Tuesday, June 26, 2018

Radicals can DELiver for DEL

You may have noticed that our work on sp3-sp3C-C coupling for DNA encoded library (DEL) synthesis was deposited on Chemrxiv about two months ago. Recently, we found a nice home for this DEL-related work. I am not talking about the keyboard.
It’s out in PNAS today. While the paper offered a detailed account of mechanistic work by the Blackmond lab alongside an assortment of working substrates, I would like to share with you some behind-the-scene stories of this “non-traditional” project. 

In a nutshell, this project can be likened to our effort to “radicalize” DEL synthesis. It all started in 2017 when our long-time industrial collaborator, Pfizer, approached us for ideas on new reaction manifolds for DEL synthesis. We learned about the stringent requirements for DEL prior to putting together a substantive proposal: 
First, a "DEL-able" reaction requires, inter alia, reagents and conditions which are compatible with DNA. We had our in-house biology expert do a group meeting on DNA chemistry – a universal verdict from his presentation appears to be that, despite thousands of years of revolution, our DNA is chemically fragile – a vast majority of reagents under the sun (or even the sun itself) can cause troubles. Second, a DEL reaction requires a solvent system comprising >20% water. While life subsists on water, to most organic reactions, irrigation breeds irreversible damages: 
 Third, the reaction temperature must not exceed 90 oC and the reaction pH must stay within the comfort zone of 4-14. Most dauntingly, the reaction concentration must be below 1mM. 
After some deliberations, we decided that perhaps radical reactions are well suited for DEL – after all, radicals are generally moisture insensitive and chemoselective (over the years we have run such reactions in many unconventional solvents). Toward this end, our lab has a long-standing interest in the radical decarboxylative cross-coupling reactions of carboxylic acids using nickel catalysis. Methods have been developed to forge carbon-carbon and carbon-heteroatom bonds. More promisingly, these reactions were compatible with solid-phase peptide synthesis. To a chemist like me, all biomolecules are perhaps equal (thus, there is a likelihood our technology will translate from peptide to DNA). (I soon learned that some biomolecules were more equal than others…). Pfizer showed strong interest in this project as it could offer access to the much desired sp3-rich space, a promised-land in the realm of DEL chemistry. Thus, the project was initiated…   
Since I have given a group meeting on “Reactions in the Presence of Water” in June of 2016, I was perhaps considered a more “hydrophilic” individual than any other lab members. Thus, I was charged with this project. Maybe destiny brought me here.
It didn’t take me too long to settle on the decarboxylative Giese reaction as the focus of the study. We started to evaluate the feasibility on small molecules at first. I was soon baffled by a seemingly trivial problem: how do I determine product yields when my reaction is 1 mM in water? NMR analysis regularly gave >100% yields (who needs conservation of mass/energy, right?). In a desperate move, I even tried to isolate the products, once obtaining 11 mg of products out of hundreds of milliliters of solvents! 
Jason came to the rescue here -- he built up the ‘Automated Synthesis Facility’ here at Scripps in 2017 and brought the analysis up to speed. I can easily get 20 reactions analyzed within an hour. More importantly, no workup was needed at all. To cheer my spirit even further, I was joined by Helena Lundberg, a really talented postdoc from the Blackmond lab. She provided extensive mechanistic insights through her kinetic studies that also provide a general protocol for converting organic reactions into DEL-compatible counterparts. With her help, we were able to get >70% yield which set the stage for evaluating functional group tolerance as well as translating to on-DNA synthesis. 
Then I was joined by another two talented chemists, Shota and Pedro, both of whom hail from a “hydrophilic” locale (Japan and La Laguna, Spain). With their help, we were able to evaluate all sorts of functional groups (thanks to Pfizer team providing suggestions and carboxylic acids) – gratifyingly, most of them were tolerated under the reaction conditions.  

However, our happiness was “ephemeral”. Our mock-DEL conditions were found to be incompatible with DNA molecules. For 8 months, we begrudgingly awaited the advent of another superhero. This time round, it was Dr. Stephen Brown, a brilliant Pfizer scientist, who discovered that by diluting the reaction by yet another order of magnitude and using DMSO as a co-solvent, the reaction worked nicely on DNA, providing the product in 40% yield. Starting from this, I was able to optimize further and demonstrate this transformation with 22 on-DNA examples.
As a historically organic synthesis-based lab, we are not equipped with any instrumentation for DNA synthesis. Thuswe are indebted to the generosity of other TSRI labs (e.g., the Romesberg lab) and Pfizer (both La Jolla and Groton) for helping us with much of the analysis.  We are also grateful to LGC for a great deal on the DNA headpiece starting materials. Our creativity also evolved during the study. For reactions that require inert atmosphere, we put the Eppendorf into a test tube and exchanged with argon. Since a stir bar is usually not an option in DEL synthesis, we bound the Eppendorf to a rotavapor and let them rotate together (inspired by Lara). For reactions that require heating, we wrap the Eppendorf with parafilm and aluminum foil, bind with a sinker and put them together into an oil bath (mimic the role of thermocycler). Fortunately, Phil didn’t ask me to demonstrate a gram-scale synthesis this time.
Doing DNA chemistry in a synthetic lab can be rather painful, especially at the very beginning. We constantly change our substrate in order to better mimic DNA functionalities as well as obtain good LC separation [LC traces could be unbelievably messy when we were using super stoichiometric reagents (>100 eq is very common in DEL)]. Setup, analysis, purification… each step seems to pose problems. Moreover, on such small scales, it is challenging to diagnose problems, let alone solve them. During the dark times, I was jealous of my colleagues – as they are able to “concentrate” on their hydrophobic transformations while I constantly battled the consequences of “low concentration” and water. Nevertheless, ultimately, I find this experience greatly fulfilling and the final protocol was reproducible and reliable. It is always a satisfying learning experience when working with a team of dedicated and talented scientists. In this case, I particularly relished collaborating with a number of brilliant people withvarying areas of expertise. The science shown in this recent publication is the result of collaboration among Baran lab, Blackmond lab, ScrippAutomated Synthesis Facility, Pfizer La Jolla and Pfizer Groton. I would like to express my heart-felt gratitude to all of them!

Lastly, I hope our DNA chemistry would one day translate into an NDA of your research program… (Special thanks to Dr. Ming Yan for linguistic suggestions and contributing to the jokes here.)
Feel free to let us know if there are any questions you may have! Thanks for reading.


Jie

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