NMR Methodological Overview

C H A P T E R

7 NMR Methodological Overview Zolta´n Szaka´cs and Zsuzsanna Sa´nta Gedeon Richter Plc, Spectroscopic Research Division, Budapest, Hungary

O U T L I N E 7.1 Introduction

7.3.6 1H,13C-HMBC 7.3.7 1H,15N-HSQC and 1H, 15 N-HMBC

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7.2 One-Dimensional (1D) NMR Measurements 7.2.1 1D 1H NMR Spectrum 7.2.2 Selective 1D NOESY (ROESY) Spectrum 7.2.3 Selective 1D TOCSY Spectrum 7.2.4 1D 13C NMR Spectrum 7.3 Two-Dimensional (2D) Methods 7.3.1 COSY 7.3.2 2D TOCSY 7.3.3 2D NOESY 7.3.4 1H,13C-HSQC 7.3.5 HSQC-TOCSY

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7.5 Diffusion-Ordered Spectroscopy (DOSY)

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7.6 Summary

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Acknowledgments

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References

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7.1 INTRODUCTION As a result of the fascinating development that NMR spectroscopy underwent in the past 60 years, today it offers the richest source of structural information for small molecules. Hundreds of NMR experiments (pulse sequences) are currently available to generate various types of NMR spectra providing complementary pieces of structural information that can be used in assembling the 3D structure of small organic molecules (typically with molecular weights below 1000 Da).

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In this chapter, our primary aim is to give a short description of what each of those experiments can “do.” Although our discussion is aimed to be accessible to the non-NMR expert, we assume that the reader has some basic knowledge about the fundamentals of applied NMR spectroscopy, and we also rely to some extent on some of the NMR concepts discussed in Part II. Based on that premise, we do not wish to explain the definitions and technicalities underlying such basic concepts as the chemical shift or chemical exchange, or to dwell into the spin-physical design or technical implementation of pulse sequences; besides the problem that this would make yet another book on NMR by itself, it would also be redundant, because all of these ideas and methodologies are thoroughly discussed in countless excellent reviews in the literature. Rather, our goal is to provide a compact overview of the most common NMR techniques used today in small-molecule structure elucidation, with focus placed on the kind of structural information that is “revealed” by each of those experiments, and with a view to serving the following two specific purposes within the context of this book. On the one hand, we mainly discuss those NMR methodologies that will come up in later chapters so as to serve as a reference for the better understanding of those case studies, and thus to make this book reasonably self-contained. Although in that respect our overview is far from being comprehensive with regard to the number of NMR methods currently available, we believe that it faithfully reflects those core methods that are the most important in small-molecule structure determination today. On the other hand, in line with the philosophy of this book, the discussed methods are illustrated with real-life examples through which we wish to show not only the utility of these experiments, but also their potential limitations and interpretational pitfalls (Mental Traps). Readers interested in the greater technical depths of the topic should consult dedicated monographs (see, e.g., Refs. 1–10), which discuss the fundamentals of NMR,5 give illustrative examples of how molecular structures are deduced from NMR spectra,2,5,6 outline the possible strategies of structure elucidation,6,8–10 offer problems for self-training,2,7,8 introduce the reader to the compelling world of implementing pulse sequences,1,4,9 or help organic chemists choose the most effective pulse sequence for a particular problem.3

7.2 ONE-DIMENSIONAL (1D) NMR MEASUREMENTS 7.2.1 1D 1H NMR Spectrum The majority of organic molecules of a synthetic or natural origin are rich in hydrogen atoms. Due to the favorable magnetic properties of this nucleus, 1H NMR spectroscopy is very sensitive as compared to the less abundant observable nuclides of “heavier” atoms such as 13 C and 15N. Thus, recording a simple 1D proton spectrum within a few minutes is usually the very first step of any NMR-based structural study, and, in favorable cases, a wealth of information may emerge from the key 1H spectral parameters discussed below. Protons in different chemical environments give rise to signals at different positions in the spectrum. The chemical shift (d in ppm) often reveals the functional group a given proton belongs to (see Fig. 7.1). Symmetrically located protons give a common signal. Applying the proper experimental settings discussed below, the integral of the signal becomes a measure of the number of protons contributing to that signal. This is a useful feature

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FIGURE 7.1 400 MHz 1H NMR spectrum of ethyl-(2E)-4-oxo-4-phenylbut-2-enoate 7.1 in DMSO-d6. Key spectral parameters are the chemical shifts, multiplicities, and integral values. The signal of tetramethylsilane (TMS) serves as the reference for the chemical shifts at d ¼ 0.00 ppm.

to be exploited during spectrum interpretation, for example, when distinguishing between a CH2 group and a CH3 group if both give singlets. In this way, not only can the number (and identity) of functional groups within the compound under study be assessed, but also, for mixtures, the constituents (reaction by-products, contaminants, solvent residues, etc.) can be identified by their characteristic chemical shifts and, at the same time, their molar ratio can be determined from the corresponding integrals (this is the realm of quantitative NMR spectroscopy)11 provided that each compound has at least one nonoverlapping signal that can be selectively integrated. The concept that the integral values are directly proportional to the number of protons contributing to the signals is only valid, among other experimental conditions, if pulsed excitation is fast on the T1 and T2 relaxation timescales. If spectrum accumulation is employed (see Chapter 2), the recycle delay (the sum of the acquisition time and an additional time allowing for T1 relaxation) between two consecutive pulses should be set long enough (>5T1) to ensure complete relaxation and thus to obtain correct integral values. This can be a difficulty if some of the protons in the molecule have (a priori unbeknownst to the analyst) unusually long T1 values (say, more than 5 s), in which case signals due to these protons will give smaller integral values in the spectrum than those belonging to the other protons with more typical relaxation times (ca. 1-2 s) if the recycle delay is optimized to the latter. In contrast, in the same spectrum, signals broadened due to rapid T2 relaxation (such as OH or NH protons) often give smaller measured integral values than the sharp signals due to protons attached to a carbon.

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1

H resonance signals typically also feature a fine structure composed of several spectral lines with a well-defined ratio of intensities. This phenomenon is due to the so-called scalar or spin-spin coupling (see Chapter 2), which is useful to detect neighborhood proton-proton constitutional and geometric relationships. The magnitude of this coupling is expressed in terms of the coupling constant J, which is measured in Hz. For “simple” multiplets, the homonuclear coupling constant over m bonds, mJHH, can be read off as the difference in the appropriate peak frequencies. If the interaction of a given proton occurs with (almost) the same coupling constant with its n proton “neighbors,” its resonance signal is split into n + 1 lines. For example, in a –CH2CH3 moiety, the methyl group gives a triplet while its CH2 neighbor a quartet as demonstrated in Fig. 7.1. This simple “n + 1 rule” holds only for the so-called first-order spin systems. With an increasing ratio of the coupling constant (J) and the chemical shift difference (Dd) between the coupled protons, the shape of the multiplet is “deformed,” and the inference of vicinity relationships may become far from obvious, or even misleading. The largest splitting is usually caused by geminal or vicinal proton partners over m ¼ 2 or 3 bonds, respectively, while the magnitude of splitting decreases steeply with the number of separating bonds. (Geminal CH2 protons split each other’s signals or give rise to cross peaks in the 2D COSY or TOCSY spectra (see below) only if they have different chemical shifts.) When the molecule contains NMR-active “heteronuclei” (such as the spin-1/2 19F or 31P), then the signal of a proton situated 2-4 bonds away from the heteroatom is further split by the heteronuclear mJHX coupling. In saturated systems, the magnitude of the vicinal 3JHH coupling constant reflects the H–C– C–H dihedral angle ’: 3

