Quantitative Synthesis of Genetically Encoded Glycopeptide Libraries Displayed on M13 Phage

Abstract

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.

Critical Evaluation and Rate Constants of Chemoselective Ligation Reactions for Stoichiometric Conjugations in Water

“…Kinetic studies of aniline-catalyzed oxime ligations with peptide substrates containing no cysteine residues have been conducted in detail, and a clear rate acceleration in the presence of aniline was observed. 27,39 In our case, oxime formation was observed in the absence of aniline, but the ligation product was readily decomposed under acidic pH. Although the hydrolysis of the oxime is a known decomposition pathway, we observed the formation of an unidentified compound.…”

Encoded libraries of chemically modified peptides

“…glycopeptides with high affinity for carbohydrate-binding proteins [17,18]. Mihara and Tsutsumi et al created glycopeptide ligands of a mannose-binding protein by conjugating mannose functionalized with pyridyldithiol to a cysteine residue within a randomized peptide by a disulfide exchange reaction (Figure 2c) [17].…”

“…Derda et al, conjugated mannose to the N-terminus of a phage peptide library [18], first oxidizing the N-terminal serine or threonine residues to aldehyde and then appending an aminoxy derivative of mannose by oxime ligation (Figure 2d). …”

Chemical modulation of M13 bacteriophage and&nbsp;its functional opportunities for nanomedicine

“…Ng et al 47 synthesized genetically encoded glycosylated peptide libraries displayed on M13 phage using N-terminal serines/threonines ( Figure 5A). Among the peptide libraries, the phages carrying N-terminal serine and threonine on pIII were oxidized to form N-terminal glyoxals, which can act as reactive handles for subsequent immobilization of glycans.…”

“…Such oxime ligation on phage-displayed peptides can have broad applications in the synthesis of custom-designed chemically modified phage libraries. 47,48 Although no detailed results of phage stability were reported, the authors claimed that this chemical modification had minimal interference on phage infectivity.…”