Toward the ideal reaction – Part 1

Having worked in the oligonucleotide area where one often wants to snap on a label such as a fluorophore in a site-specific way, I came to appreciate the need for the “ideal reaction;” things are hard enough in this area without messy labeling methods.

Here’s what an ideal conjugation reaction might look like:

  • One step; no protecting group manipulations.
  • Fast at room temperature.
  • Quantitative.
  • Specific.  No cross-reactivity at other sites, double labeling, etc.
  • No catalyst, no additive, no external stimulus.
  • No by-products hanging around that have to be removed.

In the context of bigger molecules such as polymers and biopolymers, one also has to consider compatibility with their chemistry and synthesis.  Let’s stick with oligonucleotides for the moment, though this discussion applies pretty readily to peptides.

In this and the next post, we’ll consider some methods that approach this ideal.

The traditional methods for labeling and bioconjugation are no fun.

As it stands, the classic approach to oligonucleotide modification is a challenge.  First, you synthesize an oligonucleotide that has a protected amine or thiol hanging off somewhere, namely from a nucleobase, the 3’-phosphate terminus, or the 5’-alcohol terminus.  You then deprotect it and treat the resultant amine or thiol with the desired labeling agent, e.g., an NHS ester (for amine labeling) or a maleimide or iodoacetamide (for thiol labeling).  Such reactions are quite imperfect, proceeding in modest yield and selectivity.  A tedious purification is then required to remove the desired labeled material from unreacted starting material, labeling by-products, and other oligonucleotides that somehow aren’t the right ones.  What a blast!

There has to be a better way…

The ideal reaction:  What fits the bill?  

An attractive approach is a spontaneous reaction between the label and a handle that you’ve installed synthetically (and site-specifically) on the oligonucleotide.  Let’s stick with the simplicity of addition reactions, recognizing that there are some other great approaches such as Bertozzi’s Staudinger ligation.

To start with, you would synthesize an oligonucleotide with a handle on it so you have a chance to zero in on that site and that site only.  Check.

Now we need a label that bears a moiety that will recognize the handle (and only the handle) and snap onto it quickly and without any help.  The term “spring-loaded” often gets used.

Sharpless’s concept of click chemistry readily comes to mind.  The de facto standard click reaction, virtually synonymous with the term “click,” is the copper-catalyzed Huisgen 1,3-dipolar cycloaddition between azides and alkynes, an addition reaction that leaves behind a robust triazole linkage.  This reaction has enabled some wonderful things in the world of biomolecule labeling and conjugation.  But still, there’s that copper.  Not quite ideal.  We’ll hear more about this from Jack Hodges in the next post.

Okay, what about metal-free click reactions?  There are a growing number of copper-free azide-alkyne cycloadditions, e.g., with strained cycloalkynes (Bertozzi and others); a great idea.  Again, more on that in the next post.

Inverse-Demand Diels-Alder reactions of tetrazines

Here’s another addition reaction that I like, which draws on the 50+ year-old inverse-demand Diels-Alder reaction between tetrazines and strained alkenes to give dihydrodiazines.  Okay, technically, this is an addition-elimination reaction that produces dinitrogen, but I think we can live with that as a by-product.  On the oligonucleotide front, Jäschke and co-workers  showed that norbornenes could be incorporated into the nucleic acid using normal phosphoramidite chemistry.  Subsequent reaction with various 1,2,4,5-tetrazines, e.g., the dansyl-bearing compound shown, occurred simply by mixing at room temperature in neutral aqueous solution; no catalyst, no other reagents, no help required.   (See also O’Reilly’s very recent example involving polymer functionalization.)

Things are starting to look rather ideal!

For those of you that need to modify biomolecules and polymers, what’s your favorite approach to the ideal reaction?


Hooked on heterocycles

I have a question: How did you get hooked on heterocycles?

I’ll go first:

A failed exam, cocaine, and a crusty chemist:  How I came to love heterocyclic chemistry.

I became an azaphile during my undergraduate days at UNC-Chapel Hill, a process that involved failing a final exam, becoming enraptured by the chemistry of lysergic acid, morphine, and cocaine, then being turned down for undergraduate research.  Well, initially at least.

