Assignment of homonuclear spectra

The principle process of homonuclear sequential assignment was developed by Kurt Wüthrich and coworkers. Experiments as 2D COSY and TOCSY are employed for the identification of amino acid spin systems (blue arrows). The 2D NOESY experiment is used to sequentially connect the spin systems (red arrows).

The fist step in sequential assignment is the identification of certain amino acids, with a characteristic pattern of cross signals, i.e. of glycine, alanine, threonine, valine, leucine and isoleucine.

Glycine (left picture) contains two H^alpha protons and is therefore readily identified. Valine (right picture), leucine and isoleucine can be recognized by their two methyl groups which give a characteristic row of double signals between 0 and 1.5 ppm. In the same way, alanine and threonine are identified by their single methyl groups.

In the second stage of the assignment process, the sequential contacts from the already identified amino acids to the neighboring ones are searched for in the 2D NOESY spectra. The connectivity of a given amino acid in the sequence (i) to its following one (i+1) can be observed in the NOESY because the distance of the amide proton of (i+1) to the H^alpha, H^beta or H^gamma protons of (i) is smaller than 5 A in almost every case (left picture). Therefore, sequential cross signals to H^alpha(i), H^beta(i) etc. are observed at the frequency of H^N(i+1) (right picture, dark blue signals). These interresidual cross signals can be distinguished from the intraresidual ones by comparing the 2D NOESY with the 2D TOCSY spectrum. A series of these sequential cross signals between H^alpha(i) and H^N(i+1) determines the order (i, i+1, i+2,...) of the amino acid spin systems.
Thus, dipeptides are identified and subsequently prolonged to oligopeptides by the search for further sequential contacts. Some time along the line these oligopeptides can be placed at a unique place in the primary structure by comparison with the amino acid sequence of the protein - they are sequentially assigned.
The chain of sequential connectivites is interrupted by proline residues because these have no amide proton. Therefore, no H^N(i)-H^alpha(i-1) cross signal can be observed. However, if the proline (i) is in itstrans conformation, the sequential H^N(i-1)-H^delta(i) and H^alpha(i-1)-H^delta(i) cross signals can be observed.
Another problem is, that this approach of sequential assignment breaks down for larger proteins because the vast number of signals leads to spectral overlap which hinders the identification of signals.

1H-13C COSY (HETCOR)

1H-13C COSY (HETCOR) 

1H-13C COSY is the heteronuclear correlation spectroscopy. The HETCOR spectrum is correlated 13C nuclei with directly attached protons. 1H-13C coupling is one bond. 

The cross peaks mean correlation between a proton and a carbon . If a line does not have cross peak, this means that this carbon atoms has no attached proton (e.g. a quaternary carbon atom) 

1H-1H COSY (Correlation Spectroscopy)

1H-1H COSY (Correlation Spectroscopy)

COSY

COSY (COrrelation SpectroscopY) was one of the first and most popular 2D NMR experiments to be developed. It is a homonuclear experiment that allows one to correlate different signals in the spectrum to each other. In a COSY spectrum (see Figureb), the chemical shift values of the sample’s 1D NMR spectrum are plotted along both the vertical and horizontal axes (some 2D spectra will actually reproduce the 1D spectra along the axes, along with the frequency scale in ppm, while others may simply show the scale). This allows for a collection of peaks to appear down the diagonal of the spectrum known as diagonal peaks (shown in Figureb, highlighted by the red dotted line). These diagonal peaks are simply the peaks that appear in the normal 1D spectrum, because they show nuclei that couple to themselves. The other type of peaks appears symmetric across the diagonal and is known as cross peaks. These peaks show which groups in the molecule that have different chemical shifts are coupled to each other by producing a signal at the intersection of the two frequency values.

Example of correlation spectroscopy: (a) On the left is shown a portion of a 3D or “stacked” plot of a 2D NMR COSY spectrum in which two frequency domains are plotted in two dimensions and intensity is plotted in the third. On the right is shown a contour plot, where the intensities have been depicted topographically. Spectra from Acorn NMR, Inc. (b) A spectrum of the disaccharide xylobiose (structure shown), taken from a COSY 2D NMR experiment. The red dotted line highlights the diagonal peaks. Spectrum adapted from F. Sauriol, NMR Webcourse, Department of Chemistry, Queen’s University, Ontario, www.chem.queensu.ca/facilities/nmr/nmr/webcourse/.

One can then determine the structure of a sample by examining what chemical shift values the cross peaks occur at in a spectrum. Since the cross peaks are symmetric across the diagonal peaks, one can easily identify which cross peaks are real (if a certain peak has a counterpart on the other side of the diagonal) and which are digital artifacts of the experiment. The smallest coupling that can be detected using COSY is dependent on the linewidth of the spectrum and the signal-to-noise ratio; a maximum signal-to-noise ratio and a minimum linewidth will allow for very small coupling constants to be detected.

