Nitrogen-15 nuclear magnetic resonance spectroscopy

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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]

Contents

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 strength. [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] Magnetic dipole
moment (μN) [4] 		Nuclear spin
number [4] 		Natural
abundance (%) [4] 		Gyromagnetic ratio
(106 rad s−1 T−1) [4] 		NMR frequency
at 11.7T (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
10B1.80064478(6)319.928.747-53.718
11B2.68864893/280.185.847-160.42
13C0.7024118(14)1/21.167.238-125.725
14N0.40376100(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

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

Typical N chemical shift (d) values for common organic groups where pressurized liquid ammonia is the standard and assigned a chemical shift of 0 ppm. N15 Chemical Shifts Chart.png
Typical N 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 (δNH3 = 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] [2]

Gyromagnetic ratio

The sign of the gyromagnetic ratio, g, determines the sense of precession. Nuclei such as H and C are said to have clockwise precession whereas N has counterclockwise precession. Gyromagnetic Nuclei Precession.png
The sign of the gyromagnetic ratio, γ, determines the sense of precession. Nuclei such as H and C are said to have clockwise precession whereas N 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. [3] [4]

Applications

Tautomerization

Example N chemical shifts for tautomers undergoing tautomerization. Tautomerization and 15N-NMR.png
Example N 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. [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 C and N. 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 C and N.

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

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

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

See also

Related Research Articles

In nuclear magnetic resonance (NMR) spectroscopy, the chemical shift is the resonant frequency of an atomic nucleus relative to a standard in a magnetic field. Often the position and number of chemical shifts are diagnostic of the structure of a molecule. Chemical shifts are also used to describe signals in other forms of spectroscopy such as photoemission spectroscopy.

<span class="mw-page-title-main">Nuclear magnetic resonance spectroscopy</span> Laboratory technique

Nuclear magnetic resonance spectroscopy, most commonly known as NMR spectroscopy or magnetic resonance spectroscopy (MRS), is a spectroscopic technique based on re-orientation of atomic nuclei with non-zero nuclear spins in an external magnetic field. This re-orientation occurs with absorption of electromagnetic radiation in the radio frequency region from roughly 4 to 900 MHz, which depends on the isotopic nature of the nucleus and increased proportionally to the strength of the external magnetic field. Notably, the resonance frequency of each NMR-active nucleus depends on its chemical environment. As a result, NMR spectra provide information about individual functional groups present in the sample, as well as about connections between nearby nuclei in the same molecule. As the NMR spectra are unique or highly characteristic to individual compounds and functional groups, NMR spectroscopy is one of the most important methods to identify molecular structures, particularly of organic compounds.

<span class="mw-page-title-main">Solid-state nuclear magnetic resonance</span>

Solid-state NMR (ssNMR) spectroscopy is a technique for characterizing atomic level structure in solid materials e.g. powders, single crystals and amorphous samples and tissues using nuclear magnetic resonance (NMR) spectroscopy. The anisotropic part of many spin interactions are present in solid-state NMR, unlike in solution-state NMR where rapid tumbling motion averages out many of the spin interactions. As a result, solid-state NMR spectra are characterised by larger linewidths than in solution state NMR, which can be utilized to give quantitative information on the molecular structure, conformation and dynamics of the material. Solid-state NMR is often combined with magic angle spinning to remove anisotropic interactions and improve the resolution as well as the sensitivity of the technique.