J ð’Þ ¼ Acos 2 ð’Þ + Bcos ð’Þ + C,

(7.1) 12

where A, B, and C are parameterized for the given class of compounds and solvent. The Karplus-type relationship Eq. (7.1) enables the spatial arrangement of two protons to be determined, which is useful, for example, for the stereospecific assignment of methylene protons or to distinguish between epimeric structures containing cyclohexane (or pyranose, piperidine, morpholine, tropane, etc.) rings in a chair conformation. Finally, (pro)chirality, hindered molecular motions, or intermolecular exchange processes may lead to more complicated spectra than expected, containing several sets of multiplets or broadened resonances. Such 1H spectra may often become tractable for structure determination only after some “manipulation” of the sample, such as “freezing” the nitrogen inversion of an alicyclic ring by adding strong acid, or by merging the multiple signal sets by accelerating the molecular dynamics by heating. Unless otherwise stated, all NMR spectra presented in this book were recorded near room temperature (at 25 or 30 °C). Can a 1H NMR spectrum alone be sufficient to define the constitution of a molecule? A quick survey of the 1H NMR spectrum proves or disproves the presence of most hydrogen-containing functional groups, such as the acetyl, amide, or ethoxy groups (moieties comprising exclusively noncarbon heavy atoms, such as SO2 or NO2, may be identified from the IR and MS spectra). In favorable cases, the 1H NMR spectrum may even allow the complete determination of the molecular constitution without having to resort to more time-demanding (2D) NMR experiments. This is exemplified by the following case. A synthetic chemist colleague of ours attempted to prepare the rather simple heterobicycle 7.3 (Fig. 7.2) by ring closure of the b-alanine derivative 7.2, and the purified reaction product,

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expected to have the structure 7.3, was submitted for structure verification. MS determined the nominal mass to be 148 Da, which accords with 7.3, and even the molecular formula C8H8N2O from high-resolution MS or the observed fragmentation pattern did not cast any doubt on the correctness of the proposed constitutional formula 7.3. However, the 1D 1H NMR spectrum of the sample (Fig. 7.3) exhibited no CH2 signals in the aliphatic region; hence, the structure 7.3 had to be refuted. Three characteristic multiplets between 5.6 and 6.8 ppm suggested the presence of a vinyl group, and the broad singlet at 10.70 ppm an amide moiety. Based on these facts, the formation of the unexpected amide derivative 7.4, having the same molecular weight as 7.3, could be deduced. Of course, in the majority of cases the 1H NMR spectrum does not contain all the information necessary to exclude the possible constitutional isomers or stereoisomers. To avoid or alleviate Mental Traps #4, #21, #22, #23, and #29 associated with overlooking those alternative structures that may also be consistent with the 1H NMR data, we need to uncover the throughspace and through-bond relationships (correlations) that exist, as already mentioned in Chapter 6, Section 6.2.1, between the protons in the molecule. These correlations give rise to the NOE-based and COSY/TOCSY-type experiments, respectively, which will be elaborated further in the following sections.

FIGURE 7.2 Synthetic route with the expected intermediate 7.2 and product 7.3 (above); the correct structure 7.4 was deduced from the 1D proton spectrum shown in Fig. 7.3.

FIGURE 7.3 400 MHz 1H NMR spectrum of 7.4 in DMSO-d6 (for the numbering of protons, see Fig. 7.2).

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7.2.2 Selective 1D NOESY (ROESY) Spectrum While the conventional 1H NMR spectrum displays the resonance signals of each proton in the molecule, individual signals can also be selectively excited by the combination of soft shaped pulses (which have a temporal profile other than rectangular) and gradient pulses (which manipulate the local B0 field experienced by the spins in the different volume elements of the sample; see also Section 7.5).13 If the signal of a particular proton does not overlap with other signals, the effect of its selective excitation can be transferred via dipole-dipole relaxation (cross relaxation) to the other protons in the molecule that are located close in space ˚ ). In small molecules, this transfer of magnetization manifests itself in the following (<4 A phenomenon: If the magnetization of proton A is selectively excited (say, saturated or inverted), then the integral value of a spatially close proton B will increase by a few percent of its original value (in large molecules, the signal intensity decreases). This is the nuclear Overhauser effect (NOE), which is extremely important in exploring the 3D geometry of a molecule, and as such, it is extensively used in conformational and configurational studies, but it can also be decisive in determining constitutional issues.14 The NOE on HB does not appear instantaneously, but builds up on a timescale of T1 after HA has been selectively excited. The speed with which the NOE develops depends primarily on the distance rAB between HA and HB (it is proportional to r6 AB) as well as on the average rate of molecular tumbling, expressed by the so-called rotational correlation time tc, relative to the Larmor frequency o0. There are several ways of measuring the NOE.14 In the 1D nuclear Overhauser effect spectroscopy (NOESY) method,13 the magnetization of HA is selectively inverted and there is a so-called mixing time, set by the spectroscopist, during which the NOE on the spatially close protons is allowed to develop (typical values range between 0.5 and 1 s for small molecules). There is always an optimum in choosing the mixing time: by increasing its value, there will be more time for the buildup of NOEs, but after a while the NOE intensity starts to decrease due to T1 relaxation. It is important to note that while the presence of an NOE between HA and HB is a strong indication of their spatial vicinity, the absence of an NOE does not necessarily mean that they are far apart. One important reason for this is because if o0 tc  1, the molecule will fall in between smallmolecule behavior (the so-called extreme narrowing regime with a positive NOE) and largemolecule behavior (the so-called spin-diffusion regime with a negative NOE), and so the NOE is close to zero (this is the so-called zero-crossing region).14 In that regard, the common distinction between the positive and negative NOE regions only in terms of molecular size can be misleading. In fact, the tumbling rate tc is determined by not only the size of the molecule, but also by the viscosity of the sample solution and possibly by attractive solute-solute intramolecular interactions that may slow down the rotation of the molecule. Thus, a “nominally” small molecule with a molecular weight of, say, 500 Da can fall in the zero-crossing regime or even give negative NOEs if the solvent is viscous or there are strong intramolecular attractions present. In practical terms, compounds of ca. 1 kDa molecular mass may not furnish the structurally relevant NOE peaks at 500 MHz in solvents of moderate viscosity. The zero-crossing problem can be circumvented by implementing the so-called rotatingframe spin-lock technique into the measurement of the NOE. Roughly, the idea is that the magnetization is brought onto the, say, x0 axis of the (resonant) rotating frame (cf. Chapter 2), and is “locked” there during the mixing time by applying a so-called spin-lock field Block along the x0 axis. (Technically, the spin-lock field is achieved by a special sequence of delays and “hard” RF

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FIGURE 7.4 Distinction between the regioisomers 7.5 and 7.6 using the 400 MHz 1D NOESY spectrum (bottom) in DMSO-d6. Arrows indicate the decisive NOEs. The conventional 1H spectrum is also shown (top).

pulses.) In the rotating frame Block acts as if this was the only field experienced by the spins, which then exhibit a Larmor frequency olock ¼ gBlock . Because Block is much smaller than B0, olock is also much smaller than o0, and thus the value of olocktc will be smaller than 1, so the NOEs fall safely into the positive regime irrespective of molecular weight, viscosity, or self-associations. Spin-lock NOE methods are typically less robust in terms of experimental setup and the appearance of artifacts than the conventional NOE experiments; therefore, often the latter are recommended as the first choice in exploring the NOEs in a small molecule. A typical example of the spin-lock concept used for the measurement of NOEs is the selective 1D rotating frame Overhauser effect spectroscopy (ROESY).3 As an example, the position of the methanesulfonyl moiety on either of the indazone nitrogens, N(2) or N(1) in 7.5 and 7.6 (Fig. 7.4), can be rapidly assessed by recording a 1D NOESY spectrum. Upon selective inversion of the methyl singlet at 3.76 ppm, the singlet at 8.99 ppm gives an NOE peak, which must belong to H-3 rather than H-7. Thus, the sample is proved to contain compound 7.5 besides some (related) impurities.

