Ch3 12ch2 12ch2 oh

0.96663567 mM = 1mM - 3 x 11 ^M - 3 x 121 nM - 1.33 nM

This species is invisible to 13C NMR and does not contribute at all to the 13C spectrum.

The advantage of 13C's low natural abundance can be seen clearly in this example: each carbon resonance in the spectrum represents a pure isotopomer with 13C only at that position and 12C at all other positions within the molecule. Any species with two or more 13 C atoms in the molecule is present at a concentration of at most two orders of magnitude lower than the one-13C isotopomers, so we will never see any contribution from these species in our 13C spectrum.

4.2.2 Isotopic Enrichment

You may be familiar with the use of carbon-14 as a "tracer" in biosynthetic studies: a metabolic building block such as acetate (CH3-CO-) can be prepared with 14C

(a radioactive isotope) enriched at one of the carbon positions in the molecule. Any biomolecule that is put together using this building block will end up with 14C in it, which can be detected by measuring radioactivity. We can do the same thing with 13 C, without the dangers and cumbersome precautions of working with radioactivity. For example, starting with 13CH3-12CO- (prepared synthetically), an enzyme, cell-free extract, cell culture, or a whole organism can be used to prepare a natural product. This molecule is isolated and purified, and in the 13 C NMR spectrum we would see that the peak corresponding to any carbon position that is derived from the methyl group of acetate will be 91 times more intense (abundance 100% vs. 1.1%) than the other peaks! The 14C tracer method only tells us whether the building block is incorporated or not, but the 13 C NMR method tells us exactly at which position the labeled carbon is incorporated, assuming that the peaks in the spectrum can be assigned to specific carbon positions in the molecular structure.

An even more powerful technique is to label both the positions of a two-carbon building block such as acetate (13CH3-13CO-) and mix it equally with natural-abundance molecules (primarily 12CH3-12CO-). If the two-carbon "synthon" is incorporated intact, without breaking it apart into two one-carbon pieces, we should see 13C-13C coupling due to 1JCC in all of the final molecules that contain 13C from the acetate building block. If the acetate is broken down first into one-carbon pieces and then joined together in the biosynthesis, there would only be 50% abundance of 13C at each of the two positions, and we would see a normal resonance (singlet) superimposed on a split resonance (doublet) at each position derived from the building block.

13C labels can also be used in metabolic studies to watch the breakdown of biological molecules. This can even be done in suspensions of living cells in an NMR tube, watching the progression of 13C peaks in a starting molecule (such as glucose) moving to 13C signals of breakdown products (such as ethanol). The background of natural abundance 13C is much weaker and usually does not rise above the noise level.

Finally, uniform labeling with13 C is extremely important in biological NMR. Expression of proteins in cell culture can be carried out with uniformly labeled 13C-glucose or 13C-acetate at high enrichment (95-99%) as the only carbon source. Isolation and purification of the overexpressed protein leads to an NMR sample with the potential of measuring and assigning 13C chemical shifts at all positions. We will see in Chapter 12 how 13C-13C and 13C-15N one-bond couplings can be used to build complex and sophisticated biological NMR experiments capable of determining the three-dimensional structure and residue-specific dynamics of very large (e.g., 30 kD) biological molecules.

4.2.3 1H-13C J Coupling

So far we have ignored the effect of protons on the 13C spectrum. The 1H-13C one-bond coupling (1/CH) is very large (~150 Hz), so we can expect to see very wide doublets (for methine, CH), triplets (for methylene, CH2), and quartets (for methyl, CH3) for the 13 C resonances in our spectrum. Only the quaternary carbons (Cq) would be free of this large coupling. In fact, for all but the simplest molecules, a simple pulse-and-observe 13 C experiment (relaxation delay-pulse-acquire FID) will give a forest of overlapping peaks that is very difficult to unravel and analyze. In addition, there are long-range (two-bond and three-bond) couplings between 1H and 13C. Because 1H has essentially 100% natural abundance, any coupling to 1H will show up completely and not as a small satellite. For example, consider n-propanol again. The isotopomer that gives rise to the CH3 peak in the

13 C spectrum is

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