High-throughput crystallography for rapid fragment growth from crude arrays by low-cost robotics

28 March 2023, Version 1


We demonstrate that a simple workflow of array synthesis, combining low-cost robotics with analytic techniques to deconvolute crude reaction mixtures, is an effective way to collect structural data on a binding site. Starting from the high information content of the crystallographic fragment screens on PHIP(2) (second bromodomain of the pleckstrin homology domain interacting protein), a collection of more than 1800 compounds was enumerated. Several thousand Crude Reaction Mixtures (CRMs) were synthesized on one robotic platform, an OpenTrons OT-1 liquid handler, using reaction sequences of up to 5 chemical steps. Analysis via MScheck, an algorithm-based system for finding an m/z in a CRM, significantly shortened product identification protocol times. 969 usable X-ray diffraction datasets were acquired, which resolved as 22 reaction products binding to the protein, 19 with conserved poses relative to the original fragment and 3 with a new, unexpected binding pose. The 22 crystallographic hit compounds were subsequently tested with peptide displacement alpha-screen assay and time-resolved grating-coupled interferometry-based biosensor assays, which confirmed one molecule with an IC50 = 34 μM and KD = 50 μM, from an inactive fragment. The procedures described are entirely formulaic and engineerable and the method is eminently scalable. We anticipate that this cheap, low solvent-use approach will yield vast amounts of data, enabling rapid structural SAR landscape exploration around fragments, leading to faster fragment-to-lead times.


High-throughput crystallography
Fragment growth
Crude reaction mixtures
Quality control
Automated synthesis
Parallel synthesis
High-throughput synthesis
Multi-step synthesis

Supplementary materials

Supplementary information for the main paper
The supplementary information details: - materials and methods - development of a liquid-handler applicable urea synthesis (it 1+2) - development of a multiple step liquid-handler applicable elaborated urea synthesis (it 2 +3) - development of a multiple step liquid-handler applicable elaborated sulphonamide urea synthesis (it 4) - development of a multiple step liquid-handler applicable elaborated urea synthesis (it 3.5 and 4.5) - synthesis of pure hit compounds as positive controls and for further testing - compounds synthesised and synthesis QC results - spectra - 2d structure of reaction product binding in crystal structures

Supplementary weblinks


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