Structural Chemistry Using NMR Spectroscopy, Organic Molecules*

Structural Chemistry Using NMR Spectroscopy, Organic Molecules*

Structural Chemistry Using NMR Spectroscopy, Organic Molecules Cynthia K McClure, Montana State University, Bozeman, MT, USA & 1999 Elsevier Ltd. All ...

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Structural Chemistry Using NMR Spectroscopy, Organic Molecules Cynthia K McClure, Montana State University, Bozeman, MT, USA & 1999 Elsevier Ltd. All rights reserved. This article is reproduced from the previous edition, volume 3, pp 2234–2245, & 1999, Elsevier Ltd.

Nuclear magnetic resonance spectroscopy is one of the most powerful tools that chemists use to determine the structure of compounds. Generally, NMR spectroscopy is the technique that most chemists, especially organic chemists, use first and routinely in structural analysis. In organic compounds, this non-destructive spectroscopic analysis can reveal the number of carbon and proton atoms and their connectivities, the conformations of the molecules, as well as relative and absolute stereochemistries, for example. The advent of pulsed field gradient (PFG) technology for NMR spectrometers has allowed the routine acquisition of sophisticated onedimensional (1D) and two-dimensional (2D) NMR spectra in relatively short periods of time on complex organic molecules. This in turn has revolutionized organic structure determination such that deducing the three-dimensional structure of compounds takes a fraction of the time it used to. Mention of relevant 2D experiments that can aid in structure determination will be made in the appropriate sections herein. This article is geared toward the analyses of small organic compounds, and will cover the following topics: practical tips in sample preparation; basic principles of one-dimensional 1H and 13C NMR spectroscopy and their use in organic structure determination, including chemical shifts, coupling constants and stereochemical analyses; and the application of more sophisticated 1D and 2D experiments to structure elucidation. Examples of structural analyses of organic compounds via NMR methods are ubiquitous in the literature such that it is impractical to mention more than just a few of them here. Therefore, the reader is encouraged to peruse the organic chemistry literature to find structural analyses of the specific types of organic compounds of interest. This article will deal mainly with generalities of organic compound structure elucidation, although several relevant examples will be presented.

General Practical Considerations Deuterated solvents are utilized with FT NMR spectrometers to provide an internal lock signal to compensate for drift in the magnetic field during the experiment. The more common solvents used for organic compounds are CDCl3, CD3CN, CD3OD, acetone-d6, benzene-d6,

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DMSO-d6 and D2O. Since all deuterated solvents contain some protonated impurities (e.g. CHCl3 in CDCl3), one should choose a solvent that will not interfere with the NMR peaks of interest from the sample. Tetramethylsilane (TMS) is usually added to the sample as an internal standard for both proton and carbon spectra, being set at 0.0 ppm in both cases. However, the small protonated solvent impurities also make good standards as the chemical shifts of these peaks are published in many texts and are reported relative to TMS. Protons provide the highest sensitivity for NMR observations, and therefore only small quantities of sample are needed (1–10 mg in 0.5 mL of solvent for an FT instrument). 13C NMR has a much lower sensitivity than proton NMR due to the low natural abundance of 13 C (1.1%) compared with 1H (close to 100%), and the fact that the energy splitting and hence the resonance frequency for carbon is approximately one quarter that of proton. Thus, for a spectrometer whose 1H frequency is 300 MHz, the frequency for 13C is 75.5 MHz. To obtain a carbon NMR spectrum in a timely manner, one needs to use either more sample than for a 1H NMR spectrum (420 mg of a compound with MW E150–300 g mol1), or a higher field spectrometer. 1

H NMR

As mentioned earlier, 1H NMR is a very valuable method for obtaining information regarding the molecular structure of organic compounds with any number of protons. The electronic environment, as well as near neighbours and stereochemistry, can be determined by analysing the chemical shifts and spin–spin couplings of protons. The relative number of protons can be determined by direct integration of the areas under the peaks (multiplets), as the number of protons is directly proportional to the area under the peaks produced by those protons. To obtain accurate integrations, however, the relaxation delay needs to be at least 5 times the longest T1 in the sample. Proton Chemical Shift Chemical shifts are diagnostic of the electronic environment around the nucleus in question. Withdrawal of electron density from around the nucleus will deshield the nucleus, causing it to resonate at a lower field (higher

