A platform for high-throughput screening of DNA-encoded catalyst libraries in organic solvents

Abstract

We have developed a novel high-throughput screening platform for the discovery of small-molecules catalysts for bond-forming reactions. The method employs an in vitro selection for bond-formation using amphiphilic DNA-encoded small molecules charged with reaction substrate, which enables selections to be conducted in a variety of organic or aqueous solvents. Using the amine-catalysed aldol reaction as a catalytic model and high-throughput DNA sequencing as a selection read-out, we demonstrate the 1200-fold enrichment of a known aldol catalyst from a library of 16.7-million uncompetitive library members.

Fig. 1. General strategy for the high-throughput screening of catalysts using DNA-encoded libraries in organic solvents.

Fig. 2. UV-Vis spectra of 48 nt PEGylated ssDNA in various solvents. (a) ssDNA conjugated to PEG 10k. (b) ssDNA conjugated to PEG 40k.

Fig. 3. Optimised molecular architecture of the amphiphilic DNA-encoded catalyst with aldol substrate. Ketone aldol reactant is shown attached to DNA.

Fig. 4. Streptavidin-mediated EMSA comparison of aldol catalysis with either (a) aldehyde reactant on DNA with biotinylated ketone in solution and (b) ketone on DNA with biotinylated aldehyde in solution. Reactions were performed for 5 days in DCE. POS = positive control, biotinylated DNA.

Fig. 5. EMSA experiments demonstrating selective catalysis. (a) Aldol reaction depends on the presence of a catalyst on DNA (Lane 2 vs. Lane 3). Aldol reaction is rescued by the addition of 1 mM pyrrolidine as a catalyst (Lane 4). (b) EMSA shift requires attached substrate (Lane 3 vs. Lane 5) and requires aldol reactant in solution (Lane 5 vs. Lane 6). Reaction conditions: DNA (0.5 μM) was dissolved in of DCE. Biotinylated benzaldehyde (500 μM) was added and the reaction were shaken for 5 days at room temperature; pyrrolidine (1 mM) was added when applicable. POS = positive control, biotinylated DNA.

Fig. 6. Mock selection for aldol catalysis with DNA-encoded small-molecule libraries. (a) General scheme for the in vitro mock selection of a DNA-encoded aldol catalyst. (b) Gel analysis of mock aldol selection resulting from a 500-fold dilution of DNA-encoded aldol catalyst. (c) High-throughput sequencing analysis of mock aldol selection resulting from a 2000-fold dilution of DNA-encoded aldol catalyst.