Nitrogen-15 nuclear magnetic resonance spectroscopy

Nitrogen-15 nuclear magnetic resonance spectroscopy (nitrogen-15 NMR spectroscopy, or just simply ¹⁵N NMR) is a version of nuclear magnetic resonance spectroscopy that examines samples containing the ¹⁵N nucleus. ¹⁵N NMR differs in several ways from the more common ¹³C and ¹H NMR. To lift the restraint of spin 1 found in ¹⁴N, ¹⁵N NMR is employed in samples for detection since it has a ground-state spin of ½. Since¹⁴N is 99.64% abundant, incorporation of ¹⁵N into samples often requires novel synthetic techniques. Two sources of nitrogen-15 are the positron emission of oxygen-15 and the beta decay of carbon-15.

Nitrogen-15 is frequently used in nuclear magnetic resonance spectroscopy (NMR), because unlike the more abundant nitrogen-14, that has an integer nuclear spin and thus a quadrupole moment, ¹⁵N has a fractional nuclear spin of one-half, which offers advantages for NMR like narrower line width. Proteins can be isotopically labeled by cultivating them in a medium containing nitrogen-15 as the only source of nitrogen. In addition, nitrogen-15 is used to label proteins in quantitative proteomics (e.g. SILAC).

Implementation

¹⁵N NMR has complications not encountered in ¹H and ¹³C NMR spectroscopy. The 0.36% natural abundance of ¹⁵N results in a major sensitivity penalty. Sensitivity is made worse by its low gyromagnetic ratio (γ = -27.126 × 10⁶ T⁻¹s⁻¹), which is 10.14% that of ¹H. The signal to noise ratio for ¹H is about 300 fold greater than ¹⁵N at the same magnetic field.

Physical properties

The physical properties of ¹⁵N are quite different from other nuclei. Its properties along with several common nuclei are summarized in the below table.

Isotope	Mag Moment (μ, nm)	Nuclear Spin	Natural Abundance (%)	Gyromagnetic Ratio (γ 10^6 rad s^−1 T^−1)	NMR Frequency at 500 MHz
1H	2.79284734(3)	1/2	~100	267.522	-500
2H	0.857438228(9)	1	0.015	41.066	-76.753
3H	2.97896244(4)	1/2	0	285.349	-533.32
10B	1.80064478(6)	3	19.9	28.747	-53.718
11B	2.6886489	3/2	80.1	85.847	-160.42
13C	0.7024118(14)	1/2	1.1	67.238	-125.725
14N	0.40376100(6)	1	99.6	19.338	-36.132
15N	-0.28318884(5)	1/2	0.37	-27.126	50.782
17O	-1.89379(9)	5/2	0.04	-36.281	67.782
19F	2.628868(8)	1/2	~100	251.815	-470.47
31P	1.13160(3)	1/2	~100	108.394	-202.606

Chemical shift trends

File:N15 Chemical Shifts Chart.png

Typical ¹⁵N chemical shift (δ) values for common organic groups where pressurized liquid ammonia is the standard and assigned a chemical shift of 0 ppm.

The International Union of Pure and Applied Chemistry (IUPAC) recommends using CH₃NO₂ as the experimental standard; however in practice many spectroscopists utilize pressurized NH₃(l) instead. For ¹⁵N, chemical shifts referenced with NH₃(l) are 380.5 ppm upfield from CH₃NO₂ (δ_NH3 = δ_CH3NO2 + 380.5 ppm). Chemical shifts for ¹⁵N are somewhat erratic but typically they span a range of -400 ppm to 1100 ppm with respect to CH₃NO₂. Below is a summary of ¹⁵N chemical shifts for common organic groups referenced with respect to NH₃, whose chemical shift is assigned 0 ppm.

Gyromagnetic ratio

The sign of the gyromagnetic ratio, γ, determines the sense of precession. Nuclei such as ¹H and ¹³C are said to have clockwise precession whereas ¹⁵N has counterclockwise precession.

Unlike most nuclei, the gyromagnetic ratio for ¹⁵N is negative. With the spin precession phenomenon, the sign of γ determines the sense (clockwise vs counterclockwise) of precession. Most common nuclei have positive gyromagnetic ratios such as ¹H and ¹³C.

Applications

Tautomerization

File:Tautomerization and 15N-NMR.png

Example ¹⁵N chemical shifts for tautomers undergoing tautomerization.

¹⁵N NMR is used in a wide array of areas from biological to inorganic techniques. A famous application in organic synthesis is to utilize ¹⁵N to monitor tautomerization equilibria in heteroaromatics because of the dramatic change in ¹⁵N shifts between tautomers.

Protein NMR

File:Backbone Walk for NCACX, NCOCX, and CANcoCX for SS-NMR.png

The ssNMR polarization pathways for the NCACX, NCOCX, and CANcoCX experiments respectively. In each case, all carbon and nitrogen atoms are either uniformly or partially isotopically labeled with ¹³C and ¹⁵N.

¹⁵N NMR is also extremely valuable in protein NMR investigations. Most notably, the introduction of three-dimensional experiments with ¹⁵N lifts the ambiguity in ¹³C–¹³C two-dimensional experiments. In solid-state nuclear magnetic resonance (ssNMR), for example, ¹⁵N is most commonly utilized in NCACX, NCOCX, and CANcoCX pulse sequences.

INEPT

File:INEPT Pulse Sequence.png

Graphical representation of the INEPT NMR pulse sequence. INEPT is utilized often to improve ¹⁵N resolution because it can accommodate negative gyromagnetic ratios, increases Boltzmann polarization, and decreases T₁ relaxation.

Insensitive nuclei enhanced by polarization transfer (INEPT) is a signal resolution enhancement method. Because ¹⁵N has a gyromagnetic ratio that is small in magnitude, the resolution is quite poor. A common pulse sequence which dramatically improves the resolution for ¹⁵N is INEPT. The INEPT is an elegant solution in most cases because it increases the Boltzmann polarization and lowers T₁ values (thus scans are shorter). Additionally, INEPT can accommodate negative gyromagnetic ratios, whereas the common nuclear Overhauser effect (NOE) cannot.