HMBC

As we noted in Section 10.2, HMBC is a slighdy modified version of HMQC that is designed to emphasize couplings through more than one heteronuclear bond (e.g., 13C—C—C—H). One pulse sequence for HMBC, as illustrated in Fig. 12.11, is identical to that in Fig. 12.10 with the addition of a 90° pulse and adjustment of timing and phase cycling. 

FIGURE 12.11 Pulse sequence for the heteronuclear multiple bond correlation experiment. A = 1 /(21/) and A = l/(2 7), where [J and J are spin couplings between I and S through one and n bonds, respectively.The first S pulse, marked 90, is cycled through + x and —X. See text for discussion of the state of the spin system at the times indicated. 

When collected in a phase-sensitive mode, HMBC cross peaks are found to have a mixed phase character. That is, we cannot phase HMBC cross peaks so that they are purely absorptive. The use of pulsed field gradients for the purpose of coherence selection in the HMBC experiment (gHMBC) renders a nonphase-sensitive 2-D data set. This latter method is generally preferred because phasing of the spectrum is not required. 

The HMBC experiment typically requires roughly four times more scans per tj time increment (to obtain a spectrum with a comparable signal-to-noise ratio) than do the HMQC and HSQC experiments. Thus, the HMBC experiment takes three or four times longer to acquire than the HMQC or HSQC experiment (involving the same two nuclides, e.g., H and C). This extensive scanning is required because the sensitivity of the HMBC experiment is poorer than that of the HMQC or HSQC experiments. 

It generally holds that the intensity of an HMBC cross peak drops off as the number of bonds separating the two coupled nuclides (normally H and C) increases because cross peak intensity roughly mirrors the magnitude of the J-coupling between the two species. 

Karplus diagram. A plot showing the Karplus relationship. 

Strong HMBC cross peaks are often observed between H s and C s that are two and three bonds distant, provided that the geminal and vicinal (dihedral) hond angles are not such that the coupling is at or near zero (recall the Karplus diagrams presented in Sections 6.4 and 

---

A H(detected)- C shift correlation spectrum (conmion acronym HMQC, for heteronuclear multiple quantum coherence, but sometimes also called COSY) is a rapid way to assign peaks from protonated carbons, once the hydrogen peaks are identified. With changes in pulse timings, this can also become the HMBC (l eteronuclear multiple bond coimectivity) experiment, where the correlations are made via the... 

Because of the complexity of the polyether antibiotics tittle progress has been made in stmcture determination by the chemical degradation route. X-ray methods were the techniques most successfully applied for the early stmcture elucidations. Monensin, X206, lasalocid, lysocellin, and salinomycin were included in nineteen distinct polyether x-ray analyses reported in 1983 (190). Use of mass spectrometry (191), and H (192) and nmr (141) are also reviewed. More recently, innovative developments in these latter techniques have resulted in increased applications for stmcture determinations. Eor example, heteronuclear multiple bond connectivity (hmbc) and homonuclear Hartmann-Hahn spectroscopy were used to solve the stmcture of portimicin (14) (193). East atom bombardment mass spectrometry was used in solving the stmctures of maduramicin alpha and co-factors (58). 

Other two-dimensional techniques, such as COSY (122), DEPT (123), HOHAHA, soHd state (124) etc. give varying degrees of success when apphed to the stmcture-property relationship of cellulose triesters. The recent appHcation of multiple-bond correlation (HMBC) spectroscopy for... 

Two-dimensional C//correlations such as C//COSY or HC HMQC and HSQC provide the Jqh connectivities, and thereby apply only to those C atoms which are linked to H and not to non-protonated C atoms. Modifications of these techniques, also applicable to quaternary C atoms, are those which are adjusted to the smaller Jqh and Jqh couplings (2-25 Hz, Tables 2.8 and 2.9) Experiments that probe these couplings include the CH COLOC (correlation via long range couplings) with carbon-13 detection (Fig. 2.16) and HC HMBC (heteronuclear multiple bond coherence) with the much more sensitive proton detection (Fig. 2.17)... 

