Peptoids, oligomers ofN-substituted glycines, are described as a motif for the generation ofchemically diverse libraries of novel molecules. Ramachandran-type plots were calculated and indicate a greater diversity of conformational states available for peptoids than for peptides. The monomers incorporate t-butyl-based side-chain and 9-fluorenylmethoxycarbonyl a-amine protection. The controlled oligomerization of the peptoid monomers was performed manually and robotically with in situ activation by either benzotriazol-lyloxytris(pyrrolidino)phosphonium hexafluorophosphate or bromotris(pyrrolidino)phosphonium hexafluorophosphate. Other steps were identical to peptide synthesis using a-(9-fluorenylmethoxycarbonyl)amino acids. A total of 15 monomers and 10 oligomers (peptoids) are described. Preliminary data are presented on the stability of a representative oligopeptoid to enzymatic hydrolysis. Peptoid versions of peptide ligands of three biological systems (bovine pancreatic a-amylase, hepatitis A virus 3C proteinase, and human immunodeficiency virus transactivator-responsive element RNA) were found with affinities comparable to those of the corresponding peptides. The potential use of libraries of these compounds in receptor-or enzyme-based assays is discussed.Broad screening of compound libraries, of broths grown from soil samples, and of synthetic intermediates has been a fruitful method for discovery of lead compounds in pharmaceutical and agrochemical research (e.g., ref. 1). With the advent of automated chemical methods for solid-phase peptide and nucleotide synthesis, and of molecular biological methods for protein and nucleic acid synthesis, the stage has been set for the generation of new kinds of compound libraries, namely, collections of oligomeric biomolecules (2-14). Such libraries have been used to map epitopes for antibody binding, to discern ribonucleotide sequences with specific binding or catalytic activity, and to provide initial leads in receptor-based assays. Advantages of these oligomeric molecules are an almost limitless diversity as a result of their modular structure, the ease with which they can be synthesized and sequenced, and their inherent biological relevance. On the other hand, the metabolic instability of peptides and nucleotides and their poor absorption characteristics mean that any lead sequence will require extensive modification before in vivo activity can be expected.Many of these problems could be avoided if an alternative, modular system was devised, with a basis set of "unnatural" monomers and a method for their controlled oligomerization. A host of chemically and pharmaceutically interesting subunits or modules would generate a diverse and novel set of heteropolymers. Once an interesting compound has been identified from a library of such nonpeptide polymers, it can serve as a lead for drug discovery, further along the road to a metabolically stable drug. Optimized analogs of a lead compound could then be developed rapidly due to the modular synthetic nature of th...

A series of homologous L-amino acid, D-amino acid, and both parallel and antiparallel (retro) sequence N-substituted glycine peptide and peptoid oligomers were prepared and incubated with a series of enzymes representative of the major classes of proteases. Each respective L-amino acid containing peptide sequence was readily cleaved by the appropriate enzyme, namely

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Abstract:The amplification of phage-displayed libraries is an essential step in the selection of ligands from these libraries. The amplification of libraries, however, decreases their diversity and limits the number of binding clones that a screen can identify. While this decrease might not be a problem for screens against targets with a single binding site (e.g., proteins), it can severely hinder the identification of useful ligands for targets with multiple binding sites (e.g., cells). This review aims to characterize the loss in the diversity of libraries during amplification. Analysis of the peptide sequences obtained in several hundred screens of peptide libraries shows explicitly that there is a significant decrease in library diversity that occurs during the amplification of phage in bacteria. This loss during amplification is not unique to specific libraries: it is observed in many of the phage display systems we have surveyed. The loss in library diversity originates from competition among phage clones in a common pool of bacteria. Based on growth data from the literature and models of phage growth, we show that this competition originates from growth rate differences of only a few percent for different phage clones. We summarize the findings using a simple two-dimensional "phage phase diagram", which describes how the collapse of libraries, due to panning and amplification, leads to the identification of only a subset of the available ligands. This review also highlights techniques that allow elimination of OPEN ACCESSMolecules 2011, 16 1777 amplification-induced losses of diversity, and how these techniques can be used to improve phage-display selection and enable the identification of novel ligands.

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Phage display is a powerful technology that enables the discovery of peptide ligands for many targets. Chemical modification of phage libraries have allowed the identification of ligands with properties not encountered in natural polypeptides. In this report, we demonstrated the synthesis of 2 × 10(8) genetically encoded glycopeptides from a commercially available phage-displayed peptide library (Ph.D.-7) in a two-step, one-pot reaction in <1.5 h. Unlike previous reports, we bypassed genetic engineering of phage. The glycan moiety was introduced via an oxime ligation following oxidation of an N-terminal Ser/Thr; these residues are present in the peptide libraries at 20-30% abundance. The construction of libraries was facilitated by simple characterization, which directly assessed the yield and regioselectivity of chemical reactions performed on phage. This quantification method also allowed facile yield determination of reactions in 10(9) distinct molecules. We envision that the methodology described herein will find broad application in the synthesis of custom chemically modified phage libraries.

We describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based discovery (GE-FBD) uses selection of phage-displayed glycopeptides to dock a glycan fragment at the CRD and guide selection of synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 108 glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40–50-fold enhancement in affinity over methyl α-D-mannopyranoside (MeMan). Lectin array suggested specificity: Man-WYD derivative bound only to 3 out of 17 proteins—ConA, LcH, and PSA—that bind to Man. An X-ray structure of ConA:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking, but their extra-CRD binding modes are significantly different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins.

We describe the rapid reaction of 2-amino benzamidoxime (ABAO) derivatives with aldehydes in water. The ABAO combines an aniline moiety for iminium-based activation of the aldehyde and a nucleophilic group (Nu:) ortho to the amine for intramolecular ring closure. The reaction between ABAO and aldehydes is kinetically similar to oxime formations performed under stoichiometric aniline catalysis. We characterized the reaction by both NMR and UV spectroscopy and determined that the rate-determining step of the process is formation of a Schiff base, which is followed by rapid intramolecular ring closure. The relationship between apparent rate constant and pH suggests that a protonated benzamidoxime acts as an internal general acid in Schiff-base formation. The reaction is accelerated by substituents in the aromatic ring that increase the basicity of the aromatic amine. The rate of up to 40 M(-1) s(-1) between an electron-rich aldehyde and 5-methoxy-ABAO (PMA), which was observed at pH 4.5, places this reaction among the fastest known bio-orthogonal reactions. Reaction between M13 phage-displayed library of peptides terminated with an aldehyde moiety and 1 mM biotin-ABAO derivative reaches completion in 1 h at pH 4.5. Finally, the product of reaction, dihydroquinazoline derivative, shows fluorescence at 490 nm suggesting a possibility of developing fluorogenic aldehyde-reactive probes based on ABAO framework.