This tedious and error-prone workflow has frequently yielded inconclusive or misassigned structures, leading to the heuristic “Crews rule” – a low ratio of H/C (<1:1) could complicate or even prevent unequivocal structure elucidation using the conventional suite of 2D NMR experiments (e.g., COSY/TOCSY, HSQC, HMBC, and NOESY/ROESY) 10, 11, 12, 13.

However, this limitation is problematic for proton-deficient compounds with a preponderance of quaternary carbons or heteroatoms, and has led to frequent “guesswork” to elucidate the carbon skeleton from HMBC correlations, due to the inability to differentiate the number of bonds between atoms. Typically, carbon-carbon bond connections in a C aH-C bH spin system have been established by a combination of COSY and HSQC experiments, or other COSY-based experiments such as 2 J, 3 J-HMBC or H2BC 6, 7, 8, 9. Reliable differentiation of two- and three-bond correlations in HMBC is not possible due to overlapping ranges of the magnitudes of 2 J CH and 3 J CH coupling constants: from 0 to 8 Hz. However, a fundamental limitation is the inability to differentiate two-bond correlations ( 2 J CH) from other longer-range correlations, particularly 3 J CH. HMBC can routinely detect correlations between 1H and 13C nuclei separated by two to four bonds, and occasionally even five or six bonds 2, 3, 4, 5. Since being introduced in 1986 1, HMBC quickly became a pivotal NMR experiment for structure elucidation of small molecules, including a diverse array of natural products, due to the enhanced ability of the experiment to detect long-range correlations between NMR active nuclei. Because i-HMBC overcomes the key limitation of HMBC without significant reduction in sensitivity or performance, i-HMBC can be used as a complement to HMBC when unambiguous identifications of two-bond correlations are needed. Experimental utility was demonstrated at the sub-milligram / nanomole scale with only a few hours of acquisition time required for structure elucidation of several complex proton-deficient natural products, which could not be fully elucidated by conventional 2D NMR experiments. Here we present a sensitive and universal methodology to identify two-bond HMBC correlations using isotope shifts, referred to as i-HMBC (isotope shift detection HMBC). There have been several attempts to address this issue, but all reported approaches suffer various drawbacks, such as restricted utility and poor sensitivity. HMBC is an essential NMR experiment for determining multiple bond heteronuclear correlations in small to medium-sized organic molecules, including natural products, yet its major limitation is the inability to differentiate two-bond from longer-range correlations.

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