Optimized Reaction Conditions for Amide Bond Formation in DNA-Encoded Combinatorial Libraries

Abstract

DNA-encoded combinatorial libraries are increasingly being used as tools for the discovery of small organic binding molecules to proteins of biological or pharmaceutical interest. In the majority of cases, synthetic procedures for the formation of DNA-encoded combinatorial libraries incorporate at least one step of amide bond formation between amino modified DNA and a carboxylic acid. We investigated reaction conditions and established a methodology by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1-hydroxy-7-azabenzotriazole and N,N′-diisopropylethylamine (EDC/HOAt/DIPEA) in combination, which provided conversions greater than 75% for 423/543 (78%) of the carboxylic acids tested. These reaction conditions were efficient with a variety of primary and secondary amines, as well as with various types of aminomodified oligonucleotides. The reaction conditions, which also worked efficiently over a broad range of DNA concentrations and reaction scales, should facilitate the synthesis of novel DNAencoded combinatorial libraries. KeywordsDNA-encoded chemical libraries; amide bond formation; synthesis; bioconjugation DNA-encoded combinatorial libraries (DECLs) are collections of organic compounds, individually coupled to distinct DNA fragments, serving as identification barcodes.1-4 Since DNA can be efficiently amplified by the polymerase chain reaction (PCR) and read by highthroughput DNA sequencing methods, the encoding of combinatorial libraries with DNA barcodes allows both the facile identification of specific ligands to protein targets immobilized on a solid support and the convenient handling of the libraries as mixtures of compounds. Compared to High-Throughput Screening, which relies on expensive compound Various chemical strategies have been developed for the generation of large combinatorial DECLs, including DNA-templated synthesis,10,11 DNA-encoded routing,12 hybridizationbased chemical assembling 13-17and DNA-encoded solid-phase synthesis.18 In addition, the iterative assembly of sets of building blocks in a "split-and-pool" methodology, accompanied by the stepwise addition of DNA barcodes (which unambiguously identify the chemical identity of the nascent molecules), is the most generally used strategy for DECLs construction. This strategy facilitates the preparation of DECLs containing millions or even billions of compounds, starting from just few hundred building blocks and oligonucleotides. 19,20,21 In the majority of DECLs disclosed so far, at least one synthesis step involved the formation of an amide bond.4 More than 20'000 carboxylic acids can be purchased from commercial sources at moderate costs (less than $150/g). 22 The acylation of DNA-attached amines with carboxylic acids can be performed on protected DNA structures linked to Controlled Pore Glass (CPG) supports 6 or in solution, 23 followed by deprotection and HPLC purification of the individual conjugates at early stages of library construction. Additionally, for unprotected DNA-attached amines, acylation can be performed either...

A DNA‐Encoded Chemical Library Incorporating Elements of Natural Macrocycles

“…Although EDC/HOAt/DIPEA is often the system of choice, [14] we found that coupling with DMTMM-BF 4 /NMM was better in most cases,r esulting in higher conversions and cleaner products.E DC/HOAt/DIPEA was only superior for strained cyclic secondary amino acids and tyrosine derivatives,aproblem that likely stems from the reaction of the tyrosine hydroxy group with DMTMM-BF 4 /NMM. Although EDC/HOAt/DIPEA is often the system of choice, [14] we found that coupling with DMTMM-BF 4 /NMM was better in most cases,r esulting in higher conversions and cleaner products.E DC/HOAt/DIPEA was only superior for strained cyclic secondary amino acids and tyrosine derivatives,aproblem that likely stems from the reaction of the tyrosine hydroxy group with DMTMM-BF 4 /NMM.…”

Synthesis of DNA‐Encoded Disulfide‐ and Thioether‐Cyclized Peptides

“…Several efficient strategies for the synthesis of DNA‐encoded peptides are used . Most protocols rely on Fmoc‐protected amino acids and the water‐soluble coupling reagent 4‐(4,6dimethoxy‐1,3,5‐triazin‐2‐yl)‐4‐methylmorpholinium chloride (DMT‐MM).…”

Screening of Three Transition Metal‐Mediated Reactions Compatible with DNA‐Encoded Chemical Libraries

“…[12,22,23] The construction of large DNAencoded chemical libraries crucially relies on the availability of 'robust' reactions, which can be performed in water, are DNA-compatible, [24 -26] and which accept a large number of structurally diverse substrates. One of the most widely used reactions for the construction of DNA-encoded chemical libraries is the formation of amide bonds, since excellent procedures are available, [24,27] and since thousands of building blocks (e. g., amines and carboxylic acids) can be purchased from commercial sources at moderate costs. However, even a robust reaction such as amide bond formation with DMT-MM methodology displays acceptable conversion yields only for approximately 44 % of carboxylic acids.…”

“…For these reasons, improved methodologies are constantly being explored. [27] In this work, we aimed at implementing and optimizing copper and palladium-catalyzed reactions, which could be readily used for the construction of DNA-encoded chemical libraries. [28 -31] We focused on Suzuki cross-coupling, Sonogashira cross-coupling, and copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC).…”