Chapter 6(HSQC/HMQC/HMBC)

Proton-X Nucleus Correlation Correlate proton with the carbon or other X-nucleus that the proton is bound to in 2D. Indirect detection of carbon experiment -the experiment directly detects proton (detect dimension or t2; the fid is proton) and indirectly detects carbon (indirect detect dimension or t1). Why would you want to correlate Proton chemical shift with X nucleus (usually carbon or nitrogen) chemical shift? 1) Overlap of proton resonances 2) Assignment of carbon (or nitrogen...) spectra based upon proton spectrum or assignment of proton spectrum based upon carbon (or nitrogen..) 3) Indirect detection of carbon (or nitrogen) is more sensitive than direct observation or carbon (or nitrogen).

Proton-X Nucleus Correlation What are the advantages of 1D direct detection of carbon versus 2D proton-carbon correlation? 1) Carbons, with no protons attached, do not show up in direct proton-carbon correlations. 2) Resolution of 1D carbon detect should be better than 2D indirect detect of carbon. 3) 2D takes a little longer if sample is very concentrated

Sensitivity of H-X Correlations

2D Proton-Carbon Correlation

HSQC/HMQC HSQC = Heteronuclear Single Quantum Coherence HMQC = Heteronuclear Multiple Quantum Coherence What is the difference between HSQC and HMQC? The two pulse sequences are very different, one depends upon the build up of single quantum coherence (as in INEPT), the other multiple quantum coherence (as in DEPT), but the results look nearly the same to the user. How do the experiments work? You start with magnetization on the proton and then transfer the magnetization to the carbon, record the chemical shift of the carbon, transfer back to proton, and detect the magnetization on proton.

HMQC HMQC Pulse Sequence: 1 H 90º X  180º X  Acquire X 90º X  t1  90º X Decouple  = 1/2J CH = ~1/(140*2) for aliphatic = ~1/(200*2) for aromatic

HSQC/HMQC HSQC is not affected by proton proton couplings during t1 as only X chemical shift evolves. HSQC is more complicated pulse sequence; it is more susceptible to signal loss due to incorrect pulse widths, RF inhomogeneity, and off- resonance effects.

2 Major Versions on HSQC for Small Molecules 1) Multiplicity edited HSQC (gHSQC) 2) Sensitivity Enhanced HSQC (gHSQCSE)

HSQC Parameters

HETCOR HETCOR- HETeronuclear shift CORrelation H/X correlation with detection of the X nucleus rather than 1 H, which obviously will be less sensitive due to the difference in the  for 1 H and any other nucleus The advantages of HETCOR over HMQC are: 1) Reduction in the amount of time that magnetization is in the x-y plane when it can be lost due to T2 relaxation 2) Reduction of noise in t1, but modern versions of the HMQC are not as bad as the original HMQC for t1 noise, so this point is minor 3) Resolution in 13 C is better, as 13 C is now the dimension detected (remember that np >> ni). Of course, this means that resolution in 1 H is much worse. Generally, resolution in 1 H is preferred over resolution in 13 C as there is greater resonance dispersion in 13 C than 1 H.

HETCOR

HMBC (Heteronuclear shift correlations through Multiple Bond Connectivities) Proton resonances are correlated by direct coupling through multiple bonds to carbon resonances while attempting to filter out the 1-bond proton-carbon correlations. Thus, the HMBC depends upon the magnitude of the 2 J CH, 3 J CH and 4 J CH. As in the COSY type experiments, the three-bond and four-bond scalar coupling constants are dependent upon torsion angle, in this case that is the proton-carbon torsion angle.

HMBC The one-bond carbon-proton couplings are supposed to be filtered out, but filtering of resonances is never 100%, so normally there are some weak 1-bond correlations observed between proton and carbon.

HMBC Parameters

Other Methods for Multiple Bond Correlation of 1 H and X Nuclei I. Heteronuclear COSY This is a 1 H detect experiment so it is sensitive, with antiphase multiplet crosspeaks as in DQF- COSY. 1 H 90º X Acquire X90º X —— t1—— 90º X Decouple

Other Methods for Multiple Bond Correlation of 1 H and X Nuclei The advantages of the COSY-type experiment versus the HMQC-type (HMBC) are: 1) There is no HMQC-type delay that is correlated to the coupling constant. The crosspeak intensity is mostly correlated to the magnitude of the J HX coupling constant as in a COSY, and the T2 relaxation. 2) The J HX coupling constant is easily measured. Since the 3 J HX coupling constant has torsion angle information according to the Karplus relationship, precise measurement of the 3 J HX can be useful data. 3) There is a greater loss of magnetization in the HMQC type experiment due to T2 relaxation because the HMQC delay is longer than the delays during the COSY type experiment. The primary advantages of HMBC over the heteronuclear COSY: 1) Higher sensitivity overall, as the Heteronuclear COSY is a 1 H detect experiment but the initial pulse is on X. 2) Difficult to remove 1-bond HX couplings in heteronuclear COSY

II. CIGAR (Constant time Inverse-detected Gradient Accordion rescaled long- range heteronuclear multiple bond correlation) Modified HMBC type experiment to achieve better resolution in t1 and improve the signal/noise for crosspeaks with coupling constants different than input value. CIGAR offers slight improvement on IMPEACH-MBC (IMproved PErformance ACcordion Heteronuclear Multiple-Bond Correlation) Advantages of CIGAR over HMBC: 1) Somewhat better resolution in t1 dimension 2) Improved signal/noise for crosspeaks with coupling constants different from input value 3) Can potentially observe difference in 2 J vs. 3 J Disadvantages: 1) Overall signal/noise is poorer for CIGAR 2) T2 relaxation is a bigger problem as the CIGAR pulse sequence is longer

HSQC-TOCSY First, an HSQC correlating the proton and nitrogen or carbon, then a 1H/1H TOCSY. When there are resonances with coincidental chemical shifts in proton but not carbon, you can acquire a HSQC-TOCSY and determine which resonance is which. This can also be acquired as 3D proton/carbon/proton as well.

First, HSQC, record 13 C shift Then, TOCSY, detect proton HSQC-TOCSY

HSQC-TOCSY on Sugar

HSQC TOCSY SPECTRUM/DEMO

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