DOI: 10.1002/cmdc.201200512 Efficient Stacking on Protein Amide Fragments Michael Harder, [a] Bernd Kuhn,* [b] and FranÅois Diederich* [a] Introduction Stacking interactions between p-systems are of great impor- tance in biomolecular recognition. Experimental and theoreti- cal studies on small, mainly aromatic model systems have pro- vided valuable insight into the relative strength and geometri- cal preference of different stacking partners, [1–3] and this knowl- edge is routinely used in the drug design process. The proto- type of p-stacking, the parallel interaction of two benzene rings, has been thoroughly investigated. [4] High-level quantum mechanical (QM) computations predict a favorable association energy of about 2.7 kcal mol 1 in the gas phase for the paral- lel-displaced configuration. [5] This geometry is also significantly enriched in the contact statistics of aromatic side chains in pro- tein structures. [6] In contrast, the eclipsed stacking geometry is hardly observed in protein structures, and calculations reveal an approximate 1.0 kcal mol 1 lower energy gain than for the parallel-displaced arrangement. [5] Wang and Hobza demon- strated that the binding energy of the benzene dimer can be further increased if one of the benzene rings is replaced by an isoelectronic six-membered N-heteroarene. [7] Recently, signifi- cant energy differences between parallel- and antiparallel-ori- ented pyridine dimers were reported, highlighting the influ- ence of dipole alignment in stacking interactions. [8] In contrast to purely aromatic interactions, much less is known about the stacking characteristics of amide p-systems. Spectroscopic studies on g-peptides revealed that face-to-face amide stacking with anti-aligned dipoles is competitive with intramolecular hydrogen bonding. [9, 10] QM calculations on the formamide–benzene complex suggest a slightly better interac- tion energy than the benzene dimer. [11–13] This was rationalized by a more favorable electrostatic term due to strong dipole– quadrupole interactions which overcompensate the smaller dispersion contribution. The large experimental dipole moment of 3.7 D for N-methylacetamide (NMAC), [14] which we use as a model for the amide backbone fragment, suggests that stacking to this motif can be further enhanced if its part- ner also carries a dipole moment and is properly aligned. Targeting amide groups with suitable ligand functionalities is attractive for two reasons: First, they are abundant in protein binding sites, either as part of the backbone or in asparagine and glutamine side chains. For a considerable proportion of these amides, the NH donor and CO acceptor groups are en- gaged in intra-protein hydrogen bonds, [15] while the less polar p-surface is exposed for ligand binding. Second, backbone amides are often involved in a-helix or b-sheet secondary structures, which decreases their flexibility, and thus greatly facilitates the structure-based design of a desired ligand inter- action. Examples in which stacking interactions with protein back- bone amides are important for ligand binding are the S3 pocket in cathepsin L, [16] the selectivity pocket of PDE10, [17] or the S1 pocket in factor Xa. [18] Factor Xa and related chymotryp- sin-like serine proteases expose in the S1 pocket two flat pep- tide walls, which are separated by 7–8 , and which preferen- tially bind (hetero)arenes. We previously identified two oxa- zole-containing factor Xa inhibitors with almost identical bind- ing modes, but an 11-fold difference in binding affinity (Figure 1). [18] The main difference between both X-ray struc- tures is the relative orientation of the dipoles of the oxazole linker with respect to the amide backbone of Cys191–Gln192, which is at van der Waals distance. While it is generally chal- lenging to associate individual interactions in a protein–ligand complex with energies, it is in line with our expectation that the antiparallel dipole alignment is observed in the compound with the better binding affinity to factor Xa. In this study, we focus on a quantitative characterization of the stacking energies and orientational preferences of various The less polar p-surface of protein amide groups is exposed in many receptor binding sites, either as part of the backbone or in Gln/Asn side chains. Using quantum chemical calculations and Protein Data Bank (PDB) searches on model systems, we investigate the energetics and geometric preferences for the stacking on amide groups of a large number of heteroarenes that are relevant to medicinal chemistry. From this study, we discern that the stacking energy of an aromatic ligand sub- stituent can be improved by: 1) orienting the fragment dipole vector such that it is aligned in an antiparallel fashion with the dipole of the interacting protein amide group, 2) increasing its dipole moment, and 3) decreasing its p-electron density. These guidelines should be helpful to more rationally exploit this interaction type in future structure-based drug design. [a] M. Harder, Prof. Dr. F. Diederich Laboratorium für Organische Chemie, ETH Zürich Hçnggerberg, HCI, 8093 Zürich (Switzerland) E-mail : diederich@org.chem.ethz.ch [b] Dr. B. Kuhn Discovery Chemistry, F. Hoffmann–La Roche AG Grenzacherstrasse 124, Bau 92, 4070 Basel (Switzerland) E-mail : bernd.kuhn@roche.com Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201200512. # 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 2013, 8, 397 – 404 397 CHEMMEDCHEM FULL PAPERS