Structural Chemistry Using NMR Spectroscopy, Organic Molecules

frequency or chemical shift). Higher electron density around a nucleus results in shielding of the nucleus and resonance at higher field (lower frequency or chemical shift (d)). Therefore, basic details of the molecular structure can be gleaned from analysis of the chemical shifts of the nuclei. Factors that affect the electron density around the proton in question include the amount of substitution on the carbon (i.e. methyl, methylene, methine), the inductive effect of nearby electronegative or electropositive groups, hybridization, conjugation interactions through p bonds, and anisotropic (ring current) effects. Tables of proton chemical shifts can be found in various texts, such as those listed in Further Reading. As alkyl substitution increases on the carbon that possesses the proton(s) in question, the deshielding increases due to the higher electronegativity of carbon compared with hydrogen (e.g. CHR34CH2R24CH3R), producing a downfield shift of the resonances (methine most downfield, methyl most upfield). The deshielding effect of electron-withdrawing groups depends directly upon the electronegativity of these groups, and upon whether their effects are inductive (less effective) or through resonance (more effective). This deshielding effect falls off rapidly with increasing number of bonds between the observed proton and the electronegative group. One can, therefore, estimate chemical shifts of alkyl protons by analysing the amount of carbon substitution and the effects of nearby electron-withdrawing groups. A fairly accurate calculation of chemical shifts for methylene protons attached to two functional groups (X–CH2–Y) is possible by using Shoolery’s rule, where the shielding constants for the substituents, Di, are added to the chemical shift for methane. Tables of these shielding constants can be found in most texts on NMR spectroscopy. To some extent, hybridization also influences the electron density around the proton in question by electronegativity effects. With increasing s character in a C–H bond, the electrons are held closer to the carbon nucleus. The protons consequently experience less electron density and are, therefore, more deshielded. This reasoning applies very well to protons attached to sp3 rather than sp2 carbons. For sp (acetylenic) protons, however, anisotropic effects are the dominating factors. Electron-donating or electron-withdrawing groups directly attached to aromatic or alkene sp2 carbons greatly affect the chemical shifts of aromatic or vinyl protons via p bond interactions (resonance). Thus, vinyl protons on the b-carbon of an a,b-unsaturated carbonyl system are further downfield (more deshielded) than the proton on the a-carbon due to resonance, and the opposite holds true for the b-proton(s) of a vinyl ether, as shown in Figure 1. In aromatic systems, electron-withdrawing

Figure 1

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Shielding and deshielding effects due to resonance.

Figure 2 Proton Ha is deshielded by B1 ppm due to the ring current of the nearby phenyl group.

groups deshield the protons ortho and para to it relative to unsubstituted benzene, while a group that is electrondonating by resonance will shield the ortho and para protons such that they resonate at a field higher than unsubstituted benzene (d7.27). Empirical methods for estimating the chemical shifts of protons on substituted alkenes and benzene rings have been developed (see Further Reading). It should be realized, however, that the anisotropies of aromatic and alkenyl systems are also responsible for the larger than expected downfield shifts of the protons. The large downfield shift of aldehyde protons (Bd9.5) is due in large part to the anisotropic shielding/deshielding effect (called the cone of shielding/ deshielding in carbonyls), as seen in alkenes and aromatic compounds. Shielding and deshielding effects via anisotropy caused by ring currents can also affect protons not directly attached to the alkene, alkyne, carbonyl or aromatic systems. A good example of this is shown in Figure 2. The calculated chemical shift of the methine proton Ha in the absence of any ring current effects is d4.40, while the observed chemical shift is d5.44. The low energy conformation of the molecule (from molecular modelling) has one of the phenyl rings very near the

