Quantitative Synthesis of Genetically Encoded Glycopeptide Libraries Displayed on M13 Phage

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 × 10⁸ 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 <i>via</i> an oxime ligation following oxidation of an <i>N</i>-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 10⁹ distinct molecules. We envision that the methodology described herein will find broad application in the synthesis of custom chemically modified phage libraries

Allene Functionalized Azobenzene Linker Enables Rapid and Light-Responsive Peptide Macrocyclization

In this manuscript, we describe the efficient synthesis of a bis­(allenamide) functionalized water-soluble azobenzene reagent (BSBDA) and its application as a new tool for the rapid generation of visible light-responsive macrocyclic peptides and peptide libraries displayed on the surface of bacteriophage. The allenamide functionality promotes cysteine ligation in model peptides and those displayed on phage with rates 2–3 orders of magnitude faster than the established alkyl halide containing azobenzenes

Quantitative Synthesis of Genetically Encoded Glycopeptide Libraries Displayed on M13 Phage

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 × 10⁸ 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 <i>via</i> an oxime ligation following oxidation of an <i>N</i>-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 10⁹ distinct molecules. We envision that the methodology described herein will find broad application in the synthesis of custom chemically modified phage libraries

Rapid, Hydrolytically Stable Modification of Aldehyde-Terminated Proteins and Phage Libraries

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

Synergic “Click” Boronate/Thiosemicarbazone System for Fast and Irreversible Bioorthogonal Conjugation in Live Cells

Fast, high-yielding, and selective bioorthogonal “click” reactions employing nontoxic reagents are in high demand for their great utility in the conjugation of biomolecules in live cells. Although a number of click reactions were developed for this purpose, many are associated with drawbacks and limitations that justify the development of alternative systems for both single- or dual-labeling applications. Recent reports have highlighted the potential of boronic ester formation as a bioorthogonal click reaction between abiotic boronic acids and diols. Boronic ester formation is a fast dehydrative process; however it is intrinsically reversible in aqueous medium. We designed and optimized a synergic system based on two bifunctional reagents, a thiosemicarbazide-functionalized nopoldiol and an <i>ortho</i>-acetyl arylboronic acid. Both reagents were shown to be chemically stable and nontoxic to HEK293T cells at concentrations as high as 50 μM. The resulting boronate/thiosemicarbazone adduct is a medium-sized ring that forms rapidly and irreversibly without any catalyst at low μM concentrations, in neutral buffer, with a rate constant of 9 M^–1 s^–1 as measured by NMR spectroscopy. Control experiments in the presence of competing boronic acids showed no crossover side-products and confirmed the stability and lack of reversibility of the boronate/thiosemicarbazone conjugates. Formation of the conjugates is not affected by the presence of biological diols such as fructose, glucose, and catechol, and the thiosemicarbazide-functionalized nopoldiol is inert to aldehyde electrophiles of the sort found on protein-bound glyoxylyl units. The suitability of this system in the cell-surface labeling of live cells was demonstrated using a SNAP-tag approach to install the boronic acid reagent onto the extracellular domain of the Beta-2 adrenergic receptor in HEK293T cells, followed by incubation with the optimal thiosemicarbazide-functionalized nopoldiol reagent labeled with fluorescein dye. Successful visualization by fluorescence microscopy was possible with a reagent concentration as low as 10 μM, thus confirming the potential of this system in biological applications

Platform for High-Throughput Testing of the Effect of Soluble Compounds on 3D Cell Cultures

