SUCROSE.....TOCSY, COSY ETC

SUCROSE

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.

Model of sucrose molecule

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.











COSY

COSY (Correlated Spectroscopy) was one of the first multidimensional experiments.  Cross peaks in COSY are between protons that are coupled. COSY tells you "what proton is coupled to what proton." Protons that are two, three, or sometimes four bonds apart may show cross peaks. The magnitude of the couplings affects the peak intensity. For small couplings, such as four-bond couplings, or three-bond couplings where the dihedral angle is near 90 degrees, the peak intensity will be low. The peaks along the diagonal are the projection of the one dimensional spectrum. The cross peaks are symmetrical about the diagonal.
Below is a 500 MHz spectrum of sucrose in D2O. Some cross peaks between protons that are separated by 3 bonds are emphasized. For example, the red cross peak labled 1 links proton 1 and proton 2.






COSY-DQF


The COSY-DQF (Cosy Double Quantum Filter) provides two advantages over the magnitude COSY. Higher resolution is possible and multiplet fine structure can be seen. This may allow proton-proton couplings to be measured. When there are several couplings to a given proton and the multiplet is complex, however, often the cross peak is not interpretable because of cancellation of multiplet components. The E.COSY experiment is usually necessary for coupling measurement.
The DQF version reduces the diagonal dispersive peaks of the COSY experiment but sensitivity is reduced by one half. In addition, uncoupled spins such as water are removed. This experiment is seldom used for small molecules.
Below is a 500 MHz spectrum of sucrose.

H-H Homonuclear Decoupling

During H-H Homonuclear Decoupling, a proton resonance is irradiated during acquisition. The irradiated coupling is then collapsed in its partner's mulitplet. This allows one to identify the coupling network, i.e., determine what protons are coupled. Since the multiplet is simplified, it can also allow one to measure the J value for the remaining coupling.

The proton spectrum of sucrose at 500 MHz is shown in the lower trace. In the upper spectrum, the 1 proton has been irradiated, collapsing the 2 proton (which is a doublet of doublets from J interactions with the 1 and 3 protons) into a doublet.

HSQC

The HSQC (Heteronuclear Single Quantum Coherence) experiment is used to determine proton-carbon single bond correlations, where the protons lie along the observed F2 (X) axis and the carbons are along the F1 (Y) axis.
An edited HSQC spectrum of sucrose at 500 MHz is shown below. The edited version shows CH2 peaks in blue and CH (and CH3-although there are none in sucrose) peaks in red in opposite phase. Edited HSQC provides the same information as the DEPT135 experiment but HSQC is much more sensitive.
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Selective 1D TOCSY

1D TOCSY (Total correlation spectroscopy), like 2D TOCSY gives correlations between all protons within a given spin system. In 1D TOCSY, one peak is selected and signal is transferred from it to to all J-coupled protons in a stepwise process. Instead of cross peaks, magnetization transfer is seen as increased multiplet intensity.

TOCSY spectra are especially useful for carbohydrates since each ring is a discrete spin system separated by oxygen. TOCSY transfer correlates protons within each ring. Shown below is a series of selective TOCSY spectra of sucrose where the anomeric proton (5.3 ppm) has been selected and the mixing time and thus, the number of transfers, has been changed. In the top spectrum (21 ms mixing) only 1-2 step transfer is seen. At longer mixing times, progressively more transfers are seen. The 1D version is especially useful since this whole series of spectra was taken in less than 10 minutes. (A 2D TOCSY with a single mixing time requires more than 1 hour.)

1D selective TOCSY spectrum of sucrose at 500 MHz

HSQC-TOCSY


The HSQC-TOCSY (Heteronuclear Single Quantum Coherence-Total Correaltion Spectroscopy) is a 2D TOCSY that has been resolved into the carbon dimension. This is especially useful when overlap in the proton spectrum prevents analysis since often the corresponding carbons will be resolved. Cross peaks are seen between all J coupled protons in a spin system and each carbon in that spin system.

