DNA-templated Organic Synthesis Research Articles

DNA Catalyzed Dithioacetalization in Water

The most popular hypothesis of precellular life is the world, which is utilizing rna as both genetic as well as catalytic material. the chemical similarity between rna and dna leads researchers to investigate whether dna has the catalytic function, although dna is commonly viewed as a genetic carrier with a ubiquitous double-stranded architecture in living world. nevertheless, the catalytic dna has not been discovered in nature to date. in recent years, the catalytic function of dna in nonbiological applications has aroused much interest to chemists in chemical synthesis such as dna-templated organic synthesis and dna-based asymmetric catalysis. however, the investigation of dna as a direct catalyst for organic synthesis is largely elusive. here we report that double-stranded dna from herring sperm can catalyze the dithioacetalization in water for a wide range of aldehydes under mild reaction conditions. it is proposed that the phosphate groups of dna together with the duplex architecture are responsible for the catalytic reaction.

In vitro selection of a DNA-templated small-molecule library reveals a class of macrocyclic kinase inhibitors.

DNA-templated organic synthesis enables the translation of DNA sequences into synthetic small-molecule libraries suitable for in vitro selection. Previously, we described the DNA-templated multistep synthesis of a 13 824-membered small-molecule macrocycle library. Here, we report the discovery of small molecules that modulate the activity of kinase enzymes through the in vitro selection of this DNA-templated small-molecule macrocycle library against 36 biomedically relevant protein targets. DNA encoding selection survivors was amplified by PCR and identified by ultra-high-throughput DNA sequencing. Macrocycles corresponding to DNA sequences enriched upon selection against several protein kinases were synthesized on a multimilligram scale. In vitro assays revealed that these macrocycles inhibit (or activate) the kinases against which they were selected with IC50 values as low as 680 nM. We characterized in depth a family of macrocycles enriched upon selection against Src kinase, and showed that inhibition was highly dependent on the identity of macrocycle building blocks as well as on backbone conformation. Two macrocycles in this family exhibited unusually strong Src inhibition selectivity even among kinases closely related to Src. One macrocycle was found to activate, rather than inhibit, its target kinase, VEGFR2. Taken together, these results establish the use of DNA-templated synthesis and in vitro selection to discover small molecules that modulate enzyme activities, and also reveal a new scaffold for selective ATP-competitive kinase inhibition.

Translation of DNA into a Library of 13 000 Synthetic Small-Molecule Macrocycles Suitable for in Vitro Selection

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.

ChemInform Abstract: DNA‐Templated Organic Synthesis (DTS): A Novel Approach to the Discovery of Functional Small Molecules Based on Nature′s Evolutionary Principles

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Effects of Template Sequence and Secondary Structure on DNA-Templated Reactivity

DNA-templated organic synthesis enables the translation, selection, and amplification of DNA sequences encoding synthetic small-molecule libraries. As the size of DNA-templated libraries increases, the possibility of forming intramolecularly base-paired structures within templates that impede templated reactions increases as well. To achieve uniform reactivity across many template sequences and to computationally predict and remove any problematic sequences from DNA-templated libraries, we have systematically examined the effects of template sequence and secondary structure on DNA-templated reactivity. By testing a series of template sequences computationally designed to contain different degrees of internal secondary structure, we observed that high levels of predicted secondary structure involving the reagent binding site within a DNA template interfere with reagent hybridization and impair reactivity, as expected. Unexpectedly, we also discovered that templates containing virtually no predicted internal secondary structure also exhibit poor reaction efficiencies. Further studies revealed that a modest degree of internal secondary structure is required to maximize effective molarities between reactants, possibly by compacting intervening template nucleotides that separate the hybridized reactants. Therefore, ideal sequences for DNA-templated synthesis lie between two undesirable extremes of too much or too little internal secondary structure. The relationship between effective molarity and intervening nucleic acid secondary structure described in this work may also apply to nucleic acid sequences in living systems that separate interacting biological molecules.