<i>In vitro</i> 3D culture could provide an important model of tissues <i>in vivo</i>, but assessing the effects of chemical compounds on cells in specific regions of 3D culture requires physical isolation of cells and thus currently relies mostly on delicate and low-throughput methods. This paper describes a technique (“cells-in-gels-in-paper”, CiGiP) that permits rapid assembly of arrays of 3D cell cultures and convenient isolation of cells from specific regions of these cultures. The 3D cultures were generated by stacking sheets of 200-μm-thick paper, each sheet supporting 96 individual “spots” (thin circular slabs) of hydrogels containing cells, separated by hydrophobic material (wax, PDMS) impermeable to aqueous solutions, and hydrophilic and most hydrophobic solutes. A custom-made 96-well holder isolated the cell-containing zones from each other. Each well contained media to which a different compound could be added. After culture and disassembly of the holder, peeling the layers apart “sectioned” the individual 3D cultures into 200-μm-thick sections which were easy to analyze using 2D imaging (e.g., with a commercial gel scanner). This 96-well holder brings new utilities to high-throughput, cell-based screening, by combining the simplicity of CiGiP with the convenience of a microtiter plate. This work demonstrated the potential of this type of assays by examining the cytotoxic effects of phenylarsine oxide (PAO) and cyclophosphamide (CPA) on human breast cancer cells positioned at different separations from culture media in 3D cultures

Phage Display of the Serpin Alpha-1 Proteinase Inhibitor Randomized at Consecutive Residues in the Reactive Centre Loop and Biopanned with or without Thrombin

<div><p>In spite of the power of phage display technology to identify variant proteins with novel properties in large libraries, it has only been previously applied to one member of the serpin superfamily. Here we describe phage display of human alpha-1 proteinase inhibitor (API) in a T7 bacteriophage system. API M358R fused to the C-terminus of T7 capsid protein 10B was directly shown to form denaturation-resistant complexes with thrombin by electrophoresis and immunoblotting following exposure of intact phages to thrombin. We therefore developed a biopanning protocol in which thrombin-reactive phages were selected using biotinylated anti-thrombin antibodies and streptavidin-coated magnetic beads. A library consisting of displayed API randomized at residues 357 and 358 (P2–P1) yielded predominantly Pro-Arg at these positions after five rounds of thrombin selection; in contrast the same degree of mock selection yielded only non-functional variants. A more diverse library of API M358R randomized at residues 352–356 (P7–P3) was also probed, yielding numerous variants fitting a loose consensus of DLTVS as judged by sequencing of the inserts of plaque-purified phages. The thrombin-selected sequences were transferred en masse into bacterial expression plasmids, and lysates from individual colonies were screening for API-thrombin complexing. The most active candidates from this sixth round of screening contained DITMA and AAFVS at P7–P3 and inhibited thrombin 2.1-fold more rapidly than API M358R with no change in reaction stoichiometry. Deep sequencing using the Ion Torrent platform confirmed that over 800 sequences were significantly enriched in the thrombin-panned versus naïve phage display library, including some detected using the combined phage display/bacterial lysate screening approach. Our results show that API joins Plasminogen Activator Inhibitor-1 (PAI-1) as a serpin amenable to phage display and suggest the utility of this approach for the selection of “designer serpins” with novel reactivity and/or specificity.</p></div

Panel A: Volcano plot showing enrichment of sequences in thrombin panning versus mock panning.

<p>Panel B: Enrichment of sequences with respect to naïve library. In (A) and (B), red dots describe sequences that have been enriched by factor >2 with significance level of p>0.05 (calculated by two-tail unequal variance t-test). Panel C: Plot of sequences that have been significantly enriched over two controls. Red dots represent the top 20 sequences that show the highest level of enrichment with respect to naïve library (ca. factor of 100 or more). The pentapeptide sequence of the top 20 most enriched sequences lacking stop codons is shown in Panel D, in order of enrichment. The sequence of all significantly enriched sequences (784 in total) is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084491#pone.0084491.s004" target="_blank">Table S3</a>.</p

P7–P3 variants binding thrombin in a capture assay more effectively than API M358R.

<p>P7–P3 variants binding thrombin in a capture assay more effectively than API M358R.</p

Frequency of Specific P7–P3 sequences in biopanned phage display libraries.

<p>Frequency of Specific P7–P3 sequences in biopanned phage display libraries.</p