Happy Accident Leads To Faster Synthesis | Chemical & Engineering News
  • CORRECTION: This story was updated on Jan. 12, 2016, to correct the percent conversion for the oxime reaction after three freezing and thawing cycles.
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Web Date: January 8, 2016

Happy Accident Leads To Faster Synthesis

Protein Chemistry: Power outage reveals that oxime ligation speeds up below freezing
Department: Science & Technology
News Channels: Organic SCENE
Keywords: protein label, oxime, freezing, reaction rates
The reaction of a ketone or aldehyde with an aminooxy moiety produces an oxime bond. This reaction occurs more than two orders of magnitude faster at -20 ˚C than at 37 ˚C.
Credit: Bioconjugate Chem.
A reaction scheme for an oxime reaction, with arrows indicating that at -20 ˚C the reaction achieves 91% conversion, whereas at 37 ˚C, it achieves only 2%.
The reaction of a ketone or aldehyde with an aminooxy moiety produces an oxime bond. This reaction occurs more than two orders of magnitude faster at -20 ˚C than at 37 ˚C.
Credit: Bioconjugate Chem.

When a power outage hit the labs of biochemist Tilman M. Hackeng of the University of Maastricht, his colleague Stijn M. Agten rushed to remove some samples from their ultra-performance liquid chromatography-tandem mass spectrometry machine. Agten was monitoring the progress of an oxime ligation reaction, and to give himself time to reboot the machine, he froze his samples to -20 ˚C, expecting to slow the reaction rates and allow him to salvage something from his efforts.

But when the sample thawed, Agten was shocked to see that his reaction was almost complete (Bioconjugate Chem. 2015, DOI: 10.1021/acs.bioconjchem.5b00611). “Normal oxime ligations take days to get acceptable yields of partial completion,” Hackeng says.

In oxime ligations, an aldehyde or ketone reacts with an aminooxy group on another molecule to form an oxime bond. Hackeng and his colleagues, who are investigating oxime ligations as a way to label proteins, were testing how temperature, concentration, and catalysts affect the rate of oxime formation when they made their discovery.

The team tried to shorten the freezing time by using lower temperatures: -80 and -196 ˚C. But the hour-long freeze at -20 ˚C they uncovered by accident sped up the reaction most effectively. This is probably because slow-growing ice crystals push out the reactants and concentrate them in the liquid phase, Hackeng says.

They also discovered that by freezing, they could run the reaction without a catalyst at a neutral pH and still get better results than adding aniline as a catalyst and reacting at low pH, as is typically done for oxime ligations at warmer temperatures. They then tested the technique by labeling a model protein system. After three freezing and thawing cycles, the reaction was 91% complete—a stark improvement on the usual method, which left the reaction only 10% complete after 48 hours.

Hackeng says that the notion of accelerating a reaction by freezing is “completely counterintuitive,” though scouring the literature brought up a few other examples.

Meanwhile, the serendipitous finding has had great practical effect: “We use it every day,” he says.

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Jim Davis (January 11, 2016 11:25 AM)
I wonder, too, if the freezing out of the eliminated water onto forming ice crystals contributes in a Le Chaterlier fashion to driving the nascent equilibrium system to the right...
Tilman Hackeng (January 13, 2016 11:21 AM)
That's an interesting thought, although considering the concentration of water (55.6 M), reactants should reach staggering concentrations for the reaction to be influenced by relative lowering of concentrations of water...which even then would represent a low effect on reaction rate compared to the increase caused by increasing reactant concentrations...
john tamine (January 13, 2016 2:52 PM)
that actually makes a lot more sense than the author's suggestion that it is simply a result of increasing the concentration. were it simply a matter of concentration alone, one would assume (from the sketchy details provided here) that they would have explored the effect of concentration at RT and discovered the effect previously. perhaps they might try the reaction in anhydrous solvent with a dehydrating agent present, such as 4A molecular sieves?
Ming An (January 13, 2016 4:22 PM)
the reaction faster at lower temperature effect (not the water freeze out effect in increasing reactant concentration) has been shown in 2011 in a very similar context (hydrozone formation) in this paper by Prof. Susan Bane and colleagues:


Bioconjug Chem. 2011 Oct 19;22(10):1954-61. doi: 10.1021/bc2001566. Epub 2011 Oct 4.

4-aminophenylalanine as a biocompatible nucleophilic catalyst for hydrazone ligations at low temperature and neutral pH.

Blanden AR1, Mukherjee K, Dilek O, Loew M, Bane SL.

Shouldn't it be mentioned in this article and referenced in the original paper?
M. John Perkins (January 13, 2016 4:57 PM)
Back in the early 1960s, a great friend and contemporary, A.R.Butler, (now at St Andrews) told me he was off to Cornell to work with T.C. Bruice. The plan was to take a further look at the kinetics of some reactions he had researched in England for his Ph.D. He said he was going to study them in ice. I said, "They'll go faster". I believe they did!
Brad Powell (January 13, 2016 7:43 PM)
In addition, total entropy for water may be lower after ligation.
Bradford Powell (January 13, 2016 7:53 PM)
By the way, congratulations and beautiful example of serendipity from care and diligence. Great read.
A. Chandrasekaran (January 14, 2016 12:45 AM)
I also thought that it must be an equilibrium thing. If it is concentration related problem, then we should be able to do at room temperature itself with appropriately higher concentration. That would be the most efficient way to do than either high or low temperature.

Similar counter-intuitive example is lower critical solution temperature (LCST), such as in Poly(EthyleleGlycol)-water system.

However, such cases are so rare that it is good to be reminded as often as possible. So, it is very interesting. Even more interesting is the story of finding it!
Bruce Burton (January 14, 2016 3:08 PM)
I think Jim Davis has it right. In creating aldimines or ketimines I've seen room temperature reactions stall in the 60-70% range. The removal of water via molecular sieves increases the yields considerably. The ice formation is a way of doing the same thing.
George Pomonis (January 14, 2016 5:24 PM)
At these temperatures what is the physical conformation of the protein in one of the reactants? Could it be that at warmer temperatures the protein is steric hinderance to the reaction?
dan (January 18, 2016 11:43 PM)
Seems like C&EN is really reaching for content. Kinetic Vs thermodynamic control is older than my parents.

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