Below is a spectrum of sucrose taken at 500 MHz. Notice the patterns of the two spin system groups designated in red and green representing the two sugar moieties. The oxygen between the two rings breaks up the magnetization transfer between the two groups. To interpret the HSQC-TOCSY spectrum, one should start with the direct correlation HSQC peaks. For example, in the HSQC the 1 (anomeric) proton and carbon are at 5.42 ppm and 92 ppm, respectively. TOCSY then transfers proton signal to coupled protons such as the 2, 3, 4, and 5 protons. These transfered signals appear at the 1 carbon frequency of 92 ppm. By looking at this anomeric carbon frequency, one can see all the protons coupled to the anomeric proton. There is no overlap problems as there would be in the proton-only 2D TOCSY - the 5, 6', and 6 protons are all very close.

HMBC

The HMBC (Heteronuclear Multiple Bond Correlation) experiment gives correlations between carbons and protons that are separated by two, three, and, sometimes in conjugated systems, four bonds. Direct one-bond correlations are suppressed.This gives connectivity information much like a proton-proton COSY. The intensity of cross peaks depends on the coupling constant, which for three-bond couplings follows the Karplus relationship. For dihedral angles near 90 degrees, the coupling is near zero. Thus, the absence of a cross peak doesn't confirm that carbon-proton pairs are many bonds apart.
Because of the wide range (0-14 Hz) of possible carbon-proton couplings, one often does two experiments. One optimized for 5 Hz couplings and the second optimized for 10 Hz. This gives the optimum signal-to-noise. Alternatively, a comprise value of 7-8 Hz can be used. There are also "accordion" versions that attempt to sample the full range of couplings.

The spectrum of sucrose at 500 MHz is shown below. The peak outlined in green shows the two bond correlation between the 2' carbon and the 1' proton. The peak outlined in red correlates the 6 carbon and 4 proton separated by 3 bonds. Note also that the 2' carbon correlates with the 1 proton across the glycosidic bond.






H2BC


The H2BC (Heteronuclear 2 Bond Correlation) experiment correlates carbons and protons that are two bonds away by using one-bond carbon-proton and proton-proton couplings. The H2BC is essentially an HSQC-COSY experiment or an HSQC-TOCSY with very short mixing time. The spectrum of sucrose at 500 MHz is shown below. The peaks outlined in green show two bond correlations between the 5' carbon and the 4' and 6' protons.To interpret the H2BC spectrum, one should start with the direct correlation HSQC peaks. For example, in the HSQC the 5' proton and carbon are at 3.9 ppm and 82 ppm, respectively. COSY then transfers proton signal to coupled 4' and 6' protons. These transferred signals appear at the 5' carbon frequency of 82 ppm but at the proton shifts of 4' and 6'.

A major disadvantage of H2BC is that quaternary carbons are not observed. Only carbons with attached protons are present. The H2BC is sometimes considered complimentary to the HMBC experiment since HMBC uses different types of couplings. That is, HMBC uses 2- and 3-bond carbon-proton while H2BC uses one-bond carbon-proton and proton-proton. Quaternary carbons are present in HMBC.

 

DEPT

The DEPT (Distortionless ehancement by Polarization Transfer) experiment is used to determine the multiplicity of carbon atoms, that is, whether they are C, CH, CH2, or CH3. The DEPT 135 experiment used at Columbia gives inverted CH2 and C groups. CH and CH3 groups are upright. The DEPT 90 produces inverted C groups and upright CH's. The CH2's and CH3's are nulled.

Below is a group of spectra taken of  sucrose at 300 MHz.    


