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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DRUG APPROVALS BY DR ANTHONY MELVIN CRASTO

 
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DRUG APPROVALS BY DR ANTHONY MELVIN CRASTO

Short lived metal complexes of alkanes and other weakly binding ligands

Simple saturated hydrocarbons, alkanes, are extremely poor ligands and complexes containing alkane molecules acting as discrete ligands are typically very short lived, with lifetimes less than ~100 ms at room temperature. We can observe alkane complexes and other reactive species using NMR spectroscopy by using a combination of photochemistry to generate the reactive alkane complex and low temperatures to stabilise it for sufficient time to allow characterization.

Photochemical generation of an alkane complex.

In this case, absorption of a UV photon results in the loss of a carbonyl ligand from CpRe(CO)₃. Cyclopentane replaces the CO as a ligand, forming an σ-alkane complex. These molecules are of interest both from the standpoint of basic coordination chemistry (an agostic interaction) and because alkane complexes are known intermediates in the C-H activation process, a potentially useful route to functionalising these relatively unreactive hydrocarbons found in petroleum and an intense area of research around the world.
We have characterised several types of alkane complexes including the rhenium and tungsten pentane complexes, CpRe(CO)₂(pentane-η²-C²,H²) and (η⁶-hexaethylbenzene)W(CO)₂(pentane-η²-C¹,H¹) shown below. We have extended our work on alkanes to include binding of xenon in complexes of the [CpRe(CO)(PF₃)(Xe)] type. The structures shown below are all calculated using density functional theory (DFT) methods. We are increasingly employing DFT and ab initio quantum chemical methods in ths project to aid the elucidation of the stutructure and reactivity of these fascinating compounds. Frequently, this work is done in collaboration with groups from around Australia and overseas (see references below).

Set up for an in situ photolysis experiment at low temperature using an excimer laser and a 600 MHz NMR spectrometer.

Monitoring the formation and disappearance of an alkane complex at -90 °C.

Current research is aimed at answering questions such as:

• How does the alkane bind to the metal centre?
• Can we make more stable alkane complexes?
• Can we do useful chemistry with alkane complexes?
• Can we observe more complexes with ligands that bind even more weakly than alkanes using NMR?

We are constantly seeking to expand the applicability of the in situ photolysis with NMR detection technique to new areas in inorganic and organic chemistry and welcome the opportunity to forge new collaborations in the area.
Key references:
Young, R.D.; Lawes, D.J.; Hill, A.F.; Ball, G.E. "Observation of a tungsten alkane σ-complex showing selective binding of methyl groups using FTIR and NMR spectroscopies" J. Am. Chem. Soc., 2012, 134, Article ASAP DOI: 10.1021/ja300281s.
Ball, G.E.; Darwish, T.A; Geftakis, S.; George, M.W.; Lawes, D.J.; Portius, P.; Rourke, J.P. "Characterization of an Organometallic Xenon Complex using NMR and IR Spectroscopy." Proc. Natl. Acad. Sci. USA., 2005, 102, 1853.
Lawes, D.J.; Geftakis, S.; Ball, G.E. "Insight into binding of alkanes to transition metals from NMR spectroscopy of isomeric pentane and isotopically labelled alkane complexes." J. Am. Chem. Soc., 2005, 127, 4134.
Geftakis, S.; Ball, G.E. "Direct Observation of a Transition Metal Alkane Complex, CpRe(CO)₂(cyclopentane), Using NMR Spectroscopy", J. Am. Chem. Soc., 1998, 120, 9953.

 
 amcrasto@gmail.com

http://newdrugapprovals.org/

DRUG APPROVALS BY DR ANTHONY MELVIN CRASTO

 
 amcrasto@gmail.com

http://newdrugapprovals.org/

DRUG APPROVALS BY DR ANTHONY MELVIN CRASTO

NMR characterisation of substituted fullerenes

• NMR characterisation of substituted fullerenes

- with Prof. Steve Pyne and A/Prof. Paul Keller, University of Wollongong.
Since its discovery in 1985, [60]-fullerene (C₆₀) and its homologues have shown promise for exciting new developments and applications in areas such as medicinal chemistry and materials science. The synthetic goals of this work, carried out at the University of Wollongong, involves developing methods for preparing optically active, multi-functionalized fullerene derivatives in a regioselective and diastereoselective manner and investigating their applications. At UNSW, we are using NMR spectroscopy as a robust and definitive technique for the assignment of regiochemistry in these compounds.

