Inverse HETCOR

An alternative way of acquiring the data is to observe the signal. These experiments are referred to as reverse- or inverse-detected experiments, in particular the inverse HETCOR experiment is referred to as a heteronuclear multiple quantum coherence (HMQC) spectmm. The ampHtude of the H nuclei is modulated by the coupled frequencies of the C nuclei in the evolution time. The principal difficulty with this experiment is that the C nuclei must be decoupled from the H spectmm. Techniques used to do this are called GARP and WALTZ sequences. The information is the same as that of the standard HETCOR except that the F and F axes have been switched. The obvious advantage to this experiment is the significant increase in sensitivity that occurs by observing H rather than C. 

Figure I. The methylene region of the inverse-HETCOR spectrum of A/M copolymer.

The overlapping and broad signals in the carbon and proton spectra was assigned completely to various compositional and configurational sequences with the help of inverse HETCOR and TOCSY / DQFCOSY experiments. 

GENERAL APPEARANCE OF INVERSE 2D SPECTRA 11.2.1 2D HETCOR versus 2D HSQC/HMQC... 

H multiplets observed in F2), but we have to solve the technical problems of removing the 12C-bound 1H artifact (100 times larger than the desired signal), and we would like to be able to decouple 13C. Thus, there are two important differences between HETCOR and the inverse one-bond experiments HSQC and HMQC ... 

ID ll spectrum, and crosspeaks are arranged symmetrically around the diagonal. There is only one radio frequency channel in a homonuclear experiment, the H channel, so the center of the spectral window (set by the exact frequency of pulses and of the reference frequency in the receiver) is the same in If and F (Varian tof, Bruker ol). The spectral widths should be set to the same value in both dimensions, leading to a square data matrix. Heteronuclear experiments have no diagonal, and two separate radio frequency channels are used (transmitter for F2, decoupler for F ) with two independently set spectral windows (Varian tof and dof, sw, and swl, Bruker ol and o2, sw(If), and sw(I )). Heteronuclear experiments can be further subdivided into direct (HETCOR) and inverse (HSQC, HMQC, HMBC) experiments. Direct experiments detect the X nucleus (e.g., 13C) in the directly detected dimension (Ff) using a direct probe (13C coil on the inside, closest to the sample, H coil on the outside), and inverse experiments detect XH in the To dimension using an inverse probe (XH coil on the inside, 13C coil outside). 

HETCOR has been largely replaced by the far more sensitive inverse experiments, HSQC and HMQC. Because HETCOR is a 13C-detected experiment, it is called direct ... 

For smaller quantities of compounds more sensitive inverse detected techniques are available, such as HMQC ( IH-I C one bond correlation via heteronuclear multiple quantum coherence, analogous to HETCOR) and HMBC (proton detected heteronuclear multiple bond correlation spectroscopy) (15). The last provide, in addition to the intraresidue multiple bond correlations, interresidue correlations between the anomeric carbon and the aglycone protons.We follow this general strategy for the structural determination of tri terpenoid saponins of Bupleurum fruticosum (16) andArdisia japonica (9). 

Glass et al., were the first to mention the enormous sensitivity gain achievable by H-detected Se correlation spectroscopy " They showed for a number of representative organoselenium compound that the theoretical SIN ratio enhancement factor of 72, as compared to direct Se detection, can nearly be achieved so that this technique should be applied for recording biomolecules in low concentration, e.g., in selenoproteins. If inverse detection is combined with isotopic enrichment of Se, the factor may be increased up to 800. Selenomethionine, selenocysteine, and related compounds have been recorded using Se, H HETCOR experiments. ... 

D Correlation Spectroscopy. A simple, quahtative approach has been described for the determination of membrane protein secondary structure and topology in lipid bilayer membranes." The new approach is based on the observation of wheel-like resonance patterns in the NMR H- N/ N polarization inversion with spin exchange at the magic angle (PISEMA) and H/ N HETCOR spectra of membrane proteins in oriented lipid bilayers. These patterns, named Pisa wheels, have been previously shown to reflect helical wheel projections of residues that are characteristic of a-helices associated with membranes. This study extends the analysis of these patterns to P-strands associated with membranes and demonstrates that, as for the case of a-helices, Pisa wheels are extremely sensitive to the tilt, rotation, and twist of P-strands in the membrane and provide a sensitive, visually accessible, qualitative index of membrane protein secondary structure and topology. 

Heteronuclear correlation experiments with IR detection in the direct acquisition period t2 are often called inverse detected experiments. The term "inverse" is also used to describe probeheads that are constructed with the coil as the inner coil and the rf coil for the heteronuclear frequency as the outer coil. The outer coil has a lower sensitivity because of the lower fill factor. This term may also be used to distinguish between two classes of experiments. The HETCOR and COLOC experiments belong to the class of direct detection experiments with take advantage of the coherence transfer from the sensitive nucleus to the relatively insensitive heteronucleus. This type of experiment is illustrated by the first entry in Table 5.22. However experiments in this category are no longer popular and have been superseded by inverse detected experiments, shown in the second entry in Table 5.22. In a comparison of the different types of experiments the IH detected heteronuclear correlation experiments have three distinct advantages over the detected experiment ... 

FIGURE 6.18. The 300-MHz (F2) HMQC (also called inverse detected HETCOR ) spectrum of caryophyllene oxide. 

The HMQC spectrum is a proton-detected (inverse detection) HETCOR (Chapter 6) it shows lJ CH coupling. Table 5.9 in Chapter 5 allows us to arrange the aromatic unsubstituled carbon atoms as C-6, C-4, C-3 from left to right. The HMQC spectrum confirms the same sequence for H-6, H-4, H-3. The aromatic, unsubstituted carbon atoms can now be correlated with the firmly assigned aliphatic protons. The substituted aromatic carbon atoms cannot yet be assigned. 

Clearly the development and dissemination of two-dimensional NMR techniques has had a profound impact in natural products structure elucidation. Some techniques, COSY and variants of the C-detected heteronuclear chemical shift correlation (variously referred to as HETCOR, HC-COSY, etc.) experiment, have been widely used by the natural products chemistry community. Inverse-detected heteronuclear shift correlation techniques are becoming recognized as a powerful adjunct to the COSY experiment and a replacement for their less sensitive and, in some cases, less versatile C-detected predecessor experiments (Martin and Crouch 1991). 

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