Thursday, January 2, 2020

Tryptorubin A

"It's just a peptide, how hard could it be?" That was Phil's remark in late 2017, when I set out to pursue the total synthesis of the alkaloid tryptorubin A.

Fast forward two years and a bit, and the lab has published the molecule's synthesis (Download paper here). That means it's my turn to write another blog post, on topics (at least vaguely) related to that paper.

A summary of the paper if you feel the manuscript is tl;dr, or you're behind a paywall:


  • Tryptorubin A is a neat indole alkaloid. It's isolated from an interesting ternary symbiosis: Leaf-cutter ants pile up leaves to compost into mulch. Then, the mulch they have generated goes into little mushroom farms, where the ants grow mushrooms to eat. This is their main diet. Meanwhile, on top these farmed mushrooms live a variety of defense bacteria. One of those bacteria, a Streptomyces sp., generates tryptorubin A as a metabolite. From these bacteria Clardy et al. isolated the molecule and published its fascinating structure (link to isolation).
    • Actually, none of this is in the paper but it's just cool trivia about the molecule.
  • Although nothing is known about the bioactivity or biological role of tryptorubin A, we pursued its synthesis strictly for reasons of structural interest. This is a somewhat old-school, very academic way of thinking. The project was incepted as follows:
    • <scrolling through JACS ASAPS>, "Hey, that's an interesting structure. I bet it would be hard to make."
    • Me, to Phil: "This molecule would be hard to make."
    • Phil, to me: "Great. The best projects are ones where people are slightly scared at the start. Have fun."*
    • Me: "Damnit, now I have to make it."
  • Off I set to make it. The chemistry is pretty self-explanatory (tons of peptide couplings, an Ulmann and a Friedel-crafts), so I won't go into much detail there. However, I will note that in the SI, we briefly highlight a dozen or so failed strategies (SI pages 8-10), if you're curious to see some early things we tried.
    • The extremely short summary of chemical lessons learned is (a) don't try to close highly strained macrocycles with macrolactamizations, if possible, and (b) KISS. Fancy ring contraction or other trickery never beat out simple, straightforward old school robust chemistry.
  • I made what I thought was the natural product <9 months into the project. This is where stuff got interesting. 
    • None of the NMR data matched the real natural product.
    • However, the 2D data unambiguously confirmed that we had the correct atomic connectivity and stereochemistry.
    • What the hell was going on?
  • At this point, we reached out to team Clardy and got all the original NMR data. I spent about 2 weeks simply staring at NMRs trying to understand what could possibly be the difference between what I had made and the natural product.
    • This was, by far, both the most challenging and the most rewarding couple weeks in my graduate career.
    • I don't have many photos relating to this project, but I have this one, which summarizes the era nicely. This was taken at about 4AM. Coffee in one hand, beer (unopened?) in the other. Cumulative sleep in the preceding 72 hours? Approximately 6 hours total. Structural assignment is great. And also terrible.
  • Finally, we hit the conclusion. Both natural tryptorubin A (compound 1a in the published manuscript) and what we had made (compound 1b) had identical connectivity and point chirality, but were inside-out with respect to one another. Formally, this renders them 'non-canonical atropisomers'**
    • We spent a lot of time thinking about how to communicate this isomerism most clearly, and our best efforts are represented with cartoon drawings in the paper, reproduced here:
  • We of course still had to make the natural product. Luckily, a crystal structure (compound 7) in the paper gave us a big hint: The indoline's point chirality enforced a pro-atropisomeric conformation that only allowed the bicycle to close in one way. We're not the first to use point chirality to relay to atrop-control, but to our knowledge this is the first control of such a "right-side-in vs. inside-out" molecular shape in a natural product.
  • We made the molecule, and everyone was happy. Well, I would be happy, except I haven't gotten my fricking molecule cake. Phil, I know you're reading this. Everyone who finishes a molecule gets a cake. Where's my cake? Cake please.
  • Given the interesting structural nuances of this story, we desperately attempted to get solid-state structures of the respective isomers. I launched a collaboration with Hosea Nelson @ UCLA to try to do MicroED, and also with James Nowick @ UCI to try high-throughput crystallography screening, but unfortunately, never got any structures. So, we're still reliant on NMR for all conclusions.
  • In parallel with finishing the synthesis, we were also lucky to tie in with the Clardy group who was further elucidating the biosynthesis of this molecule. That's an interesting story in itself, but not mine to tell -- the extremely short version is this molecule is a RiPP, not an NRPS as originally believed, and there is some very cool CYP machinery to close the necessary macrocycles. More to come on this side of the story, I hope.
Fundamentally, this is a synthesis paper that's not about synthesis -- it's about understanding molecular conformation and supramolecular isomerism, and about how we can control said isomerism in a flask. We hope it's an enjoyable read.