These two-dimensional CH shift correlations indicate CH relationships through two and more bonds (predominantly Jch and Jch connectivities) in addition to more or less suppressed Jch relationships which are in any case established from the CH COSY contour diagram. Format and analysis of the CH COLOC or HMBC plots correspond to those of a C//COSY or HSQC experiment, as is shown for a-pinene (1) in Figs. 2.14 - 2.17. 

Figure 2.17. HC HMBC experiment of a-pinene [ CDCb, 5 % v/v, 25 °C, 125 MHz for C, 500 MHz for H, 16 scans, 256 experiments, contour plot]. This experiment gives the same information as Fig. 2.16 within 24 min instead of 8 hrs required for the CH-COLOC in Fig. 2.16 due to higher sensitivity because of proton detection and stronger magnetic field. Deviations of proton shifts from those in Fig. 2.16 arise from the change of the solvent. The methylene protons collapsing in Fig. 2.16 at Sh =2.19 (200 MHz) display in this experiment an AB system with Sa = 2.17 and Sb = 2.21 (500 MHz)...

CH or HC COSY (HMQC) CH bonds CH COLOC or HC HMBC. Jch and Jch relationships between carbon and protons... 

Carbon atoms and protons are assigned by means of the proton-carbon connectivities as identified in the HC HSQC and HMBC experiment (b and c). The latter also permits the derivation of the connection of the ethyl groups to the porphyrin ring. The cross signals in the relevant part a of the HH COSY plot (a) are used to connect the methyl and methylene subunits to the ethyl groups. 

HMBC Heteronuclear multiple bond correlation, inverse CH correlation via long-range CH coupling, same format and information as described for ( C detected) CH COLOC but much more sensitive (therefore less time-consuming) because of H detection... 

Proton resonances for aU residues were assigned using a combination of COSY, TOCSY, HSQC, and HMBC experiments. The large values of y(NH/H-C(/9)) and the small values of J(H-C(a)/H-C(y9) were indicative of antiperiplanar and synclinal arrangements respectively, around those bonds. In addition, medium-range NOE connectivities H-C(/ )i/NH +i, H-C(a)i/NH, + i, NHi/NH +i were consistent... 

Heteronuclear Multiple-Bond Connectivity (HMBC) Spectra... 

H/ C Long-Range Interactions from the HMBC Spectrum... 

The HMBC spectrum of podophyllotoxin is shown. The cross-peaks in the HMBC spectrum represent long-range heteronuclear H/ C interactions within the same substructure or between different substructures. Interpretation should start with a readily assignable carbon (or proton), and then you identify the proton/s (or carbon/s) with which it has coupling interactions. Then proceed from these protons, and look for the carbon two, three, or, occasionally, four bonds away. One-bond heteronuclear interactions may also appear in HMBC spectrum. 

The H-NMR and C-NMR chemical shifts have been assigned and substractures have been deduced on the basis of COSY-45° (Problem 5.13) and other spectroscopic observations. Interpret the HMBC spectrum and identify the heteronuclear long-range coupling interactions between the H and C nuclei. 

The HMBC spectrum of vasicinone along with the H-NMR assignments are shown. Determine the H/ C long-range heteronuclear shift correlations based on the HMBC experiment, and explain how HMBC correlations are useful in chemical shift assignments of nonprotonated quaternary carbons. 

Like the HMBC, the COLOC experiment provides long-range hetero-nuclear chemical shift correlations. The COLOC spectrum, H-NMR, and C-NMR data of 7-hydroxyfrullanolide are presented here. Use the data to assign the quaternary carbons. 

The HMBC spectrum of vasicinone displays long-range heteronuclear shift correlations between the various H/ C nuclei. These correlations are very helpful to determine the C-NMR chemical shifts of quaternary carbons and allow the interlinking of the different substructures obtained. 

Figure 7.14 Pulse sequence for the HMBCS (heteronuclear multiple-bond correlation, selective) experiment, which uses advantageously a 270° Gaussian pulse for exciting the carbonyl resonances. It is also called the semisoft inverse COLOC. (Reprinted from Mag. Reson. Chem. 29, H. Kessler et al., 527, copyright (1991), with permission from John Wiley and Sons Limited, Baffins Lane, Chichester, Sussex P019 lUD, England.)...

---