Hydrogen bonding asymmetries

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Have you ever wondered why the Rule of 5 (Ro5) specifies hydrogen bond (HB) thresholds of 10 acceptors but only 5 donors? This is, perhaps, the prototypical example of what I'll call a 'hydrogen bonding asymmetry' and it is sometimes invoked in support of the folklore that HB donors are somehow 'worse' than HB acceptors in drug design. I have, on occasion, tried to track down the source of this folklore but that trail has always gone cold on me. In any case, I don't think the HB asymmetry in Ro5 has any physical significance since HB acceptors (especially as defined for Ro5) tend to be more common in chemical structures of interest to medicinal chemists than HB donors. This was discussed in our correlation inflation article and the bigger Ro5 question for me is why the high polarity limit is defined by counts of HB donors and acceptors while the low polarity limit is defined in terms of lipophilicity. As may become a blogging habit, I'll include some random photos (these are from a visit to India late in 2013) to break up the text a bit. 

Drum fest at Buland Darwaza

It was this article in JCAMD about the 'polarized' nature of protein-ligand interfaces that got me thinking again about hydrogen bonding asymmetries. The study found that proteins donate twice as many HBs as they accepted. While the observation is certainly interesting, I do think that the authors might be over-interpreting it. For example, the authors suggest that it appears to be an underlying explanation for Ro5 and they may find that there are significant differences in their definitions of HB acceptors and those used to apply Ro5. The authors also state "Peptidyl ligands, on the other hand, showed no strong preference for donating versus accepting H-bonds". This observation would more be consistent with 'polarization' of protein-ligand interfaces being determined by nature of the ligand.

The authors assert that "lone pairs available to accept H-bonds are actually 1.6 times as prevalent as protons available to donate, both on the protein and ligand side of the interface." While it is appropriate to count lone pairs in situations where only one lone pair accepts an HB (e.g. when considering 1:1 hydrogen bonded complexes in low polarity solvents), I would argue that it is not appropriate to do so when considering biomolecular recognition in aqueous media because the acceptance of an HB by one oxygen lone pair makes the other lone pair less able to accept an HB. You can see this effect using molecular electrostatic potential as discussed in this article (see polarization effects section and Table 4). Put another way, how often is a carbonyl oxygen observed to accept two HBs from a binding partner? How many docking tools would explictly penalize a pose in which a carbonyl oxygen accepted two HBs?

As I see it, a typical protein is more likely to have a surplus of HB donors under normal physiological conditions. Some parts (e.g. serine, threonine, tyrosine and histidine side chains and the backbone) of a protein can be regarded as having equal numbers of HB donor and acceptor atoms. While the anionic side chains of aspartate and glutamate cannot donate HBs, the cationic side chains of arginine and lysine have five and three donor hydrogen atoms respectively while lacking HB acceptors. The tryptophan side chain has only a single HB donor (although its p-system is likely to be able to accept HBs) while each side chain of aspargine and glutamine has two donor hydrogen atoms and one acceptor oxygen atom. The histidine side chain is sometimes observed to be protonated in X-ray crystal structures which means that it should be considered to be more HB donor than HB acceptor in the constext of protein-ligand recognition. The tyrosine hydroxyl would be expected to be a stronger HB donor (and weaker HB acceptor) than the hydroxyls of either serine or threonine.  

A magical place

The study considers the "possibility is that nature avoids the presence of chemical groups bearing both H-bond donor and acceptor capacity, such as hydroxyl groups, in the binding sites of proteins or ligands" although it is not clear what glycobiologists would have to say about this. Let's think a bit about what happens when a hydroxyl group donates its hydrogen atom. Let's suppose you've spotted a nice juicy hydrogen bond acceptor at the bottom of a deep binding pocket that is otherwise hydrophobic. The ligandability is eye-wateringly awesome (the ligandometer is beeping loudly and appears to have gone into dynamic range overload). Even the tiresome Mothers Against Molecular Obesity (MAMO) are impressed and have recommended that you deploy a hydroxyl group since this will be great for property forecast index (PFI). What could possibly go wrong?

The main problem is that the hydroxyl HB donor comes with baggage. In order to donate an HB to the acceptor at the bottom of that pocket, you're going to need to force an HB acceptor into contact with the non-polar part of that binding pocket. Although this contact is not inherently repulsive, it is destabilizing. Another factor is that donation of an HB by the hydroxyl group is likely to increase the HB basicity of the oxygen (which will exacerbate the problem). You can think of other neutral HB donors (e.g. amide NH) but the vast majority of them come with baggage the form of an accompanying HB acceptor. Exceptions such as NH in pyrrole (not renowned for stability) and indole (steric demands) come with baggage of their own. In contrast, the drug designer has access to a diverse set (e.g. heteroaromatic N, nitrile N, tertiary amide O, sulfoxide O, ether O) of HB acceptors that are not accompanied by HB donors. If you use one of these, you don't have the problem of having to also accommodate a ligand HB donor.

This is a good place to wrap up. In the next post, I'll talk about a completely different type of hydrogen bonding asymmetry, but for now, I'll leave you with some photos from an afternoon spent admiring asses in the Rann of Kutch. 

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