Wednesday, March 22, 2017

Thermodynamic Peptide Macrocyclization

Frequenters of the Open Flask may have noticed the increased occurrence of peptide substrates in some of our recent methods reports. Nearly two years ago, we were joined by an exceptionally talented post-doctoral researcher, Dr. Lara Malins; her expertise in the field of peptide synthesis has proven a boon for the group, and her contributions can be found scattered throughout many of our recent papers. “Peptide Macrocyclization Inspired by Non-Ribosomal Imine Natural Products” (J.Am. Chem. Soc. 2017, ASAP) however, represents the Baran Lab’s first true foray into the field of peptide chemistry.

While the project has been rewarding, it was not without its practical challenges. As an historically total synthesis-based group, we are not equipped with the standard supplies and instrumentation found in even the smallest of peptide synthesis laboratories; instead we have relied on both the generosity of other TSRI groups (shout out to the Ghadiri lab!) and our own creativity to accomplish this study. Reactions that required heating—specifically, the loading of amino aldehydes onto our manually prepared Rink TG resin—posed a unique problem, as our orbital shakers are limited to room temperature agitation. As such, we were forced to use rubber bands and rotavaps, sans vacuum, to simultaneously stir and heat the reaction vessels. While this was the best method available, it wasn’t great… We fished soaking syringes out of the water bath on more occasions than we care to remember. We were also on the receiving end of judgement from labmates about (a) what on earth we thought we were doing and (b) whether this was really the best use of a rotavap…

“I think your peptide is dry, Justine…” – fellow labmate after rotovap resin-loading for over 5 h…
It is also worth mentioning that the majority of substrates were made without the aid of an automated peptide synthesizer, relying instead on our own two (four) hands. Scale-up for initial reaction screening meant running multiple 300–400 μmol scale reactions in parallel; tennis elbow has nothing on peptide elbow!

When Phil asks for a gram-scale reaction, you find a way…
After all of the work (and swollen elbows), we were thrilled to submit a JACS communication in the fall of 2016, wherein we described a novel approach to thermodynamic peptide macrocyclization. This unconventional method allows for late-stage diversification of high-value substrates by virtue of an inherently reactive imine intermediate, a process inspired by a non-ribosomal peptide synthetase/reductive release mechanism. Incorporation of isotope labels and bioorthogonal functional handles proved facile, as did further manipulation of the installed moieties. Additionally, four distinct natural products and associated analogues were synthesized, each in a time frame of about 2 days (start to finish). As per Baran Lab dogma, the reactions require no protecting groups and are tolerant of all proteinogenic functionality.

While much of the initial feedback was positive, we received a few valid critiques to address prior to resubmission. The first point raised concerns over whether conformational predisposition of the chosen amino acid sequences contributed to the robust protocol. Given that many of our sequences were inspired by natural products, we held similar concerns; in the full paper (out today), we opted to use an online random sequence generator to minimize the possibility of practitioner bias in sequence selection. We were pleased to discover no notable difference in reaction rate or yield between our original substrates and those picked at random. Our observations were further corroborated by variable temperature NMR studies done by our friends at Bristol-Myers Squibb (thanks, Kevin and Paul!). Unsurprisingly, Kevin was able to identify hydrogen-bonding networks in the four natural products synthesized, which likely contribute to the facility of cyclization in these cases. In contrast, the other substrates investigated showed few, if any, of these interactions. While we cannot claim that conformational bias plays no role, we have determined that it is not a strict prerequisite.

A second, but related, matter raised by the referees was the origin of selectivity in sequences that possess an internal lysine residue (i.e., cyclization at the terminal amine versus the lysine ε-NH2). In our original report, we had included only one example with an internal lysine, which—as pointed out by one of the reviewers—was an consideration that should be further explored. We agreed, and have now expanded our study to include six examples, one of which was generated at random, and are confident that reaction conditions, regardless of protocol, can be successfully tuned to favor terminal amine cyclization. Once again, VT-NMR studies suggest no internal bonding networks in the lysine-containing peptides analyzed, indicating that conformational bias is not the sole contributing factor.  

We are immensely grateful for the insightful criticism received from our first submission—the suggestions provided helped guide our revision process, and served as a prime example of peer-review at its finest. We now present a thorough, and much expanded, study on our approach to thermodynamic peptide macrocyclization. Enjoy!


