Dynamic combinatorial chemistry employing boronic acids/boronate esters leads to potent oxygenase inhibitors.

The application of dynamic reactions is a promising approach for the discovery of small-molecule ligands for proteins. To date, however, this method is limited by the few appropriate reactions and the techniques used for the analysis of protein– ligand complexes. “Dynamic” functional group interconvertions that have been employed include the conversion of thiols to disulfides, the aldol reaction, and the addition of nucleophiles to ketones and aldehydes. The reaction of boronic acids with diols to form boronate esters is attractive for dynamic-library formation, because it is reversible in aqueous solution in a pH-dependent manner. The dynamic boronic acid/boronate ester system has been used to form supramolecular switches, some of which have been used for sugar detection. 5] However, this system has not been used for the identification of protein ligands. Proof of principle work with proteases, which react reversibly with boronic acids, suggests that boronic acid/boronate ester systems might be useful for the identification of enzyme inhibitors. One issue with the application of reversible reactions for ligand identification is the need to analyze labile complexes that are derived from mixtures. High-resolution techniques, such as NMR spectroscopy and X-ray crystallography, are applicable, but these are time-consuming. Our research group and that of Poulsen, have used non-denaturing protein mass spectrometry to identify protein–ligand complexes formed from equilibrating mixtures of thiols/disulfides and aldehydes/hydrazones. The dynamic-combinatorial mass spectrometry (DCMS) technique has the advantages of being efficient and providing information on mass shifts, which can be used for assigning structures to the ligands that bind preferentially. Herein we demonstrate that boronic acid/boronate ester dynamic systems coupled with protein mass spectrometry analysis are useful for the identification of protein inhibitors (Scheme 1). Our target model enzyme was prolyl hydroxylase domain isoform 2 (PHD2), which is a Fe and 2-oxoglutarate (2OG) oxygenase that regulates the human hypoxic response. PHD2 inhibition is of therapeutic interest for the treatment of anemia and ischemia-related diseases. DCMS experiments were carried out using “support ligands” 2 and 3 (Scheme 2), which were designed to participate in Fe chelation in the active site and, through the incorporation of a boronic acid moiety, participate in boronate ester exchange. We selected the 2-(picolinamido)acetic acid scaffold because, based on crystal structures of PHD2, it is predicted to fit into the active site through its chelation with Fe. The low potency of 2-(picolinamido)acetic acid (IC50> 1 mm) enabled the effect of boronate ester substitution to be monitored. Modeling studies suggested that whereas the boronic acid group in support ligand 2 would fit into the active-site subpocket, that of 3 would clash with the active-site wall. Hence, it was envisaged that the reactivity of 3 might serve as a control to investigate possible non-specific binding. The analysis of mixtures of 2 or 3 with PHD2·Fe through the use of non-denaturing ESI-MS led to the observation of a new peak at 27 887 Da (187 2 Da shift), corresponding to a small molecule/protein adduct, in which the OH groups of the boronic acids moiety are cleaved. We have previously observed, through the use of non-denaturing ESI-MS, analogous apparent fragmentation of boronic acids complexed with other enzymes. Notably, the mixture of boronate ester 4 and PHD2·Fe gave the same mass shift (187 2 Da) as that observed with 2 and 3 at a cone voltage of 80 V. However, when a lower cone voltage was used (30 V), the mass shift corresponding to an adduct of 4 with the protein, without fragmentation, was apparent (358 2 Da), demonstrating that boronate ester formation can be observed when sufficiently mild ionization is used. Both 2 and 3 compete with the 2OG analogue N-oxalylglycine (NOG) for the 2OG binding site of PHD2. To ensure that boronate ester formation involving 2 and 3 was favorable under the conditions used (NH4OAc [*] M. Demetriades, I. K. H. Leung, Dr. R. Chowdhury, M. C. Chan, Dr. M. A. McDonough, Dr. K. K. Yeoh, Dr. T. D. W. Claridge, Prof. C. J. Schofield Chemistry Research Laboratory, University of Oxford 12 Mansfield Road, Oxford, OX1 3TA (UK) E-mail: christopher.schofield@chem.ox.ac.uk