Variations of COSY

Although COSY is very useful, it does have its disadvantages. First of all, because the anti-phase structure of the cross peaks, which causes the spectral lines to cancel one another out, and the in-phase structure of the diagonal peaks, which causes reinforcement among the peaks, there is a significant difference in intensity between the diagonal and cross peaks. This difference in intensity makes identifying small cross peaks difficult, especially if they lie near the diagonal. Another problem is that when processing the data for a COSY spectrum, the broad lineshapes associated with the experiment can make high-resolution work difficult.

In one of the more popular COSY variations known as DQF COSY (Double-Quantum Filtered COSY), the pulse sequence is altered so that all of the signals are passed through a double-quantum coherence filter, which suppresses signals with no coupling (i.e. singlets) and allows cross peaks close to the diagonal to be clearly visible by making the spectral lines much sharper. Since most singlet peaks are due to the solvent, DQF COSY is useful to suppress those unwanted peaks.

ECOSY (Exclusive COrrelation SpectroscopY) is another derivative of COSY that was made to detect small J-couplings, predominantly among multiplets, usually when J ≤ 3 Hz. Also referred to as long-range COSY, this technique involves adding a delay of about 100-400 ms to the pulse sequence. However, there is more relaxation that is occurring during this delay, which causes a loss of magnetization, and therefore a loss of signal intensity. This experiment would be advantageous for one who would like to further investigate whether or not a certain coupling exists that did not appear in the regular COSY spectrum.

GS-COSY (Gradient Selective COSY) is a very applied offshoot of COSY since it eliminates the need for what is known as phase cycling. Phase cycling is a method in which the phase of the pulses is varied in such a way to eliminate unwanted signals in the spectrum, due to the multiple ways which magnetization can be aligned or transferred, or even due to instrument hardware. In practical terms, this means that by eliminating phase cycling, GS-COSY can produce a cleaner spectrum (less digital artifacts) in much less time than can normal COSY.

Another variation of COSY is COSY-45, which administers a pulse at 45° to the sample, unlike DQF COSY which administers a pulse perpendicular to the sample. This technique is useful because one can elucidate the sign of the coupling constant by looking at the shape of the peak and in which direction it is oriented. Knowing the sign of the coupling constant can be useful in discriminating between vicinal and geminal couplings. However, COSY-45 is less sensitive than other COSY experiments that use a 90° RF pulse.

 

1H-1H COSY is used for clearly indicate correlation with coupled protons. A point of entry into a COSY spectrum is one of the keys to predict information from it successfully. 

Relation of Coupling protons is determined by cross peaks(correlation peaks) and in the COSY spectrum.

In other words, Diagonal peaks by lines are coupled to each other. Figure  indicates that there are correlation peaks between proton H1 and H2 as well as between H2 and H4. This means the H2 coupled to H1 and H4. 

..............

Below is a portion of a 1H-1H COSY-45 spectrum for an unknown compound. The COSY data exhibits 5 correlations: 3 diagonal and 2 off-diagonal. The 2 off-diagonal correlations at (1.45, 2.36) and (2.36, 1.45) ppm indicate 4 possible 1H-C-1H structural configurations between the protons at 1.45 and 2.36 ppm. The issue is compounded by the fact that for each off-diagonal correlation, there are 4 possibilities to consider when trying to build a fragment(s). Keep in mind that COSY data by itself may not constitute adequate information to narrow down the possibilities. However, noting the multiple possibilities will ensure that nothing is overlooked.

 

Note: the intensity of the off-diagonal correlations, judged by the number of contours in respect to the diagonal correlations, may provide a clue in eliminating some of the possibilities. However, this is dependent on how the data is collected and/or on the dihedral angles of the coupled protons.

One of the trickiest parts of interpreting a 1H-1H COSY experiment is deciding how to classify the correlations. The goal of this puzzle is to assess the COSY correlations and narrow down a set of fragments that support the data.

An expansion of the aromatic region of a 1H NMR spectrum shows four methine multiplets at 6.61, 7.15, 7.22 and 7.58 ppm with coupling constants less than 2.5 Hz. The 1H-1H COSY spectrum below points to two off-diagonal correlations: between 6.61 and 7.15 ppm and another at 7.22 and 7.58 ppm. Are the correlations on the COSY experiment relating to 3JHH or 4JHH or 5JHH coupling responses?

