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.^{[1] 15}N 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.^[2] Two sources of nitrogen-15 are the positron emission of oxygen-15 and the beta decay of carbon-15.^[3]

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.^[4]

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[5]	Mag Moment (μ, nm)[4]	Nuclear Spin[4]	Natural Abundance (%)[4]	Gyromagnetic Ratio (γ 10^6 rad s^−1 T^−1)[4]	NMR Frequency at 500 MHz[4]
^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

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.^[6]

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.^[6]

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.^[2][4]

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. ^[2][4]

Applications

Tautomerization

Example ¹⁵N chemical shifts for tautomers undergoing tautomerization.^[6]

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.^[1]

Protein NMR

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

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.^[2]

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.

References

1. Witanowski, M (1974). “Nitrogen N.M.R. Spectroscopy”. Pure and Applied Chemistry. 37, pp. 225-233. doi:10.1351/pac197437010225
2. M H Levitt (2008). Spin Dynamics. John Wiley & Sons Ltd. ISBN 0470511176.
3. Haynes, William (2013). CRC handbook of chemistry and physics : a ready-reference book of chemical and physical data. Boca Raton, Florida: CRC Press, Taylor & Francis Group. ISBN 1466571144.
4. Arthur G Palmer (2007). Protein NMR Spectroscopy. Elsevier Academic Press. ISBN 012164491X.
5. Stone, Nicholas J (2005). “Table of nuclear magnetic dipole and electric quadrupole moments”. Atomic Data and Nuclear Data Tables. 90 (1), pp. 75-176. doi:10.1016/j.adt.2005.04.001
6. Mooney, E F; Winson, P H (1969). “Nitrogen Magnetic Resonance Spectroscopy”. Annual Reports on NMR Spectroscopy (2), pp 125-152. doi:10.1016/S0066-4103(08)60321-X