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] 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.[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]
1H2.79284734(3)1/2~100267.522-500
2H0.857438228(9)10.01541.066-76.753
3H2.97896244(4)1/20285.349-533.32
10B 1.80064478(6)319.928.747-53.718
11B 2.68864893/280.185.847-160.42
13C 0.7024118(14)1/21.167.238-125.725
14N 0.40376100(6)199.619.338-36.132
15N -0.28318884(5)1/20.37-27.12650.782
17O -1.89379(9)5/20.04-36.28167.782
19F2.628868(8)1/2~100251.815-470.47
31P1.13160(3)1/2~100108.394-202.606

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 CH3NO2NH3 = δCH3NO2 + 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. 1 2 Witanowski, M (1974). “Nitrogen N.M.R. Spectroscopy”. Pure and Applied Chemistry. 37, pp. 225-233. doi:10.1351/pac197437010225
  2. 1 2 3 4 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. 1 2 3 4 5 6 7 8 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. 1 2 3 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
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