Special events

I have been in Florida over the past four days, doing a bunch of things that loosely relate to peptide science. In fact, Orlando is where the American Peptide Society Meeting will be held in 2015. I found Miami to be particularly hot, and I suspect that Orlando will be even more so when our conference is held there in June 2015. But at least we will have a first class program for all of you peptide aficionados!

When I got home and sat in front of my computer, the first thing I noticed was a very cool JACS paper by Professor Shu-Li Yu. The authors describe a palladium system that allows for highly regioselective allylation of substituted pyrroles at the C-2 position. While the regioselectivity is largely driven by the relative steric congestion at the more likely allylation site (the NH), the overall dearomatization of the pyrrole core deserves attention and is very impressive. The products of this transformation are chiral aza-dienes that will almost certainly generate attention in the synthesis community. Noting unusual selectivity is often a sure recipe for a productive research project, and Yu’s paper is no exception. The best thing is that complexity clearly increases in this reaction, which always pleases one’s eye (and is not always what we see in the literature these days).



Getting there virtually

β-Lactams have long been the antibiotics of choice in the fight against S. aureus infections, but resistance to these molecules has emerged, causing alarm bells. MRSA, or methicillin-resistant staphylococcus, has been a growing concern for a long time due to the so-called PBPs (Penicillin-Binding Proteins). β-Lactams are known to irreversibly acylate the active-site serine of PBPs, resulting in bacterial death. In contrast, PBP2a is refractory to inhibition by essentially all commercially available β-lactams. Below you see a generalized β-lactam structure and its PBP2 nemesis.


There have been many ways to approach the problem of creating new antibiotics, particularly non-β-lactam types (to circumvent resistance). Inevitably, these methods call for screening some large collections of molecules. I was intrigued by a paper in JACS published by Chang and co-workers from the University of Notre Dame. Looking for potential inhibitors, the authors screened 1.2 million compounds from the ZINC database against the X-ray structure of PBP2a of MRSA. The ZINC database was created by the Shoichet lab at the UCSF (Brian is now at the University of Toronto, and is also a member of the SAB of Encycle Therapeutics, a company I started in 2012). The complexes obtained using this method were scored using DOCK, Gold, FlexX, and ChemScore. Subsequently, 29 molecules were synthesized and/or purchased. The lead compound shown below was generated using a comparatively small-scale synthetic campaign, which is the main attractive feature here. This substituted oxadiazole is an exciting entity that offers a new avenue for exploring non-β-lactam inhibitors of PBPs. What is the main lesson here, you might ask? I think it is a clear and demonstrable promise of addressing important problems using modern docking algorithms. Papers such as this underscore the power of screening virtual collections.



Not an imitation

Here’s a fairly nasty question from the cumulative exam I gave earlier this month. It really got the graduate students confused, but that’s ok – it is indeed a bizarre reaction. I learned about it several years ago, when I visited Professor Chiba’s lab in Japan. What you see is a process that amounts to radical cation-driven metathesis reaction. This is a good example of a process that goes by an unusual mechanism and accomplishes something that is well familiar to many. There is no need to compare the scope of this process to what we are accustomed to seeing in ruthenium catalyzed chemistry. This is not the point. We should still be thrilled when we get to see nifty tricks emerge in less conventional areas that are relatively underpopulated (I refer to electrosynthesis). This is the quintessential “outside the box” sort of stuff!




About a year and a half ago, I was asked to put together a special issue of Chemical Reviews dedicated to small ring heterocycles. This project has taken a lot of organizational effort, but it has been rewarding to assemble a team of researchers who have pushed back the frontiers of this area over the past 10-15 years. I am going to post more information pertaining to this special issue as the project nears completion, so stay tuned. I just want to mention one name from this compilation: Professor Daniel Romo of Texas A & M University. The reason I want to talk about some of his classic work tonight is because it serves as an example of going after molecules which, on paper, have little reason to exist, yet are surprisingly stable even to column chromatography. Below you see Romo’s spiroepoxy-beta-lactones. Their synthesis is deceptively simple: add DMDO to a diketene. That’s it. The resulting molecules look wild, but many of them are isolable and stable to column chromatography. In these structures, you might imagine some really interesting manifestations of the anomeric effect. Romo talks about the crystal structure of a representative member of this series in his JACS report and discusses stereoelectronics there. There are also some nice synthetic applications of spiroepoxy-beta-lactones demonstrated in this manuscript. One disadvantage of these molecules is the symmetry of the ketene dimer precursors. Oh well, life can’t be perfect (my lab knows it all too well when we deal with an occasional reaction that does not fully destroy the aziridine aldehyde dimer).



