I was under the weather last night, so there was no chance to publish my Friday post. But I am back now, sitting in our living room and marveling at the copious amount of snow that has fell upon us over the past 24 hours. I think it is easily 25cm, although I have not heard the official forecast. Now… Hydrogen bonds hold together the snowflakes I am looking at. The impressive cooperativity of these weak forces is responsible for the snowflake formation. In a totally unrelated domain, the cellular permeability of peptides and amino acid-derived small molecules is also related to hydrogen bonds. Peptides are typically awful in their ability to traverse non-polar cellular membranes, save for a few exceptions. Cyclosporine A (shown in inset A below) is among the better ones. The network of hydrogen bonds that you see provides for a fairly lipophilic conformation of this molecule and helps it go through cellular membranes by passive diffusion. Well, at least this is what people think now (you never know when we will all flip our minds upon discovery of a predominantly active pathway that involves a protein transporter for cyclosporine A). The lessons offered by the internally satisfied hydrogen bonds here extend to other areas. The inset B below shows a marvelous case from a paper by Jacobson and colleagues: http://pubs.acs.org/doi/abs/10.1021/jm201634q. The two molecules clearly have identical polar surface areas, yet the one on the right is 4 times more cell-permeable by virtue of the hydrogen bond marked in red.

So what should those, who want to design cyclic peptides with improved cellular permeability, take from all of this? I think the main lesson is that we need to continue our hard-target search for hydrogen bond pairs and they do NOT need to be restricted to what you see in the case of cyclosporine. The classical NH-O hydrogen bonds are cool, but we need to go way beyond that. Here is a paper that talks about glycine’s ability to engage its alpha C-H bonds in polar contacts (inset C) that lead to stabilization of inter-helical motifs in proteins: http://www.pnas.org/content/98/16/9056. Does this mean that we want to see more glycines in cyclic peptides in order to make them have better chances of being cell-permeable? I am not sure. But I do know that glycine has two alpha C-H bonds and this is the reason why the likelihood of forming the “right” connection is simply higher for glycine. It follows that perhaps d-amino acids should be considered more often if these unusual hydrogen bond motifs are to be captured. Lastly, I will present another case that is well familiar to protein chemists – that of serine (inset D). Here is a paper showing that serine is a residue par excellence in “reaching over” and forming hydrogen bonds using its OH group: http://www.biomedcentral.com/1471-2148/10/161. Someone has to take all of this protein chemistry knowledge and translate it into cyclic peptides. We may then indeed start to see trends that will emerge from “unusual suspects” for hydrogen bond formation in macrocycles. I do want to end by saying that these less common hydrogen bonds are fairly weak, but they sure have their place under the sun!

Molecular diversity is a term that is familiar to synthetic chemists. A diverse collection of molecules is characterized by a broad representation of functional groups, chemotypes, ring sizes, and so on. How do we judge how rich is the conformational diversity of a given collection? My lab’s efforts in the area of peptide macrocyclization have made me think quite a bit about this matter. It is particularly enticing to make big gains without too much synthetic effort.

Time and again, I look for papers that provide straightforward mechanisms of controlling accessible conformational space. In this regard, cyclic peptides offer an unprecedented opportunity, particulalrly once proline is considered. Proline is known for its relatively high barrier for cis/trans interconversion, which makes the X-Pro motif (X is an amino acid) an interesting structural feature to explore. A paper by Rabenstein and co-workers cited below provides a marvelous demonstration of capturing cis-amide bonds in disulfide macrocycles. The authors have found that the corresponding linear peptides contain Cys-Pro bond in its predominantly trans form. However, cyclic peptides display periodicity in cis/trans geometry of the Cys-Pro bond (marked red in the graphic below). It turns out that macrocycles containing an odd number of amino acid residues are predominantly cis at the Cys-Pro juncture, whereas those with an even number of amino acids are predominantly trans. This finding is significant as it offers a tool to sample various conformations by changing the number of residues in a macrocycle. I’m afraid the precise nature of this intriguing phenomenon is unknown.

http://onlinelibrary.wiley.com/doi/10.1002/anie.200503470/abstract

Diazotransfer from activated azides to primary amines is a fascinating process that provides a useful route to organic azides using a fairly accessible reagent – (triflyl)azide. The mechanism of the reaction remains somewhat of an enigma. There are several metals, including zinc and copper, that catalyze this process. Prof. Chi-Huey Wong has used diazotransfer chemistry in several of his elegant approaches to azasugars and put forth a mechanism that involved 5-membered intermediates. Below I am showing a representative case. You will note that protected threonine is used in this process.

http://pubs.acs.org/doi/pdf/10.1021/ja0264605

The resulting azido version of threonine is a useful building block. In fact, the reason for this post is that I have been looking at ways to circumvent some of the troubles encountered in really hindered amide couplings. One way to deal with this challenge is to mask the alpha-amino group by way of an azide, which is not sterically demainding (compared to some N-protected versions). Notably, this amino acid derivative is configurationally stable (to an extent, of course). Nicolaou and co-workers have put this versatile building block to good use in their synthesis of thiostrepton (http://pubs.acs.org/doi/abs/10.1021/ja0529337).

