Identification and structure elucidation of organic molecules are generally done by 2D NMR, which can provide extensive structural, conformational and dynamic information. However, such experiments are often time-consuming as they in general cover a large frequency range and provide information about all spin systems present. In many cases, it is just information originating from a single system that is needed to solve the problem at hand. Here 1D 1H selective experiments can often extract the key information required for structural and conformational analysis in a much shorter time. Such experiments are indeed often employed and can be immensely useful. Unfortunately, though, in 1H NMR the narrow range of chemical shifts often causes multiplets to overlap, so that traditional selective excitation methods cannot select a single chemical site. Potential solutions include the use of doubly selective experiments such as 1D COSY-TOCSY and HSCQ-TOCSY, but these require prior knowledge about the spin systems and/or have a steep cost in sensitivity. The chemical shift selective filter (CSSF)1 is a more general solution and does allow selection of a single chemical shift in an overlapped region, but is time-consuming because multiple increments are required to achieve selectivity.
Here, a new family of 1D ultra-selective experiments, GEMSTONE2 (gradient-enhanced multiplet-selective targeted-observation NMR experiment), is described and demonstrated. GEMSTONE allows selective excitation of a single multiplet even in the presence of severe overlap and requires only a single scan to achieve multiplet selectivity, saving significant time compared to CSSF. GEMSTONE versions of a range of 1D selective methods will be presented2,3, providing unambiguous through-space and through-bond correlations for individual selected signals.
1D GEMSTONE TOCSY
Selective 1D TOCSY experiments are powerful tools e.g. in the elucidation of organic molecules, providing through-coupling information for an entire spin system. The ultra-selective GEMSTONE TOCSY experiment will be illustrated with examples for which conventional selective 1D experiments are unhelpful. These include:
The aminoglycoside antibiotic amkacin has four overlapping proton multiplets in the region 3.2-3.3 ppm, from three different six-membered rings. While the result from standard selective TOCSY is ambiguous, the GEMSTONE experiment provides clean, readily interpretable information.
A mixture of diastereomers from the flavone glycosides hesperedin and naringin contains four components with very similar structures, leading to severe overlap in most parts of the spectrum. Four heavily overlapping signals at 2.6-2.8 ppm are resolved by GEMSTONE, unlocking their coupling information.
1D GEMSTONE NOESY
NOESY is unparalleled in providing through-space information for structural and conformational elucidation of molecules. GEMSTONE NOESY will be demonstrated here on a range of problems.
The oestrogen steroid hormone 17-estradiol has overlapped multiplets from three different rings at 1.7-1.9 ppm. A conventional 1D NOESY experiment does not distinguish the three protons, while the GEMSTONE experiment cleanly selects each separately to allow verification of conformation.
A diastereomeric carbocyclic product of an asymmetric synthesis4 contains a doublet and two methyl triplets in a small region of the spectrum around 0.9 ppm. This region also contains the methyl signal from a minor side product; the methyl signal here is particularly important for determining configuration due its closeness to the chiral centre. GEMSTONE is able to excite this signal only, allowing unambiguous assignments. In another region of the spectrum, around 1.2 ppm, there are several overlapping signals from the main carbocycle. Again, the GEMSTONE experiment is able to pull out the contributions from the individual protons.
1D GEMSTONE ROESY
A novel ultra-selective GEMSTONE-ROESY adaptation will also be presented, aiding structural and conformational analysis where the NOE provides little or no intelligible data. The benefits of the new method will be demonstrated in the structural analysis of lacto-N-difucohexaose I, a structurally complex oligosaccharides present in human breast milk.
1D GEMSTONE CLIP COSY
1D COSY methods are not very popular, due to the difference in phase between diagonal and cross peaks in conventional COSY and to a lesser extent in multiple quantum filtered COSY. However, the more recent CLIP-COSY experiment 5 provides pure phase spectra and is therefore a more attractive option for 1D experiments. The GEMSTONE CLIP-COSY experiment will be demonstrated on a complex mixture, e.g. sage essential oil, where there are many overlapping signals from species of different concentrations.
1D pure shift GEMSTONE TOCSY
In some cases overlapping signals make even the spectrum of a single spin system difficult to interpret. One attractive option is then to use a pure shift6 version of GEMSTONE to collapse the multiplets measured, further simplifying such complex spectra. One example is a mixture of estradiols, where the structural similarities between these steroids cause severe overlap and where the component spectra are highly complex in themselves.
The NMR pulse sequence element GEMSTONE uses simultaneous pulsed field gradients and swept-frequency pulses to extract single NMR chemical shifts in a spectrum even when multiplets overlap. It provides unprecedented selectivity in a single scan, and can be implemented in any selective excitation experiment without increasing the minimum experiment time required. The impressive performance of GEMSTONE should allow a greater uptake of selective 1D methods, in lieu of time-consuming multi-dimensional experiments.
Session #3: Metabolomics/Mixture analysis: to isolate or not to isolate … lets go with not!