Tuesday, August 11, 2015

Hey! We used C-H activation to make a natural product!

Once upon a time, we were interested making fumitremorgin A...

Phil's interest in verruculogen and fumitremorgin A has been long standing (see: Austamine and Okaramine), but strangely the endoperoxide-containing indole alkaloids haven't seen any attention despite the long history of syntheses of related natural products, including the (spiro)trypostatins, brevianamides, and other tryptophan-proline-diketopiperaizne alkaloids. Around '03 or '04, while I was toiling away in high school, a graduate student, Ben Hafensteiner, did a few reactions on the project before falling deep into the hole of a related family, the stephacidins. So the fumitremorgin project lay dormant for quite some time, before I took it over in 2011 (or was it '12? I don't even remember).

So in this post, I mainly want to talk about weird stuff, or stuff that didn't work; things not in the paper. Some are elaborations that were included in my thesis. I'm also much more likely to talk about things I already have schemes for. Its my blog post. I do want I want. 

As I wound down my work on a previous project, I was looking around for something to work on for my last years of graduate school... after a few forays into bad ideas, including a comically convoluted, terrible, imidazolidine synthesis and a proposal to work on cubane, Phil suggested taking a look at fumitremorgin A. From the start, this molecule wasn't love at first sight. It was nothing like any of the molecules I was attracted to:  typically compact, caged, multi-stereocentered, terpenes (e.g. vinigrol or maoecrystal). I chose to work on it anyway. 

Although it was not quite to my tastes, it was intriguing: the peroxide was neat. There's a medium sized ring; and the peroxyhemiaminal-thing is funky. Hmm... late stage oxidation probably won't work in the presence of the indole.  Oh... you can't buy 6-methoxytryptophan? That complicates things too. So I agreed to work on it. I estimated we'd be done in about a year. No big deal, this class of molecules had seen a lot of work.

I started my journey with Will Gutekunst, a fourth year graduate student who worked on a multitude of successful projects. We worked on a disconnection for the molecule that looked approximately like this:

Unfortunately, that retrosynthetic disconnection failed completely. We were never able to realize this transformation from compound A to the pentacycle without over-oxidation of the molecule. What that looks like in practice is a whole lot of reactions that were not characterizable by NMR (so called "nonspecific decomposition"), as well as the following scheme:

I thought this next oxidation was kind of neat. I once made these two isolable rotomers, which were separable by PTLC and could be characterized fully by NMR. The fact that they convert during heating, but not with base, was suggestive of rotomers. I never really fully explored this, since I made only a few mg of material. Oh well, I still think its cool. 

Eventually, Will moved on to brighter pastures, and I turned my attention to one of the bigger problems, the endoperoxide. From the get-go, we had though late stage oxidation would be an issue -- and the work Will and I conducted made this seem likely. We assumed that the peroxide could be installed early from O2 and a Mukaiyama oxidation, or perhaps through the addition of peroxide into an aldehyde. Ultimately, we found that the Mukaiyama peroxidation of prenyl aldehyde diethyl acetal was a high yielding, and reproducible method for forming a "prenyl peroxide." We did explore other conditions for this transformation involving a variety of metals (notably Co, Mn, and Fe), as well as the use of H2O2  with (and without) acid. Ultimately, the Mukaiyama reaction, one of the first we looked at for peroxide installation, worked well. During this time, the blast shield was kept on-hand, along with peroxide test strips and a lot of reducing agent. There were never any issues working with peroxides in our hands, but as always, take care, don't concentrate to dryness, and know what you're doing, please.

Here's a scheme I'm self plagiarizing from my thesis (deal with it). The first reaction was one of Mukayiama's own. Then we slowly worked our way to the final reaction (to make 4.37). In the paper, you'll notice we had to swap the TES- for a TBDPS-group. Its worth mentioning that you can put other silanes into this peroxidation (like TBSi-H) and achieve the same reaction. However, bulkier silanes (TIPSi-H and TBDPSi-H) failed to produce peroxide in our hands... hence the protecting group swap. The TBDPS-peroxide functioned well in the Pictet-Spengler reaction as well as the PhNO/ZrCl4 mediated unsaturation, so we needed it. Protecting groups, yay!

