Tandem Wittig/Diels–Alder diversification of genetically encoded peptide libraries

Abstract

In this paper, we developed a tandem of two carbon–carbon bond-forming reactions to chemically diversify the libraries of peptides displayed on a bacteriophage. The Wittig reaction of a biotin-ester from a stabilized phosphorane ylide with model peptides containing N-terminal glyoxal exhibits reaction rates of 0.07 to 5 M⁻¹ s⁻¹ in water at pH 6.5–8. The log(k) scaled linearly with pH from pH 6 to 8; above pH 9 the reaction is accompanied by the hydrolysis of the ester functionality. Capture of the phage displaying the biotinylated product by streptavidin beads confirmed the rate of this reaction in a library of 10⁸ peptides (k = 0.23 M⁻¹ s⁻¹ at pH = 6.5) and also confirmed the regioselectivity of this modification. Olefins introduced into the Wittig reaction can act as Michael acceptors: addition of glutathione, cysteamine, and DYKDDDDKC (“FLAG-Cys”) peptide occurs with k = 0.12–4.1 M⁻¹ s⁻¹ at pH 7.8. Analogous reactions with the DYKDDDDKC peptide take place on phage-displayed peptides modified via the Wittig reaction. This reaction is manifested as a progressive emergence of a FLAG-epitope on the phage and detected by the capture of this phage using an anti-FLAG antibody. Olefins introduced into the Wittig reaction also act as dienophiles in the Diels–Alder reaction with cyclopentadiene. The conversion of the dienophile to norbornene-like adducts on the phage was observed by monitoring the disappearance of the thiol-reactive olefin on the phage. This report broadens the reaction scope of genetically-encoded peptide libraries displayed on the phage, expanding the structural diversity of these platforms and increasing their potential to be used in screening against important protein targets. The possibility of monitoring tandem reactions by the use of different labels illustrates the feasibility of obtaining highly functionalized peptides with chemical motifs impossible to achieve using conventional translational machinery.