benchtop system and explore factors to consider when purchasing or customizing a robot, such as automation benefits, vendor selection, and current system limitations. Issues involving system specification, software design, and hardware customization are then discussed. Additionally, system optimization, validation, and support are detailed. Given a properly designed and implemented system, the combinatorial laboratory can markedly increase compound synthesis and purification.

Computers↗
PubMed1995

A single-bead decode strategy using electrospray ionization mass spectrometry and a new photolabile linker: 3-amino-3-(2-nitrophenyl)propionic acid.

A new linker that employs a photosensitive 3-amino-3-(2-nitrophenyl)propionyl functionality (ANP-resin) has been developed for the preparation of C-terminal carboxamides. A wide range of carboxamides were prepared and identified using the ANP-resin and electrospray ionization mass spectrometry. A single bead containing tripeptide Fmoc-Asp-Arg(Tos)-Val-NH2 was isolated, photocleaved and the peptide was characterized by tandem mass spectrometry, thereby verifying a library decode strategy that avoids complex tagging procedures.

Amino Acid Sequence↗
PubMed1995

Multiple simultaneous synthesis of phenolic libraries.

A series of analogous arrays of small, non-peptidyl, non-oligomeric compounds were synthesized on polystyrene resin. With the aid of a functionally differentiated phenolic scaffold, the batch preparation of unique benzamide and urea resins was accomplished, which were further derivatized in modified 96-well plates. An efficient cleavage reaction of the phenyl benzoate link enabled the isolation of more than 600 phenolic compounds in milligram quantities that were suitable for direct biological screening. The technology described herein represents a facile, economical approach to non-peptidyl chemical diversity.

Chemistry, Organic↗
PubMed1995

Mapping the detailed specificity of a calcium-dependent monoclonal antibody through the use of soluble positional scanning combinatorial libraries: identification of potent calcium-independent antigens.

The detailed specificity of monoclonal antibody M1, which has been reported to bind in a calcium-dependent manner to the 'FLAG' sequence DYKDDDDK-NH2, was examined using soluble hexa- and decapeptide positional scanning synthetic combinatorial libraries (PS-SCLs) made up of 52 x 10(6) and 4 x 10(12) different sequences, respectively. To study the influence of calcium on the specificity of this antigen-antibody interaction, each PS-SCL was screened in the presence and absence of calcium using a competitive ELISA. Overall, peptide mixtures had greater inhibitory activity against mAb M1 binding to FLAG in the absence of calcium. A total of 16 individual hexapeptides were identified, all of which contained the motif-DYK_K_(-), and were recognized by mAb M1 in the absence of calcium with 50- to 100-fold higher affinity than the FLAG octapeptide (IC50 = 273 nM). On average, the same set of peptides bound 10-fold less effectively in the presence of calcium. Upon screening the decapeptide PS-SCL in the absence of calcium, lysine was also more active in the fifth position than the original aspartic acid. Based on the screening results, 24 individual decapeptides were prepared and were found to have activities 10- to 100-fold higher than the FLAG octapeptide in the absence of calcium. The specificity of lysine at the fifth position in the antigen-antibody interaction was further examined by synthesizing and assaying substitution analogs at this position for the octapeptide and hexapeptide forms of the FLAG sequence, as well as for two hexapeptides identified from the PS-SCL. Truncation analog analysis was also carried out on the FLAG octapeptide to determine optimal antigen length for antibody binding. Overall, lysine at the fifth position could be substituted with ornithine with no significant loss in activity, and peptide length was not a critical factor for antibody binding in the absence of calcium. Also, the octapeptide having lysine at the fifth position in place of the aspartic acid had the same activity in the presence or absence of calcium. This study demonstrates the ease and effectiveness of PS-SCLs over individual peptide analogs for the examination of the degree of cross-reactivity for a given monoclonal antibody as well as for the identification of novel, high-affinity peptides.

Amino Acid Sequence↗
PubMed1995

Libraries of random-sequence polypeptides produced with high yield as carboxy-terminal fusions with ubiquitin.

Libraries of random-sequence polypeptides have been shown to be valuable sources of novel molecules possessing a variety of useful biologic-like activities, some of which may hold promise as potential vaccines and therapeutics. Previous random peptide expression systems were limited to low levels of peptide production and often to short sequences. Here we describe a series of libraries designed for increased polypeptide length. Cloned as carboxy-terminal extensions of ubiquitin, the fusions were produced in E. coli at high levels, and were purified to homogeneity. The majority of the extension proteins examined could be cleaved from ubiquitin by treatment with a ubiquitin-fusion hydrolase. The libraries described here are appropriate sources of novel polypeptides with desired binding or catalytic function, as well as tools with which to examine inherent properties of proteins as a whole. Toward the latter goal, we have examined structural properties of random-sequence proteins purified from these libraries. Quite surprisingly, fluorescence emission spectra of intrinsic tryptophan residues in several purified fusion proteins, under native-like and denaturing conditions, often resemble those expected for folded and unfolded states, respectively. The results presented here detail an important expansion in the range of potential uses for random-sequence polypeptide libraries.

Amino Acids↗
PubMed1995

A combinatorial method for constructing libraries of long peptides displayed by filamentous phage.

We describe the construction and screening of a random peptide library displayed by filamentous phage. The peptides are expressed in multiple copies on the filamentous phage M13 as amino-terminal fusions with the major coat protein, the product of gene VIII. These libraries are efficiently screened for reactive peptides, using a combination of panning in solution followed by a plaque lift assay. Advantages of this system are that both high- and low-affinity phage clones are simultaneously identified and the analysis of non-reactive phage is minimized. The vector system utilized to construct this library enables it to be used for the construction of peptide libraries employing a combinatorial cloning strategy. This feature makes it especially suitable for construction of peptide libraries using codon-based oligonucleotide synthesis. The vectors also allow rapid optimization and modification of lead peptides by codon-based mutagenesis. A 20-amino acid long random peptide library of 1 x 10(9) members was constructed and screened for peptides that bound to (i) a monoclonal antibody recognizing the amino-terminus of beta-endorphin; (ii) a monoclonal antibody recognizing a peptide epitope derived from the v-ros oncogene product; and (iii) the constant region of murine IgG2b. The approach described here provides a means for the construction of customized libraries that can be screened with a variety of target molecules.

Amino Acid Sequence↗