I want to close out by thanking the incredible team I was lucky to work with throughout this process, most notably Yang Gao, who is an all around killer chemist. Thanks also to Jon Clardy and his talented postdocs Allison, Eric, and Emily for being a pleasure to collaborate with. And Phil, of course, is an amazing boss to work with. Even when he forgets to buy cake.

Y. Gao (left) and S.H. Reisberg. The Baran lab student subset of the tryptorubin A team. 

                                                                            

*TANGENTIAL SIDEBAR #1: "Why the heck do we still do total synthesis in the modern era?"
I want to start this sidebar with the statement that opinions here (and throughout this blog post) are strictly my own and may not represent Phil or the lab's overall perspective. For Phil's opinion on the utility of total synthesis in a modern context, it's worth reading his recent perspective.

With that note, here is my hot take on total synthesis in the abstract. My viewpoint is that the goal of a total synthesis must be considered with intentionality, and that the total synthesis, once completed, should be scored against that intentionally-designed goal. In my view, any of the following are perfectly reasonable drivers of a synthesis:


  • Scalable access to material is needed
  • SAR of unnatural analogs needs to be probed
  • The robustness of a method for a key step is being tested, and the synthetic target is simply an arbitrary vehicle on which to test a new methodology
  • Drivers for new methods are desired, and the total synthesis is being pursued to illuminate "which bonds are difficult to make with existing methods" with the explicit plan of spinning out new methods projects
  • Blind faith that with structural complexity comes chemical insight, and that some serendipitous discovery occurs
  • Sheer aesthetic work (directly analogous to a concert violinist's profession; creating beauty for the sake of creating beauty)
I feel that synthesis done under any of these umbrellas is useful. The problems start to arise when the declared goal is not actually enabled in the synthesis. It drives me up a wall when people introduce synthesis as 'a tool to make scalable amounts of material' and then make <1 mg. Because, at that point, you're not actually progressing solutions to the problem you're trying to solve. Likewise, if someone has a declared goal of exploring SAR, and subsequently make a single analog, it amounts to snakeoil salesmanship.  These kinds of farces are where the field bears risk for stagnation.

So, I encourage everyone considering a synthesis to spend some deep introspection asking, "Why should I make this molecule," and subsequently throughout the project enforcing that all strategic decisions are well-aligned with the answer to that 'why.


                                                                            
**TANGENTIAL SIDEBAR #2: "What's in a name?"

One issue that is worth discussing is the complexity around naming the isomeric relationship between 1a and 1b. The easy layman's explanation that 'one is inside-out relative to the other' is somewhat difficult to translate into formal language. Originally (i.e., before the peer review process), we referred to these compounds as topoisomers. If you poke around for the formal definition of topoisomer, the two definitions that pop up are "two compounds with identical connectivity but non-identical molecular graphs" and "two compounds with identical connectivity and point chirality, but that can only be interconverted via scission and reformation of bonds." Now, according to the former definition, 1a and 1b are definitely not topoisomers. According to the latter definition, things begin to get a little hairy, because if one ignores the physical limitations of bond lengths, 1a and 1b can interconvert (see Figure 4), but it's a very unphysical transformation.