Carbon-13 (C13) nuclear magnetic resonance is the application of nuclear magnetic resonance (NMR) spectroscopy to carbon. It is analogous to proton NMR and allows the identification of carbon atoms in an organic molecule just as proton NMR identifies hydrogen atoms. 13C NMR detects only the 13
C
 isotope. The main carbon isotope, 12
C
 does not produce an NMR signal. Although ca. 1 mln. times less sensitive than 1H NMR spectroscopy, 13C NMR spectroscopy is widely used for characterizing organic and organometallic compounds, primarily because 1H-decoupled 13C-NMR spectra are more simple, have a greater sensitivity to differences in the chemical structure, and, thus, are better suited for identifying molecules in complex mixtures. At the same time, such spectra lack quantitative information about the atomic ratios of different types of carbon nuclei, because nuclear Overhauser effect used in 1H-decoupled 13C-NMR spectroscopy enhances the signals from carbon atoms with a larger number of hydrogen atoms attached to them more than from carbon atoms with a smaller number of H's, and because full relaxation of 13C nuclei is usually not attained, and the nuclei with shorter relaxation times produce more intense signals.

<span class="mw-page-title-main">Proton nuclear magnetic resonance</span> NMR via protons, hydrogen-1 nuclei

Proton nuclear magnetic resonance is the application of nuclear magnetic resonance in NMR spectroscopy with respect to hydrogen-1 nuclei within the molecules of a substance, in order to determine the structure of its molecules. In samples where natural hydrogen (H) is used, practically all the hydrogen consists of the isotope 1H.

Nuclear magnetic resonance spectroscopy of proteins is a field of structural biology in which NMR spectroscopy is used to obtain information about the structure and dynamics of proteins, and also nucleic acids, and their complexes. The field was pioneered by Richard R. Ernst and Kurt Wüthrich at the ETH, and by Ad Bax, Marius Clore, Angela Gronenborn at the NIH, and Gerhard Wagner at Harvard University, among others. Structure determination by NMR spectroscopy usually consists of several phases, each using a separate set of highly specialized techniques. The sample is prepared, measurements are made, interpretive approaches are applied, and a structure is calculated and validated.

The heteronuclear single quantum coherence or heteronuclear single quantum correlation experiment, normally abbreviated as HSQC, is used frequently in NMR spectroscopy of organic molecules and is of particular significance in the field of protein NMR. The experiment was first described by Geoffrey Bodenhausen and D. J. Ruben in 1980. The resulting spectrum is two-dimensional (2D) with one axis for proton (1H) and the other for a heteronucleus, which is usually 13C or 15N. The spectrum contains a peak for each unique proton attached to the heteronucleus being considered. The 2D HSQC can also be combined with other experiments in higher-dimensional NMR experiments, such as NOESY-HSQC or TOCSY-HSQC.

Two-dimensional nuclear magnetic resonance spectroscopy is a set of nuclear magnetic resonance spectroscopy (NMR) methods which give data plotted in a space defined by two frequency axes rather than one. Types of 2D NMR include correlation spectroscopy (COSY), J-spectroscopy, exchange spectroscopy (EXSY), and nuclear Overhauser effect spectroscopy (NOESY). Two-dimensional NMR spectra provide more information about a molecule than one-dimensional NMR spectra and are especially useful in determining the structure of a molecule, particularly for molecules that are too complicated to work with using one-dimensional NMR.

Insensitive nuclei enhancement by polarization transfer (INEPT) is a signal enhancement method used in NMR spectroscopy. It involves the transfer of nuclear spin polarization from spins with large Boltzmann population differences to nuclear spins of interest with lower Boltzmann population differences. INEPT uses J-coupling for the polarization transfer in contrast to Nuclear Overhauser effect (NOE), which arises from dipolar cross-relaxation. This method of signal enhancement was introduced by Ray Freeman in 1979. Due to its usefulness in signal enhancement, pulse sequences used in heteronuclear NMR experiments often contain blocks of INEPT or INEPT-like sequences.

In nuclear chemistry and nuclear physics, J-couplings are mediated through chemical bonds connecting two spins. It is an indirect interaction between two nuclear spins that arises from hyperfine interactions between the nuclei and local electrons. In NMR spectroscopy, J-coupling contains information about relative bond distances and angles. Most importantly, J-coupling provides information on the connectivity of chemical bonds. It is responsible for the often complex splitting of resonance lines in the NMR spectra of fairly simple molecules.

