Nitrogen-15_nuclear_magnetic_resonance_spectroscopy Knowpia

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]^[2] ¹⁵N NMR differs in several ways from the more common ¹³C and ¹H NMR. To circumvent the difficulties associated with measurement of the quadrupolar, spin-1 ¹⁴N nuclide, 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.^[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 edit

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 strength.^[4]

Physical properties edit

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]	Magnetic dipole moment (μ_N)^[4]	Nuclear spin number^[4]	Natural abundance (%)^[4]	Gyromagnetic ratio (10⁶ rad s⁻¹ T⁻¹)^[4]	NMR frequency at 11.7T (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

From these data, one can see that at full enrichment, ¹⁵N is about one tenth (-27.126/267.522) as sensitive as ¹H.

Chemical shift trends edit

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]^[2]

Gyromagnetic ratio edit

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.^[3]^[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.^[3]^[4]

Applications edit

Tautomerization edit

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 edit

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.

Investigation of nitrogen-containing heterocycles edit

¹⁵N NMR is the most effective method for investigation of structure of heterocycles with a high content of nitrogen atoms (tetrazoles, triazines and their annelated analogs).^[7]^[8] ¹⁵N labeling followed by analysis of ¹³C–¹⁵N and ¹H–¹⁵N couplings may be used for establishing structures and chemical transformations of nitrogen heterocycles.^[9]

INEPT edit

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.^[3]

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 edit

^ ^a ^b Witanowski, M (1974). “Nitrogen N.M.R. Spectroscopy”. Pure and Applied Chemistry. 37, pp. 225-233. doi:10.1351/pac197437010225
^ ^a ^b J. H. Nelson (2003). Nuclear Magnetic Resonance Spectroscopy. Prentice-Hall. ISBN 978-0130334510.
^ ^a ^b ^c ^d M H Levitt (2008). Spin Dynamics. John Wiley & Sons Ltd. ISBN 978-0470511176.
^ ^a ^b ^c ^d ^e ^f ^g ^h Arthur G Palmer (2007). Protein NMR Spectroscopy. Elsevier Academic Press. ISBN 978-0121644918.
^ 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
^ ^a ^b ^c 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
^ Shestakova, Tatyana S.; Shenkarev, Zakhar O.; Deev, Sergey L.; Chupakhin, Oleg N.; Khalymbadzha, Igor A.; Rusinov, Vladimir L.; Arseniev, Alexander S. (2013-06-27). "Long-Range 1H–15N J Couplings Providing a Method for Direct Studies of the Structure and Azide–Tetrazole Equilibrium in a Series of Azido-1,2,4-triazines and Azidopyrimidines" (PDF). The Journal of Organic Chemistry. 78 (14): 6975–6982. doi:10.1021/jo4008207. hdl:10995/27205. ISSN 0022-3263. PMID 23751069.
^ Deev, Sergey L; Paramonov, Alexander S; Shestakova, Tatyana S; Khalymbadzha, Igor A; Chupakhin, Oleg N; Subbotina, Julia O; Eltsov, Oleg S; Slepukhin, Pavel A; Rusinov, Vladimir L (2017-11-29). "15N-Labelling and structure determination of adamantylated azolo-azines in solution". Beilstein Journal of Organic Chemistry. 13 (1): 2535–2548. doi:10.3762/bjoc.13.250. ISSN 1860-5397. PMC 5727827. PMID 29259663.
^ Deev, Sergey L.; Khalymbadzha, Igor A.; Shestakova, Tatyana S.; Charushin, Valery N.; Chupakhin, Oleg N. (2019-08-23). "15 N labeling and analysis of 13C–15N and 1H–15N couplings in studies of the structures and chemical transformations of nitrogen heterocycles". RSC Advances. 9 (46): 26856–26879. Bibcode:2019RSCAd...926856D. doi:10.1039/C9RA04825A. ISSN 2046-2069. PMC 9070671. PMID 35528595.