Modifications on the go

The correct choice of a ligand in transition metal catalysis is often the difference maker. Besides the steric and electronic parameters inherent to a given ligand structure, an interesting facet of structure/function relationship lies in the chemical modification of the ligand core during the reaction. This feature relates to a general question that is seldom addressed: how do you know that what you think is the ligand structure is actually responsible for the reaction under scrutiny? There are many examples where one can propose in situ modifications of the ligand core, yet one does not always monitor what happens to his/her catalyst during and after the reaction. I am sure there are many additional insights that might be obtained from such studies. There are, in fact, good examples showing the significance of in situ ligand modification. The one I want to cover today comes courtesy of Professor Pat Walsh at Penn. In his JACS report, Pat and his students describe the NiXantphos ligand that is deprotonated during the reaction. As a result, the authors observe facile oxidative addition of aryl chlorides to palladium coordinated to a bidantate ligand, which is unprecedented. Take a look below – there is no reaction with Xantphos, but 91% is obtained with NiXantphos. This is a striking example of how a “deprotonatable” ligand site plays the decisive role.



Weak coordination

I was looking at my Papers3 collection today (those of you who have a Mac and do not have this software – get it right now, while those of you who have a PC – well, I am sorry…) for some non-coordinating anion chemistry. I came across an old paper by my good friend Sergey Kozmin of the University of Chicago, who explored the powerful properties of HNTF2 acid in catalysis. The reaction you see below is one of the examples in the fascinating segment of synthesis where triflamide anion plays its pivotal role. Incidentally, the chloride substituent in the product does come from dichloromethane, so check out the mechanism.



Lewis acid catalysis is another area where triflamide anions have been put to good use. The link below describes the work of Niggemann and co-workers, who have developed a very unusual set of conditions that call for the use of calcium triflamide in conjunction with tetrabutylammonium hexafluorophosphate (this combo is now available from Sigma-Aldrich, so I want to check it out in our own work). The central role of the non-coordinating triflamide anion is on display in this reaction as well. What else might we think about? Perhaps electrophilic fluorinating reagents of the Tf2NF class come to mind, plus some other applications? The bottom line is that non-coordinating anions offer a rich area for exploration because they do not meddle in polar reactions that occur in their vicinity.



In praise of complex macrocycles

Today I will talk about microcystin and its mode of action. This molecule is one of my all-time favourites because of its unique reactivity. Below you can see a view I created using 1FJM entry from the Protein Databank. This picture shows the electrophilic warhead of microcystin and identifies the surface-exposed nucleophilic Cys-273 residue of PP1 phosphatase. This cysteine irreversibly interacts with the electrophilic acrylamide portion of microcystin.

My lab has been after some complex peptide macrocycles equipped with electrophilic aziridine residues. We do not yet have any significant stories to tell in our efforts to covalently inhibit cysteine-bearing protein targets, but we do have the methodological makings of an interesting approach in collaboration with Ben Cravatt of Scripps. So far our molecules seem to be inert against cysteines, which is somewhat of a surprise, yet gives us confidence that we might eventually find something really selective. Back to microcystin: this nanomolar phosphatase inhibitor is a nasty beast that was isolated from cyanobacteria. The corresponding blue-green algae contaminate drinking water and have long been known to be the cause of animal deaths.


On good and bad privileges in chemistry

The notion of privileged scaffolds originated in the pharmaceutical industry. The term relates to disproportionately high occurrence of a given scaffold among drugs and other bioactive molecules. One excellent review dealing with this subject comes from Snyder and Stockwell:


Piperazine is a well-known example of a privileged structure. The are two troubles with this presumably worthwhile status: a. it is tough to carve out a good intellectual property niche because there are just too many molecules that contain a privileged scaffold (by definition) and b. many privileged scaffolds are associated with promiscuous binding to their biological targets. Thus, you might not only have trouble obtaining good claims in your patent, but you might also have to deal with molecules that lack target selectivity. The piperazine case is actually not that bad, but a mere mention of a urea to someone in drug discovery might make him/her cringe.


