As you were likely notified via Twitter, our work on the second-generation synthesis of the arylomycins was recently published in JACS. To tell the truth, saying “our work” is a little complicated and maybe even misleading if you found your way to this post via baranlab.org. As some of you may have noticed, the author list for this publication is a little unusual. Why are there two TSRI professors listed, yet only one student? Whose lab did this work, and where is the inspiration for this project coming from? I hope to answer these questions as well as some other more scientific ones people may have in this blog post about the work and my journey (so far) as a jointly advised graduate student.
|Scalable Synthesis of Arylomycin|
It all started in 2014 when I was at a symposium where Prof. Floyd Romesberg was speaking about his lab’s work in antibiotic discovery, specifically regarding a class of molecules called the arylomycins. At the time, I was grad school bound, wanting to do synthetic chemistry yet also having a newly found penchant for the field of microbiology. You can’t always get what you want, but sometimes you do, and I was provided with the opportunity for my graduate studies to involve doing synthetic chemistry in the Baran lab with the purpose of working on antibacterial and microbiological research in the Romesberg lab. The first goal set for this collaborative work was to come up with a more efficient way to make the arylomycins, a still active interest in the Romesberg lab for the use as a chemical probe. On day one of my time at TSRI Floyd, Phil, and I met and the idea of oxidative coupling was born. On day two I had compiled a list of methods available for the oxidative couplings of phenols, and on day three I was in the hood making substrates. The real reason that this came together so fast is that there were so many methods available to perform such a coupling, making the outlook for this project good (at least to a bright-eyed graduate student on day three).
The plan moving forward was to make the desired product of the macrocyclization via the original Suzuki coupling route to have a standard, that once in hand would allow the rapid screening of the plethora of oxidative coupling conditions at our disposal to identify hits. This ultimately ended up providing me with the clearest picture of why this project would be so impactful to the synthesis of the arylomycins. Making the macrocycle via the original route was not only time consuming but extremely low yielding, and the boronic ester substrates that need to be handled turn out to be very sensitive. If banging your head against the wall was considered a synthetic step, it would definitely be included in our first-generation synthesis. Ultimately, the limiting factor (by a large margin) in me starting the screening became the synthesis of the product standard, not the synthesis of the oxidative coupling substrates! I should mention at this point though that the impact of the first-generationsynthesis should never be minimized. It has, up to this point, provided ALL of the material used in ALL of the work that has made the arylomycins the center of an ongoing antibacterial drug discovery campaign.
Once the standard was made, the screening began with really no agenda. We were agnostic to the nature of the oxidant, catalytic or stoichiometric reactions, and operational ease. Early on the usual results were “complete and rapid decomposition” or “no reaction.” You can read more about the discovery of copper being a competent oxidant in the manuscript, but what it won’t tell you is the battle I didn’t know that I was having with ambient water after the 10% yield obtained with complex Nakajima originally reported. A lot of screening was done around this reaction to no avail. Nothing I seemed to do improved the yield, though I did find a few good ways to decrease the yield. Through all of this, I had been working under the assumption that it was totally fine to run the reaction open to air in a “no precautions” manner, since that is what had been reported by Nakajima. When I finally got fed up with having nothing good to write home about I decided to put good old Schlenk technique to work and ran the reaction in an “every precaution I could think of” manner. The result: 65% yield. I honestly didn’t believe the result so I ran it two more times before telling anyone. Doing experiments in which I added water I confirmed that it was the culprit. The rest of the optimization from there you can find in the supporting information.