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Structural Chemistry Using NMR Spectroscopy, Organic Molecules

proton Ha. Therefore, it appears that the ring current of this phenyl group is deshielding this proton by B1 ppm. In organic molecules possessing protons with very similar chemical shifts, such as steroids or carbohydrates, it would be advantageous to be able to simplify the spectrum by eliminating all spin–spin splittings, thereby allowing the determination of resonance frequencies by only chemical shift effects. An improved method has been developed to produce this type of ‘chemical-shift spectrum’, and is illustrated in Figure 3. Overlapping resonances are resolved into singlets, and this allows for a more straightforward structural assignment of the resonances. Near neighbours, coupling constants and relative stereochemistries can be determined by other spectral editing techniques and experiments (see below). Through-Bond Coupling: Determination of Near Neighbours and Stereochemistry The analysis of through-bond spin–spin coupling (scalar or J coupling) allows for ready determination of the number of neighbouring protons, as well as the relative stereochemistry in certain cases. See the texts listed in Further Reading for more in-depth discussions of spin–spin coupling. In short, spin–spin couplings occur between magnetically nonequivalent nuclei (here, protons) through intervening bonding electrons, and decrease with increasing number of intervening bonds. Protons that are chemically equivalent (interchangeable by a symmetry operation) are magnetically equivalent if

they exhibit identical coupling to any other nucleus not in that set. However, protons with the same chemical shift do not split each other even when the coupling constant between them is non-zero. Rapid rotation about a C–C single bond, such as with a CH3 group, results in an average environment for each methyl proton, and hence, equivalence. Interacting protons with very different chemical shifts are weakly coupled if the difference in chemical shifts between the coupled protons, Dd, is large compared to the coupling constant J, i.e. Dd/J 410. The multiplets resulting from this weak coupling are considered ‘firstorder’ patterns, and can be interpreted easily. The multiplicity is governed by the (2nI þ 1) rule, where n is the number of magnetically equivalent coupled protons and I is the spin of the nucleus. In first-order systems, the multiplicities and peak intensities of coupled protons can be predicted using Pascal’s triangle. For example, a proton split by two magnetically equivalent neighbours will be a triplet with peak intensities of 1:2:1. The frequency difference between the lines of the multiplet is the coupling constant, J, reported in Hz, and is invariant with changes in the strength of the magnetic field. The greater accessibility to higher NMR field strengths has enabled the interpretation of many proton spectra as first-order. In symmetrical spin systems, these simple rules do not apply and a more rigorous analysis is needed. The Pople spin notation system is generally utilized to indicate the degree of difference among nuclei. Thus, in a two spin system, AX indicates a molecule with two

Figure 3 Chemical shift spectra of 4-androsten-3,17-dione obtained from (a) the reflected J spectrum; (b) the purged J spectrum (the additional response near d1.7 is from the residual water signal); and (c) the z-filtered J spectrum. The conventional 1H spectrum is shown in (d). Reprinted with permission from Simova S, Sengstschmid H, and Freeman R (1997) Proton chemical-shift spectra. Journal of Magnetic Resonance 124: 104–121.

Structural Chemistry Using NMR Spectroscopy, Organic Molecules

nuclei where the chemical shift difference is much larger than the coupling between them (weakly coupled system, first-order analysis possible), whereas AB indicates a molecule containing two strongly coupled nuclei with similar chemical shifts. An A2BB0 notation indicates a set of two equivalent nuclei (A) interacting with two nuclei (B, B0 ) that are chemically, but not magnetically, equivalent. For proton NMR, the most diagnostic couplings are 2-bond (2J, geminal), 3-bond (3J, vicinal), and 4-bond (4J, W-type) couplings. Geminal couplings can be quite large, but may not be evident due to the symmetry associated with the carbon and protons in question. As mentioned above, the lack of appearance of geminal coupling is due to the identical chemical shifts of the protons involved. Vicinal 3-bond proton–proton couplings tend to be the most useful when determining stereochemistry, although coupling beyond three bonds can be important in systems with ring strain (small rings, bridged systems) or bond delocalization, as in aromatic and allylic systems. For simple organic molecules, pattern recognition of multiplets can simplify structure determination. For example, the presence of an upfield triplet due to three protons and a more downfield quartet due to two protons with the same coupling constant is most probably due to an ethyl group (X–CH2CH3). Therefore, it is useful to look for common patterns. Many preliminary assignments can be made in a standard 1D 1H spectrum due to the reciprocity of coupling constants (JAB ¼ JBA). With more complex patterns due to coupling to several magnetically non-equivalent protons, interpretation can be done via first-order analysis only if no two of the spins within an interacting multispin system have Dd/J r 6. Multiplets, such as doublet of doublets (dd), doublet of triplets (dt), triplet of doublets (td), doublet of quartets (dq), doublet of doublets of doublets (ddd), etc., can usually be analysed by first-order techniques, especially if the spectrum was run at a fairly high magnetic field. A very useful and practical guide to first-order multiplet analysis that utilizes either a systematic analysis of line spacings or inverted splitting trees to determine the couplings is listed in Further Reading. Measurement of coupling constants can usually be done directly from the 1D spectrum with well-resolved multiplets, or aided by simple 1D homonuclear decoupling experiments where irradiation of one of the weakly coupled nuclei (i.e. nuclei with very different chemical shifts) simplifies a multiplet by eliminating that spin–spin interaction. Several two dimensional techniques also help to determine the coupling network, and determine and assign coupling constants. A 2D COSY (correlated spectroscopy) spectrum is a homonuclear experiment, and provides a map of the proton–proton J-coupling network in the molecule. The spectrum contains a set of auto-correlated peaks along the diagonal