However, a very thoughtful reviewer (thanks!) was kind enough to point out that physicality should be ignored in the topoisomer definition, and thus we can't think of 1a and 1b as topoisomers. For this reason, they should actually be called atropisomers. This risks swinging too far in the other direction, though: 1a and 1b's isomerism ('inside out') is categorically different from a prototypical atropisomer (e.g., binaphthyl) -- tryptorubin's isomerism has way more going on than simple sigma bond torsion.

To give a sense of just how confusing all this nomenclature can be, we decided to run a small social experiment. Phil tweeted a poll about lasso peptide isomerism:

Now, before we view the results of this poll, note that lasso peptides are just like tryptorubin: They are formally topologically trivial, but have a very defined set of shape-based isomers. 

Go to twitter to read some of the fascinating commentary, but the short version is this: Even Twitter experts (people with the doctoral degree TweeHD?) have diverse and contradictory views on what this isomerism should be called. 

Combining all of this, we coined the term 'non-canonical atropisomerism' to encapsulate tryptorubin's (and, for that matter, lasso peptides') shape-based isomerism. For me, the upshot of the entire nomenclature story is a classic 'a rose by any other name would smell as sweet' -- what we call the isomerism basically doesn't matter; the important part is that it's communicated clearly, and hopefully the manuscript (and especially structural graphics) make everything comprehensible.

Monday, September 9, 2019

Decarboxylative etherification

Our most recent work in electrochemistry was published today: A method to make hindered ether compounds from abundant carboxylic acids. Given the extensive information in the >400 pages of SI, we are not going through the details of the work in this blog post, but instead decided to present some details from behind the scenes.

As a project that required massive work, you might be wondering how it got started. Actually, Phil always got the consulting question that how to construct hindered ether bonds effectively from the pharmaceutical industry because they are quite important in drug discovery. It surprised us that in the 21st century, people still rely on acid promoted hydroalkoxylation to make such bonds, which often fails in the real-world cases due to a lack of chemoselectivity and sluggish reactivity. Being aware of this, we decided to tackle this problem.

By digging into the literature, we found an interesting reaction called the Hofer-Moest reaction which has a >100-year old history. This reaction enables generation of carbocations that can be intercepted by solvent quantities of alcohol to form the ether products. Without even understanding the details in the original paper published in 1902 (written in German), we started our journey towards the hindered ether synthesis. Carefully studying the follow up literature, we found this reaction is extremely limited in terms of the scope of both carboxylic acids and alcohols. In order to develop a practical method that can be used by the industry, we thought the key was to find a suitable solvent and electrolyte that enabled us avoiding use solvent amount of alcohol. After extensive evaluation (>1000 experiments), we identified a new set of conditions for this old chemistry. To make a long story short, here is an example of just how simplifying this chemistry is, in the context of synthesis of biologically active intermediates. Using compound 1 which is an intermediate of aurora kinase modulator as an example, previously, a multi-step route was employed requiring over 6 days of reaction time (<4% overall yield), wherein the key C–O bond-forming reaction—the treatment of methylenecyclobutane with BF3•Et2O—provides the ether in only 11% yield. Now with our electrochemical method, it can be accessed in only 2 steps, 51% yield and most impressively 15h.



While we think the step-count and time savings largely speak for themselves, our colleagues who are industrial process chemists are much more rigorous about efficiency characterization.






Process chemists tend to use “PMI,” or Process Mass Intensity, as a quantitative measure of a synthetic route’s efficiency. Our friends at BMS have conveniently developed an app, the “PMI Prediction Calculator” (Click here to play with the app yourself), that enables people quickly to know the efficiency and PMI of a certain process. We used this app to calculate the PMI of the old vs. new route to 1. The result: Our route to synthesis of compound 1 has a PMI of 252, versus 1304 for the previous one. This result clearly demonstrates the high efficiency, robustness and greenness of our new method.