                                     
 


ADEQUATE
The ADEQUATE (Adequate Sensitivity Double-Quantum Spectroscopy) experiment correlates two directly bonded carbons with a proton directly bonded to one of the carbons. With this experiment, one obtains connectivity information betwen carbons and protons that are two bonds away.
One problem with the HMBC experiment is that correlations are seen between protons and carbons that are two, three, and sometimes four or more bonds away. This failure to establish the number of bonds between correlated nuclei often limits the ability to distinguish between different molecular structures. In the ADEQUATE experiment, carbons and protons that are ONLY two bonds apart are correlated. This is becuase one-bond carbon-carbon and one-bond carbon-proton couplings are used.
Shown below is an ADEQUATE spectrum of  sucrose at 500 MHz. In this version, cross peaks are interpreted like the familiar HMBC cross peaks. For example, the anomeric proton, 1, has a cross peak (lableled 2-1) at the carbon frequency of 2. Likewise, the proton 2 has a cross peak (labeled 1-2) at the carbon frequency of 1.
Since one-bond carbon-carbon couplings are used, signal only arrises from molecules containing two, adjacent 13 C nuclei. For natural abundance work, this affects the sensitivity drastically since only 0.01% of molecules are seen. The original INADEQUATE experiment that used carbon detection was so insensitive that almost pure samples were necessary. The ADEQUATE is proton-detected and thus, has much higher, although still poor, sensitivity. Typically, 35 -50 mg of a small molecule are necessary for an overnight accumulation at 500 MHz with a standard probe.








 T1 Measurement


The spin-lattice relaxation time constant, T1, is a measure of how quickly equilibrium magnetization is re-established.

Show below are some proton T1 results for an inversion recovery experiment of sucrose at 300 MHz.




MASS











IR



















1H NMR



















13C NMR









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NMR PREDICTIONS

H-NMR spectral analysis

CAS NO. 57-50-1, D(+)-Sucrose H-NMR spectral analysis

C-NMR spectral analysis

CAS NO. 57-50-1, D(+)-Sucrose C-NMR spectral analysis

 
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http://newdrugapprovals.org/

DRUG APPROVALS BY DR ANTHONY MELVIN CRASTO

TOCSY and ROESY

These experiments are characterized by a mixing time during which the magnetization of a spin A, submitted to a radio wave frequency field Bl is transferred to a spin linked to A either by a dipolar coupling or by a scalar coupling. During the mixing time the magnetizations are locked according to one direction of the space defined by the direction of the field Bl and the spin frequency shifting versus the main component. The block in the spin system is commonly named spin-lock.

TOCSY ou HOHAHA :

This method allows to demonstrate scalar couplings. The pulse sequence scheme is given Fig. 23. The first 90°x impulsion rotates the magnetization vectors in the xOy plane, in which they freely evolve during the time tl, under the influence of their chemical shift and the scalar coupling constants. We apply then a spin lock on the x axis, that means a low power impulsion or set of impulsions whose duration represents the mixing time tm. During this time, the spin coherence of the system is exchanged. By increasing the mixing time we increase the number of the visible transitions. That is to say that within the same experiment, we get the informations of a COSY, of a LR COSY and of a relayed COSY. The pulse sequence widely used is base upon the MLEV-17 sequence which contains a set of 17 pulses.

Fig. 29 : Saccharine molecule

Fig. 23 : The pulse sequence TOCSY or HOHAHA

The spectrum 10 shows the result of three different mixing time for the saccharose (Fig.29). These are given in milliseconds (20, 60, l00 ms). For each mixing time, we see the appearance of new correlation spots. There is thus an increase of the number of relays with the spin lock time. These ones correspond then to long range correlations.

Spetrum 10: 20ms, 60 ms, 100ms.

Tocsy 20

Tocsy 60

Tocsy 100

ROESY ou CAMELSPIN :

The aim of the dipolar correlation experiments in two dimensions is to take advantage of the vicinity in space of some nuclei. The result of this kind of experiment is a 2D map in which the signals outside of the diagonal arise from the Over Hauser enhancement effect between two space coupled nuclei(Fig.24).

Fig. 24 : The ROESY pulse sequence

In this case, we apply a spin-lock onto the y axis. The correlation peaks arising from the ROE effect are on the opposite sign compared to the diagonal one their intensity is different from 0. However, the correlation peaks coming from a chemical exchange are of the same sign than the crossing one. The ROESY experiment allows by this way the separation of the contributions coming from the exchange and those coming from the dipolar interactions. The ROESY sequence is thus complementary of the NOESY one and it is more often used for the structural determination of the small molecules.

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/.