ＤＮＡテンプレート有機合成去（ＤＴＳ）：生命原理に学ぶ機能性分子創製・探索への新規アプローチ

ＤＮＡテンプレート有機合成去（ＤＴＳ）：生命原理に学ぶ機能性分子創製・探索への新規アプローチ

DNA‐Templated Synthesis in Organic Solvents

DNA-templated organic synthesis (DTS) enables new modes of controlling chemical reactivity and allows evolutionary principles to be applied to the discovery of synthetic small molecules, synthetic polymers, and new chemical reactions. The structures that can be accessed in a DNA-templated format thus far have been limited to those that can be generated in aqueous solvents. Here we report efficient and sequence-specific DTS in a variety of organic solvents with low or minimal water content. These methods expand the scope of DTS by providing access to reagents that are insoluble in water as well as reactions in which the participation of water inhibits product formation. Previous studies by Okahata and others suggest that quaternary ammonium salts can associate with DNA phosphates to form complexes that are soluble in organic solvents. We hypothesized that short (10–30 bp) DNA duplexes formed in aqueous solution and then transferred to an organic solvent containing low concentrations (mM) of quaternary ammonium salts might retain their double-stranded structure and mediate DNA-templated organic synthesis. Although we found that DNA-templated chemistry could indeed take place efficiently and sequence-specifically in organic solvents in the presence of alkyl ammonium salts (see Supporting Information), we further speculated that at the extremely low concentrations required for DTS (nM), alkyl ammonium salts might not be necessary for the solubilization of duplexes preformed in aqueous solution (Figure 1A). To evaluate the ability of preformed duplexes to support DTS in primarily organic solvents, we first investigated three known DNA-templated chemistries in four distinct contexts (Figure 1B): i) in a simple end-of-helix architecture with juxtaposed reactants (E1), ii) in a long-distance end-of-helix architecture with ten intervening nucleotides between the hybridized reactants (E10), iii) in the “omega” architecture with a five-base loop (W5), and iv) with reactants linked to noncomplementary (mismatched) oligonucleotides. Products were characterized both by denaturing PAGE analysis and by MALDI mass spectrometry (see Table 1 and Supporting Information for details). DNA-templated amine acylation mediated by 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC) and N-hydroxysulfosuccinimide (sNHS) has been well characterized in aqueous solution and is known to take place efficiently even when reactive groups are separated by many intervening nucleotides. To carry out DNA-templated amine acylation in organic solvent, template and reagent oligonucleotides (Table 1) were prehybridized in a small volume of aqueous 70 mM NaCl. Amine acylation was initiated by the addition of organic solvent containing

Reviews in Angewandte Chemie: Influence, Language, Competition

Reviews in <i>Angewandte Chemie</i>: Influence, Language, Competition

DNA-Templated Functional Group Transformations Enable Sequence-Programmed Synthesis Using Small-Molecule Reagents

DNA-templated organic synthesis (DTS) has previously been used primarily to direct coupling reactions between two DNA-linked reactants. In some cases, reactants are difficult or impossible to tether to DNA oligonucleotides. The development of strategies that enable non-DNA linked small-molecule reagents to participate in sequence-programmed synthesis therefore would significantly expand the capabilities of DTS. We developed efficient DNA-templated functional group transformations of template-linked azides into corresponding amines, carboxylic acids, and thiols. The application of these reactions to a single-solution mixture of four template-linked organic azides enabled each azide to be transformed sequence specifically into a sulfonamide, carbamate, urea, or thiourea using small-molecule sulfonyl chloride, chloroformate, isocyanate, or isothiocyanate reagents not tethered to DNA. Only the four desired products were observed, without formation of any of the 12 possible undesired cross-products. Our results represent a new approach to small molecule diversification in a DNA-programmed manner.

DNA-templated organic synthesis: nature's strategy for controlling chemical reactivity applied to synthetic molecules.

In contrast to the approach commonly taken by chemists, nature controls chemical reactivity by modulating the effective molarity of highly dilute reactants through macromolecule-templated synthesis. Nature's approach enables complex mixtures in a single solution to react with efficiencies and selectivities that cannot be achieved in conventional laboratory synthesis. DNA-templated organic synthesis (DTS) is emerging as a surprisingly general way to control the reactivity of synthetic molecules by using nature's effective-molarity-based approach. Recent developments have expanded the scope and capabilities of DTS from its origins as a model of prebiotic nucleic acid replication to its current ability to translate DNA sequences into complex small-molecule and polymer products of multistep organic synthesis. An understanding of fundamental principles underlying DTS has played an important role in these developments. Early applications of DTS include nucleic acid sensing, small-molecule discovery, and reaction discovery with the help of translation, selection, and amplification methods previously available only to biological molecules.

