Yale Professor of Chemistry Mark Johnson and his group have recently developed a new technique called cryogenic infrared (IR) spectroscopy. “This technique can provide a stop-action picture of what is happening through a series of stages,” Johnson explains. The lab has demonstrated the power of the technique in a recent Science paper by elucidating hydrogen-bonding interactions in a supramolecular complex held together by hydrogen bonds. Understanding the pattern of hydrogen bonding is exceedingly important in the contexts of enzyme-substrate and drug-protein interactions.
According to Johnson, the new technique provides the resolution and sensitivity of mass spectrometry coupled with structural characterization by FT-infrared spectroscopy. The molecules of interest are ionized using electrospray ionization and then frozen into stable configurations by cooling to around 10 kelvins. The cryogenic temperature traps some of these molecules while they are in otherwise short-lived states. An atmosphere enriched in D2 is introduced, causing a D2 molecule to associate with each of the trapped species. Then the desired D2 adduct can be separated by its heavier mass and exposed to the IR radiation. The temperature increase caused by absorption of IR energy causes the D2 molecule to evaporate from the adduct. By detecting the lighter fragment arising from D2 loss as the frequency is scanned, the vibrational IR spectrum is obtained with great sensitivity.
This technique was successfully used to investigate hydrogen bonding in a simple peptide catalyst-biaryl substrate system developed by Professor Scott Miller of the Yale Chemistry Department. Hydrogen bonding between carbonyl (C=O) and amine (N-H) moieties is very important in biochemistry. The pattern of these linkages effectively determines the complex formed by two molecules, but there are typically a wide variety of different possible conformations. Information provided by the cryogenic spectroscopy experiment dramatically narrows the computational search for the correct complex by indicating which C=O and N-H groups are active in a particular binding pair. The critical information was obtained using single-site isotopic substitution. The relevant isotopes (13C and 15N) increase the mass of the oscillator, causing a reduction in its resonant frequency. Often, careful comparison will show that only a single peak is significantly shifted in the isotope-labeled spectrum. This peak can then be unambiguously identified as representing the frequency of the bond where the substitution has occurred. The value of this frequency is strongly dependent on the local hydrogen-bonding environment of the oscillator. The existence of a hydrogen bond with hydrogen results in a slight red shift of the resonance to a lower frequency.
According to Johnson, “This technique is powerful because it provides a way to gain access to chemical events in the midst of bond rearrangement, which is the essence of chemistry.” He believes that this new technology has great potential to become a commonly used spectroscopic technique. The Johnson lab plans to extend the work to areas of interest in energy chemistry such as water splitting and CO2 activation.
