High-Throughput Quality Control Assay for the Solid-Phase Synthesis of DNA-Encoded Libraries of Macrocycles*

Abstract

There is considerable interest in the development of libraries of scaffold-diverse macrocycles as a source of ligands for difficult targets, such as protein-protein interaction surfaces. A classic problem in the synthesis of high-quality macrocyclic libraries is that some linear precursors will cyclize efficiently while some will not, depending on their conformational preferences. We report here a powerful quality control method that can be employed to readily distinguish between scaffolds that do and do not cyclize efficiently during solid-phase synthesis of thioether macrocycles without the need for tedious liquid chromatography/mass spectrometry analysis. We demonstrate that this assay can be employed to identify linear impurities in a DNA-encoded library of macrocycles. We also use the method to establish a useful quality control protocol for re-synthesis of putative macrocyclic screening hits.

Keywords: DNA-encoded libraries; macrocycles; peptidomimetics.

Figure 1.

Structures of control compounds and FACS analysis of beads displaying these molecules after staining. Beads displaying compound 1 were not stained with either dye, whereas beads displaying compounds 2 or 3 were stained with both dyes.

Figure 2.

Monitoring the progress of macrocylization of DDA. A) Reaction conditions used for the experiment. B) Flow cytometer histograms showing the level of staining of 10 μm TentaGel beads with mBBr at the times indicated (post-deprotection). B1 shows beads displaying 3 (the methionine control) and B2 shows beads displaying 2 (the cysteine control; see Figure 1). B3–B10 show mBBr staining of beads displaying DDA. C) Progress of DDA cyclization on 160 μm TentaGel ebads as monitored by LC-mS analysis after alkylation of free thiol with benzylbromide and release of the compound from the bead. D) mBBr staining and FACS analysis of 10 μm TentaGel beads displaying DD-OAc, a molecule that cannot cyclize. Even 16 h post-deprotection, the thiol is robustly alkylated, by mBBr, showing that oxidation of the sulfur over this time period is not significant.

Figure 3.

Analysis of the efficiency of macrocyclization of 64 PICCO scaffolds created by parallel solid-phase synthesis. A) Structures of the building blocks employed (box) and the protocol employed for macrocylization. Diamine K was always employed following acid B, while amine L was always used following all of the other acids. This is because acylation of N-alkylated alanines is difficult.^[19] Cy5-azide dye was attached to alkyne handle of the linker, the thiol protection was removed and the beads were stained with mBBr eight hours later. Bead fluorescence was then analyzed using a flow cytometer. B) Summary of ring-closure status of 64 scaffolds from FACS & LCMS analysis. Cells in yellow indicate scaffolds that had between 5–40% linear material after 8 hours while red indicates scaffolds that had more than 40% linear material present. The rest of the scaffolds did not show any detectable linear material. C) FACS plots for the control molecules 2 and 3 (see Figure 1). D) FACS plots for selected scaffolds that showed a high degree of cyclization after eight hours. E) Selected scaffolds that showed incomplete cyclization after eight hours (top row of FACS plots) but completed macrocyclization by 16 h (bottom row of FACS plots).

Figure 4.

Staining in the presence of DNA encoding tags to monitor on-resin cyclization. The FACS plots show the level of staining of beads eight hours after removal of the thiol protecting group.

Figure 5.

Monitoring the progress of macrocyclization in the context of an OBOC DEL. A) Flow cytometry plot of library beads stained with Cy5 and mBBr as described in the text. The plots shown are for aliquots of library beads stained 8, 12, and 16 hours following exposure of the thiol group. B) Collection of beads with the highest level of staining by mBBr. The first two plots are beads displaying control molecules 3 (methionine) and 2 (cysteine), respectively. These are provided for comparison. The third plot is a blow-up of the stained library beads 16 h after deprotection of the thiol. The rectangle represents the gate set to collect the beads (ca. 6500) with a level of green fluorescence significantly higher than that of the methionine control. The encoding tags on these beads were amplified and deep sequenced. C) Building blocks used to create the library (left) and the general structure of the library and protocol for the experiment. Note that acids H and I were used only at position X3 in amounts such that about 4% of the beads would display molecules unable to cyclize.

Scheme 1.

Representation of the fluorescence-based assay for monitoring the efficiency on on-resin macrocyclization via thioether formation.