Methyl resonance

The other peaks demonstrate the power of NMR to identify and quantitate all the components of a sample. This is very important for die phannaceutical industry. Most of the peaks, including a small one accidentally underlying the methyl resonance of paracetamol, arise from stearic acid, which is connnonly added to paracetamol tablets to aid absorption. The integrals show diat it is present in a molar proportion of about 2%. The broader peak at 3.4 ppm is from water, present because no attempt was made to dry the sample. Such peaks may be identified either by adding fiirther amounts of the suspected substance, or by the more fiindamental methods to be outlined below. If the sample were less concentrated, then it would also be... 

Figure Bl.11.14. NOE-difFerence spectrum (see the text) of aspirin, with pre-saturation at the methyl resonance, proving that the right-hand confomration is dommant.

Under conditions where the rotation about the C-N bond of dimethylformamide is slow relative to the NMR time scale, the two methyl resonances will be separate singlets. Conversely, if the rotation is made to be very fast, the two methyl groups will be chemically equivalent. Their resonance will then appear as a sharp singlet. In between these extremes, kinetic information can be extracted from the line shapes. In most systems the parameter that is changed to go between these limits is the temperature. In some systems, pH or pressure has the same effect. 

The fine structure of the spectrum is the splitting of the resonance into sharp peaks. Note that the methyl resonance in ethanol at 8 = 1 consists of three peaks with intensities in the ratio 1 2 1. The fine structure arises from the presence of other magnetic nuclei close to the protons undergoing resonance. The fine structure of the methyl group in ethanol, for instance, arises from the presence of the protons in the neighboring methylene group. 

Chemical shifts are sometimes expressed in parts per million, such as 8 = 1 ppm for the methyl resonance ignore the ppm in calculations. 

Integration of the H-NMR spectrum shows 55% of tetramer proportions of individual isomers were estimated from GC/MS data. Tetramer mixtures show an asymmetrical, vinyl resonance centered at S 5.40, and asymmetrical, methyl resonances centered at 8 1.70 ( vs. Me4Si in CS2 solution). Integration of the H-NMR spectrum. 

One way in which to determine whether one part of the molecule may influence the structure about the N-terminus, or whether the assignments of the [ C]methyl resonances in the C-n.m.r. spectra of fully reductively [ CJmethylated glycophorins A and A are correct is to isolate the various glycophorin glycopeptides that have been produced by enzymic or chemical means. 

Not all of the spectral data are given, The C-enriched-methyl resonance of the di[ C]methylSer residue overlaps with this resonance. 

Figure 13. Effect of varying concentrations of the NMR shift reagent Eu(fod), on methyl resonances of soyasapogenol B phenyl borate. Euffodjg was dissolved in a minimum amount of acetone-dg and added to a 2 ml solution of soyasapogenol B phenyl borate in the same solvent. Spectra were obtained at 360 MHz.

Figure 4. Ratio of the relative amounts of m and r isomers of DCP remaining after reduction by (n-Bu)3SnH,as measured by the carbon-13 methylene (see Figure 2) and methyl resonances.

Oxostephasunoline (4) was isolated from the roots of Stephania japonica(4). The UV spectrum of oxostephasunoline (4) showed an absorption maximum at 286 nm, and the IR spectrum depicted bands at 3550,3500, and 1670 cm, indicating the presence of a hydroxyl group and a y-lactam. The mass spectrum (Table VI) exhibited the most abundant ion peak at m/z 258, and the H-NMR spectrum (Table II) revealed the presence of three methoxyl and one N-methyl group. The downfield shift (53.06) of the JV-methyl resonance indicated that oxostephasunoline (4) was a y-lactam, which was further supported by the IR band at 1670 cm 1, significant features of the mass spectrum (Table VI), and the 13C-NMR spectrum (Table III). On exhaustive H-NMR analysis similar to the case of stephasunoline (17), the structure of oxostephasunoline (4) including the stereochemistry was practically proved (4). 

Oxostephabenine (15) was isolated from the fruits of Stephania japonica (7). The UV spectrum of 15 showed absorption maxima at 294 and 230 nm, and the IR spectrum depicted bands at 1700, 1680, and 1600 cm-1. Its mass spectrum revealed a molecular ion peak at m/z 493 (17%), the most abundant ion peak at m/z 241 (C14HuN03), and another significant ion peak at m/z 242 (60%, C14H12N03). The H-NMR (Table II) and 13C-NMR (Table III) spectra exhibited close similarity to those of stephabenine (13) (10) except for W-methyl resonance (7). 

This peak is broadened and contact shifted down field by the unpaired electron (Fig. 24). A spectrum of a mixture of methylcobinamide and free nitroxide shows broadening of the methyl resonance but no shift in resonance position. Thus the nitroxide must remain attached to the cobalt atom in solution. 

When the pD of a D2O solution of methyl cobalamin is lowered, the resonance position for the C(20)-methyl shifts progressively to lower field and the intensity of the peak remains constant (50). Thus rather than observing two C(20)-methyl resonances corresponding to the base-on... 