At some point in the aforementioned ramblings, was joined on the project by Feng Yu and Jochen Zoller, a postdoc and visiting graduate student, respectively. Feng was tasked with something I had barely touched: a better 6-methoxytrpytophan synthesis. Until this point in time, I had only been using known methods to form my methoxytryptophan, and while each of these worked, they were time consuming syntheses in their own right (see scheme below for synthetic detail). So Feng really dug into the literature to come up with something better. After a while, he came up with a proposal for a C-H oxidation methodology that, in the end, looked a lot like what was published in our paper. I tip my hat to Feng for the dedication to stick with this method, because I was busy playing with the peroxide while Feng was taking multiple steps off the synthesis and developing the method fully! I've got a decent comparison of Feng's method to to other known literature methods (below), but you should check the paper for the full method details. He'll likely chime in in the comments if anyone has any questions. BTW, 47% yield to methoxytryptophan more than doubles the literature standard (~20%)!

While Feng was busy with methoxytryptophan, Jochen and I were working out the final sequence of our synthesis of demethoxyverruculogen (4.18 below). I'll let the following scheme speak for itself, but suffice it to say, we tried almost ever possible iteration to achieve a sequence that worked.
The reason for a single (or close to it) diastereomer of the 8-membered endoperoxide forming remains unknown. I played with a lot of models and drew a lot of schemes to try and rationalize it. Here are some ideas. Maybe you have a better one?

With that final result (demethoxyverruculogen) in hand, Feng, Jochen, and I pushed the last remaining bit of material (methoxytryptophan) forward to complete the synthesis of verruculogen. The NMR was completely terrible (think: 600 MHz cryoprobe, proton NMR, over the weekend, impure), even after purification by PTLC the size of a TLC plate (2 cm x 4 cm). Actually, the only way we were able to even find the natural product on TLC, NMR, or LC/MS is because I previously had grown the fungus in our lab for natural product isolation and stability studies. That's right, the Baran Lab does "biology." Sort of. Many thanks to Van Vu of the Marletta Lab and Erik Wold of the Felding Lab for helping me successfully total biologize.

This was last summer! Then I graduated and peaced out of San Diego.

Since that time, Leah Simke and Shigenobu Umemiya joined the project and Feng Yu has remained working on it. They optimized the entire sequence and tweaked the final steps to make them work better (switch reagents and order). In addition, fumitremorgin A was synthesized (verruculogen+prenyl group, see the first scheme).

What I mean to say is, a lot of people deserve credit for work done here. I mean to thank them. Finally, I would like to thank coffee, without you I would have probably slept more.

Monday, August 3, 2015

The Baran Lab as Mangos

Top (from left to right): Matt, Dr. Oberg, Phil, Evan Horn
Bottom (from left to right): Julian, Jacob, Rohan, Ming

By Hang Chu, (one day) Ph.D.

Saturday, August 1, 2015

Introducing....The Baran Lab Snack Room!!!

That's right...we now have a new snack room in the Baran Lab. Phil took us on a Costco shopping spree to get all of our favorite goodies. And yes, he does make us wear diapers when we are running big columns!

So far, the favorite items the barnowls are protein bars and shakes, beef jerky, and oreo cookies. Shouldn't every PI do this? I think so. I have noticed an uptick in productivity especially when the cafeteria is closed and there is nowhere to get a quick bite. 

And yes, Phil money tastes VERY GOOD!!!!!

Friday, June 19, 2015

A Baran lab cycle of discovery: hydromethylation of unactivated olefins

If the Baran lab cycle of discovery was a baseball field, total synthesis is like the home plate where much of the action takes place. Just like a baseball player trying to score a run, a graduate student who joins the lab has to go through the first base of making a natural product, the second base of designing new strategies, the third base of finding new methodologies and back to home plate to complete the discovery cycle.

In my last project, I barely made it to the first base with the synthesis of some steroid skeletons. When I was about to continue “the cycle” with some ideas for a further development, Phil told me not to, and convinced me to start a seemingly non-related project, a method for an overall addition of a methane molecule to unactivated olefins.

How did we start?

  During the preparation for the manuscript of the last project, I went to Phil to get his opinion on how to describe the figure (below) more convincingly.

