Extended Flip-back Schemes for Sensitivity Enhancement in Multidimensional HSQC-type Out-and-back Experiments

In many NMR experiments, only polarisation of a limited sub-set of all protons is converted into observable coherence. As recently shown by the “longitudinal” TROSY implementation (Pervushin et al. (2002) J. Am. Chem. Soc., 124, 12898–12902) and SOFAST-HMQC (Schanda and Brutscher (2005) J. Am. Chem. Soc., 127, 8014–8015), recovery of unused polarisation can be used indirectly and unspecifically to cool the proton lattice and, thus, accelerate re-equilibration for the selected proton subset. Here we illustrate transfer of this principle to HSQC-based multi-dimensional out-and-back experiments that exploit only polarisation of ¹⁵N-bound protons. The presented modifications to the pulse sequences can be implemented broadly and easily, extending standard flip-back of water polarisation to a much larger pool of protons that may comprise all non−¹⁵N-bound protons. The underlying orthogonal separation of H^N polarisation (selected by the main transfer path) from unused H^u polarisation (flipped-back on the recovery path) is thereby achieved through positive or negative selection by J-coupling, or using band-selective pulses. In practice, H^u polarisation recovery degrades mostly through cumulative pulse imperfections and transverse relaxation; we present, however, strategies to substantially minimise such losses particularly during interim proton decoupling. Depending on the protein’s relaxation properties and the extended flip-back scheme employed, we recovered up to 60% H^u equilibrium polarisation. The concomitant cooling of the proton lattice afforded substantial gains of more than 40%, relative to the water-only flip-back version, in the fast pulsing regime with re-equilibration delays τ much shorter than optimal (τ_opt = 1.25 · T₁(H^N)). These would be typically employed if resolution requirements dominate the total measurement time. Contrarily, if sensitivity is limiting and optimal interscan delays τ_opt can be set (optimal pulsing regime), the best of the presented flip-back schemes may still afford up to ca. 10% absolute sensitivity enhancement.