Teaching an Old DA New Tricks

Javier first introduced me to this project in December 2015.  Around this time I had just finished published my methodology project and had been working on a total synthesis of (±)-sunken costataxine.  It didn't hurt that the new project was funded by MedImmune, which meant I wouldn't have to grade lab reports/exams!

The idea was simple:

  1. Antibody–drug conjugates are mostly made through a thio-Michael addition between a thiol and maleimide
  2. The thiol–maleimide bond are reversible (like velcro)
  3. The same maleimides could react with diene through a strong Diels–Alder adduct (like superglue)
My thiol-maleimide = velcro metaphor falls apart because these shoes look awesome. CC Image courtesy of ashleigh290 on Flickr

My thiol-maleimide = velcro metaphor falls apart because these shoes look awesome.
CC Image courtesy of ashleigh290 on Flickr

Maleimides attached to powerful drugs are common (I count at least 10 in the pipeline).  They're synthesized on large scale, and there's more on the way.  Many groups are making maleimides with new combinations of toxic drugs, solubilizing PEGs, cleavable linkers, and self-immolative spacers.
This part of project reminded me of the proverb, "If all you have is a hammer [thiol], everything looks like a nail [maleimide]".  Now we have many maleimides so:

If you have a lot of nails, you should make a really good hammer
— Andre

Enter the Diels–Alder reaction!

I know what you're thinking: "The Diels–Alder reaction has been around for 90 years, surely there's nothing new you can do with it."

That's a good point, and it's one reason why this project was hard to sell.  The recent literature is full of useful examples of the inverse electron-demand Diels–Alder reaction (I consider this to be New Coke), but we want to bring back the normal electron-demand Diels–Alder reaction (Coca-Cola Classic).  The main concerns were that good dienes aren't stable and that the rates wouldn't be fast enough.

Maleimide is an amazing dienophile and we're not the only ones working on this:
Dr. Molly Shoichet used the Diels–Alder reaction between furan and maleimide to make hydrogels or conjugates.  This is very cool work but it doesn't quite meet our requirements - the rates aren't known and the conversions needed to form gels/conjugates don't need to be quantitative.
Dr. Anna Grandas Sagarra's group has a lot of cool papers, but my favourite is the reaction between a trans-3,5-hexadiene + maleimide to produce conjugates.  The reaction is in water, which accelerates the reaction, and the yield is 90—95%!  The kinetics aren't known and they use concentrations too high for antibody chemistry (1 mM vs 30 umol).

Turbodiene CC Image courtesy of  j4p4n  on openclipart and modified by me in ChemDraw

CC Image courtesy of j4p4n on openclipart and modified by me in ChemDraw

Emboldened by these examples we chose some turbo-charged dienes from the literature.  The dienes need to be stable in water, start from cheap starting materials, and easy to make (this makes it scalable/accessible to other labs - it's not laziness!)

After the literature search phase I ended up making 3 different cyclopentadienes, and all have a special place in my heart.  Mono-substituted cyclopentadiene tends to dimerize, it's kind of the wildcard of the group.  Spiroheptadiene was easy to make and has a rich history in total synthesis, so it's the brainy one.  Pentacyclopentadiene (Cp*H) smells like buttered popcorn, so it's the fun one.

I was also inspired by some very cool 3-alkoxyfurans developed by Dr. Tom Sheppard's lab at University College London (the old London, not London Ontario).  This super powerful furan is an excellent dienophile becau — OK YOU GOT ME, I just wanted to do some gold chemistry.  I share an office with Dr. Liming Zhang's students and they make it look so cool!  I was able to trade a drum of acetone for a few milligrams of gold and the long-ish synthesis was so satisfying.  Since it's the only furan in the series, it's the black sheep of the lot.

I'm not going to go over the details, but attaching these dienes to antibodies was easy and they reacted really well with maleimide!  The Diels–Alder bond is more stable than the thiol–maleimide, so I think we'll be seeing much more normal electron-demand Diels–Alder bioconjugations in the future.

This whole project has been a dream!  We had a bioconjugation problem and a clever solution.  I was given pretty much free rein on designing the dienes, which meant I read a lot of DA papers in bioconjugate chemistry, polymer chemistry, and regular old organic synthesis.  We shipped the good ones across the country to my fantastic collaborators at MedImmune, where they took care of all of the biochemistry stuff.

So many fields of chemistry use the DA reaction as molecular glue, hopefully our work makes it a bit easier.