3D Heteronuclear long‐range 1H–13C scalar correlation at natural abundance: application to oligosaccharide analysis

A 3D ¹H–¹³C–¹H refocused INEPT transfer experiment is proposed in which the initial coherence transfer of ¹H longitudinal to ¹³C transverse magnetization is tuned to the long‐range ¹H, ¹³C couplings while the reverse INEPT component transfers the magnetization to the directly bonded ¹H. Integration of a constant time ¹H evolution period into the long‐range coherence transfer interval provides absorption mode signals for each dimension. A ¹³C purge component at the beginning of the sequence selects for ¹²C‐bound ¹H magnetization that is then transferred to a ¹³C‐bound hydrogen, thus strongly suppressing the diagonal signals. This experiment is expected to be of particular value for situations in which resonance overlap in the ¹³C dimension renders 2D long‐range heteronuclear correlation data ambiguous. In combination with a diagonal‐suppressed 3D ¹H–¹³C–¹H TOCSY‐HSQC experiment, complete assignment of the ring resonances of the Lewis‐b hexasaccharide was obtained on a 4.2 mM sample using a conventional 500 MHz probe (0.1% ethylbenzene signal‐to‐noise ratio of 600), suggesting its applicability to sub‐millimolar samples using cryoprobe technology. Copyright © 2002 John Wiley & Sons, Ltd.