Personally, I get annoyed when those who do synthetic methodology talk about a new method of making a certain sub-structure and tout the method’s significance because there is a lot of molecules out there that contain that structural element. If you are interested in discovering new bioactive molecules, this prevalence should be a good reason for NOT making the darn thing, but I let go of this argument when I talk to students who get excited about producing a sub-structure that is present in 3 thousand other molecules. Why spoil the party? After all, we are in the business of education and there is a sense of synthetic accomplishment if you make a molecule that contains some omnipresent core, particularly if there is an interesting mechanism involved. We even mention this in our own papers…

There are other kinds of privileged structures that extend beyond drug discovery. I think Eric Jacobsen was the first to bring this term to asymmetric catalysis (see the link below). Here, there are some wonderful molecules that indeed possess seemingly magical properties in terms of enabling better control of chiral space around a given catalytic centre. BINOL is a good example. You might argue that some researchers used to over-interpret the significance of C2 symmetry in relation to its catalytic prowess, but, without a doubt, in the case of BINOL we have a really good reason to call it a privileged core.


I suppose there is some irony in all of this. The term “privileged” came from drug discovery, yet I think there are major negative connotations to the “privileged” status of many bioactive structures, which is certainly not the case for catalysis, where we do not judge molecules on the basis of their interaction with proteins and DNA. As a result, there are some highly desirable attributes in a select few cores.

Melting proteins in cells

I had a busy Friday, but it was a good day. I attended three different lectures, including one by Rebecca Courtemanche, my MSc student who presented her thesis work. Rebecca has been working on an ongoing project with SGC (Structural Genomics Consortium), in which we are using chemical synthesis in order to develop probes for epigenetic reader proteins. We use a range of methods to tackle this problem (a paper describing some of our advances is now being written in collaboration with the folks at SGC). From my lab’s perspective, this has been an exhilarating experience because my students see first-hand how physicochemical methods such as X-ray crystallography enable them to gain a molecular-level glimpse of how small molecules interact with complex protein targets. There are many methods we use and, while I was listening to Rebecca’s talk, I kept thinking about a paper that really amazed me not too long ago. Dr. Raymond Hui of SGC made me aware of this work. Here is a link to this work:


A short background is in order: when proteins in their native conformation are denatured, they quickly lose higher-order structures, transitioning to the unfolded states. A thermal shift assay allows you to measure how the melting temperature (Tm, or the temperature at which 50% of the protein is denatured) changes when small molecules interact with proteins. The stronger the binding, the larger is the value of Tm shift (it makes sense, because binding typically stabilizes the folded conformation). This thermal melt assay is one of the central experiments in understanding small molecule/protein binding, especially if your protein has no enzymatic function. The corresponding measurements are typically performed using isolated proteins that are well-behaved and characterized. In the 2013 Science report by Nordlund and colleagues (above), the thermal melt experiment was performed in within cells. This methodology is label free, which is to say there is no need to interfere with the chemical composition of the molecule being investigated, introduce any kinds of linkers, and do anything special for that matter. The method is called cellular thermal shift assay (CETSA). In brief, the authors took aliquots of lysates of mammalian cells, treated them with a drug versus control and heated cells to various temperatures. As the temperature increased, cells were lysed, and denatured protein aggregates (they are insoluble) were separated from their soluble counterparts by centrifugation. Now comes the best part: it was possible to assess the levels of target protein remaining in solution at each temperature by means of Western blotting. If you plot the relative band intensity of the soluble target protein against temperature, the melting temperature of the protein is derived for the free versus the drug-bound. The difference corresponds to the affinity of the interaction. The authors showed that the thermal stability of the target protein was increased when the drug was added in a dose-dependent manner. This is what I call a powerful method.

What’s in a Ph.D.

There’s this question about what constitutes the optimal set of individual qualities that lead to a successful PhD in science. This is a loaded question as there are so many interrelated considerations. People are different and everyone has his/her preferences of how to work in the lab. If I were to comment on which individual quality I respect, the ability to stand one’s ground and be assertive in defending a viewpoint is probably on the very top of my list. This trait speaks to one’s intellectual maturity and capacity to take a stand when it matters. In every PhD there is a time when a student has to take control over a field he/she feels passionate about, circle the wagons, and have the guts to voice a strong view. In this regard, I found David Evans’ answer to the question “Which advice would you give to someone who is embarking on a career in science now?” both eloquent and instructive. This talk was recorded at the 2013 National Organic Symposium. Please take a look – the question/answer are at around 1:15:49 in the link below.