2-Heptanone, , CPD and DEPT-35

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KALAHARI DESERT

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The Kalahari Desert (in Afrikaans Kalahari-woestyn) is a large semi-arid sandy savannah in southern Africa extending 900,000 square kilometres (350,000 sq ...

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The town of Tsabong

 

A view of a Ghanzi street

COSY VS TOCSY

COSY (COrrelation SpectroscopY) and TOCSY (TOtal Correlation Spectroscopy) experiments are very common. Although both experiments provide a diagonally symmetric two dimensional contour plot with the one dimensional spectrum on the diagonal and correlations off of the diagonal, they have different information content. A COSY spectrum will have off-diagonal correlations between coupled spins whereas a TOCSY spectrum will have off-diagonal correlations between all spins in a spin system. For example, consider a spin system where A is coupled to B, B is coupled to C, and C is coupled to D. A COSY spectrum will have correlations between A and B, B and C, and C and D, whereas a TOCSY spectrum will have correlations between all of the spins. This is illustrated in the figure below for 3-heptanone. In this case, there are two spin systems (one on either side of the carbonyl group). The COSY spectrum shows correlations between adjacent spins whereas the TOCSY spectrum shows correlations between all of the spins in each of the two spin systems.

NOESY and ROESY

NOESY (Nuclear Overhauser Effect SpectroscopY) is an NMR experiment that can detect couplings between nuclei through spatial proximity (< 5 Å apart) rather than coupling through covalent bonds. The Nuclear Overhauser Effect (NOE) is the change in the intensity of the resonance of a nucleus upon irradiation of a nearby nucleus (about 2.5-3.5 Å apart). For example, when an RF pulse specifically irradiates a proton, its spin population is equalized and it can transfer its spin polarization to another proton and alter its spin population. The overall effect is dependent on a distance of r-6. NOESY uses a mixing time without pulses to accumulate NOEs and its counterpart ROESY (Rotating frame nuclear Overhauser Effect SpectroscopY) uses a series of pulses to accumulate NOEs. In NOESY, NOEs are positive when generated from small molecules, are negative when generated from large molecules (or molecules dissolved in a viscous solvent to restrict molecular tumbling), and are quite small (near zero) for medium-sized molecules. On the contrary, ROESY peaks are always positive, regardless of molecular weight. Both experiments are useful for determine proximity of nuclei in large biomolecules, especially proteins, where two atoms may be nearby in space, but not necessarily through covalent connectivity. Isomers, such as ortho-, meta-, and para-substituted aromatic rings, as well as stereochemistry, can also be distinguished through the use of an NOE experiment. Although NOESY and ROESY can generate COSY and TOCSY artifacts, respectively, those unwanted signals could be minimized by variations in the pulse sequences. Example NOESY and ROESY spectra are shown in Figure.

Nuclear Overhauser effect spectroscopy: 

(a) An expanded portion of a 1H NOESY spectrum of clarithromycin (structure of which is shown in 

(c). Figure from F. G. Vogt, Analytical Instrumentation Handbook, edited by J. Cazes,

 3rd, Marcel Dekker, New York, (2005). (b) Part of a homonuclear 1H ROESY spectrum of a triterpenoid saponin extracted from the root 

of the Acanthophyllum gypsophiloides Regel (Turkestan soap root). (d) A few important ROESY correlations in the saponin, depicted by dashed arrows in (b). 

Figures (b) and (d) from E. A. Khatuntseva, V.M. Men’shov, A.S. Shashkov, Y.E. Tsvetkov, R.N. Stepanenko, R.Y. Vlasenko, E.E. Shults, G.A. Tolstikov, T.G. Tolstikova, D.S. Baev, V.A. Kaledin, N.A. Popova, V.P. Nikolin, P.P. Laktionov, A.V. Cherepanova, T.V. Kulakovskaya, E.V. Kulakovskaya, and N.E. Nifantiev, Beilstein J. Org. Chem. 2012, 8, 763.