Ideality is another way to quantify the parameters of efficiency for a synthetic sequence. We calculated the ideality% for the 12 real-word applications based on the definition published a few years ago (this paper). Amazingly, for 9 examples, the current methods have an ideality of 100% which are much higher than the reported routes.





Lastly, it is a tradition for our lab to showcase the limitations of the methodology that we have developed, to give the readers a better understanding of the reaction. Some of the limitations for the decarboxylative etherification and hydroxylation are shown below. In general, the secondary acids without any stabilizing effect such as benzyl or hetero atom will be problematic for this etherification reaction, due to the relatively instability of the corresponding carbocations. We hope this may be helpful for those who decide to try this reaction on similar substrates.



Let us know if you have any questions or comments regarding the work! Thanks for reading!

Ming, Jinbao and the etherification team

Tuesday, July 9, 2019

Cyclofeign?

We came across an interesting compound in Natural Product Reports Hot off the Press today:

 This rather precarious-looking [3.2.2] bicyclic compound was originally isolated from Bacillus sp., in a 2015 report, and the same compound was re-isolated separately from Melocanna baccifera in a new 2018 publication. Curious about this unique structure, we did what any good chemist would do, and attempted to build a model of it. After breaking some plastic we quickly realized that this compound, if it existed, would have to adopt a bent-arene conformation wherein the pi-system of the arene is bent out of conjugation (see haouamine for an example of a real cyclophane that exhibits this behavior).
 Seeming to contradict this unconjugated structure were the reported 13C NMR shifts (published earlier by Zeng et al), which were within the expected range of an unstrained para-substituted phenol. Additionally, the reported HMBC data reported by Zeng and coworkers lacked any correlations spanning across the supposed ethereal oxygen (e.g., between C-5 and H-3), which would have provided key evidence for the bicyclic structure. The reported mass [M+H] = 121.0645 is consistent with an [M+H] ion of the disclosed structure; however, it would also be consistent with an [M - H2O] ion from 4-hydroxyphenethyl alcohol…

Fortunately, we had a bottle of the latter compound hanging around the lab. A quick NMR confirmed our suspicions – the proton and carbon chemical shift data (see below) for this primary alcohol are exactly the same as those reported by the authors for the "bicyclic" compound. Furthermore, after shooting our commercial material on the LCMS we observed a major ion peak at 121.1 ([M - H2O]), with none of the 139.0754 one would expect for M+H of 4-hydroxyphenethyl alcohol. (EDIT: A kind reader pointed out to us that the much larger peak observed at 107.1 corresponds to [M - MeOH], via C-C bond homolysis to give the benzylic radical cation)
Further, a careful observer of the MS data could have known this off-the-bat: In the 2018 report, the authors ionize the compound on negative mode, and see a large peak at 137.06, corresponding to [M-H] of the (correctly assigned) compound.
Here's our data for commercial 4-hydroxyphenethyl alcohol:



We thus reassign the proposed structure to the much simpler 4-hydroxyphenethyl alcohol.
Our conclusion? If something looks really unusual, check all the reasonable hypotheses before you propose something exotic. Sometimes, things are zebras, but if you hear hoofbeats...it's probably a horse.

– Kyle M. and Sol

Monday, April 1, 2019

You heard it here first

La Jolla, California -- Sources close to Professor Phil "Strongman" Baran inform us that he his quietly announced his surprise early retirement. Says Baran, "I'm really tired of organic chemistry, and want to move forward in pursuing my passion project -- artisanal organic free-range grass-fed bodybuilding supplements. Given the immense responsibility of running an ethically-sourced supplement company, I just can't juggle being a PI any more."