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(o1 ¼ o2), which is the original spectrum. For those spins that exchange magnetization due to J coupling, o2ao1 and off-diagonal peaks appear. The diagonal peaks that correspond to J-coupled spins are connected by symmetrical pairs of off-diagonal peaks. In general, strongly coupled protons are handled better in a COSY experiment than with conventional 1D homonuclear decoupling. However, in molecules that have overlapping resonances, it can be difficult to accurately assign the cross peaks. Computer programs have been developed to provide automated processing and assignment of the data (see Further Reading). Further simplification of the spectrum can be attained by utilizing a DQF-COSY (double quantum filtered COSY) experiment, where singlets are essentially eliminated from the spectrum. Coupling constants can be attained from a COSY spectrum, but it is not a trivial process. The J values measured from a COSY spectrum also tend to be slightly larger than the actual J value. The two-dimensional experiment, homonuclear Jresolved spectroscopy, is utilized to accurately measure the scalar coupling constants. This method can readily resolve overlapping signals, as well as strongly coupled systems. From the contour plot of the spectrum, multiplets are resolved along the y-axis (Hz), and the coupling constants are read directly along this axis. Projection of the multiplet onto the x-axis (d-axis) provides a single resonance line for each distinct spin system without the effects of coupling (i.e. is proton-decoupled), and accurate values of d (ppm) can be attained. Stereochemical Assignments Accurate stereochemical assignments are generally only possible in rigid or ring systems where free rotation about carbon–carbon bonds is hindered or not possible. As mentioned above, vicinal, three-bond couplings (3J) can be quite diagnostic of the stereochemical relationship between the coupling protons. The Karplus equation (eqn [1]) can predict the vicinal coupling constant 3 JH–C–C–H with reasonable accuracy if the H–C–C–H dihedral angle is known. Thus, dihedral angles near 01 or 1801 have the largest coupling constants, while a dihedral angle of 901 has a coupling constant near 0 Hz. 3 3

J ¼ A cos 2 f þ C 0

2

J ¼ A cos f þ C

0

ðf ¼ 02901Þ ðf ¼ 9021801Þ

½1

Use of this relationship in alkenes and ring (or bridged) systems works very well to predict stereochemistry. For alkenes, trans coupling is in the range of 12–18 Hz, and cis coupling is 6–12 Hz. See the texts listed in Further Reading for tables listing coupling constants in various alkenyl systems. Trans, diaxial protons on a six-membered carbocyclic ring have a dihedral angle

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Structural Chemistry Using NMR Spectroscopy, Organic Molecules

of B1801 and a J value of 6–14 Hz (typically 8–10 Hz), whereas protons oriented axial–equatorial or equatorial– equatorial with dihedral angles near 601 have coupling constants of 0–5 Hz (typically 2–3 Hz). In five-membered rings, vicinal trans and cis coupling constants may be similar in magnitude due to the reduced conformational flexibility of the ring relative to six-membered rings. Five-membered rings will adopt a twist conformation to relieve eclipsing interactions. For example, in the oxazo-

Figure 4 Oxazolidinones exhibiting coupling constants of 3 J4–5 cis ¼ 7.2 Hz and 3J4–5 trans ¼ 5.9 Hz.