At some point, anything with a nitrogen atom in it became fascinating, especially if the nitrogen was in a ring.  Everything else started to look… bare.

How did this happen?  I can trace it back to Professor Robert L. McKee, a “heterocyclic chemist’s heterocyclic chemist” at UNC.  He’s no longer with us, but he’s worth a tribute, as you’ll see.

You know those dreams where you’re not prepared for the final exam?

I had Professor McKee for two classes, one that proved traumatic, the other enlightening, both being important to my baptism into heterocyclic chemistry.

First, the trauma.

I walked into the final exam for McKee’s second semester organic chemistry class with a strong A average and a lot of confidence.  I loved the class and had studied hard for the final.  But my confidence quickly faltered as I found out that he had made two final exams, one for those with an A average and one for everyone else.  Huh?

I sat down and looked at it, and there were all these crazy rings with nitrogen atoms sprinkled all over them, none of which I’d never seen.  And the questions were unintelligible.

I walked to the front of the room to ask what was going on, and Professor McKee told me that the “A” students were supposed to have read Chapter 10 on Nucleic Acids on their own, for the exam, which would be entirely on that topic.  I had somehow missed this announcement, despite attending every single lecture.

I sat back down and tried, but I couldn’t answer a single question.  I got maybe ten points.

My “A” became a “B”, which McKee told me was generous.

I still get sweaty thinking about this.

Nonetheless, it exposed me to purines, pyrimidines, nucleosides, and nucleic acids, prime denizens of the world of heterocyclic chemistry.

Ironically, I would end up as VP of a company that specializes in nucleic acids chemistry some twenty-five years later.

I’d like to see that exam again.

Onward to enlightenment…


With my tail between my legs, I enrolled in UNC’s advanced organic chemistry course, taught by none other than my beloved Professor McKee.

Things went much better this time.

We used Noller’s Advanced Organic Chemistry, an old textbook even back then.  I loved it because it had plenty of history and was rather descriptive in its approach.  It was also heavily slanted towards heterocyclic chemistry.

What I remember most were McKee’s lectures on the chemistry of lysergic acid, morphine, and cocaine.  These were beautiful molecules and came with such rich chemistry.  I can’t say I remember much else from the class, but that was enough; I was hooked on heterocycles.

It felt empowering to know something about the drugs that were making the rounds back then.  Even more exciting was the realization that powerful physiologically-active substances can actually be synthesized.  From scratch!

Pearson/Mans synthesis of (+)-cocaine

I remember sitting on the bed in my dorm room at UNC with a ball-and-stick molecule of cocaine, thinking, “I wonder how I might make this?”  Not because I wanted to use it or sell it, mind you; just because it was beautiful.

Fast forward to 2004, when Douglas Mans, a talented graduate student in my research group at the University of Michigan, synthesized (+)-cocaine, the enantiomer of natural (-)-cocaine.  Full circle.

Do you really want to do undergraduate research with this guy?

Sometime during this period, my budding interest in heterocyclic chemistry led me to stop by McKee’s office.  It was attached to a one-man lab, and since he was getting near retirement, he no longer had graduate students.

I was struck by the dark, archaic lab crowded with hundreds of vials of crystals, each labeled with hand-drawn structures of unique heterocycles.  I could swear there was also a retort containing some bubbling liquid.  I wanted to get in there and make something!

A few things concerned me, though.

First, he smoked a pipe.  In the lab.  Not good.

Second, he tasted all of his compounds.  Even as a neophyte, I knew this couldn’t be a good thing, and I quickly made an association between this practice and the various growths I could see on his skin.  He was old-school though, and taste was part of compound characterization.

Despite my misgivings, I asked this kindly, crusty man if I could work for him.  His response was quick:  “If you want to do research and continue on to graduate school, you need to go work for someone other than me.”

He suggested Ernest Eliel, whose area was stereochemistry, a topic I also loved.  I joined Eliel’s lab, where I managed to get some heterocyclic chemistry in:  We published the first example of neighboring group participation involving a four-membered ring sulfur intermediate, a thietane.

And we used Barry Trost’s method for making the thietane intermediates, relevant in that I ended up getting my Ph.D. with Barry at Wisconsin, working on… things with lots of nitrogens!

So, how did you get hooked on heterocycles?