Friday, February 10, 2017

The Hitchhiker’s Guide to RAE Cross-Coupling

The Answer to the Ultimate Question of Carboxylic Acid, Heterocycle and Everything

We are excited to announce that our paper “Alkyl-(Hetero)Aryl Bond Formation via Decarboxylative Cross-Coupling: A Systematic Analysis” (in the following referred to as the Guide) has today been published in AngewandteChemie International Edition!
I joined the Baran lab as a visiting student around six months ago. When I had my first meeting with Phil, he had two important messages for me. First of all he introduced me to the project and one of the things he showed me was the sales statistics of N-hydroxytetrachlorophthalimide (TCNHPI). This reagent was commercialized as a typical activating agent used in cross-couplings of redox-active esters. Impressively, all major pharmaceutical companies world-wide could be found in the list of buyers – RAE cross coupling was already being applied broadly and its adoption was proceeding at blistering pace less than a year after the invention of this reaction. At this point four different reaction types had been published, and we were using several different RAEs and various procedures for their generation. We had gathered rich in-house knowledge by that time and after running thousands of reactions my lab mates and I had developed a "feeling" how to get almost any substrate to work. As we did not want it to be lost forever, we were asking ourselves how can we pass this valuable information on to all the chemists out there? The answer was rather simple – we were going to create the Guide.

Owing to my German origin Phil's second advice was rather practical: American highways are not the Autobahn, and if I was speeding I should do it carefully - "there is a science to speeding!"
Starting to work in the lab I was certainly not lost in time and space, but still in need for the friendly advice DON’T PANIC. There was a maze of four publications with more than 900 pages of SI and more unpublished material in front of me. The first challenge was to get an overview of all the published and unpublished procedures, and to arrange them in a simple and clear manner. Our team developed a matrix chart, in which four different catalytic systems are fixed on the x-axis, while four different modes of activation are fixed on the y-axis giving a total of 16 different procedures (Figure 1). Combined with a visual identification of the success of the reactions by a simple color code the Heart of the Guide was born.

Figure 1. a) Activating agents, nucleophiles and catalytic systems in RAE cross-coupling. b) Simple classification combined with visual identification.
Next, Matt and I selected nine different substrates covering a broad chemical space. The Guide offers a selection of variously functionalized primary, secondary and tertiary carboxylic acids including α-amino acids, heterocycles and bioisosteres (Figure 2a). Each of these substrates was subjected to our 16 different reaction conditions and the obtained data was clearly arranged in a one-page table (Figure 2b). Looking at the number of red squares, we were definitely exploring the edges of our methodology here…! However, in each case we found at least one set of conditions to yield the desired product in more than 60% yield! This underlines how being able to fall back on many different conditions combined with a Guide can be a real asset when tackling challenging synthetic problems!
Figure 2. a) Selection of substrates for b) the Guide.   
In the second part of my stay, I tried to expand the scope of the Guide to new areas of RAE cross-coupling. What we were really excited about was applying more heterocycles in our chemistry. At this point we had mainly explored the nature of the alkyl carboxylic acid, as previous investigations suggested that the influence of the aryl nucleophile on the reaction outcome was rather small, except for heteroarenes. After some experimentation we discovered that methylpyrazole magnesium bromide undergoes simple Fe-Kumada coupling in synthetically useful yield! Encouraged by this result we decided to widen the scope of our Kumada, Negishi and Suzuki couplings to medicinally relevant heterocyclic nucleophiles (Figure 3).

Figure 3. Heterocyclic nucleophiles explored in the Guide.

Except for furan and thiophene all of the heteroarenes explored in the Guide contain basic nitrogens (“real heterocycles”). Searching the literature for instances where these heteroaryl nucleophiles were used in cross-coupling reactions revealed what we had already suspected: despite their importance in medicinal chemistry and other chemical industries cross-coupling of N-heterocycles is severly underdeveloped. During our investigations we found that the best choice of method for RAE cross coupling is strongly depending on the electron-density of the metalated carbon atom. While electron rich heteroarenes easily undergo Fe-Kumada coupling, Ni-Suzuki is the best choice for electron deficient heterocycles. Interestingly, we observed 2-pyridyls to be most challenging with our methodology. While the 2-pyridineboronic acids are known to be unstable, Kumada coupling turned out not to be feasible either. We think that the homo-coupling byproduct (bipy) is the culprit and deactivates the Fe catalyst. Fortunately, we could solve these issues by applying a modified Fe-Negishi protocol.
A good guide should provide you with overview and main directions at a glance, but also with detailed information where you need it. That’s why we have put together a Supporting Information which contains clear, exact and easy-to-follow experimental procedures to make the application as straightforward as possible. Besides graphical descriptions of the reactions and detailed characterizations of the products, our Supporting Information includes an overview of all reaction conditions in one scheme (Figure 4). We were delighted that this important part of the Guide was also acknowledged by the referees:
“The supporting information is of high quality […]”

“The Supporting Information, to the best of my ability to determine, is spectacular, and includes pictures detailing each step. Only a full-length motion picture could be better. The products are adequately characterized and look pristine.”

Figure 4. Overview of all 16 different reaction conditions.
Unfortunately, Angewandte did not print the words DON’T PANIC in large and friendly letters on its cover, but I still hope that the Guide will help many organic chemists to find their way through RAE cross-coupling – in our lab it already finds regular application.

Fred and the team of the Guide

We apologize to all those who expected 42 to be the answer to everything – we could at least confirm it in three cases.