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Figure 1.22

pattern (inset, Fig. 1.22) shows two nearly equal large couplings (J = 9.9 and 10.9 Hz) due to the axial-axial relationships to H-2 and H-6ax. Because these two couplings are not equal, the double-triplet (1:1:2:2:1:1) pattern is distorted, widening the two center peaks and making them shorter (less than twice the height of the four outer peaks). This is an example of an unresolved splitting: we should be seeing eight peaks, but we see only six because the separation of the third and fourth peaks (and of the fifth and sixth) is comparable to the peak width. This separation is about 1.0 Hz (10.9-9.9) and the peak width (measured at half-height) of the outer peaks is 1.3 Hz. Later on we will see how resolution enhancement can be used to make the peaks sharper and at least begin to see the separation of this multiplet into eight peaks. The third coupling of the double-doublet-doublet (ddd) is 4.3 Hz, due to the interaction with H-6eq. This coupling is axial-equatorial (gauche relationship), so it is smaller, in the middle range of observed couplings.

The peak at 2.14 ppm is a double septet, with an intensity ratio 1:1:6:6:15:15:20: 20:15:15:6:6:1:1 and J couplings of 7.0 Hz for the septet and 2.6 Hz for the doublet. The only proton with six coupling partners is the CH proton of the isopropyl group, H-7. A J coupling near 7.0 Hz is typical of a vicinal coupling with free rotation (of the methyl group) averaging the dihedral angle effects. The additional coupling of 2.6 Hz is due to its interaction with H-2. The outer peaks of the septet are only one twentieth of the intensity of the center peaks, so unless you have very good signal-to-noise you might miss these peaks and mistake it for a quintet. The intensity ratio for this "quintet" is 1:2.5:3.3:2.5:1, instead of the expected 1:4:6:4:1. The remaining resolved single-proton peaks ("l" and "h") cannot be assigned without advanced experiments. The strong, sharp peaks at the right-hand side of the spectrum correspond to the methyl groups. All three methyl groups are attached to CH carbons ("methine" carbons) so they will appear as doublets. One doublet ("a") is separate

Figure 1.23

from the other two ("b" and "c"), but we cannot make the assumption that it represents the "lone" methyl group H-10. Because this is a chiral molecule, the isopropyl group can have distinct environments and widely different chemical shifts for the two methyls. The J couplings for these three doublets are all around 7.0 Hz due to free rotation of the C-C bond, although one is slightly lower (6.6 Hz) and this corresponds to the CH-CH3 group, C-10. Chemical shifts for the methyl groups are a bit less than 1 ppm, typical for methyl groups in a saturated hydrocarbon environment, far from any functional group. The same is true for the four proton signals buried in the overlapped region between 0.75 and 1.15 ppm: they are shifted downfield of the methyl groups slightly because of the higher degree of substitution (CH2 and CH), but they are not close to any functional group.

The 1H-decoupled 13C spectrum of menthol (Fig. 1.23) has ten peaks in addition to the three solvent peaks. All we can say about it is that the most downfield peak ("j") corresponds to the carbon with the alcohol oxygen: C-1. We can see a bit of a gap between this peak and the rest of the peaks, and we expect singly oxygenated sp3 carbons in the range 50-90 ppm, with methine carbon (CHOH) typically in the range 70-80 ppm. Every time we replace an H with C we add about 10 ppm to the chemical shift, so compared to CH3O (50-60 ppm) we can add about 20 ppm to get the range of CHOH. The rest of the carbons can only be assigned if we can assign the attached protons and then correlate the 13C shifts with the 1H shifts by a 2D spectrum such as HSQC.