I mentioned that my sabbatical is coming to an end in a couple of weeks, which is somewhat sad, given that there are many things I would still like to try. But let’s be honest: things are better left to our graduate students, who will carry the torch forward. As a matter of fact, this was one of my stated goals before I started my protein work at SGC – to try things out, but eventually put it into the right hands. In this regard, it was strategic on my part to stay in Toronto for my sabbatical. I am really happy to see that my student Victoria (shown below) has gained some valuable experience under Elena’s supervision. Victoria now knows the whole process – from cloning to protein production, purification, and crystallization. In addition, Victoria is busy with her synthesis projects. I tip my hat off to her tenacity and ability to pick up new experimental tricks. This is what it’s all about! As for me – it is time to leave protein projects in the hands of experts and not some fluffy amateurs such as myself. I know I will miss my first hand experience, but I will soon need to put back my grant writing/teaching/research advisor hat on.

Proline has a number of distinct features. They range from its effect on the formation of turn structures in proteins to proline’s involvement in organocatalytic processes that are rooted in enamine chemistry (inset A). In our lab, we resort to proline when we run peptide macrocyclization: when used as an N-terminal residue in a linear peptide, proline plays an enabling role in the macrocyclization process. We have tried many other secondary amines, yet proline stands out. Interestingly, this observation mirrors organocatalysis in that proline’s core – its five-membered pyrrolidine ring – is a rather special enamine/iminium ion precursor. Notably, piperidine (a six-membered ring) pales in comparison as far as efficiency of iminium ion generation. The other day I was pondering over what makes proline so unique in our reactions and realized that I need to re-read some of the classic papers by Eschenmoser. His excellent Helvetica Chimica Acta work cited below describes X-ray structures of several enamines, including the two shown in inset C. The tau angle is what we want to focus on. Tau is a measure of nitrogen pyramidalization as it describes an average of two dihedrals marked by the small letters in inset B. According to this X-ray study, the pyrrolidine-derived enamine is more or less flat, whereas the piperidine congener is highly pyramidalized. This important crystallographic finding shows that pyrrolidine enamines have a shorter =C–N bond, leading to a higher C-N double bond character compared to their piperidine counterparts. The greater nucleophilicity of pyrrolidine enamines toward C-alkylation becomes clear and also explains why piperidine enamines are more prone to N-alkylation than pyrrolidine enamines.

http://onlinelibrary.wiley.com/doi/10.1002/hlca.19780610839/abstract

The volume of activation… This is one of those parameters we rarely think about… It represents the difference in the partial molar volume of the transition state compared to that of the reagents. The value of volume of activation provides information about the structural changes that take place in the transition state. Here is a good example: the Baylis-Hillman reaction.

Baylis-Hillman reactions can be really slow (a week to go to completion is not unusual, which is really atrocious). The calculated volume of activation in a prototypical Baylis-Hillman process is approximately -70 cm³/mol. This is a fairly high negative number, indicating a volume reduction when reagents are transformed into the transition state. It can be inferred that an increase in pressure would result in rate acceleration. Indeed, an increase in pressure from 1 bar to 1000 bar provides approximately 15-fold increase in the reaction rate. Please note that no gases are employed here (and pressure still matters)!

One can anticipate that reactions that are accompanied by an increase in the volume of activation should be accelerated when performed in vacuo. Elimination reactions belong to this class. We typically don’t think of vacuum as a means of driving reactions to completion, save for a few rare cases. By the way, volume of activation can be easily measured. Here is a classic paper:

http://pubs.acs.org/doi/abs/10.1021/ja01093a019

Synthetic chemists are good at creating conditions under which unstable molecules or intermediates can be generated and observed. George Olah’s 1994 Nobel Prize was largely due to his finding that low nucleophilicity media based on antimony chloride provides an environment where carbocations can live long enough to be detected. Under any other conditions, solvents are too nucleophilic to be innocent by-standers.

Now let’s talk about organoboron compounds, which are isoelectronic to carbocations. I just marvel at biological examples that demonstrate how electrophilic functional groups familiar to chemists are prevented from showing their expected properties. Boron in its trivalent form is electrophilic, which is the reason boron inhibitors developed by Anacor work as protease inhibitors. I blogged about these molecules a while back, on August 24. Earlier today I was interested to see a paper describing a recently disclosed Anacor’s structure that defies logic in that it shows a case where boron persists in its trivalent form and is caught in the act of inhibiting a ROCK2 kinase. While the binding mode at the hinge region is common for a kinase inhibitor, I am not aware of a synthetic variant of such hydrogen-bonded pattern involving a boron-containing functionality. As it stands, the fact that boron remains tricoordinate within the kinase active site, is quite unusual.

http://jpet.aspetjournals.org/content/347/3/615.full.pdf+html