7.2.3 Selective 1D TOCSY Spectrum Let us concentrate on the protons within a typical small organic molecule. As already noted in Section 7.2.1, the protons “sense” each other via through-bond J coupling such that the magnitude of the coupling diminishes rapidly with the number of bonds separating the coupling partners. Typically, a given proton senses its close neighbors that are within a distance of 2-4, or sometimes 5 bonds, but does not “see” more distant protons. Thus, the protons in a molecule form an overall 1H-1H coupling network within which we typically find smaller clusters of protons that see each other well, but are more or less isolated from other members of the whole network. These smaller spin “families” are often called spin systems. Most III. SMALL-MOLECULE STRUCTURE ELUCIDATION BY NMR AND MS

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typically, 1H spin systems comprise –CHk–CHl–CHm– (etc.) groups that are isolated from each other in terms of coupling by a quaternary carbon or a heteroatom. Note however that NHm or OH groups may also become the coupling partner in a spin system if the rate of proton dissociation (chemical exchange) is low, which is typical when, for example, DMSO-d6 is used as the solvent or for amide protons in D2O at acidic pHs. Spin systems are characteristic of the different moieties in a molecule, and thus their identification is an essential part of the structural assignment procedure. However, identifying the individual spin systems is often far from easy, especially if we have a complicated coupling network giving congested signals in the 1H spectrum. A very useful technique through which we can identify spin systems is called total correlation spectroscopy (TOCSY). In the 1D 1H TOCSY experiment a given proton is selectively perturbed and its magnetization is transferred to the neighboring protons. The 1D TOCSY experiment also has an adjustable mixing time during which a spin-lock field is applied. The mixing time controls how far the magnetization transfer will propagate within a spin system. A low (20-25 ms) setting of mixing time allows magnetization to be transferred only to the immediately adjacent CHn group(s), while longer mixing times (70-90 ms) enable the detection of all of the signals of a spin system. As an example of the utility of the 1D TOCSY experiment, consider the 1H NMR spectrum of 5-b-androstanedione 7.7 in which 22 methine and methylene protons resonate between 2.8 and 1.1 ppm, several of them giving overlapping signals even at 800 MHz (Fig. 7.5a). For instance, the multiplet near 1.87 ppm consists of the overlapping signals of H-5b and H-12b. In order to observe the multiplet of H-5b alone, a 1D TOCSY spectrum was recorded by the selective excitation of the adjacent H-4a proton at 2.68 ppm. As shown in Fig. 7.5b, magnetization was transferred within a mixing time of 50 ms from H-4a to H-4b at 2.05 ppm, to H-5b and to a lesser extent to both of the H2-6 methylene protons at 1.33 and 1.93 ppm. The now

FIGURE 7.5 (a) Partial 800 MHz 1H spectrum of 5-b-androstanedione 7.7 recorded in CDCl3. (b) 1D TOCSY spectrum with 50 ms mixing time upon selective excitation of the H-4a signal at 2.68 ppm.

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clearly observable multiplet of H-5b at 1.87 ppm exhibits the following coupling constants: 13.5, 4.8, 4.8, and 2.1 Hz. From the Karplus-type relationship Eq. (7.1) and the molecular model shown in Fig. 7.5, the largest vicinal coupling of H-5b is with H-4a, because H-5b and H-4a are in an anti-position; the two medium-sized (4.8 Hz) couplings are with H-6a and H-6b, reflecting their gauche arrangement relative to H-5b; the smallest (2.1 Hz) coupling is with H-6b, in line with the dihedral angle ’ between H-5b and H-6b being nearly 90°. The stereochemistry of H-5b is thus confirmed with these arguments. Although the complete 1H signal assignment of this steroid relies on additional NMR experiments, most notably the 2D 1H,13C-HSQC (see below), this example should illustrate well the benefits of 1D 1H TOCSY: after selectively exciting a proton signal, its coupling network can be gradually explored. One can thus assess the multiplicity and the coupling constants (which carry valuable stereochemical information) in a spin system, since this technique retains the superior digital resolution of the conventional 1D proton spectrum (this will not necessarily be the case in the direct dimension of the 2D TOCSY variant discussed in Section 7.3.2). It can also be extremely useful to apply 1D 1H TOCSY in impurity profiling. If a multiplet of the impurity can be selectively excited, this technique will expose its whole spin system, including the signals obscured by those of the main component. Such a case is demonstrated in Fig. 7.6,

FIGURE 7.6 (a) Partial 1H spectrum of an aromatic compound (its huge peaks are clipped vertically for clarity) with impurities present in less than 0.5 mol%. (b-d) Selective 1D 1H TOCSY spectra started from a nonoverlapping peak (denoted by a spark) of each impurity (X, Y, and Z) in order to identify the substitution pattern of its aromatic ring. The asterisks denote subtraction artifacts created during elimination of the intense signals of the main component. III. SMALL-MOLECULE STRUCTURE ELUCIDATION BY NMR AND MS

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where the task was to determine whether three impurities of the sample exhibit the same splitting pattern as the main component: two doublets, a doublet of doublets and a broad singlet, characteristic of a 1,3-disubstituted phenyl ring. The 1D 1H TOCSY spectra proved that all the impurities share the same splitting pattern; thus, their structure contains the same 1,3-substituted phenyl ring as the main component.

7.2.4 1D

13

C NMR Spectrum

Since the 12C nucleus is magnetically inactive, only the naturally much less abundant ( 1%) spin-½ 13C nucleus can be subject to NMR observation. The gyromagnetic ratio g of the 13C nucleus is one-fourth that of 1H; therefore, in a given B0 magnetic field the Larmor precession frequencies of the 13C nuclei within a molecule are also nearly one-fourth of the 1 H frequencies. This means that for a magnet in which protons resonate at, say, 500 MHz, the 13C nuclei will resonate at nearly 125 MHz, and consequently the proton and carbon frequencies can be excited separately by hard RF pulses of the pertinent frequencies. The low gyromagnetic ratio of 13C also contributes to the fact that 13C spectroscopy is much less sensitive than the 1H measurement. To achieve an appropriate signal-to-noise ratio, either a more concentrated sample solution or longer accumulation is needed. The latter may last several hours when the sample amount is limited (<1 mg) unless cryogenically cooled probeheads and/or very high field strengths are used (see Chapter 6, Section 6.3.4). A conventional 13C NMR spectrum is shown in Fig. 7.7. The 13C chemical shift is indicative of the chemical environment of the 13C nuclei such that there are characteristic ranges for the

FIGURE 7.7 100 MHz 13C spectrum, with partial signal assignment, of compound 7.1 in DMSO-d6, showing characteristic spectral positions of the carbons belonging to different chemical environments.

