Extending the scope of singlet-state spectroscopy.

Different decoupling sequences are tested-using various shaped radio-frequency (RF) pulses-to achieve the longest possible lifetimes of singlet-state populations over the widest possible bandwidths, that is, ranges of offsets and relative chemical shifts of the nuclei involved in the singlet states. The use of sinc or refocusing broadband universal rotation pulses (RE-BURP) for decoupling during the intervals where singlet-state populations are preserved allows one to extend the useful bandwidth with respect to prior state-of-the-art methods based on composite-pulse WALTZ decoupling. The improved sinc decoupling sequences afford a more reliable and sensitive measure of the lifetimes of singlet states in pairs of spins that have widely different chemical shifts, such as the two aromatic protons H(5) and H(6) in uracil. Similar advantages are expected for nucleotides in RNA and DNA. Alternative approaches, in particular frequency-modulated decoupling sequences, also appear to be effective in preserving singlet-state populations, even though the profiles of the apparent relaxation rate constants as a function of the offset are somewhat perturbed. The best decoupling sequences prove their utility in sustaining longer lifetimes of singlet states than previously achieved for the side-chain tyrosine protons in bovine pancreatic trypsin inhibitor (BPTI) at 600 MHz (14.1 T), where the differences of chemical shifts between coupled protons are a challenge.