Presaturation NOESY

Figure 6J8 (a) The chromatogram from a 0.75 mg injection of an impure steroidal drug substance. Peaks 1-5 are at levels of 2-3% each. The individual peaks were stored in BPSU loops, (b) NMR spectra were acquired with NOESY presaturation with an acquisition time of 10 h on a Bruker DRX500 fitted with a 4-mm SEl z-gradient EC probe with a 120 pi active cell volume. 

Solvent suppression is a particular problem in LC/NMR and has been a theme throughout its development. Early methods for suppression of the pro-tonated solvent signals which otherwise dominate the NMR spectrum made use of binomial pulse sequences [124-126]. Methods in use today either use fully deuterated solvents, or make use of solvent suppression schemes such as the NOESY presaturation technique [127], WATERGATE [28,128], WET [29,129], or excitation sculpting [30,130,131]. These methods have for some time made it possible to study relatively low-level (several %) impurities [132,133]. The need... 

Pre-saturation In this technique prior to data acquisition, a highly selective low-power rf pulse irradiates the solvent signals for 0.5 to 2 s to saturate them. No irradiation should occur during the data acquisition. This method relies on the phenomenon that nuclei which have equal populations in the ground and excited states are unable to relax and do not contribute to the FID after pulse irradiation. This is an effective pulse sequence of NOESY-type pre-saturation that consists of three 900 pulses RD - 900 - tx - 900 - tm - 90° - FID, where RD is the relaxation delay and t and tm are the presaturation times. 

Figure 6.36 500 MHz H NMR spectra obtained during a stop-flow LC-NMR experiment on a 1 mg injection of a crude sample of a drug compound, (a) LC chromatogram, (b) spectram corresponding to the parent bulk drug compound acquhed for 64 transients and (c) to the impurity peak RRT 0.87 (—3% by area), acquhed for 1024 transients. NOESY-type presaturation was used to suppress the solvent resonances. Bruker DRX500 H/ C 4-mm z-gradient probe with a 120 pi active cell volume.

ID NOESY spectra (b) and (c) were acquired using the pulse sequence of fig. 1(a). A 59.2 ms half-Gaussian pulse was applied to proton H-ld. Mixing time was 25 ms in (b) and 200 ms in (c). Water presaturation was applied during the relaxation delay and the NOE mixing... 

Results similar to those shown in the slice of Fig. 8.22 can be obtained with the so-called NOE-NOESY sequence [36]. Here a hyperfine shifted signal, e.g. I2-CH3 of the above compound, is selectively saturated, and then the NOESY pulse sequence is applied. The NOESY difference spectrum obtained by subtracting a NOESY spectrum without presaturation of the I2-CH3 signal is shown in Fig. 8.23. Here, some more cross peaks are evident with respect to the 3D NOESY-NOESY experiment because secondary NOEs develop much more when the primary NOEs from the I2-CH3 signal evolve in a steady state experiment like the NOE-NOESY rather than in a transient-type experiment like the NOESY-NOESY. In Fig. 8.23, dipolar connectivity patterns are apparent among protons... 

The principle of presaturation relies on the phenomenon that nuclei which are unable to relax, because their population in the ground state a and the excited state (3 is the same, do not contribute to the free induction decay after pulse irradiation. Prior to data acquisition, a highly selective low-power pulse irradiates the desired solvent signals for 0.5 to 2 s, thus leading to saturation of the solvent signal frequency. During data acquisition, no irradiation should occur. NOESY-type presaturation is an effective pulse sequence of presaturation. The pulse sequence consits of three 90° pulses (similar to the first increment of a NOESY experiment) ... 

All NMR spectra were collected on a Varian Unity 500 MHz spectrometer ( H frequency 499.8 MHz) equipped with a 5 mm inverse detection probe. Sample concentrations were typically 1 -2 mM and sample temperatures maintained at 25 C (unless otherwise noted). Sample pH was typically 3.5 - 4.0. Onedimensional H data were acquired with a H sweepwidth of 6000 Hz and an acquisition time of 2.3 seconds. The residual water signal was suppressed by presaturation. H DQF-COSY, NOESY and TOCSY (15) spectra were collected and processed using standard methods. All chemical shifts were referenced relative to internal DSS. 

Fig. 5. Various presaturation-based suppression sequences (A) presaturation, (B) ID NOESY, (C) FLIPSY and (D) SCUBA. TTie phase cycling for the ID NOESY and FLIPSY sequences can be found elsewhere. The tt pulse in the SCUBA sequence can be replaced with a composite tt pulse to ensure more complete inversion.

Many different ways of effecting water suppression in the NOESY sequence have been implemented, for example, presaturation, jump and return, l-T-echo, WATERGATE, SS pulses and excitation sculpting. The basic NOESY sequence using presaturation and two variations using WATERGATE are illustrated in Fig. 24. In the basic NOESY sequence (Fig. 