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most important functional groups. Usually, a single resonance line (singlet) is observed for each carbon, even for CHn groups, instead of the multiplets expected due to 1 J13 C 1 H coupling. This simplification is accomplished by the continuous irradiation of the whole 1 H spectral range by using a second RF field. This method is called broadband 1H decoupling (denoted as 13C{1H}) because it eliminates (decouples) all of the 13C-1H couplings in the 13C spectrum. Obviously, because broadband 13C{1H} decoupling collapses the 13C multiplets into singlets, it also increases the S/N ratio. As an added bonus, 13C{1H} decoupling also generates a positive heteronuclear NOE on protonated carbons, which gives a further sensitivity gain. Since the influence of relaxation may also vary from carbon to carbon in the molecule under study, unlike in 1H NMR, the signal intensities or integrals are not evaluated in routine 13 C spectra. Because a 13C spectrum gives one singlet for each carbon, this means that the number of chemically nonequivalent carbon atoms present in the molecule can usually be easily enumerated from a high-quality 13C spectrum. Molecular symmetry may reduce the number of resonances, while slow chemical exchange processes (on the 13C chemical shift timescale) lead to more lines than expected. The spectroscopist should also bear in mind that the decoupling of protons does not eliminate the splitting of the 13C resonances caused by other abundant NMR-active “heteronuclei” such as 19F or 31P (Fig. 7.8). In such cases, the magnitude of the observed n J19 F 13 C or n J31 P 13 C coupling may help locate the “heteroatom” on the carbon skeleton.

FIGURE 7.8 125 MHz 13C spectrum of triphenylphosphine oxide 7.8 in CDCl3, with splittings due to n J31 P 13 C couplings.

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7.3 TWO-DIMENSIONAL (2D) METHODS While one single FID is recorded and Fourier-transformed to yield a 1D NMR spectrum, 2D NMR involves the acquisition of a series (dozens or hundreds) of FIDs. This “data matrix” is Fourier-transformed along two time dimensions, so the signal intensity becomes a function of two chemical shifts, f(d1, d2). The resonance signals in a 2D spectrum can be interpreted as if being “mountains” on a surface and are usually visualized as a contour plot in which they become “spots,” called cross peaks. The spectrum is framed by two orthogonal chemical shift axes, and the cross peaks indicate a homo- or heteronuclear scalar or dipolar correlation (depending on the type of the experiment—see below) between the corresponding chemical shifts. The first axis, called the “direct dimension” or F2 axis, is created by the first Fourier transform of the FIDs; thus, it has a higher digital resolution. Since the detection of 1H is preferred for sensitivity reasons on most modern probeheads, the direct dimension is usually the 1 H chemical shift. The second axis, the “indirect dimension” or F1 axis, is created by the second Fourier transform and may comprise 1H (in the case of homonuclear 2D spectra) or heteronuclear (13C, 15N, etc.) chemical shifts (in the case of heteronuclear 2D spectra). In this second dimension the digital resolution is determined by the spectral width to be covered and the number of recorded FIDs. Increasing the latter however proportionally increases the time demand of the experiment. Proton-proton correlations are the most sensitive 2D experiments; thus, they can be acquired in a reasonably short time even for dilute (
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FIGURE 7.9

Summary of structural information emerging from the most widely used 2D NMR techniques (see the text for the explanation of the acronyms). From the arbitrarily chosen proton denoted by a solid circle, magnetization flows via either chemical bonds or space to the other spin(s) denoted by dotted circles, which manifests itself in the corresponding 2D spectrum as correlation peak(s) in the respective row (or column) at the chemical shift of the encircled starting proton.

7.3.1 COSY The general purpose of correlation spectroscopy (COSY) is to identify neighboring CHn-XHm moieties with X ¼ C, N, O, etc. The simplest variant of COSY (Fig. 7.10) contains the autocorrelation peaks (practically, those of the 1D proton spectrum) in its diagonal, while the cross peaks in the off-diagonal area indicate a spin-spin coupling between the corresponding protons. As expounded in Section 7.2.1, the most intensive correlation peaks can usually be attributed to geminal CH2 or vicinal CH-CH proton pairs. Since geminal protons are easily identifiable from an additional heteronuclear 1H,13C-HSQC spectrum (see below), two- or three-bond connectivities can usually be readily discerned. Weak(er) cross peaks indicate long-range coupling of protons separated by more than three bonds. In these cases, the determination of “covalent distance” has some ambiguity, especially when cross peaks are observed between seemingly distant protons in the case of polyaromatic compounds (certain geometric arrangements can promote the appearance of rather long-range spin-spin couplings that may be too small to cause a splitting in the 1D 1H NMR spectrum, but their presence can be detected in the COSY spectrum). A simple example of how a COSY spectrum is interpreted is shown in Fig. 7.10 for compound 7.9. Starting from the diagonal signal of H-1 of the phenolic ring at 6.86 ppm, the cross peak in the same row (or column) identifies its neighbor H-2 at 7.00 ppm, which is in turn coupled to H-3 at 7.23 ppm. The latter signal resides within the highly congested region of 7.20-7.28 ppm, which contains additional signals from the separate spin system of the other phenyl ring. The latter ring can be assigned in a straightforward way by starting from its clearly resolved H-6 proton signal.

7.3.2 2D TOCSY The 2D variant of the 1D TOCSY experiment discussed above reveals in one single measurement the coupling network that any proton is involved in. This feature is useful if the 1D proton spectrum happens to be too crowded to start a 1D TOCSY spectrum by the selective excitation of the signals of interest (this is typical of oligosaccharides or oligopeptides), or if

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FIGURE 7.10 Structure of tolterodine isobutyrate 7.9 and the aromatic part of its 500 MHz COSY spectrum in DMSO-d6. Excerpts of the 1H spectrum are shown as top and right-side projections.

we do not have any a priori knowledge about which correlations may turn out to be crucial for structure elucidation (see Chapter 6, Section 6.3.4) and therefore it is worthwhile to obtain abundant information about the spin systems in one go (at the expense of sacrificing some resolution).3 If short mixing times (20-30 ms for small molecules) are applied, the 2D TOCSY spectrum provides practically the same information as COSY but has more favorable line shapes, which make it easier to discern close correlation peaks and the cross peaks of broadened signals. In this regard, TOCSY is often more robust and reliable in identifying the coupling partners. As a demonstrative example, the 2D TOCSY spectrum of the already introduced steroid 7.7 was chosen (Fig. 7.11). The four methylene protons attached to C(1) and C(2) form an isolated spin system, giving rise to the correlation peaks in the 3rd row from the bottom. The last TOCSY row reveals how magnetization flows from H-4a to H-4b and H-5b, even reaching to some extent H-6b during the mixing time of 40 ms applied in this experiment. From H-16b magnetization is transferred to all protons within the distance of four covalent bonds, thus going as far as the H-14 methine proton. The remaining rows of the 2D TOCSY spectrum either repeat this information (for protons belonging to the already mentioned spin systems) or can be interpreted for the separate spin systems similarly.

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FIGURE 7.11

Part of the 500 MHz TOCSY spectrum (with 40 ms mixing time) of 5-b-androstanedione 7.7 recorded in CDCl3. Correlation peaks in the three bottom rows are labeled with assignments of the coupling protons (the same information emerges from the respective columns). Excerpts of the high-resolution 1H spectrum are shown as top and right-side projections.