A trans-4 difunctionalized fullerene and an INADEQUATE NMR experiment used to identify the substitution pattern.

References:
Ball, G.E.; Burley, G.A.; Chaker, L.; Hawkins, B.C.; Williams, J.R.; Keller, P.A.; Pyne, S.G. "Structural reassignment of the mono- and bis-addition products from the addition reactions of N-(Diphenylmethylene)glycinate esters to [60]fullerene under Bingel conditions." J. Org. Chem., 2005, 70, 8572.

Structure, mechanism and exchange processes in molecules

Structure, mechanism and exchange processes in molecules

Using NMR spectroscopy as our primary tool, backed up wuth computational methods, structures of compounds and the mechanism of fluxional processes is explored. A couple of examples one each from inorganic and organic chemistry are shown below.

• Hydrides and dihydrogen complexes

Metal hydride complexes are well known to be excellent catalysts for hydrogenation reactions and have the potential to act as hydrogen storage materials. Dihydrogen complexes contain a hydrogen molecule that is essentially intact but acting as a ligand. We have an ongoing interest in these classes of compounds. We are interested in answering questions such as can we accurately determine the location of the hydrogen atoms in such molecules and interatomic distances? This can be difficult using crystallographic methods.

Hydride and dihydrogen complexes also often show interesting mechanisms of fluxionality, i.e., the hydrogen atoms interchange their positions within the complexes. NMR is ideally suited to revealing how these molecular reorientations take place.

Studying exchange in a polyhydride complex

• Photochromic organic compounds

Building on some high profile work led by the CSIRO, we have studied certain merocyanine molecules, similar to the type found in shade-changing photochromic lenses of spectacles. We have shown that a relatively rapid isomerization of different isomers can occur and that this process is catalyzed by acid.

Elucidating an acid catalysed isomerization process in a photomerocyanine compound at -80 °C.

We are currently investigating several other photochemically generated organic reactive intermediates with NMR spectroscopy at low temperatures.
References:
Yee, L. H.; Hanley, T.; Evans, R. A.; Davis, T. P.; Ball, G. E. "Photochromic Spirooxazines Functionalized with Oligomers: Investigation of Core-Oligomer Interactions and Photomerocyanine Isomer Interconversion Using NMR Spectroscopy and DFT" J. Org. Chem. 2010, 75, 2851-2860.
Evans, R.A; Hanley, T.L.; Skidmore, M.A.; Davis, T.P.; Such, G.K.; Yee, L.H; Ball, G.E.; Lewis, D.A. "Lubrication and Control of Nano-Mechanical Processes in Polymers." Nature Materials, 2005, 4, 193.

 
 amcrasto@gmail.com

http://newdrugapprovals.org/

DRUG APPROVALS BY DR ANTHONY MELVIN CRASTO

 
 amcrasto@gmail.com

http://newdrugapprovals.org/

DRUG APPROVALS BY DR ANTHONY MELVIN CRASTO

The Carlomagno group uses NMR spectroscopy in combination with biochemical and biophysical techniques to study the structure and dynamics of biomolecular complexes.

Figure 2: Schematic representation of the principle of the INPHARMA NOEs.

READ AT
The Carlomagno group uses NMR spectroscopy in combination with biochemical and biophysical techniques to study the structure and dynamics of biomolecular complexes.
http://www.embl.de/nmr/  LINK

Figure 1: Structure of the RNA-methylating machinery Box C/D RNP shows that only one pair of proteins (blue) can add methyl groups to the RNA (red) at a time (Lapinate et al., 2013).

 
 amcrasto@gmail.com

http://newdrugapprovals.org/

DRUG APPROVALS BY DR ANTHONY MELVIN CRASTO

 
 amcrasto@gmail.com

http://newdrugapprovals.org/

DRUG APPROVALS BY DR ANTHONY MELVIN CRASTO