   Me: Since I did not make the methyl in the C,D-ring junction, how should I describe it?
   Phil: (Looks at the figure in 3 seconds)…why don’t you make it. 
   Me: uhh???(Thinking about his “irrelevant suggestion”!!! Com’on Phil!I just want to PUBLISH it).
  Phil: You can make an olefin in the D ring using Hans’ method, and add a methyl group to it…maybe useing our reductive iron coupling.
   Thinking about Phil’s suggestion more carefully, I actually liked it because it would fulfill my dream of making five different classes of steroids from the same intermediate, thus completing the idea of making a “steroid pyramid” using the idea of two-phase synthesis. So I decided to give it a try.  I quickly observed some C-C bond formation with N,N-diphenylhydrazones and O-benzyloximes while trying to wrap up the previous project.

Quentin made the first tosyl hydrazone in the lab based on studies reported by Kim and coworkers. However, after two weeks of investigation, we only got some undesired rearrangements but not C-C bond forming product. Based on the previous result with other hydrazones and oximes, I had a strong belief that the C-C bond between the olefin and tosyl hydrazones should also work. In addition, a quick search showed the feasibility of reductive C-N bond cleavage under basic conditions. Putting these things together, a two-step sequence could be envisioned. It took me a few days to prove that the C-C bond formation with a tosyl hydrazone was indeed feasible; however it took me more than a month to achieve the first C-C bond adduct with formaldehyde tosyl hydrazone, The rate-determining steps in this process were finding the conditions for in situ formation of the hydrazone and determination and suppression of the byproducts. Importantly, the C-N bond cleavage was also obtained just by heating the reaction mixture after C-C bond formation to 60 oC in methanol. Chao then joined the project, and in only a week he quickly modified conditions to a standard two-portion conditions to solve some reproducibility issues as well as improve the yield.
   During a discussion, Phil mentioned that we should call our methodology “a molecular editing method”. So two criteria for substrate scope were to have substrates with a “real name” and substrates with complexity. The good thing about the formal criterion is the diversity of the repertoire of commercially available compounds, as we basically just went through Sigma-Aldrich and TCI catalogs and ordered substrates with olefins. Commercially available terpenes such as carvone oxide, limonene oxide, dihydrocarvone, carene, rose oxide, dihydromyrcenol… all worked under our conditions but we could not separate the desired hydromethylated product from the hydrogenated byproduct; this problem is by far the biggest issue of the current work

While most of the work was completed at TSRI, in the other side of the country, Brad Maxwell at BMS tried to apply our method in radiochemistry. I told Brad that it would be my dream if he could make it because I don’t think I will have a future occasion to have radioactivity data in a publication that I co-author, and he did it amazingly with a complex terpene called rotenone using a very low concentration (ca. 1.5%) of [14C]formaldehyde. Here is what Brad taught us about the origin of 14C:

Typically, 14C is not naturally in enriched form since it is not abundant enough to make the process feasible. Instead, 14C is produced by irradiating solid beryllium, aluminum nitride or saturated ammonium nitrate solution with neutrons for 1–3 years in a 14N (n,p)14C reaction within a nuclear reactor. Longer irradiation times produce higher specific activity of 14C. In the process, the 14N is converted to 14C. After the irradiation, the material is dissolved in H2SO4 and the effluent gases are oxidized by an appropriate catalyst.  The resulting  [14C]CO2(g) is then passed through NaOH(aq) and it is eventually precipitated as Ba[14C]CO3 from which all 14C-labeled products are prepared. The only current producers of 14C are located in Russia.” 
   Looking back on what we had made in term of (radio)labeled compounds with the current method, Phil might be right when he said: “Since there is no other way to do this sort of methyl editing, even if it is a long reaction time and a complex reaction system, people still have to use it.”
   Like a baseball player who undergoes the process of bating (hitting the ball/failing/trying to hit again), running to the next base, getting tagged out by the opponent and repeating the process again until scoring. Total synthesis requires a lot of trial and error, with a lot of time spent in going back and changing the strategy. In this project, the most important moment might be the lessons that we learnt during the previous synthesis (of steroid skeletons), gave us hints to the discovery of hydromethylation of olefins. Personally, I enjoyed my experience in the lab; however, one thing I still could not fully capture is how our “coach” makes his decisions. Most of the time just in the blink of an eye, and sometimes it just comes from nowhere…but it actually works (at least in my two projects).