Figure 5

lidinones in Figure 4, it was found that 3J4– 5cis ¼ 7.2 Hz, and 3 J4–5trans ¼ 5.9 Hz. Where no coupling is possible because of a quaternary centre or if the coupling constants fall on the borderline between two possible orientations nuclear Overhauser effect (NOE) measurements may need to be taken in order to definitively establish the relative stereochemistry. The use of pulsed field gradients (PFGs) in the acquisition of NOE enhancement spectra now allows one to avoid the need to compute difference NOE spectra, as was done in the past. With NOE difference spectra, it was hard to avoid subtraction artifacts, and thus difficult to obtain accurate NOE values, especially small (o1%) enhancements. Utilizing PFGs, the only resonances now seen in the spectrum are those from spins which are cross-relaxing with the irradiated spin. The stereochemical assignments for the oxazolidinones in Figure 4 were confirmed by the NOE enhancements of 13% between H4 and H5 in the cis isomer, and only 2% between H4 and H5 in the trans isomer. There was also an NOE enhancement of 5% between the CH2 a to the phosphonate and H5 in the trans isomer. In the examples shown in Figure 5, the

NOE enhancements of two diastereomeric cyclic carbonates to confirm relative stereochemistries.

Structural Chemistry Using NMR Spectroscopy, Organic Molecules

vicinal coupling constants were all small (0–3 Hz), thus indicating that all the methine protons were in axial– equatorial or equatorial–equatorial relationships. The NOE measurements indicated in Figure 5 enabled final assignments of the relative stereochemistries. 13

C NMR

Information regarding the number and types of carbons in an organic compound can be provided by carbon NMR spectroscopy. Since the chemical shift range is greater for carbon than for proton, a greater dispersion of signals is seen. Different functional groups that contain at least one carbon, such as ketone, ester and amide carbonyls, alkenes, alkanes, alkynes, nitriles, imines, etc., can generally be readily distinguished by 13C NMR spectroscopy. The chemical shifts of aromatic and alkene carbons, however, are in the same chemical shift range, and at times cannot be differentiated. This is in contrast to aromatic and alkene protons which exhibit different chemical shift ranges. In general, the factors that affect the chemical shifts of carbons are the same as for protons (i.e. electron density around the nucleus in question, and anisotropy effects). Carbon chemical shifts can be readily calculated from tables of shift effects found in many texts. However, unlike protons attached to sp2 carbons, sp3 carbons attached to sp2 carbons exhibit only a small shift difference. There are also few good substituent parameters available for calculating the chemical shifts of alkene carbons bearing polar groups, unlike the calculation of 1 H NMR chemical shifts near polar groups. However, in systems where resonance is present, some predictions can be made of relative shift differences in the carbons (see Figure 1). Carbon–proton connectivities can be determined using several methods. The number of protons directly attached to the carbon in question will split the carbon resonance according to the 2nI þ 1 rule seen in proton NMR. There tends to be, however, much overlap of the

Figure 6 results.

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multiplets in fully proton-coupled carbon spectra, sometimes such that it is very difficult to distinguish between the various multiplets. Routine carbon spectra are therefore measured fully proton decoupled for simplicity. Information regarding the exact number of protons attached to the carbons can be acquired from APT, DEPT or INEPT experiments. In APT spectra, the carbons bearing an odd number of protons (CH, CH3) can be distinguished from carbons with no or two attached protons (quaternary C, CH2). DEPT and INEPT experiments can distinguish between all four types of carbons (primary, secondary, tertiary and quaternary). Heteronuclear 2D J-resolved spectroscopy can also be used to obtain the multiplicities of the carbons, as well as 1JC–H. A complete mapping of the protons to the carbons they are attached to is possible via a HETCOR (heteronuclear chemical shift correlation) experiment. This method correlates the peaks of the proton spectrum of a compound with the peaks of its carbon spectrum. A contour plot of the spectrum has a cross peak at the intersection of the vertical line drawn from a carbon resonance (plotted along the x-axis) with the horizontal line drawn from a proton peak (plotted along the y-axis). However, this method is relatively insensitive as it suffers from the low natural abundance of 13C atoms in the molecule. Utilization of the inverse detection method, HMQC (heteronuclear correlation through multiple quantum coherence), can alleviate this problem as 13C responses are observed in the 1H spectrum. Normally, 1H–13C coupling information is included in the 1H dimension, although proton decoupling from carbon is possible. Quaternary carbons, however, will not be present in a HMQC spectrum. A method to aid in assignments of quaternary carbons, as well as carbon and proton connectivities, is the HMBC (heteronuclear multiple bond correlation) experiment. In these spectra, cross peaks are observed connecting the 13C signals to 1H signals two or more bonds away. The INADEQUATE experiment is designed to map out the entire carbon skeleton of a molecule by providing