How to interpret 2D NMR spectra

Much of the interpretation one needs to do with 2D NMR begins with focusing on the cross peaks and matching them according to frequency, much like playing a game of Battleship®. The 1D spectrum usually will be plotted along the axes, so one can match which couplings in one spectrum correlate to which splitting patterns in the other spectrum using the cross peaks on the 2D spectrum (seeFigure).

An annotated 2D COSY spectrum of xylobiose (same spectrum as in Figureb). 

By matching up the two couplings that intersect at the cross peaks, one can easily 

determine which atoms are connected to which (shown by the blue dashed lines). 

The diagonal peaks are highlighted by the red line for clarity – the real COSY information is within the cross peaks.

Also, multiple 2D NMR experiments are used to elucidate the structure of a single molecule, combining different information from the various sources. For example, one can combine homonuclear and heteronuclear experiments and piece together the information from the two techniques, with a process known as Parallel Acquisition NMR Spectroscopy or PANSY. In the 1990s, co-variance processing came onto the scene, which allowed scientists to process information from two separate experiments, without having to run both experiments at the same time, which made for shorter data acquisition time. Currently, the software for co-variance processing is available from various NMR manufacturers. There are many possible ways to interpret 2D NMR spectra, though one common method is to label the cross peaks and make connections between the signals as they become apparent. Prof. James Nowick at UC Irvine describes his method of choice for putting the pieces together when determining the structure of a sample; the lecture in which he describes this method is posted in the links above. In this video, he provides a stepwise method to deciphering a spectrum.

Heteronuclear experiments

HETCOR (Heteronuclear Correlation) refers to a 2D NMR experiment that correlates couplings between different nuclei (usually 1H and a heteroatom, such as 13C or 15N). Heteronuclear experiments can easily be extended into three or more dimensions, which can be thought of as experiments that correlate couplings between three or more different nuclei. Because there are two different frequency domains, there are no diagonal peaks like there are in COSY or TOCSY. Recently, inverse-detected HETCOR experiments have become extremely useful and commonplace, and it will be those experiments that will be covered here. Inverse-detection refers to detecting the nucleus with the higher gyromagnetic ratio, which offers higher sensitivity. It is ideal to determine which nucleus has the highest gyromagnetic ratio for detection and set the probe to be the most sensitive to this nucleus. In HETCOR, the nucleus that was detected first in a 1H -13C experiment was 13C, whereas now 1H is detected first in inverse-detection experiments, since protons are inherently more sensitive. Today, regular HETCOR experiments are not usually in common laboratory practice.

The HMQC (Heteronuclear Multiple-Quantum Coherence) experiment acquires a spectrum (see Figurea) by transferring the proton magnetization by way of 1JCH to a heteronucleus, for example, 13C. The 13C atom then experiences its chemical shift in the t1 time period of the pulse sequence. The magnetization then returns to the 1H for detection. HMQC detects 1JCH coupling and can also be used to differentiate between geminal and vicinal proton couplings just as in COSY-45. HMQC is very widely used and offers very good sensitivity at much shorter acquisition times than HETCOR (about 30 min as opposed to several hours with HETCOR).

However, because it shows the 1H -1H couplings in addition to 1H -13C couplings and because the cross peaks appear as multiplets, HMQC suffers when it comes to resolution in the 13C peaks. The HSQC (Heteronuclear Single-Quantum Coherence) experiment can assist, as it can suppress the 1H -1H couplings and collapse the multiplets seen in the cross peaks into singlets, which greatly enhances resolution (an example of an HSQC is shown in Figureb). Figure shows a side-by-side comparison of spectra from HMQC and HSQC experiments, in which some of the peaks in the HMQC spectrum are more resolved in the HSQC spectrum. However, HSQC administers more pulses than HMQC, which causes miss-settings and inhomogeneity between the RF pulses, which in turn leads to loss of sensitivity. In HMBC (Heteronuclear Multiple Bond Coherence) experiments, two and three bond couplings can be detected. This technique is particularly useful for putting smaller proposed fragments of a molecule together to elucidate the larger overall structure. HMBC, on the other hand, cannot distinguish between 2JCH and 3JCH coupling constants. An example spectrum is shown in Figured.