Reaction discovery enabled by DNA-templated synthesis and in vitro selection.

Current approaches to reaction discovery focus on one particular transformation. Typically, researchers choose substrates based on their predicted ability to serve as precursors for the target structure, then evaluate reaction conditions for their ability to effect product formation. This approach is ideal for addressing specific reactivity problems, but its focused nature might leave many areas of chemical reactivity unexplored. Here we report a reaction discovery approach that uses DNA-templated organic synthesis and in vitro selection to simultaneously evaluate many combinations of different substrates for bond-forming reactions in a single solution. Watson-Crick base pairing controls the effective molarities of substrates tethered to DNA strands; bond-forming substrate combinations are then revealed using in vitro selection for bond formation, PCR amplification and DNA microarray analysis. Using this approach, we discovered an efficient and mild carbon-carbon bond-forming reaction that generates an enone from an alkyne and alkene using an inorganic palladium catalyst. Although this approach is restricted to conditions and catalysts that are at least partially compatible with DNA, we expect that its versatility and efficiency will enable the discovery of additional reactions between a wide range of substrates.

DNA-templated organic synthesis and selection of a library of macrocycles.

The translation of nucleic acid libraries into corresponding synthetic compounds would enable selection and amplification principles to be applied to man-made molecules. We used multistep DNA-templated organic synthesis to translate libraries of DNA sequences, each containing three "codons," into libraries of sequence-programmed synthetic small-molecule macrocycles. The resulting DNA-macrocycle conjugates were subjected to in vitro selections for protein affinity. The identity of a single macrocycle possessing known target protein affinity was inferred through the sequence of the amplified DNA template surviving the selection. This work represents the translation, selection, and amplification of libraries of nucleic acids encoding synthetic small molecules rather than biological macromolecules.

Translating DNA into synthetic molecules.

Translating DNA into synthetic molecules.

Translation of DNA into synthetic N-acyloxazolidines.

The translation of DNA into synthetic molecules enables their manipulation by powerful evolution-based methods previously available only to proteins and nucleic acids. The development of increasingly sophisticated DNA-templated small-molecule syntheses is crucial to broadening the scope of this approach. Here, we report the translation of DNA templates into monocyclic and bicyclic N-acyloxazolidines using multistep DNA-templated organic synthesis. Second-generation template architectures, used for the first time in a multistep DNA-templated synthesis, together with reactions and linker cleavage strategies not previously described in a DNA-templated format, were crucial to the successful translation. The products generated in this work represent the most complex small molecules to date synthesized in a DNA sequence-programmed manner and provide the basis for DNA-templated synthetic heterocycle libraries.

Stereoselectivity in DNA-Templated Organic Synthesis and Its Origins

DNA-templated synthesis is a surprisingly general strategy for controlling chemical reactivity that enables synthetic products to be manipulated in ways previously available only to biological macromolecules. The chiral nature of the DNA template raises the possibility that DNA-templated synthesis can proceed stereoselectively. Here, we show that DNA-templated substitution reactions can exhibit stereoselectivity without the assistance of chiral groups other than those present in DNA. By characterizing changes in stereoselectivity as a result of systematic changes in the structure of the template-reagent complexes, we begin to reveal the origins of the observed stereoselectivity. We propose that the conformations of nucleotides adjacent to the reactants are largely responsible for stereoselectivity. Indeed, template and reagent sequences that can adopt either a left-handed Z-form DNA helix or a normal right-handed B-form DNA helix generate opposite stereoselectivities in the Z-form and B-form even though they share the same covalent structure and the same absolute stereochemistry. Our findings establish ways in which the chirality of an information carrier can be transmitted to the stereochemistry of encoded products through templated synthesis.

Directing Otherwise Incompatible Reactions in a Single Solution by Using DNA-Templated Organic Synthesis

Diversification without spatial separation can be achieved using DNA-templated synthesis. Twelve reactants with functional groups of similar reactivity linked to different DNA sequences (R, R′) were subjected to several different reaction types within the same solution and only six sequence-programmed products out of the more than 28 possibilities were formed (see scheme).