Chemical reactions rarely go from start to finish in a single step, as intermediates form during the transition between starting materials and final products. For example, when our bodies synthesize proteins, a new amino acid doesn’t just merge into a growing chain of amino acids. Instead, the formation of the bonds between amino acids in a protein (a peptide bond) involves a complex coordinated effort among different RNAs and enzymatic sites. If you’ve ever taken a course in organic or physical chemistry, you’ve seen firsthand that we expend as much effort on studying how a reaction occurs as we do on figuring out its eventual outcome.

Reactions aren't movies that you can freely pause or slow down to observe sequential steps, so chemists have employed a variety of clever techniques to work with the limitations. Scientists can radioactively label molecules to track reactions, or they can make it easier to monitor reaction progression by slowing down molecular motion through temperature reduction or placing chemicals in a kinetically impeding molecular capsule, just to name a few options. Building on past techniques, Makoto Fujita’s laboratory at the University of Tokyo combined X-ray crystallography with reactions trapped in cavities to directly capture structural data of reaction intermediates. The authors use this to create an X-ray snapshot of a rarely seen intermediate in this week's issue of Nature.

While crystals appear nonporous to the naked eye, crystalline networks have minute spaces that could confine chemicals and even act as a reaction flask for certain chemical transformations. Fujita’s group reasoned "the pores of a crystalline network would also provide kinetic stabilization and hence enable direct observation by X-ray crystallography of trapped intermediates, providing structural and mechanistic information." In other words, as a reaction progresses in a crystalline pore, an intermediate stage could be stabilized and observed before the reaction goes to completion to become the final product. (X-ray crystallography can determine the identity and arrangement of atoms in a compound, providing a picture of the chemical structure. )

To test their idea, the authors decided to study the formation of a Schiff base, a textbook reaction that is important for many biochemical processes. During the synthesis of a Schiff base (also known as an imine for the chemists out there), an unstable intermediate called a hemiaminal is formed, but only exists very briefly, making it difficult to examine.

Fujita’s group formed this intermediate in a zinc iodide based crystal by reacting the two starting materials, an amine and an aldehyde, within the crystalline pores. They first formed the crystal with an amine as a guest molecule that lies within the pores and then placed the crystal in the X-ray instrument. They cooled the crystal down to 215 K (-58?C) and gently flowed an aldehyde solution over it for 10 minutes.

To trap the intermediate that was formed, they further cooled the crystal to 90 K (-183 ?C) and collected structural data using the X-ray instrument, obtaining a snapshot of the hemiaminal intermediate. After gathering the hemiaminal configuration, the authors warmed the crystal to 270 K (-3 ?C) and allowed the reaction to advance for 30 minutes. After cooling the crystal to 90 K again, they collected data and acquired the structure of the Schiff base, the final product.

The structure of the hemiaminal intermediate is rarely observed, but it has been studied before by trapping it in an enzyme pocket that stabilized it and extended its life. But enzymes are very specific and difficult to adapt for different molecules, while crystals are likely to be more generally useful. Thus, Fujita’s group has provided an important demonstration that utilizing crystalline pores can successfully provide the structure of a reaction intermediate.

With that said, there are still limitations to what can be monitored using Fujita’s method. For example, they can only study reactions that can be adapted to work in a crystalline pore without destroying the crystal host, and don't experience altered reactivity in the constrained space. Nevertheless, quite a few reactions would fit the necessary criteria, and other types of crystals can be developed to further increase the range of this technique.

Nature, 2009. DOI: 10.1038/nature08326

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