Possible products from the rearrangement of structure [1] in water. Bratis AD, Bruch MD, and Murray RK Jr unpublished

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Structural Chemistry Using NMR Spectroscopy, Organic Molecules

Figure 7 (a) APT 13C spectrum of compound [2]; (b) INADEQUATE spectrum of compound [2], with the connectivities of C1 to C7, (see Figure 6) C8 to C14 and C3 to C5 shown. Bratis AD, Bruch MD, and Murray RK Jr unpublished results.

Structural Chemistry Using NMR Spectroscopy, Organic Molecules

Figure 8

Possible products from the photolysis of compound [5]. Kiessling AJ and McClure CK, unpublished results.

Figure 9

COSY spectrum of compound [6].

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Structural Chemistry Using NMR Spectroscopy, Organic Molecules

carbon–carbon connectivities, and offers great possibilities for organic structure determination. However, it suffers severely from the low natural abundance of covalently bound 13C–13C pairs. Several groups have offered modifications of the pulse sequence to try to overcome this limitation. It remains to be seen, however, if these new programmes will produce the higher sensitivities needed for this experiment to become a routine analytical procedure. Even without the newer modifications to the pulse sequence, the INADEQUATE experiment can be an invaluable method for structure determination in some cases, as illustrated in the example in Figure 6. The rearrangement of the cage hydrocarbon diazonium ion [1] in the presence of water could lead to three possible alcohol products [2], [3] and [4]. Only two alcohol products were obtained from this reaction in isolated yields of 38% and 7%. The structures of these two products could only be determined by the 2D INADEQUATE experiment due to the hydrocarbon nature of their structures, and therefore, the lack of any distinguishing details in the proton spectra. Each compound also had the same number of methylene, methine and quaternary carbons, thus precluding the utilization of structure determination by simple 1D 13C spectroscopy. The INADEQUATE and APT spectra for the major product are shown in Figure 7. In an INADEQUATE spectrum, the pairs of adjacent carbons, and hence the

connectivity, can be mapped out similarly to a COSY spectrum. The major difference here is that the original spectrum is not on the diagonal in an INADEQUATE spectrum (as in a COSY spectrum), but is in the x-axis direction (¼ normal 13C frequencies) along the line n1 ¼ 0 (residual single quantum signals). The y-axis is the frequency n1, the double quantum frequency that is the sum of the frequencies of the two coupled nuclei referenced to a transmitter frequency at zero. The peaks arising from two coupled nuclei (here adjacent carbons) with shifts na and nb have coordinates of ((na þ nb), X), where X is the frequency of the carbon in a single quantum coherence spectrum (1D spectrum). At the double quantum frequency (n1) for each pair of adjacent carbons, doublets will occur at the coordinates of ((na þ nb), na) and ((na þ nb), nb). Thus, the midpoint between each pair of signals lies on a line with slope of 2, and helps to distinguish the real peaks from artifacts. The (C7–C1) – (C1–C7), (C14–C8) – (C8–C14) and (C3–C5) – (C5–C3) peak pairs are illustrated on the spectrum (B) in Figure 7. In the example illustrated in Figure 6, it may be noted that compound [3] would require the grouping –CH2CH2– to be present, which is not seen in the INADEQUATE spectrum. For the major compound to be structure [4], the connectivity of carbon 1 would have to be to carbon 7 and the quaternary carbon, q. The connectivity found for carbon 1 was to carbon 7 and the methine carbon 12. Therefore, only structure [2] fully

Table 1 Comparison of calculated proton distances, dihedral angles and estimated J couplings, with J couplings and NOE results for compound [6]

Protons

Ha–Hb Hb–Hc Hb–Hd Hc–Hd Hc-He Hc-Hf Hd–He Hd–Hf He–Hf Hg–Hh

Molecular Mechanics

Experimental

Distance (A˚)

Angle (1)

J coupling (Hz, estimated)

J coupling (Hz)

NOE

2.71 2.47 2.92 1.81 2.37 2.77 3.09 2.48 1.79 1.8

7.1 13 108 109 32 87 154 35 107 108

8 7.5 1 10–20 6 o1 8 6 10–20 10–20

4.9 6.5 0 14.0 7.0 0 NA 6.2 12.3 18.0

yes yes ND yes yes ND ND ND yes yes

NA ¼ not available; ND ¼ no NOE detected.