1.2.9 Cholesterol

Cholesterol (Fig. 1.20) is a steroid, the same rigid five-ring backbone used for the mammalian sex hormones. There are only two functional groups: an olefin (C-5, C-6) and an alcohol (C-3). The bulk of the molecule can be described as saturated hydrocarbon. There are five methyl groups: two are attached to quaternary carbons so they should appear as singlets; and three are attached to CH carbons so they should appear as doublets. Most of the protons in the A, B, and C rings can be described as "axial" or "equatorial" due to the rigid,

Figure 1.24

locked cyclohexane ring structure. The 600 MHz 1H spectrum is shown in Figure 1.24. The total integration adds up to 48.89 protons, a bit high for the molecular formula C27H46O but consistent with the fact that the resolved peaks in the upfield part of the spectrum integrate several percent above the expected integer values. The olefin functional group (C-5 and C-6) has a single proton, H-6, which we expect in the region 5-6 ppm. Thus the one-proton signal at 5.35 ppm (peak "i") can be assigned to H-6 (only the resolved peaks are identified with letters). The other functional group is an alcohol, and we expect the H-C-OH proton at 3-4 ppm; we can assign the one-proton signal at 3.52 ppm (peak "h") to H-3. The splitting pattern of H-3 can be described as a triplet of triplets, with a small triplet coupling of 4.6 Hz and a large triplet coupling of 11.2 Hz (Fig. 1.24, proton h inset). Because the OH group is equatorial, H-3 is axial and is split by its two equatorial neighbors, H-2eq and H-4eq. Because both the relationships are axial-equatorial (gauche), the couplings are identical and in the medium range (4.6 Hz). H-3 is also split by its two axial neighbors, H-2ax and H-4 ax. Each of these relationships is axial-axial (anti), so the couplings are identical and large (11.2 Hz). Taken together, we get a large triplet (1:2:1 intensity ratio, J = 11.2), with each of the three arms split into a smaller triplet (1:2:1 ratio, J = 4.6). These coupling relationships are shown in the partial structure in Figure 1.25.

Moving from left to right, the next resolved peak is a two-proton multiplet at 2.20-2.32 ppm (peaks "g" and "f"). The most likely assignment for these peaks would be H-4ax and H-4eq, since C-4 lies between the two functional groups and we expect the minor downfield-shifting effects of both groups to add together, pulling the H-4 resonances out of the "pack" of saturated hydrocarbon peaks (0.6-1.7 ppm). We cannot be absolutely sure of this assignment until we see two-dimensional data, but this is a reasonable guess. Looking at the fine structure of these two peaks (inset, Fig. 1.24) and ignoring the smaller couplings, we

A Ring

A Ring

Figure 1.25

see a triplet on the right (peak "g") and a doublet on the left (peak "f"). These two peaks are "leaning" toward each other, with the outer peaks reduced in intensity and the inner peaks increased relative to a "standard" doublet (1:1) or triplet (1:2:1). This distortion of peak intensities is a common feature when the chemical shift difference (in Hz) is relatively small compared to the J coupling between the two protons. In this case, the chemical shift difference is 0.053 ppm or 32 Hz and the large geminal (2JHH) coupling is 13.0 Hz, leading to a large distortion of peak intensities. The basic doublet and triplet patterns are further split by smaller couplings: each side of the doublet is split into a double doublet (J = 5.0 and 2.1 Hz) and each of the three peaks of the triplet on the right is split into a quartet (J = 2.8 Hz). Ignoring the "small" couplings, we can ask how many large couplings each proton experiences and in this way count the number of geminal and axial-axial relationships. The "doublet" peak ("g") has only the geminal (2JHH) coupling, which is always large for saturated (sp3 hybridized) carbons. So it must be the equatorial proton, H-4eq. The "triplet" peak ("f") has the geminal coupling and one axial-axial coupling, so it must be the axial proton, H-4ax, which has an axial-axial coupling to H-3. The smaller couplings can be explained as follows: H-4eq has one equatorial-axial coupling (5.0 Hz) to H-3ax and one "W" coupling (4Jhh) to H-2 ax (2.1 Hz). A "W" coupling occurs in a series of saturated carbons when the H-C-C-C-H network is rigidly aligned in a plane in the form of a "W." H-4 ax has small long-range couplings to H-6, H-7ax, and H-7eq, all around 2.8 Hz. These long-range couplings will be discussed later, but you can think of the C=C double bond as a kind of "conductor" for J couplings that allows these small interactions to occur over four or five bonds as long as the double bond is in the path: H-C-C=C-H ("allylic coupling") and H-C-C=C-C-H ("bis-allylic" coupling). In each case, if you remove the C=C from the path, you have a close bonding relationship of two bonds ("geminal") or three bonds ("vicinal").