7.3.3 2D NOESY This is a 2D variant of the 1D NOESY experiment discussed in Section 7.2.2; thus, all the experimental circumstances and settings to be considered coincide with those of the 1D counterpart. The rationale behind recording a 2D NOESY instead of its selective 1D variant includes the motives discussed in Section 7.3.2. In the following example, 2D NOESY data are used to determine the relative configuration of the tropane derivative 7.10 and its stereoisomer 7.11 formed by epimerization at C(3). Based on literature data,15,16 we could assume that in 7.10 the 6-membered “piperidine” ring favored a chair conformation such that the ethylene bridge takes an axial position (Fig. 7.12). In this geometry two of the bridging methylene protons point toward the inside of the skeleton while the two others face outside. To determine the position of the aromatic ring attached to C(4), a 2D NOESY spectrum was acquired. H-4 (resonating at 2.90 ppm in 7.10 and at 3.10 ppm in 7.11) showed correlations with the bridging endo protons (at 1.60 and 1.70 ppm in 7.10 and at 1.69 and 1.90 ppm in 7.11) in both compounds (see Figs. 7.12 and 7.13), suggesting that H-4 occupied the endo (axial) position while the aromatic ring the exo III. SMALL-MOLECULE STRUCTURE ELUCIDATION BY NMR AND MS

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Structure of 7.10, showing the 1H assignments (in ppm) and the key NOE contacts that prove the stereochemistry from the 800 MHz NOESY spectrum (recorded in CDCl3 with 500 ms mixing time).

FIGURE 7.12

FIGURE 7.13 Structure of 7.11 showing the 1H assignments (in ppm) and the key NOE contacts that prove the stereochemistry from the 500 MHz NOESY spectrum (recorded in CDCl3 with 500 ms mixing time).

(equatorial) position. Comparing now the NOESY correlation patterns of H-3, it showed a correlation with the N-methyl group in 7.11 in contrast to 7.10, where this NOE was missing and a close proximity with one of the bridging endo protons was observed instead. In addition, in 7.10 the aromatic protons exhibited an NOE only with the axial H-5ax proton (1.83 ppm), while in 7.11 the answer is ambiguous because the chemical shifts of H-3 and H-4 are too close to be resolved in the NOESY spectrum (with H-3 being in an axial position,

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we should observe an NOE correlation between the aromatic ring and H-3ax as well). Although in principle the absence of a correlation should not be decisive when choosing the right structural isomer, in this case, where both isomers are in hand and their correlation patterns can be directly compared, the absence of an NOE can be of diagnostic value. In this way, the relative configuration of the C(3) and C(4) stereogenic centers could be established to be cis for 7.10 and trans for 7.11.

7.3.4 1H,13C-HSQC Heteronuclear single quantum coherence spectroscopy (HSQC) is used to correlate the chemical shift of protons (displayed on the F2 axis) to the 13C chemical shift (on the “indirect,” F1 axis) of their directly attached carbons via the 1JCH coupling. Taking the natural abundance of 13C into account, roughly every 100th molecule responds in the HSQC experiment. A particularly useful, so-called phase-sensitive or multiplicity-edited HSQC variant enables making a distinction between carbons bearing an even (CH2) or odd number (CH or CH3) of hydrogens. This difference is encoded into the sign (color) of the correlation peak (Fig. 7.14). (Note that a similar classification of carbons can also be accomplished by recording 13 C-detected (thus, more time-consuming) 1D DEPT (distortionless enhanced polarization transfer) or APT (attached proton test) spectra (not discussed further here).) The advantage of HSQC as compared to the 1D carbon sequences is twofold: firstly, it is a proton-detected experiment, consequently it is more sensitive and less time-consuming to acquire; secondly, it is richer in information since it simultaneously allows the list of directly bound 1H-13C pairs to be

FIGURE 7.14 Partial 500 MHz phase-sensitive HSQC spectrum of dehydroepiandrosterone 7.12 recorded in DMSO-d6 (CH and CH3 correlation peaks are labeled and colored in gray).

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assembled. Thus, with the recording of an HSQC spectrum, the time-consuming acquisition of a 1D 13C spectrum can often be escaped, unless the identification of quaternary carbons is indispensable for the structure elucidation problem (see also HMBC below). Furthermore, making use of the dispersion along the 13C chemical shift axis, HSQC spectra can easily disseminate crowded 1H multiplets (Fig. 7.14). In certain classes of compounds (such as steroids), this is the sine qua non of a reliable signal assignment. HSQC may also enable the quick distinction between constitutional isomers. For instance, the isobutyl methylene group exhibits highly similar 1H chemical shifts in 7.13 and 7.14 ( 4.2 ppm, Fig. 7.15); thus, the O- or N-alkylation remains indeterminate on the basis of the 1H NMR spectrum. The HSQC spectra easily resolve this ambiguity: 13C chemical shift of the methylene group is 48 ppm in 7.13, indicating N-alkylation, while the 72 ppm observed for 7.14 clearly confirms the presence of the O-alkyl isomer in hand. An important delay parameter in the HSQC pulse sequence that the user can control when setting up the experiment depends on the magnitude of the one-bond carbon-proton coupling 1 JCH. One-bond C-H couplings typically span the range of 120-160 Hz for most CHn groups, so this delay time is routinely set according to an average 1JCH value of 140 Hz. However, in some cases this value falls away from the optimum, and the HSQC spectrum will contain signals with twisted or even (misleadingly) opposite phase. This anomalous behavior is common for functional groups such as the ethinyl group, or in the case of methylenes in 3-membered rings, where 1JCH  140 Hz. Acquiring an additional HSQC spectrum with a delay time set

FIGURE 7.15 Distinction between the N- and O-isobutyl isomers 7.13 and 7.14 on the basis of the methylene 13C chemical shift from their respective 500 MHz HSQC spectra recorded in DMSO-d6 (top: excerpts of the corresponding 1 H spectra).

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according to a more appropriate 1JCH value (180-250 Hz) can resolve this issue, and correlations will appear with the correct phase (multiplicity). An example is presented here through the structure elucidation of an unknown product of the reaction shown in Fig. 7.16. According to the 1H NMR data, the product was obviously not the expected compound 7.16, since the signals characteristic of the acetoacetyl moiety were missing. The HSQC spectrum acquired with routine parameters (Fig. 7.16b) suggested the presence of three methine groups, two of which (suspiciously) appeared at identical 13C chemical shifts. Regarding the reaction, no structure with the presence of three methine groups could be rationally proposed. Supposing that the HSQC spectrum may contain two methine peaks with a misleading phase, we could immediately suggest the aziridine derivative 7.17, which was in accordance with the reaction. Finally, in line with the structural proposition, the HSQC spectrum acquired with a setup using the value of 1JCH ¼ 181 Hz yielded the aziridine methylene signals in the correct negative phase (Fig. 7.16c).

FIGURE 7.16

(a) Reaction equation with the intended product 7.16 and the identified compound 7.17. (b) 500 MHz HSQC spectrum recorded with the usual 1JCH ¼ 140 Hz setting, showing correlations in a misleading positive phase for the aziridine protons in 7.17. (c) 500 MHz HSQC spectrum recorded by using the value 1 JCH ¼ 181 Hz, where the aziridine methylene peaks appear with the correct negative phase.