Analysts at BBT (Baran Bodybuilding Technologies) announce their excitement about the newly-developed supplements: "We've developed a secret formula that maximizes development of so-called vanity muscles (pecs, biceps, and abs), while minimizing any undesired hypertrophy of annoying functional muscles (e.g., quads). Finally practitioners can achieve that 'ripped-torso, chicken-legs' look that everyone strives for."

Baran doesn't have an estimate of when his product will go to market, saying, "I've got to graduate all these pesky PhD candidates in my lab first. Then, the real work can begin."

You heard it here first, folks.



Happy April 1.

Thursday, February 21, 2019

E-Birch

Our work on electrochemical Birch reduction was published today. In case you don't want to read the manuscript, here's the Cliff notes:

  • Pfizer approached us, saying traditional Birch is bad news for med chemists (alkali metals are spooky; condensing ammonia is gross; the whole thing is a lot of effort). They asked us to make a better Birch.
  • We developed some conditions to effect the Birch transformation with electrochem. Basically, the conditions are mix substrate, LiBr, electricity, DMU, a key phosphoramide additive (TPPA), and THF. No ammonia, no alkali metals, no cryogenics.
  • Intuitively, one might naively assume that what's going on here is that the LiBr is reduced to Li(0), and from there the reaction goes through a standard chemical Birch mechanism. We assumed that, naively (at first).
  • Turns out, that mechanism is not at all the case, as demonstrated by much experimental and computational mechanistic study. Instead, the substrate is reduced directly on the electrode. Further, Li(0) is never generated at all. Safe stuff. Cool beans.
  • We went back to Pfizer with this. They seemed pretty happy.
  • Oh, also the reaction works in flow, on >100 g scale.
  • Oh, also, the key phosphoramide additive, that makes everything work, came from the Li-ion battery literature.
A huge shout-out has to go to our collaborators: Shelley Minteer and her group (led on the student side by postdoc David) for electroanalytical work, Matt Neurock and his group (led on the student side by grad student Sagar) for computational work, and our longtime buddies at Asymchem (led by Longrui) for scaleup and flow work. We should also note that this whole collaboration is driven by the CCI electrochemistry consortium, which has been a tremendous way to put people from various subfields of electrochemistry in touch with one another.

Anyway, normally, this is the part of the blog where we'd tell some funny anecdotes about the project. Unfortunately, the whole team is way too excited to get back in the hood for subsequent projects, so we didn't write anything. In lieu of anecdotes, please accept a few vaguely-electrochemical-Birch-related poems:

Limerick:

We set out to Birch some arenes;
to make a scary reaction more clean.
It turned out lack of lithium
gave us complete freedom
to safely Birch on our Electrosyeeeens

Haiku:

Birch in Winter frost
with Electrochemistry
No lithium, good.

AABBCC:

Poor Arthur Birch
did much research.
He was always sad,
for his reaction was bad.
Now we made it better
but alas, he is deader.

Oh, also we made a video, objectively comparing the time it takes to run a classical, chemical Birch, to the new electrochemical protocol. We hope you enjoy it:



- Electrochemical Birch team




Monday, January 14, 2019

Technique primer: Test tube columns

As many of you remember, a little over a year ago Phil did a AMA on reddit. On that AMA, a bunch of people requested that we do a post on how to build/run disposable test tube columns:

Well, writing up this blog post somehow slipped through the cracks for a year (Sorry!!), but without further delay, here's how we do the technique.

Step 1: Building the glassware. 

We use these columns on crude reaction mixtures from ~10 to ~500 mg scale. For smaller scale than this, we use prep TLC; larger, just a standard reusable column.

To build the column, for 10-50 mg, we just use a Pasteur pipette as the column. For 50-200 mg, we build it out of a 16 X 150mm tube. For 200-500 mg, we build it out of a 25 X 150 mm tube.

For the latter two, the first step is to pull a bottom spout from the test tube. Basically, you heat the tube with a Benzomatic torch, until you see the bottom glow orange and "wilt" slightly. At this point, you grab the glass with a pair of tweezers and steadily pull to make a long stem. It's hard to explain, but easy to demonstrate:

It's a bit tricky the first time, but gets simple with practice.