Structural Chemistry Using NMR Spectroscopy, Organic Molecules

supports all the spectral data for the major isolated product from this rearrangement.

Putting It All Together The following is an outline of the basic procedure that a practising organic chemist follows when deducing the structure of an organic compound via NMR spectroscopy. First, standard 1D 1H and 13C spectra are

Figure 10

(a)

13

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acquired and analysed. If needed, a ‘chemical shift spectrum’ can provide a straightforward assignment of the d value of all the resonances, including overlapping multiplets. Proton spin–spin couplings and near neighbours are either determined directly from the 1D 1H spectrum, or assisted by homonuclear decoupling experiments. If these experiments are not conclusive due to overlapping resonances, changing the solvent or utilization of a shift reagent can on occasion resolve the overlapping multiplets. A map of the J-coupling network

C spectrum of compound [6] (see Figure 2) in CDCI3/benzene-d6; (b) HETCOR spectrum of compound [6].

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Structural Chemistry Using NMR Spectroscopy, Organic Molecules

in the molecule is available through 2D COSY or TOCSY experiments. Programs to assist in assignment of the cross-peaks in complicated COSY or TOCSY spectra are now available. Utilization of the DQF-COSY experiment eliminates all the singlets from the spectrum. The homonuclear 2D J-resolved spectrum allows for the separation of overlapping resonances and, therefore, accurate measurement of the coupling constants and chemical shifts. A number of 13C experiments, such as APT, DEPT, INEPT, INADEQUATE, and heteronuclear 2D J-resolved experiments, can be run to assist in determining proton–carbon attachment, carbon–carbon attachments for carbon skeleton determination, and values of 1JC–H. A 2D HETCOR spectrum will confirm the proton–carbon connectivities. Various NOE experiments can assist in determining stereochemical information whereas coupling constants cannot. The following is an example that illustrates how several of the experiments listed above, as well as molecular mechanics calculations, can be used to deduce the structure of the compound produced by a photochemical rearrangement. The photochemical irradiation of the bicyclic compound [5] was run under sensitized conditions (acetophenone). Two possible products, [6] and [7], are theoretically possible, and are shown in Figure 8. The 1,3-acyl shift product [7] normally arises from photolysis under non-sensitized conditions, but can be formed from certain compounds under sensitized photolysis conditions. The oxa-di-pi-methane rearrangement product [6] was the desired compound. From 1D 1 H and 13C NMR spectra, the only product isolated from the photochemical rearrangement did not appear to contain an olefin, as would be seen in the a,b-unsaturated ester [7]. Thus, the rearrangement most likely went via the oxa-di-pi-methane rearrangement, and not by the 1,3-acyl shift mechanism. Further proof that the structure of the photoproduct was indeed [6] is as follows. From the standard COSY spectrum (Figure 9), all the proton–proton coupling networks could be established. The proton responsible for the peak at d4.22 (d, J ¼ 4.8 Hz) was coupled only to a proton at d3.06 (dd, J ¼ 6.5, 4.9 Hz), which in turn was coupled to only one other proton at d2.25. Of the protons Ha–Hf, only proton H a was expected to be coupled to only one other proton, namely Hb. The chemical shift of d4.22 was also reasonable for Ha. The multiplet at d3.06 was therefore assigned to Hb, and was coupled to one other proton at d2.25. This other proton could be either Hc or Hd. In order to assist in the assignments, the tricyclic structure [6] was submitted to molecular mechanics calculations to estimate the dihedral angles between the protons, and thus approximate the coupling constants using eqn [1] (see Table 1). According to these calculations, Hb and Hc had a dihedral angle of B131 and thus