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7.3.5 HSQC-TOCSY As its name suggests already, an HSQC-TOCSY spectrum contains the horizontal (F2) rows of a conventional 2D TOCSY spectrum (see Section 7.3.2), but these rows appear at the chemical shift of the directly attached carbon since the indirect axis (F1) displays 13C chemical shifts. The HSQC-TOCSY experiment is particularly useful when the TOCSY spectrum happens to be crowded due to 1H spectral overlaps, because the HSQC-TOCSY gives additional signal dispersion in the 13C chemical shift dimension. Similar vicinity information can be gained from the H2BC sequence,4 but the latter is based on COSY magnetization transfers; thus, only correlations due to the coupling of the adjacent CHn-CHm groups are detected. The utility of HSQC-TOCSY is illustrated here through the structure verification of compound 7.18 shown in Fig. 7.17. In the proton spectrum of the expected N-benzylic reaction product, an anomalous anisochrony of the benzylic methylene protons was detected which

FIGURE 7.17 (a) The expected structure 7.18 and the identified correct structure 7.19 with key 1H and

13

C (in italics) assignments (ppm). (b) Part of the HSQC spectrum showing the CH2 and CH groups having coincident 1 H chemical shifts. (c) Part of the HSQC-TOCSY spectrum showing the NCH2 peak at 4.69 ppm, which correlates with the methylenes bound to the carbon at 34.9 ppm, while the methylene protons at 2.10 and 2.63 ppm couple with the CH proton of dC 34.3 ppm. The most intense peak is the HSQC correlation, while the remaining ones are TOCSY correlations.

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could not be explained with structure 7.18. Due to the coincident chemical shifts of two methylene protons and the methine proton at 1.97 ppm, the analysis of the correlations detected in the 2D TOCSY spectrum could not resolve this ambiguity. By making use of the additional dispersion offered by the HSQC-TOCSY spectrum, the coupling partners could unambiguously be identified (see Fig. 7.17c). These however suggested that instead of 7.18, the reaction product was 7.19. With this structure, the anomalous nonequivalence of the benzylic protons is evident, since a stereogenic center is present in the molecule.

7.3.6 1H,13C-HMBC The heteronuclear multiple-bond correlation (HMBC) experiment enables the detection of correlations between protons and carbons separated by 2-4 chemical bonds. The purpose of this experiment is to help assemble proton-containing molecular moieties (isolated spin systems) to each other (especially those not linked by NOE contacts or separated by heteroatoms or quaternary carbons). The intensity of an HMBC cross peak depends on the relation of the instrumentally preset and the real physical value of the multiple-bond nJCH coupling constant in the investigated compound. The usual (routine) setting is 8 Hz, since this coupling constant yields correlations over three covalent bonds in most organic molecules. However, the 3JCH coupling constant may (significantly) differ from this value in different molecular environments, resulting in the absence of the expected 3-bond correlations and possibly the presence of 2- or 4-bond correlations. This “fuzzy” nature of the experiment always has to be taken into account during the interpretation of HMBC correlations, otherwise one could easily fall into Mental Traps #25 and #30 when assembling the molecular fragments. It is often the case that the vast majority of HMBC peaks are consistent with a given structural proposition, but a few correlations (or the lack of them) seem not to be explicable. In most of these cases the structural hypothesis is in fact false. Even if we risk wasting precious time, it is advisable in these cases to fully reconsider the interpretation of correlations in order to avoid the don’t-look-any-further effect (Trap #21), or the danger of rejoicing before finding the full solution (Trap #22). This latter situation is exemplified with the structures shown in Fig. 7.18. In this case, the expected reaction product was 7.20, but the 1H spectrum contradicted this proposition and suggested the olefinic analog 7.21 instead. 2D HMBC was also recorded in order to accomplish the full 13C assignment of this molecule. Surprisingly, a strong 4-bond (in 7.21) correlation was observed between the ketone carbon (192 ppm) and the cyclic methylene protons (4.05 ppm). As a second sign of warning, the olefinic proton near 6.75 ppm was expected to show at least one correlation in the region of aromatic carbons, but none was detected. These ambiguities questioned the validity of structure 7.21. A thorough reconsideration of the HMBC data finally led to an isomeric structural suggestion, 7.22, having a five-membered ring and an exocyclic double bond. This proposition was in accordance with all of our spectroscopic observations.

7.3.7 1H,15N-HSQC and 1H,15N-HMBC Most of the molecules subjected to structural analysis in a pharmaceutical environment contain heteroatoms other than carbon as well. From an NMR spectroscopic perspective,

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FIGURE 7.18 1H,13C-HMBC spectrum of the compound initially believed to be 7.21 but proposed to be 7.22. The anomalous and missing correlations are highlighted.

one of the most important of these is nitrogen. Unfortunately, due to its low natural abundance (0.37%) and unfavorable magnetic properties, the spin-½ 15N isotope is practically inaccessible from a normal 1D spectrum. However, it can be measured indirectly using 2D HSQC (for NHm groups) or HMBC sequences (for CHm–N or CHm–C–N moieties).17 Because n J15N1H coupling constants vary on a much larger scale than nJ13C1H, these measurements are less robust than their 13C counterparts. Nevertheless, 15N chemical shift values are

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characteristic of nitrogen-containing functional groups or of a given N-positional isomer of heterocycles18; thus, it is often inevitable to determine them in order to distinguish between constitutional isomers. Real-life examples of using the 1H,15N-HMBC experiment will be given in Chapter 11, Section 11.1, and Chapter 12, Section 12.3.

7.4 AN NMR-BASED STRATEGY FOR THE STRUCTURE ELUCIDATION OF SMALL MOLECULES In this section, we intend to show on a selected compound how the previously mentioned 1D and 2D NMR methods can be used in a concerted fashion to derive and characterize the structure of a small molecule. Our model compound will be vinpocetine, a semisynthetic derivative of the natural vinca alkaloid vincamine (vinpocetine is one of the most renowned and successful drug substances marketed by Gedeon Richter since 1978, used worldwide as a cerebral blood-flow-enhancing and neuroprotective agent). The (lower-field) NMR characterization of vinpocetine is well documented in the literature.19,20 Our rationale behind presenting here a full set of 1D and 2D NMR spectra recorded on a VNMRS spectrometer with 800 MHz proton frequency is, partly, to show (for the first time) an almost fully resolved 1H NMR spectrum of vinpocetine and partly because such high resolution enables a better-discernible assignment of the 2D correlation peaks. Since the way in which each type of 2D spectra has to be interpreted has been expounded in the preceding sections, the reader is invited to follow the steps of the assignment on the spectral figures below. The key milestones in the process of assembling the atomic connectivity information will be given in Tables 7.1 and 7.2, serving as checkpoints. (The structure elucidation strategy presented here is a typical one used in our pharmaceutical R&D environment, where we need to have a relatively fast but sufficiently information-rich and robust generic approach to be able to deal with a large number of samples submitted daily without compromising on the accuracy of the results (see Chapter 6). This is certainly not an easy task because it requires walking on a fine line between going into time-consuming details and providing a highthroughput service.) For the sake of generality, we pretend as if the structure of vinpocetine were unknown at the beginning of our analysis. Thus, knowledge of the correct elemental composition (a trustable “leg” of the problem-spider (cf. Chapter 1, Pillar 21)) is an indispensable prerequisite for a successful structural characterization. High-resolution MS (see Chapter 8, Section 8.2, for details) recorded in our laboratory yielded for vinpocetine the molecular formula of C22H26N2O2 as the starting point of our analysis. A first glimpse at the 1H spectrum (Fig. 7.19) immediately reveals that both aromatic and aliphatic carbon-bound protons are present in this molecule. The sum of integral values corresponds to the figure of H26 in the elemental composition, so no major organic contaminant is detected in the sample. The triplet signal at 0.97 ppm identifies CH3 of an ethyl group, and the aromatic splitting pattern is also indicative of an ortho-disubstituted benzene ring for the trained eye. However, at this level of spectral complexity, the full analysis of each 1H multiplet in terms of extracting from them the coupling constants so as to gain proton-proton connectivity

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TABLE 7.1 Collection of All Carbon and Hydrogen Atoms Present in Vinpocetine, Showing the Type and the One-Bond Carbon-Hydrogen Connectivity for Each Group as It Can be Derived from the Edited 1H,13C-HSQC Spectrum; the Rows Are Extended with the Corresponding COSY-Type Correlations No.