After letting the tube cool to rt, snapping off the bulk of the stem gets you a nice looking column:

(If you're using a Pasteur pipette, you get to skip to this stage)

Step 2: Building/running the column.

From here, it's just a matter of packing a small chunk of Kimwipe into the bottom, and tamping it down, followed by adding a bit of sand, and then silica gel. With modern silica gel, we have found that even <3" column height is still plenty to do reasonable separations. After equilibrating with a column volume or two of solvent, here's what it looks like, ready to load and use as usual:


One nice thing about the column sizes is that the 16mm tube (loosely) accommodates a 14/20 inlet adapter, while the 25mm tube (loosely) takes a 24/40 adapter. This way, you can run it with positive air pressure exactly as you would any other flash column. This is a 16mm tube, and as you can see, the small inlet adapter fits it nicely:


When the column is finished, here at Scripps, our EH&S allows us to dispose of the whole thing, tube and all, into our silica waste stream. Check with your EH&S for what to do at your institution.

Regardless, the best part of this technique is there are no dishes to wash!

Hope that this helps aspiring chromatographers,

Baran Lab


Sunday, January 13, 2019

11-Step Total Synthesis of Teleocidins B-1–B-4

Our paper describing the total synthesis of Teleocidins B-1–B-4 is now published in JACS! I started working on this project in November 2017 and completed the synthesis in June 2018. I then spent another 6 months optimizing the route to increase the yield and decrease the step count.


In the biosynthesis of these molecules the indolactam core is transformed into Teleocidin by a Friedel-Crafts type reaction with a terpene fragment. However, nature’s synthetic strategy provides a mixture of Teleocidins B-1-B-4 which is something we wanted to avoid. When I started to evaluate our own synthetic strategy, I immediately identified the introduction of the two quaternary carbons to the indolactam core in a stereocontrolled fashion as the major challenge (even one sterically hindered quaternary carbon can be difficult!). I knew we would have to establish the relative stereochemistry independently because the terpene fragment and amino acid moieties are quite distal. 


I also realized we needed to install a functional handle on the indole moiety in a highly regioselective fashion to facilitate coupling with the terpene fragment.With a bit of luck (and a lot of hard work) we were able to overcome these challenges and you can find the details in the paper/SI. I wanted to use this blog post to provide further insight into some of the key reactions which led us to success.

In the beginning, we had to fight against nature…


The first step in our synthesis is an electrochemical amination between our indole scaffold and valine. After optimization, I tried to scale up the reaction using some conventional large glassware which I bought from our local Japanese supermarket. However, almost no desired product was observed and even worse, I lost 6 grams of starting material along with the nickel, ligand and my time. I was so shocked that I couldn't even find my tongue! Phil suggested to try again but this time using the ElectraSyn carousel. The switch in setup facilitated reactivity and with a little optimization the desired product was formed in 51% yield.


I also want to highlight one of the unexpected problems we faced in this synthesis which is not described in the paper. While the desired indolactam is commercially available, it costs $274 per mg (Sigma) so it was a bit outside of our budget! We first tried to make the 9-membered ring by hydrolysis of the valine methyl ester followed by condensation using HATU, but the desired product was isolated in just 19% yield. However, after plenty of screening we found that the use of LDA was crucial for this reaction and we managed to obtain 2.1 grams of the protected indolactam! That was a really great day in the lab.


In April 2018 I was joined by Kosuke, a very talented visiting student from Osaka (Japan), who really helped me to optimize the route to what is now shown in the paper. Yuzuru, an amazing graduate student, also helped me by optimizing a key reaction and preparing a late-stage intermediate. I want to thank the team and especially Phil who was always ready with a lot of suggestions. I can definitely say he never compromises in the quest for an ideal total synthesis. 

Hugh and Teleocidin team