Fig. 24. Various NOESY-water suppression combinations. (A) NOESY using presaturation in the relaxation delay and mixing times, ffi) WATERGATE NOESY and (C) the water flip-back WATERGATE NOESY. ...

In the first example, 2D NOESY spectra were used to define the stereochemistry in the synthetic cycloadduct 8.14 [7], a potential biomimetic precursor to the naturally occurring marine-sponge alkaloid Keramaphidine B, 8.15. This problem is essentially the same as that addressed for 8.7 above using the NOE difference experiment, but in this case the additional unsaturated sidechains caused extensive overlap in the proton spectrum and precluded the use of selective presaturation. Sufficient characteristic NOEs present in a 600 ms NOESY spectrum gave conclusive proof of the endo stereochemistry, as shown. Only positive NOEs were observed, consistent with a molecule of mass 436 daltons in chloroform. NOESY spectra have also been successfully applied to the structure elucidation of molecules for considerably greater mass and complexity, as illustrated by the cytotoxic macrolide cinachyrolide A, 8.16 [35], also from a marine sponge. The structure of the molecule was determined through extensive 600 MHz 2D NMR experiments, of which NOESY played... 

The 2D sequence [80,81], referred to as HOESY (heteronuclear Overhauser spectroscopy. Fig. 8.52b), avoids the need for selective proton presaturation but naturally suffers from low sensitivity. The sequence parallels that of NOESY,... 

Figure 9.23. Solvent suppression schemes based on presaturation (a) presaturation alone, (b) ID NOESY and (c) FLIPSY, Sequence (b) makes use of the conventional NOESY phase cycle whereas FLIPSY uses EXORCYCLE on one (or both) of the 180° pulses (i.e. pulse = X, y, —X, —y, receiver = x, —x,...

The order of review was intended to be alphabetical with the exception of presaturation and water-eliminated Fourier transform (WEFT)/NOESY due to the importance of these sequences and comparisons to subsequent developments. Presaturation and WEFT (commonly referred to as ID-NOESY) remain, for many groups, the gold standard to which all other forms of suppression are compared. Suppression techniques prior to or during acquisition are considered. Suppression in the solid state and post-acquisition mathematical suppression techniques (e.g. WAVEWAT and others °) are beyond the scope of this review. 

Figure 6 Presat, WEFT/NOESY, and FLIPSY. The typical (A) presat, (B) WEFT/NOESY and (C) FLIPSY. Any 90 and 180 hard pulses are identified, respectively, by narrow and wide hollow rectangles, respectively, with relative phases indicated inside the shape (where appropriate). Outlined Gaussian shapes indicate selective pulses while gradients are represented on their own horizontal line by filled shapes. For full phase cycle discussions please refer to the referenced manuscripts. The 45 phase shifted pulse discussed in the text is indicated by the asterisk in the NOESY sequence and an V in the FLIPSY shows the variable flip angle pulse. The modified FLIPSY contains a homo-spoil gradient just after the presaturation period.

The WEFT/NOESY style experiment provides substantially better water suppression than simple presaturation with four transients, which is the minimum for the phase cycle to take effect. With proper optimization of the mixing period the WEFT/NOESY still yields suppression even when considering only the first transient, and before pulsed field gradients were used in the mixing period. Therefore, while important, volume selection alone cannot be the sole answer. 

Neuhaus et al. first introduced FLIPSY (flip angle adjustable one-dimensional NOESY) in 1996 and re-evaluated the pulse sequence in 2008 for use with meta-bonomics projects. The modified FLIPSY is similar to the original (Figure 6C) with the exception of a small homospoil gradient prior to the flip-angle controlled pulse and just after the presaturation period. 

A) Presaturation with optimized carrier position and pulse width that was used for all subsequent hard pulses, (B) Presat90 with the same parameters, (C) PresatlSO with an independently optimized 180° pulse length, (D) WEFT/NOESY with the same carrier and pulse width as optimized in the previous experiments and a 100 ms mixing time, (E) WET (118 us inter-pulse delay) with optimized... 

Fig. 4.9 (A)-(C) H spectra recorded at 500 MHz on a 4 mM solution of a compound in a 60 40 (v/v) mix of D2O and CH3CN. (A) Simple pulse and collect ID H spectrum showing the C satellites of acetonitrile (B) NOESY presat spectrum with presaturation of the MeCN signal apphed during the relaxation delay (2 s) and during the mixing time (200 ms) using a field of 90 Hz (C) Excitation sculpting sequence using a 1 s presaturation of water and a selective proton JC pulse of 4.25 ms. Compare the spectral region around the residual acetonitrile resonance in (B) and (C) - excitation sculpting results in the obliteration of far less of the spectrum near the suppressed solvent resonance, which in this case contains many solute resonances.

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