Spectra of heteronuclear experiments: 

(a) A spectrum of xylobiose (structure shown in Figureb) taken from a 1H-13C HMQC 2D NMR experiment. 

(b) A spectrum of codeine taken from an HSQC 1H-13C 2D NMR experiment. 

Spectrum from Acorn NMR, Inc. c) The chemical structure of codeine. 

d) Another spectrum of xylobiose taken from a 1H-13C HMBC 2D NMR experiment. 

Panels (a) and (d) from F. Sauriol, NMR Webcourse, Department of Chemistry, Queen’s University, Ontario, www.chem.queensu.ca/facilities/nmr/nmr/webcourse/.

Side-by-side comparison of an HMQC spectrum (a) and an HSQC spectrum (b). 

The HSQC experiment offers better resolution than the HMQC as well as sharper peaks. 

HSQC helps solve the problem of overlapping peaks, which is often seen in NMR experiments. 

The sample in both spectra is codeine. Spectra from Acorn NMR, Inc.

TOCSY

TOCSY

TOCSY (Total Correlation Spectroscopy) creates correlations between all protons within a given spin system, not just between geminal or vicinal protons as in COSY. Correlations are seen between distant protons as long as there are couplings between every intervening proton. This is extremely useful for identifying protons on sugar rings or amino acids: All protons on a given sugar ring will have a correlation with all other protons on the same ring but not with protons on different rings.
Magnetization is transferred successively over up to 5 or 6 bonds as long as successive protons are coupled. Transfer is interrupted by small or zero proton-proton couplings. The presence of hetero-atoms, such as oxygen, usually disrupts TOCSY transfer. The number of transfer steps can be adjusted by changing the spin-lock time. A short time such as 20ms will give only one-step transfers and its TOCSY spectrum will be very similar to its COSY spectrum. More usefully, a long spin-lock time such as 80ms or 120ms will give up to 5 or 6-step transfers. The number of transfers depends on exact coupling details. A useful paper detailing TOCSY transfer in various sugars is Gheysen, K. et. al., Chem. Eur. J. 2008, 14, 8869-8878.

Shown below is a 400 MHz spectrum of sucrose. The red circles show the connections between proton 4 and all the other protons of the glucose ring.

TOCSY (TOtal Correlation SpectroscopY) is very similar to COSY in that it is a homonuclear correlation technique. It differs from COSY in that it not only shows nuclei that are directly coupled to each other, but also signals that are due to nuclei that are in the same spin system, as shown in Figure below. This technique is useful for interpreting large, interconnected networks of spin couplings. The pulse sequence is arranged in such a way to allow for isotropic mixing during the sequence that transfers magnetization across a network of atoms coupled to each other. An alternative technique to 2D TOCSY is selective 1D TOCSY, which can excite certain regions of the spectrum by using shaped pulses. By specifying particular chemical shift values and setting a desired excitation width, one can greatly simplify the 1D experiment. Selective 1D TOCSY is particularly useful for analyzing polysaccharides, since each sugar subunit is an isolated spin system, which can produce its own subspectrum, as long as there is at least one resolved multiplet. Furthermore, each 2D spectrum can be acquired with the same resolution as a normal 1D spectrum, which allows for an accurate measurement of multiplet splittings, especially when signals from different coupled networks overlap with one another.

An example of a spectrum of xylobiose (structure shown in Figureb) taken from a TOCSY 2D NMR experiment. The diagonal peaks are again highlighted by the red dotted line. Figure from F. Sauriol, NMR Webcourse, Department of Chemistry, Queen’s University, Ontario, www.chem.queensu.ca/facilities/nmr/nmr/webcourse/.