<span class="mw-page-title-main">Phosphorus-31 nuclear magnetic resonance</span> Spectroscopy technique for molecules containing phosphorus

Phosphorus-31 NMR spectroscopy is an analytical chemistry technique that uses nuclear magnetic resonance (NMR) to study chemical compounds that contain phosphorus. Phosphorus is commonly found in organic compounds and coordination complexes, making it useful to measure 31- NMR spectra routinely. Solution 31P-NMR is one of the more routine NMR techniques because 31P has an isotopic abundance of 100% and a relatively high gyromagnetic ratio. The 31P nucleus also has a spin of 1/2, making spectra relatively easy to interpret. The only other highly sensitive NMR-active nuclei spin 1/2 that are monoisotopic are 1H and 19F.

<span class="mw-page-title-main">Fluorine-19 nuclear magnetic resonance spectroscopy</span> Analytical technique

Fluorine-19 nuclear magnetic resonance spectroscopy is an analytical technique used to detect and identify fluorine-containing compounds. 19F is an important nucleus for NMR spectroscopy because of its receptivity and large chemical shift dispersion, which is greater than that for proton nuclear magnetic resonance spectroscopy.

<span class="mw-page-title-main">Nuclear magnetic resonance</span> Spectroscopic technique based on change of nuclear spin state

Nuclear magnetic resonance (NMR) is a physical phenomenon in which nuclei in a strong constant magnetic field are perturbed by a weak oscillating magnetic field and respond by producing an electromagnetic signal with a frequency characteristic of the magnetic field at the nucleus. This process occurs near resonance, when the oscillation frequency matches the intrinsic frequency of the nuclei, which depends on the strength of the static magnetic field, the chemical environment, and the magnetic properties of the isotope involved; in practical applications with static magnetic fields up to ca. 20 tesla, the frequency is similar to VHF and UHF television broadcasts (60–1000 MHz). NMR results from specific magnetic properties of certain atomic nuclei. Nuclear magnetic resonance spectroscopy is widely used to determine the structure of organic molecules in solution and study molecular physics and crystals as well as non-crystalline materials. NMR is also routinely used in advanced medical imaging techniques, such as in magnetic resonance imaging (MRI). The original application of NMR to condensed matter physics is nowadays mostly devoted to strongly correlated electron systems. It reveals large many-body couplings by fast broadband detection and it should not to be confused with solid state NMR, which aims at removing the effect of the same couplings by Magic Angle Spinning techniques.

Triple resonance experiments are a set of multi-dimensional nuclear magnetic resonance spectroscopy (NMR) experiments that link three types of atomic nuclei, most typically consisting of 1H, 15N and 13C. These experiments are often used to assign specific resonance signals to specific atoms in an isotopically-enriched protein. The technique was first described in papers by Ad Bax, Mitsuhiko Ikura and Lewis Kay in 1990, and further experiments were then added to the suite of experiments. Many of these experiments have since become the standard set of experiments used for sequential assignment of NMR resonances in the determination of protein structure by NMR. They are now an integral part of solution NMR study of proteins, and they may also be used in solid-state NMR.

<span class="mw-page-title-main">Paramagnetic nuclear magnetic resonance spectroscopy</span> Spectroscopy of paramagnetic compounds via NMR

Paramagnetic nuclear magnetic resonance spectroscopy refers to nuclear magnetic resonance (NMR) spectroscopy of paramagnetic compounds. Although most NMR measurements are conducted on diamagnetic compounds, paramagnetic samples are also amenable to analysis and give rise to special effects indicated by a wide chemical shift range and broadened signals. Paramagnetism diminishes the resolution of an NMR spectrum to the extent that coupling is rarely resolved. Nonetheless spectra of paramagnetic compounds provide insight into the bonding and structure of the sample. For example, the broadening of signals is compensated in part by the wide chemical shift range (often 200 ppm in 1H NMR). Since paramagnetism leads to shorter relaxation times (T1), the rate of spectral acquisition can be high.