13

1

C

H

Neighbor

13

No.

1

C

H

Neighbor

2

ALIPHATIC

AROMATIC, OTHER sp

1

8.5 CH3

0.97t

1.85

12

108.1 C

—

—

2

13.9 CH3

1.34t

4.35

13

112.3 CH

7.16dd

7.08

3

15.8 CH2

2.43ddd and 2.95dddd

3.15 and 3.23

14

117.9 CH

7.42dd

7.05

4

19.8 CH2

1.35m and 1.61qt

0.84 and 1.49; 2.46 and 2.54

15

119.9 CH

7.06ddd 7.40

5

26.5 CH2

1.86m

0.96

16

121.4 CH

7.08ddd 7.14

6

28.4 CH2

0.85td and 1.50d

1.34 and 1.60

17

127.6 CH

6.10s

7

37.1 C

—

—

18

127.7 C

—

8

44.4 CH2

2.47td and 2.55d

1.34 and 1.60

19

128.6 C

—

9

50.7 CH2

3.16ddd and 3.24dd

2.42 and 2.94

20

130.9 C

—

10

54.9 CH

4.07s

—

21

134.9 C

—

11

61.4 CH2

4.35m and 4.39m 1.33

22

162.6 C

—

—

TABLE 7.2 List of the Correlations Observable in the HMBC Spectrum of Vinpocetine That Are the Most Important for the Assembly of the Different Moieties Determined Previously 1

Assignment

13

Assignment

13

Assignment

0.85

CH2(6) (B)

127.6

4.35

CH2(11)O

162.6

C¼O (22)

0.97

CH3(1)

37.1

C(7)

6.10

CH¼ (17)

54.9 127.7 162.6

CH(10) C(18) C¼O (22)

1.86

CH2(5)

54.9 28.4 127.6

CH(10) CH2(6) (B) CH¼ (17)

7.08

CH(16)

133.4

C(21)

3.16 and 3.24

CH2(9)N (A)

44.4 54.9 108.1

CH2(8)N (B) CH(10) C(12)

7.06

CH(15)

128.6

C(19)

2.43 and 2.95

CH2(3) (A)

108.1 130.9

C(12) C(20)

7.16

CH(13)

128.6

C(19)

4.07

CH(10)

28.4 44.4 108.1 130.9

CH2(6) (B) CH2(8)N (B) C(12) C(20)

7.42

CH(14)

133.4 108.1

C(21) C(12)

H

C

Assignment

1

CH¼ (17)

H

C

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FIGURE 7.19

281

1

H spectrum of vinpocetine (DMSO:CDCl3 4:1, 800 MHz) with integrals; the aliphatic part is enlarged for enhanced clarity (upper spectrum).

information is a much too time-consuming process, so at this stage we prefer turning our attention to heteronuclear 2D spectra. The purity and amount of substance in the current vinpocetine sample permitted us to collect all relevant 2D spectra on the 800 MHz spectrometer within a reasonable time according to the “holistic” GRAPS strategy outlined in Chapter 6. The 13C spectrum (Fig. 7.20) contains 22 lines in accord with the figure of C22 that we know from the elemental composition, so there are no symmetrically positioned carbon atoms in vinpocetine. The 13C chemical shifts are listed and numbered in ascending order in Table 7.1. From this pool of carbon atoms the multiplicity-edited 1H,13C-HSQC spectrum in Fig. 7.21 unambiguously identifies the CH, CH2, and CH3 groups, even those with overlapping 1H signals (Table 7.1). The remaining carbon atoms with no bound protons are quaternary carbons. Since every proton was found to be connected to carbon in the HSQC spectrum, the presence of NH or OH groups in the structure can be excluded. The TOCSY spectrum in Fig. 7.22 (alternatively, a COSY could also have been shown) enables the connection of the already identified CHn groups to each other (see the “neighbor” columns in Table 7.1). From the TOCSY-based connectivities the following moieties can be established with high certainty: ethyl, ethoxy, ortho-disubstituted aromatic ring, CH2–CH2N (spin system “A”), CH2–CH2–CH2N (spin system “B”), and an olefinic ¼ CH. Our experience shows that the HMBC spectrum in Fig. 7.23 provides the most straightforward and robust tool to assemble these groups and also the quaternary carbons together, so we followed this strategy. The most decisive HMBC correlations are collected in Table 7.2. We encourage the reader to construct

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FIGURE 7.20 13C spectrum of vinpocetine (DMSO:CDCl3 4:1, 201 MHz); the numbers depicted refer to the corresponding rows in Table 7.1.

FIGURE 7.21 Multiplicity-edited 1H,13C-HSQC spectrum of vinpocetine (DMSO:CDCl3 4:1, 800 MHz); the numbers depicted refer to the corresponding rows in Table 7.1.

the 2D structural formula of vinpocetine from these correlations. A key step and the result of the structural assembly are depicted in Fig. 7.24. At this stage of the analysis the constitution of the molecule has already been established. However, the structure elucidation is still incomplete without clarifying the stereochemistry of the ring annellation, that is, the relative configuration of the two stereogenic centers. This information is readily derived from the 2D NOESY spectrum shown in Fig. 7.25: a strong NOE correlation is observed between the methine proton at 4.07 ppm to both methylene protons (at 1.86 ppm) and the methyl protons (at 0.97 ppm) of the ethyl group, indicating the

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FIGURE 7.22 2D TOCSY spectrum of vinpocetine (DMSO:CDCl3 4:1, 800 MHz, 30 ms mixing time), aliphatic (left) and aromatic (right) parts. The arrows show the correlations listed in the corresponding rows in Table 7.1.

cis-annellation of the rings. Although this result could have been obtained from a simpler 1D NOESY spectrum by selective inversion of the methine proton signal, having a full 2D NOESY spectrum in hand is beneficial beyond a certain level of structural complexity, since it provides a means to quickly check the spatial connections between several other pairs of protons. To this end, we generated a 3D molecular-mechanics optimized model of vinpocetine (see Fig. 7.26). The observed NOE contacts corroborate the proposed 3D structure of the molecule which can be considered as the final result of our structure elucidation, apart from the fact that the performed NMR experiments did not shed light on its absolute configuration (the enantiomer of vinpocetine would yield the same NMR spectra, and thus, chiroptical spectroscopy would be needed to distinguish between these enantiomers). The above-described general methodology seems straightforward, and similar assignment strategies have been published with illustrative examples in the literature.6,8–10 We note that usually we do not need the whole arsenal of 2D methods for identifying simpler structures. On the other hand, in more difficult cases, for example, when investigating heterocycles with numerous possible isomers regarding heteroatom positions and a limited number of hydrogen atoms, or if the spectra are “fuzzy” (peaks are broad, 2D correlations are missing, the signals of the unknown compound cannot be unambiguously

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FIGURE 7.23 1H,13C-HMBC spectrum of vinpocetine (DMSO: CDCl3 4:1, 800 MHz). The most important correlations are highlighted (cf. Table 7.2).