an estimated coupling constant of 7.5 Hz, while Hb and Hd had a dihedral angle of B1101 and an estimated coupling constant of 1 Hz. The measured J value between signals at d3.06 and d2.25 was 6.5 Hz, closely matching the calculated coupling constant between Hb and Hc. Therefore, Hc was assigned to the signal at d2.25. This multiplet (ddd) exhibited three coupling constants of 14.0, 7.0 and 6.9 Hz. The large coupling of 14 Hz would be consistent with geminal coupling to proton Hd. The calculations predicted that in addition to Hb, Hc would couple to He with a dihedral angle of 321 and an estimated coupling constant of 6 Hz. Hf was predicted to be nearly orthogonal to Hc, and thus have little or no coupling to Hc. From this data, Hd was assigned to the multiplet at d1.99 and He to the multiplet at d3.32. From the COSY spectrum, the multiplet at d1.99 was further coupled to the multiplet at d2.79, which was assigned to Hf . The multiplet (dd) at d2.79 had two coupling constants of 12.3 and 6.2 Hz. The large coupling constant was geminal coupling with He. The other coupling constant was consistent with the calculation of the dihedral angle of 351 between Hf and Hd. The protons Hg and Hh were coupled only to each other, and the exo proton Hg was assigned as the downfield doublet of doublets at d3.65. The distances between the protons of the proposed structure were also calculated by molecular mechanics and are summarized in Table 1. The photoproduct was submitted to NOE experiments to verify the spatial relationships. The signals assigned to Ha, Hb and He yielded meaningful data, and the NOE results are shown in Table 1. Results of the NOE experiments are in agreement with the proposed structure, where Ha, Hb, Hc and He are shown to be in a cis relationship. The 13C and HETCOR spectra in Figure 10a and Figure 10b, respectively, further verified the proposed structure [6]. No carbon signals were detected in the Table 2

HETCOR data for compound [6]

Carbon

13

Proton(s)

1

C-1 C-2 C-3 C-4 C-5 C-6

63.7 43.7 26.5 64.3 68.8 53.1

Ha Hb Hc,d He,f Hg,h Hi

4.23 3.06 2.25, 1.99 3.32, 2.79 3.64, 2.84 3.75

Cd

Hd

Structural Chemistry Using NMR Spectroscopy, Organic Molecules

alkene region of the spectrum, consistent with the lack of alkene protons. The only carbonyl peak detected was at d207.6, consistent with the ketone in [6]. The HETCOR data is summarized in Table 2. See also: Chemical Exchange Effects in NMR, Chemical Shift and Relaxation Reagents in NMR, ChromatographyIR, Applications, Enantiomeric Purity Studied Using NMR, Magnetic Field Gradients in High Resolution NMR, NMR Data Processing, NMR Pulse Sequences, Nuclear Overhauser Effect, Structural Chemistry Using NMR Spectroscopy, Peptides, Structural Chemistry Using NMR Spectroscopy, Pharmaceuticals, 13C NMR, Methods, 13C NMR, Parameter Survey, Two-Dimensional NMR.

Further Reading Bourdonneau M and Ancian B (1998) Rapid-pulsing artifact-free double-quantum-filtered homonuclear spectroscopy. The

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2D-INADEQUATE experiment revisited. Journal of Magnetic Resonance 132: 316--327. Bruch MD (ed.) (1996) NMR Spectroscopy Techniques, 2nd edn. New York: Marcel Dekker. Derome AE (1987) Modern NMR Techniques for Chemistry Research. Oxford: Pergamon. Hoye TR, Hanson PR, and Vyvyan JR (1994) A practical guide to firstorder multiplet analysis in 1H NMR spectroscopy. Journal of Organic Chemistry 59: 4096--4103. Lambert JB, Shurvell HF, Lightner DA, and Cooks RG (1998) Organic Structural Spectroscopy. New York: Macmillan. Sengstschmid H, Heinz S, and Freeman R (1998) Automated processing of two-dimensional correlation spectra. Journal of Magnetic Resonance 131: 315--326. Silverstein RM, Bassler GC, and Morrill TC (1998) Spectrometric Identification of Organic Compounds, 6th edn. New York: John Wiley. Simova S, Sengstschmid H, and Freeman R (1997) Proton chemicalshift spectra. Journal of Magnetic Resonance 124: 104--121. Stonehouse J, Adell P, Keeler J, and Shaka AJ (1994) Ultrahigh-quality NOE spectra. Journal of the American Chemical Society 116: 6037--6038.