DPC™ synthesis occurs under mild aqueous reaction conditions, highly analogous to the conditions under which enzymatic reactions occur within a cell. When two chemical precursors are brought together by the hybridization between two DNA strands, a range of chemistry becomes possible between them. We have successfully demonstrated numerous chemical transformations including amide formation, reductive aminations, Wittig reactions, aldol condensations, azide reductions, Heck reactions, epoxide openings, oxazolidine and thiazolidine formation, Huisgen 1,3 dipolar cycloadditions, and SN2 displacements. With the range of chemistry available, it is possible to make large numbers of diverse and drug-like molecules.

After each reaction, at intermediate and at final product stage, a purification step separates products from unreacted starting materials. This method is effective whether a single compound or a mixture of thousands is being prepared simultaneously in the same reaction.

At both intermediate and final product stage, LC-MS is used to indicate the presence of all of the chemical components. DNA is digested, and the macrocycle (with a vestigial phosphate group) is chromatographed and then analyzed by time-of-flight mass spectrometry. Proprietary Ensemble software permits the correlation of observed ions against expected products, confirming the successful Ensemblin synthesis.

Ensemble screens its macrocyclic compounds for biological activity through a rapid, highly sensitive and efficient affinity selection process. Drug discovery protein targets are incubated with an entire DPC mixture in one experiment, and compounds that bind are isolated and collected. This is a highly material-efficient process as only 10 femtomoles of each compound are required for an assay with excess (around 150 picomoles) of the protein target.

As the DPC synthetic process ensured a direct one-to-one correlation between compound structure and the attached DNA sequence, DNA amplification by PCR and sequencing permits structural decoding. Thus in one selection experiment it is possible to identify any number of compounds that bind to a protein target. Unlike traditional high throughput screening, we are able to detect a wide dynamic range of compound affinities, from the nanomolar down to millimolar, and this allows the derivation of ‘Instant Structure Activity Relationships (Instant SAR)’, by which the contributions to binding of every building block can be ascertained. This information has been used to identify single macrocyclic compounds for resynthesis as well as design further iterative compound collections to optimize the potency of binding macrocycles.

Incubating entire macrocyclic libraries with protein targets isolates binding compounds that are identified by subsequent DNA amplification and decoding.	Plotting the active compounds from macrocycle libraries in three-dimensional structural space can reveal structure-activity relationship information.

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