The five methyl groups of cholesterol give rise to tall, sharp peaks in the upfield region of the 1H spectrum (inset, Fig. 1.24, peaks a-e). We can see two singlet methyl signals ("a" and "e") that correspond to the "angular" methyls attached to the quaternary carbons at the A-B and C-D ring junctures (C-18 and C-19). Later on we will use an NOE experiment to assign these two peaks specifically, taking advantage of the proximity of CH3-19 to the H-4ax proton. There are also three doublet methyl signals ("b," "c," and "d") that correspond to the three methyl groups in the side chain attached to CH carbons: C-21, C-26, and C-27. Specific assignments for these signals will require two-dimensional experiments such as HSQC and HMBC.

The 125 MHz 1H-decoupled 13C spectrum of cholesterol is shown in Figure 1.26. Because the 13 C nuclear magnet is only about one fourth as strong as the 1H nuclear magnet, the 13C resonant frequency is always about one fourth of the 1H frequency in the same magnetic field. Thus on a "500 MHz" NMR spectrometer (i.e., an 11.74 T Bo field in which 1H resonates at 500 MHz) the 13C frequency is about 125 MHz. The CDCl3 peaks (a 1:1:1

triplet at 77.0 ppm) appear at the center of the spectrum. Note that there is a small peak due to CHCl3 at 77.21 ppm (upper left inset, Fig. 1.26). This may be residual CHCl3 in the CDCl3 (0.2%) or CHCl3 residue in the solid cholesterol sample. Such a small amount of CHCl3 is visible in the spectrum due to the effects of relaxation and decoupling. Because the 1H nuclear magnet is about seven times stronger than the 2H nuclear magnet, the 13C in CHCl3 relaxes faster than the 13 C in CDCl3 and thus gives a stronger NMR peak. In addition, due to 1H decoupling there is only one peak for CHCl3, and this makes for a taller peak than these for CDCl3, whose 13C intensity is divided into three peaks. Note also that there is a deuterium isotope effect on the 13C chemical shift: CHCl3 appears 0.21 ppm downfield ofCDCl3.

In addition to these solvent peaks, we can count 26 peaks in the spectrum. Because there are 27 carbons in the cholesterol molecule (three are lost in the biosythesis from a triterpene precursor), there must be one peak that accounts for two carbons. The tallest peak (labeled "l, m") in fact corresponds to two different carbons with nearly identical chemical shifts. The most downfield peaks ("aa" and "z") are in the olefin/aromatic region of the 13 C spectrum (120-140 ppm), so they must correspond to C-5 andC-6. Peak "aa" is less intense ("shorter") than all of the other peaks because of slow relaxation: it must be a quaternary carbon. We will see that the proximity of protons is the primary means of relaxation of 13 C nuclei, so carbons lacking a proton relax much more slowly and give less intense peaks, especially if the relaxation delay is short (in this case the recycle delay was only 1.74 s (1.04 s acquisition time and 0.7 s relaxation delay). So we can assign peak "aa" (140.75) to C-5 and peak "z" (121.69) to C-6. Note also that the more substituted carbon, C-5 (three bonds to carbon) is shifted downfield relative to C-6 (two bonds to carbon) due to the steric crowding effect.

Peak "y" (71.78 ppm) is in the "alcohol" region (C-O) in the range expected for methine carbon (CH-O), so it can be assigned to C-3. The next three carbons (peaks "x," "w," and "v," 50-57 ppm) could be methoxy (CH3O) groups, but because we have accounted for all the functional groups of cholesterol they must be either close to these functional groups (inductive effect) or shifted downfield due to steric crowding. The inductive effect (electron withdrawing and donating groups) is most important for 1H chemical shifts, so let us consider the steric effects. The most sterically crowded carbons in the cholesterol structure are the methine (CH) groups next to an sp3-hybridized quaternary carbon: C-9, C-14, and C-17. These three carbons account for this group of downfield-shifted peaks. The rest of the 13C peaks (a-u) lie in the region of saturated hydrocarbon (sp3 carbon with no functional groups) and cannot be assigned without more advanced experiments such as DEPT and 2D HSQC/HMBC.

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