Nuclear magnetic resonance chemical shift re-referencing is a chemical analysis method for chemical shift referencing in biomolecular nuclear magnetic resonance (NMR). It has been estimated that up to 20% of 13C and up to 35% of 15N shift assignments are improperly referenced. Given that the structural and dynamic information contained within chemical shifts is often quite subtle, it is critical that protein chemical shifts be properly referenced so that these subtle differences can be detected. Fundamentally, the problem with chemical shift referencing comes from the fact that chemical shifts are relative frequency measurements rather than absolute frequency measurements. Because of the historic problems with chemical shift referencing, chemical shifts are perhaps the most precisely measurable but the least accurately measured parameters in all of NMR spectroscopy.

Protein chemical shift re-referencing is a post-assignment process of adjusting the assigned NMR chemical shifts to match IUPAC and BMRB recommended standards in protein chemical shift referencing. In NMR chemical shifts are normally referenced to an internal standard that is dissolved in the NMR sample. These internal standards include tetramethylsilane (TMS), 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) and trimethylsilyl propionate (TSP). For protein NMR spectroscopy the recommended standard is DSS, which is insensitive to pH variations. Furthermore, the DSS 1H signal may be used to indirectly reference 13C and 15N shifts using a simple ratio calculation [1]. Unfortunately, many biomolecular NMR spectroscopy labs use non-standard methods for determining the 1H, 13C or 15N “zero-point” chemical shift position. This lack of standardization makes it difficult to compare chemical shifts for the same protein between different laboratories. It also makes it difficult to use chemical shifts to properly identify or assign secondary structures or to improve their 3D structures via chemical shift refinement. Chemical shift re-referencing offers a means to correct these referencing errors and to standardize the reporting of protein chemical shifts across laboratories.

Vanadium-51 nuclear magnetic resonance is a method for the characterization of vanadium-containing compounds and materials. 51V comprises 99.75% of naturally occurring vanadium. The nucleus is quadrupolar with I = 7/2, which is not favorable for NMR spectroscopy, although its quadrupole moment and thus the linewidths are unusually small, while its magnetogyric ratio is relatively high, so that 51V has 38% receptivity vs 1H. Its resonance frequency is close to that of 13C.

<span class="mw-page-title-main">Platinum-195 nuclear magnetic resonance</span>

Platinum-195 nuclear magnetic resonance spectroscopy is a spectroscopic technique which is used for the detection and characterisation of platinum compounds. The sensitivity of the technique and therefore its diagnostic utility have increased significantly starting from the 1970s, with 195Pt NMR nowadays considered the method of choice for structural elucidation of Pt species in solution.

<span class="mw-page-title-main">Cross-polarization</span> Spectroscopy technique

Cross-polarization (CP), originally published as proton-enhanced nuclear induction spectroscopy is a solid-state nuclear magnetic resonance (ssNMR) technique to transfer nuclear magnetization from different types of nuclei via heteronuclear dipolar interactions. The 1H-X cross-polarization dramatically improves the sensitivity of ssNMR experiments of most experiments involving spin-1/2 nuclei, capitalizing on the higher 1H polarisation, and shorter T1(1H) relaxation times. It was developed by Michael Gibby, Alexander Pines and Professor John S. Waugh at the Massachusetts Institute of Technology.

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 J. H. Nelson (2003). Nuclear Magnetic Resonance Spectroscopy. Prentice-Hall. ISBN   978-0130334510.
  3. 1 2 3 4 M H Levitt (2008). Spin Dynamics. John Wiley & Sons Ltd. ISBN   978-0470511176.
  4. 1 2 3 4 5 6 7 8 Arthur G Palmer (2007). Protein NMR Spectroscopy. Elsevier Academic Press. ISBN   978-0121644918.
  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
  7. ↑ 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.
  8. ↑ 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.
  9. ↑ 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.
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