When the data in this table are plotted, the graph shown in Fig. 33 is obtained. From this one can calculate a pKa of 2.85 for displacement of benzimidazole in D2O. In addition, since room temperature is above the coalescence temperature, it is possible to set a lower limit on the exchange rate between coordinated and uncoordinated benzimidazole of 3.1 X 102 sec-1. From Fig. 33 one can, by extrapolation, calculate the C(20)-methyl resonance of the base-on and "base-off forms to be 0.41 and 1.05 respectively. These numbers can be used, with the assumption of fast exchange, to determine the relative amounts of "base-on and base-off" species from the observed C(20)-chemical shift for any arbitrary sample. Such information would be useful, for instance, when investigating the displacement of benzimidazole by other Lewis bases. Thus for the simple case of benzimidazole displacement we have shown that NMR provides a method for studying the molecular conformation of vitamin B12. 

Fig. 33. Change in C(l)-methyl resonance position in methylcobalamin as a function...

Figure 21 Expansion of the aliphatic region of the HSQC-1,1-ADEQUATE spectrum of the CDK-2 inhibitor dinaciclib (48). The connectivity network is traced out for the 2-(P-hydroxyethyljpiperidine moiety contained in the structure. Methylene resonances are inverted and plotted in grey methine and methyl resonances have positive intensity and are plotted in black.

Figure 30 Standard full-width (200 ppm) HMBC (A), and the corresponding 10-ppm HMBC spectrum of cyclosporine (B). The excerpts (C) and (D) show the region comprising the methyl resonances of cyclosporine and the ambiguous resonance discussed in the text is highlighted with a dashed circle. Both experiments have been recorded using the same parameters, with the exception of the, 3C spectral width, which was set to 10 ppm for recording (B).

As seen from Table 10, both the methyl resonance in dimethyl cyclopropenone (7.75 r) and the separation of CH2 units a and 0 to the three-ring in di-n-propyl cyclopropenone (0.85 ppm) compare well to corresponding values for the covalent cyclopropene derivatives, but differ strongly from those of the positively charged cyclopropenium species. 

The dimethyl substituted triafulvenes 21 la-e (Table 11) show methyl resonances considerably shifted downfield from dimethyl cyclopropenone. The same... 

Fig. 11 Plot of the 13C signal intensity as a function of contact time for two distinct methyl resonances of two polymorphic forms of a developmental drug substance.

VT NMR showed that N3-[3]polynorbomane 164 existed as an equilibrium mixture of the syn-atropisomer 164a and anti-atropisomer 164b (ratio 1 1.7). NMR spectroscopy allowed distinction between the isomers on the basis of symmetry. The syn-isomer 164a exhibited two well-separated ester methyl resonances (8 3.67, 4.05) as predicted for the isomer with Cs-symmetry, whereas the anft -isomer 164b displayed a single ester methyl resonance (8 3.85) in accord with that expected for a compound with C2-symmetry. It was not possible to isolate the separate atropisomers in this system since the energy barriers governing rotation were too low. 

Of the multitude of ID 13C NMR experiments that can be performed, the two most common experiments are a simple broadband proton-decoupled 13C reference spectrum, and a distortionless enhancement polarization transfer (DEPT) sequence of experiments [29]. The latter, through addition and subtraction of data subsets, allows the presentation of the data as a series of edited experiments containing only methine, methylene and methyl resonances as separate subspectra. Quaternary carbons are excluded in the DEPT experiment and can only be observed in the 13C reference spectrum or by using another editing sequence such as APT [30]. The individual DEPT subspectra for CH, CH2 and CH3 resonances of santonin (4) are presented in Fig. 10.9. 

Fig. 10.9. Multiplicity edited DEPT traces for the methine, methylene and methyl resonances of santonin (4). Quaternary carbons are excluded in the DEPT experiment and must be observed in the 13C reference spectrum or through the use of another multiplicity editing experiment such as APT.

Using strychnine (1) as a model compound, a pair of HSQC spectra are shown in Fig. 10.16. The top panel shows the HSQC spectrum of strychnine without multiplicity editing. All resonances have positive phase. The pulse sequence used is that shown in Fig. 10.15 with the pulse sequence operator enclosed in the box eliminated. In contrast, the multiplicity-edited variant of the experiment is shown in the bottom panel. The pulse sequence operator is comprised of a pair of 180° pulses simultaneously applied to both H and 13C. These pulses are flanked by the delays, A = l/2(xJcii), which invert the magnetization for the methylene signals (red contours in Fig. 10.16B), while leaving methine and methyl resonances (positive phase, black contours) unaffected. Other less commonly used direct heteronuclear shift correlation experiments have been described in the literature [47]. 

Fig. 10.16. (A) GHSQC spectrum of strychnine (1) using the pulse sequence shown in Fig. 10.15 without multiplicity editing. (B) Multiplicity-edited GHSQC spectrum of strychinine showing methylene resonances (red contours) inverted with methine resonances (black contours) with positive phase. (Strychnine has no methyl resonances.) Multiplicity-editing does have some cost in sensitivity, estimated to be 20% by the authors. For this reason, when severely sample limited, it is preferable to record an HSQC spectrum without multiplicity editing. Likewise, there is a sensitivity cost associated with the use of gradient based pulse sequences. For extremely small quantities of sample, non-gradient experiments are preferable.

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