Translation of DNA into a library of 13,000 synthetic small-molecule macrocycles suitable for in vitro selection

Abstract

DNA-templated organic synthesis enables the translation, selection, and amplification of DNA sequences encoding synthetic small-molecule libraries. Previously we described the DNA-templated multistep synthesis and model in vitro selection of a pilot library of 65 macrocycles. In this work, we report several key developments that enable the DNA-templated synthesis of much larger (>10,000-membered) small-molecule libraries. We developed and validated a capping-based approach to DNA-templated library synthesis that increases final product yields, simplifies the structure and preparation of reagents, and reduces the number of required manipulations. To expand the size and structural diversity of the macrocycle library, we augmented the number of building blocks in each DNA-templated step from 4 to 12, selected 8 different starting scaffolds which result in 4 macrocycle ring sizes and 2 building-block orientations, and confirmed the ability of the 36 building blocks and 8 scaffolds to generate DNA-templated macrocycle products. We computationally generated and experimentally validated an expanded set of codons sufficient to support 1728 combinations of step 1, step 2, and step 3 building blocks. Finally, we developed new high-resolution LC/MS analysis methods to assess the quality of large DNA-templated small-molecule libraries. Integrating these four developments, we executed the translation of 13,824 DNA templates into their corresponding small-molecule macrocycles. Analysis of the resulting libraries is consistent with excellent (>90%) representation of desired macrocycle products and a stringent test of sequence specificity suggests a high degree of sequence fidelity during translation. The quality and structural diversity of this expanded DNA-templated library provides a rich starting point for the discovery of functional synthetic small-molecule macrocycles.

Figure 1

The previously reported scheme for the synthesis of a DNA-templated macrocycle library, where R is −NHCH₃ or tryptamine, and Ar is −(p-C₆H₄)–. In contrast with the capping-based method described in the present work, each DNA-templated reaction in this scheme requires a bond formation step, a streptavidin bead capture step, a washing step, and a product elution step.

Figure 2

A capping-based strategy for DNA-templated macrocycle synthesis. The use of a capping reagent (here, acetic anhydride) prevents unreacted or improperly reacted material from participating in further reactions. The final streptavidin bead capture and macrocyclization steps achieve effective purification of the final product, obviating the need to purify after each DNA-templated reaction as in Figure 1 and reducing the total number of required manipulations.

Figure 3

Denaturing PAGE and MALDI-TOF analysis of the capping method applied to DNA-templated macrocycle precursor synthesis.

Figure 4

Method for the computational generation, modeling, and screening of a codon set suitable for DNA-templated library synthesis.

Figure 5

DNA-templated reaction yields for all possible matched and mismatched combinations of templates and reagents used in this work. Product yields arising from sequence-matched reactivity are shown on the diagonals of the matrices; mismatched reactivity yields are shown in off-diagonal cells. Cells are colored by percent yield from highest (red) to lowest (green) in spectral order.

Figure 6

High-resolution LC/MS analysis of macrocycle 8b after S1 nuclease digestion. Macrocycle 8b was synthesized using the capping-based approach from template 1b using the new codons 1A, 2A, and 3A.

Figure 7

A systematic study of building-block compatibility with DNA-templated macrocycle synthesis. Using macrocycle 8a as a benchmark of “normal” reactivity, we selected a set of six distinct amino acids for substitution, one at a time, into the macrocycle at each of the three building block positions (R₁, R₂, and R₃). Macrocycle series 11 (substitution at R₁), 12 (substitution at R₂), and 13 (substitution at R₃) were synthesized using the corresponding reagent sets and starting materials 1a, 5a, and 6a, respectively. Percent yields of each macrocycle as analyzed by PAGE using internal standards are shown in the table.

Figure 8

Eight scaffolds for DNA-templated macrocycle syntheses. The tartaramide group (aldehyde precursor) can be placed on either the α-amine or side-chain (“s”) amine, resulting in two different orientations of building blocks within the resulting macrocycles. The eight scaffolds (denoted by the circled S) were transformed into their corresponding macrocycles using the same reagents used in the synthesis of macrocycle 8b from scaffold 1b; the respective overall percent yields for final purified macrocycles are shown in the table above.

Figure 9

Three-dimensional models for two sets of MM2 energy-minimized macrocycles. The macrocycles within each set incorporate identical building blocks, but use a different scaffold (either the Lys-s or Dpr-α scaffold). The different scaffolds can result in very different preferred conformations.

Figure 10

Amino acid building blocks for macrocycle libraries 21, 22, and 23.

Figure 11

Template and reagent sequences that mediate the synthesis of macrocycle libraries 21, 22, and 23. The optimized template codons and corresponding reagent anticodons resulting from the method shown in Figure 4 are shown.

Figure 12

LC/MS analysis of 12-membered macrocycle libraries 21, 22, and 23 following S1 nuclease digestion. In total, 34 of the 36 expected macrocyclic species (94%) are visible.

Figure 13

High-resolution LC/MS analysis of the 12-membered (25) and 1,728-membered (27) DNA-templated macrocycle sublibraries. From library 25, a stringent test of sequence specificity, 10 of 12 expected macrocycle masses were detected, and only 8.6% of masses corresponding to sequence-mismatched products were observed. For library 27, 94% of expected masses were observed. The macrocycles eluted over a total of ∼2,400 seconds; the representative mass spectrum shown here is of a 35-second elution window. Expected masses are in red and observed masses are in black.

Figure 14

Synthesis of a 13,824-membered macrocycle library (32) from DNA template library 28. Eight template library-linked scaffolds were processed in a single solution through three DNA-templated reactions, each using 12 building blocks each, to produce a final macrocycle library with 13,824 members. The synthesis was performed in triplicate, with an average overall purified yield of 1.6% as determined by PAGE (one example is shown in the gel above). In total, > 375 pmol of library was produced, sufficient material for hundreds of in vitro selections.