The optimized conditions led us to scale it up. The large amount of the macrocycle this provided us (see above) naturally led us to want to make analogs. The Romesberg lab had not been involved in making arylomycin analogs since 2013. Since then, RQx (a Romesberg lab derived start-up based on developing the arylomycins as antibiotic therapeutics) had a multi-year research collaboration with Genentech, who combined had presumably made many hundreds of analogs and knew far more about SAR than we did. The only way to not date ourselves would be to go digging in their patents for any hints as to what our new starting point should be. This digging made it clear that they had done a whole lot of work and had probed modification ideas that were both inside and outside of the box. Unfortunately, finding our new starting point from this was not so easy. At the time, we were interested in making analogs with Gram-negative activity, and unbeknownst to us, Genentech had already accomplished this goal and described it in a patent that had not yet become available to the public. With only the older patents to work with, the only Gram-negative MIC data to go off was for a permeablized strain of E. coli. We decided to take a gamble and chose the analog that had the most activity against this permeablized strain, knowing very well this was not necessarily an indicator of having real Gram-negative activity. Luckily this gamble paid off and the set of compounds we based on the Genentech analog have activity against regular Gram-negatives, the data for which you can find in the manuscript.
The one and only thing that was clear from the arylomycin patents was that overall, there was a lack of diverse chemistry being done for derivatization at the C-terminal region of the arylomycins. Great structural work done by Dr. Mark Paetzel allowed me to spend lots of time scratching my head at this region of the molecule and the way in which it interacts with the active site of SPase. The native carboxylate of the arylomycins have distant interactions with the active site Ser-Lys dyad of SPase that are on the verge of not being H-bonds at all. This isn’t surprising when taken with the fact that the arylomycins are substrate mimics for a protease with Ala-X-Ala recognition and seem to end at Ala-X. I went to Floyd with the idea of extended C-terminal analogs to mimic the missing alanine residue. He was excited and wanted whatever we could put out there. I went to Phil with the idea and he wanted to do new chemistry. We ended up with the best of both worlds and decided on the decarboxylative Giese addition that was being developed in the Baran lab at the time. This would presumably be a far cry from something RQx or Genentech would have already done and would provide new chemical connectivity in this region. While the analogs this gave weren’t the best ones around, it provided valuable information with regards to the SAR, mainly being that this area of the molecule is not inviolate.
LepB (SPase) bound to Arylomycin A2
At the end of all of this, we patented our new technologies and reached out to Genentech who is actively working on developing the arylomycins. Their interest in our work turned into a licensing agreement and collaboration that has so far been a very positive and powerful for both sides. As if being a joint student between two labs at TSRI wasn’t enough for me, I have now added the industry collaboration as well. However, if the added dimension is anything like what I have experienced being a jointly advised student, it will prove to be extremely beneficial to both the science being done and the education of the student; me in this case.
Being a joint student comes with its own unique challenges: going to twice the meetings, presenting the same work to two advisors with different preferences, working on two advisors’ ideas, dealing with twice the personalities in the lab, the list goes on. There was even a dark time at the beginning, which has now passed, when I had to drive to get from one lab to the other. Ultimately, the benefits greatly outweigh the costs and often I find that the challenges listed above are in fact perks. On certain days, I may even go as far as to call them blessings. What the benefit of it really comes down to is the greater number of brilliant people with varying areas of expertise that I get to work with. The science you see in the recent publication is the result of helpful guidance and input from both of my advisors and from both of my sets of labmates. The acknowledgements section of our publication only begins to demonstrate that as members (current and former) of both labs are duly listed. So, if you are still wondering which lab did this work, or which lab is this guy even in, the answer is definitively both.
|My Two Families|
If you have any questions or comments about the chemistry, the arylomycins, or my unique set of challenges/blessings please let us know. Thanks for reading!
Have you guys considered using this methodology on vancomycin? Might be a nice collaboration with Boger labReplyDelete
It’s a good idea! Hopefully one day we can look at that natural product family.ReplyDelete
Its very intereting that none tried this approach earlier. btw what about chirality at the biphenyl axis? Did you observe both atroposomers or just one due to them being connected via amino acid chain?ReplyDelete
Great question. The Cu-mediated macrocyclization yielded a mixture of interconverting atropisomers, however appending an amino acid to the hydroxyphenylglycine residue gave only a single atropisomer matching that of the natural products.ReplyDelete
Very interesting drug and docking study: did you ever consider making this derivative? https://imgur.com/a/qjs9b the binding site really makes this look like it will workReplyDelete
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