FIGURE 7.24 Assembly of the moieties derived from the analysis of different 2D spectra to yield structure 7.23 with 1H and 13C assignments are shown in black and gray (ppm), respectively. The numbering on the fragments refers to the data listed in Table 7.1.

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FIGURE 7.25 2D NOESY spectrum of vinpocetine (DMSO:CDCl3 4:1, 800 MHz, 500 ms), aliphatic part. The circles show the correlations diagnostic of the cis-isomer. Cross peaks and the ones in the diagonal have opposite phase. FIGURE 7.26 3D molecular model of vinpocetine 7.23; some key NOE correlations are depicted to corroborate the proposed geometry.

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differentiated from those of other compounds in a mixture, the spectrum is overly crowded, etc.), the interpretation of the spectra and the structure elucidation can become extremely difficult.

7.5 DIFFUSION-ORDERED SPECTROSCOPY (DOSY) DOSY stands apart from the other techniques discussed above in that it does not directly provide information on the topology of the spins within a molecule. Rather, DOSY essentially serves to separate the NMR signals of molecules that have different diffusion coefficients (different molecular sizes) within a mixture. Nevertheless, this feature of DOSY sometimes renders it to be a decisive technique in structure elucidation, as will be exemplified in Chapter 11, Section 11.2. From the 1H NMR spectrum of a mixture DOSY restores the spectra of the individual compounds on the basis of differences in their translational diffusion coefficients. The measured diffusion coefficients can subsequently be converted to hydrodynamic radii, thus providing a handle to assess different molecular sizes or molecular masses by applying appropriate physicochemical models. The key element of any DOSY pulse sequence is the so-called gradient pulse, which intentionally “spoils” the otherwise spatially highly homogeneous B0 static external magnetic field within the NMR sample tube. To understand this perturbation, let us define the longitudinal (vertical) axis of the sample tube as the Z direction, which coincides with the direction of the B0 vector. Note that in order to ensure that the RF coil will “see” an entirely homogeneous sample solution, the NMR sample is routinely prepared and positioned such that the solution extends significantly below and above the lower and upper edges of the coil along Z. Not the whole population of spins in the NMR tube is affected by an RF pulse, but only those residing in the so-called active volume of the RF coil (ca. 200 ml), which corresponds approximately to the middle third part of the entire sample solution length.21 The gradient pulse adds to the B0 field another field component B(Z), which varies strictly linearly as a function of the Z coordinate. Consequently the spins residing in the active volume experience an effective field B0 + B(Z) such that their individual Larmor frequencies defined by Eq. (2.8) become the function of their actual position along Z within the NMR tube. Since this effective field is active for the whole duration of the gradient pulse (usually a few milliseconds), each spin accumulates a phase shift that becomes a function of its vertical position. This is the basic idea how the actual position of a molecule can be “labeled” and its displacement caused by translational diffusion along the Z direction can be “tracked” by subsequent elements of the pulse program. For further theoretical or technical details and applications of DOSY experiments, the interested reader is referred to the literature.22,23 The simplest variant of DOSY is demonstrated below on the artificial mixture of a linear polymer with two crown ethers in CDCl3. Figure 7.27 shows a pseudo 2D spectrum with the spectra of individual compounds arranged in separate rows at the ordinate values of the respective diffusion coefficient. The magnitude of diffusion coefficients increases in the following order of compounds: 7.24  7.25 < 7.26  CHCl3, clearly reflecting major differences between the solvated sizes of these molecules.

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7.6 SUMMARY

287

FIGURE 7.27 Molecular formulas for poly(butylene oxide) 7.24, dibenzo-30-crown-10 7.25, and 18-crown-6 7.26. The 400 MHz 1H spectrum of their mixture in CDCl3 is displayed as horizontal projection above the pseudo-2D DOSY spectrum.

7.6 SUMMARY NMR offers today the most detailed information to aid structure elucidation of small organic molecules. The most widely used pulse sequences have been surveyed in this chapter, trying to familiarize the reader with their key functions as well as some interpretational pitfalls. Beyond the primary levels of constitution and configuration defining a “structure,” aspects regarding molecular dynamics (conformation), self-association, or interactions with other molecules can also be studied by solution-phase NMR. Nevertheless, this plethora of information usually comes encoded into the observed spectral parameters like chemical shifts, correlation peaks, coupling constants, NOEs, and relaxation times. The transposition of NMR spectral data into structural constraints or identified moieties as building blocks for the unknown molecule seems to be a fairly straightforward task in certain cases (a general strategy was discussed in Section 7.4). Structures seem often to be identifiable already from a sole 1D proton spectrum. We have demonstrated by our own real-life examples that, paradoxically, these “deceptively simple” situations are most susceptible to drive

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7. NMR METHODOLOGICAL OVERVIEW

the spectroscopist into Mental Traps. An AA-conscious attitude of the spectroscopist in the spirit of the GRASP approach (see Chapter 6) may induce running additional 2D spectra to exclude all the structural alternatives with full certainty, which is indispensable in competitive research environments such as the pharmaceutical industry. On the other hand, spectra of certain “NMR-unfriendly” samples (e.g., those with heavily broadened signals or large signals of contaminants) may hide the relevant chemical information to such an extent that even a thorough spectroscopic expertise and chemical intuition are insufficient to solve the problem without physically changing the sample (solvent, temperature, acidification, etc.) and rerunning the corresponding 1D and/or 2D experiments with the hope of gaining better interpretability (see Chapter 12 for a case study illustrating this point). Since the current strategies of structure elucidation are heavily biased to 1H-based measurements (due to sensitivity reasons), the very limited number of protons present in certain classes of heterocycles may lead to spectroscopic underdetermination, another possible pitfall for structural misinterpretations. Only a holistic approach aiming at the full heteronuclear NMR characterization (assigning all 13C, 15N, 19F or 31P, etc., chemical shifts if applicable for the given molecule) can guarantee an unambiguous structure identification in such problematic cases. However, despite the tremendous advancements achieved by instrument manufacturers, NMR remains the least sensitive technique in organic analysis (especially in comparison with MS), so its full “heteronuclear armory” can hardly be exploited for highly diluted or contaminated samples such as metabolites or unisolated process impurities, calling for human expertise and ingenuity for choosing and carrying out the most prospective strategy of structure elucidation.

Acknowledgments ´ gai-Csongor, Zsuzsanna Kurucz-Ribai, Sa´ndor
A The authors are indebted to Katalin Szo˝ke, Dr. Krisztina Vukics, Eva Garadnay, Jo´zsef Neu, Dr. Gyula Be´nyei, Borba´la Farkas-Juha´sz, Dr. Ga´bor Sza´nto´, Dr. No´ra Felf€ oldi, Ferenc Sebo˝k, Dr. Gy€ orgy I. Tu´ro´s, and Pe´ter Oravecz for providing their synthesized samples. Magdolna Nagy is acknowledged for the technical assistance in the NMR measurements while Dr. Zolta´n Be´ni for valuable discussions and a nice steroid example. Last, but not least, we are most grateful to Prof. Csaba Sza´ntay, Jr. for his valuable comments on the manuscript.

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