2-Bromobutane CH3CH2CHBrCH3

  COSY OF 2 BROMOBUTANE

 


2-Bromobutane
CH₃CH₂CHBrCH<sub>3



1H NMR

 



IR

 








MASS

 




13 C NMR

 



 2 BROMOBUTANE</sub>














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RES-TOCSY: a simple approach to resolve overlapped 1H NMR spectra of enantiomers

Chiral auxiliaries are used for the NMR spectroscopic study of enantiomers. Often the presence of impurities, overlap of peaks, line broadening and the multiplicity pattern restrict the chiral analysis in the 1D ¹H NMR spectrum. The present study introduces a simple 2D ¹H NMR experiment to unravel the overlapped spectrum. The experiment separates the spectra of enantiomers, thereby allowing the unambiguous assignment of all the coupled peaks and the measurement of enantiomeric excess (ee) from a single experiment even in combinatorial mixtures.

http://pubs.rsc.org/en/content/articlelanding/2014/ob/c3ob42087f#!divAbstract

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A stereochemical perspective from endo and exocyclic chiral centres

Diastereoselective syntheses of 3-aryl-(S/R)-6-methyl-1-[(S/R)-1-phenylethyl)]-2-thioxotetrahydro pyrimidin-4(1H)-ones were achieved in good yields by the condensation of aryl isothiocyanates with ethyl 3-(1-phenylethylamino)butanoate in a one-pot reaction. Benzylationof these substrates illustrated that the orientations of the exocylic and endocylic groupsdetermine the stereochemical outcome of the product formed.

http://pubs.rsc.org/en/content/articlelanding/2010/ob/c0ob00230e#!divAbstract

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The Cocaine Diastereoisomers

Table 1.
Melting point data °C.

Compound	Findlay4	Sinnema et al.5	Willstätter et al.7	Merck9	This Work
(-)-Cocaine	-	-	-	98	96-97
(+)-Cocaine	-	-	98	-	-
(±)-Cocaine	79-80	-	79-80	-	74-75
(-)-Cocaine HCl	-	-	192	195	194-196
(±)-Cocaine HCl	187	-	187	-	186-188
(+)-Pseudococaine	-	-	43-45	47	-
(±)-Pseudococaine	81.5	-	81.5	-	oil
(+)-Pseudococaine HCl	-	-	205	210	208-210
(±)-Pseudococaine HCl	205.5	-	205-206	-	202-204
(±)-Allococaine	93-95	95-97	-	-	94-96
(±)-Allococaine HCl	oil	oil	-	-	oil
(±)-Pseudoallococaine	82-84	83-84	-	-	oil
(±)-Pseudoallococaine HCl	201.5	209-210	-	-	203-205

\\\

Proton Nuclear Magnetic Resonance

¹H-NMR Spectra
(in CDCl₃)

Fig. 4.	Cocaine
Fig. 5.	Pseudococaine
Fig. 6.	Allococaine
Fig. 7.	Pseudoallococaine

Proton NMR spectra of the diastereoisomeric cocaines are significantly different. Major differences are seen in the chemical shifts associated with the respective C-3 protons. In addition, the coupling patterns that arise from the vicinal coupling of C-2 and C-4 protons with the C-3 proton are first order and relate nicely to the Karplus equation⁵.

That the diastereoisomeric cocaines, can easily be distinguished by observation of the chemical shifts and coupling patterns associated with the C-3 proton does not imply that other differences do not exist. In fact, virtually every proton in these molecules exhibits a different chemical shift or coupling pattern

COCAINE

1. Findlay, S. P., J. Am. Chem. Soc., 76, 2855-2862 (1954)
2. Findlay, S. P., J. Org. Chem., 21, 711 (1956)
3. Findlay, S. P., J. Org. Chem., 22, 1385-1394 (1957)
4. Findlay, S. P., J. Org. Chem., 24, 1540-1550 (1959)
5. A. Sinnema, L Maat, AJ vd.Gugten, HC Beyerman, Rec. Trav. Chim. Pays-Bas 87, 1027-1041 (1968)
6. Robinson, R., Journal of the Chemical Society (London), 111, 762-768 (1917)
7. Willstätter, R., Wolfes, O., and Mäder, H., Justus Liebigs Annalen der Chemie, 434, 111-139 (1923)
8. Fulton, C. C., Modern Microcrystal Tests for Drugs, Wiley, New York, 1969, pp. 21-24.
9. The Merck Index, 9th ed., 1976, Merck and Co., Rahway, N.J.
10. Fulton, C. C., Modern Microcrystal Test for Drugs, Wiley, New York, 1969, pp. 318-19.
11. Clarke, E. G. C., Isolation and Identification of Drugs, Pharmaceutical Press, London, 1971, pp. 139-41.
12. Fulton, C. G., Modern Microcrystal Tests for Drugs, Wiley, New York, 1969, p. 301.
13. Cooper, D. A. and Allen, A. C., "A Mechanistic Interpretation of the Mass Spectrum of Cocaine",
    31st Annual Meeting of the American Academy of Forensic Sciences, Atlanta, Georgia, Feb. 1979.
14. Eliel, E. L., Stereochemistry of Carbon Compounds, McGraw-Hill, New York, 1962, pp. 43-47.

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