For these novel internally encoded water suppression PSYCHE-SAPPHIRE experiments (Figure ), hard 180° (π) pulses which evolve the homonuclear J couplings are replaced with either excitation sculpting or WATERGATE-5 elements during τ1 and τ3. Both operate in a similar fashion: while a 180° pulse inverts all signals of the spectrum, the water spins are flipped by 360°. Pulse field gradients flanking either iES or iW5 element (G1 and G3) defocus any magnetization that was not inverted 180°. In iES, this is achieved by combining hard and selective (soft) 180° pulses, while in iW5, this is achieved with a series of composite pulses separated by equal delays. In the conventional WATERGATE experiment, only three pulses (90°x, 180°x, 90°-x) are used for this suppression, while in WATERGATE-5, each 90° pulse is replaced by five composite pulses, which provides a narrower inversion profile. Data acquisition and processing of SAPPHIRE data follow the same steps as described by It is important to note that pure shift methods like those described here work by refocusing the effects of homonuclear couplings at the cost of SNR. The SNR reduction is because only active spins are observed while passive spins are used for refocusing couplings. However, the use of modern cryogenically cooled probes can alleviate this penalty. Pulse sequences for SAPPHIRE-iES (top) and SAPPHIRE-iW5 (bottom) experiments. Narrow-filled and wide white rectangles represent hard 90° and 180° pulses, respectively. White-shaped RF pulses are selective 180° refocusing pulses used to flip the solvent magnetization. Either RSNOB or REBURP pulses are optimal for solvent-selective pulses. W5 represents the WATERGATE-5 pulse block, with bandwidth and delay for binomial water suppression set to 20 kHz and 125 µs, respectively. Trapezoids with cross-diagonal arrows are low-power chirp pulses of small flip angle β, set to 15° in all experiments for improving signal-to-artifact ratio due to strong coupling. G1, G2, G3, and GS are set to 41.2, 26.2, 33.7, and 1.2 G cm -1 , respectively. All gradient pulses, represented by grey-shaped pulses, with 1 ms duration, were followed by a recovery delay of 1 ms. t1 is incremented at every chunk for the pure shift dimension, reconstructed from the acquisition of a series of 50 chunks of 10 ms (τC) each. τ1 and τ3 are used to modulate the point of J refocusing during each chunk used for the interferogram, keeping a constant duration in comparison to each SAPPHIRE increment. Four SAPPHIRE increments were used in all experiments, but good chunk artifact suppression can also be achieved with only two increments. Full experimental details are provided in the ESI. ‡ Figure shows a comparison of three SAPPHIRE water suppression methods for a sample of the macrocyclic peptide Aureobasidin A (AbA, Figure ), dissolved in 10% H2O / 90% CD3CN mixture: NOESY-pre-saturation (Fig. ), iW5 (Fig. ), and iES (Fig. ). Aureobasidin A, a cyclic depsipeptide antifungal antibiotic, was chosen as a stringent test case, since in solution it exists as a mixture of trans and cis-proline (Pro) amide bond conformers,[18] greatly complicating NMR spectra as most protons yield two sets of resonances. Several aliphatic protons of AbA are near the water peak, and it is immediately noticeable that both optimized NOESY-pre-saturation and iW5 strongly attenuated these proton resonances. The pre-saturation pulse in the former was optimized for slightly stronger power, as a low power saturation tends to leave unwanted water magnetization, which causes severe baseline distortion. Although iW5 completely eliminated the water peak, SAPPHIRE-iES was the only method to retain nearby desirable resonances as close as 0.1ppm from the water peak while adequately suppressing it. No chunking artifacts were observed from any of these methods, yielding clean spectra. Because presaturation requires a long water saturation period, the overall experimental duration is approximately twice as long as for the internally encoded water suppression experiments. Fig. . Aliphatic region of 1H pure shift NMR spectra of aureobasidin A in 10% H2O / 90% CD3CN mixture. Application of SAPPHIRE-NOESY-pre-saturation (A) generated a spectrum with a large residual solvent peak (green box) and significantly reduced the intensity of nearby analyte signals. SAPPHIRE-iW5 (B) led to complete suppression of the residual water peak but also suppressed neighboring analyte signals. SAPPHIRE-iES (C) demonstrated the best H2O suppression selectivity by preserving the observation of analyte signals in immediate proximity to the suppressed frequency. Note that peak assignments are for the trans-Pro rotamer of AbA, which is the major conformer under these solvent conditions. Unlabelled minor peaks correspond to the cis-Pro rotamer of AbA.