problems, artifacts and solutions in the inadequate nmr ... · problems, artifacts and solutions in...

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Research ArticleReceived: 03 March 2010 Revised: 07 April 2010 Accepted: 26 May 2010 Published online in Wiley Interscience: 29 June 2010

(www.interscience.com) DOI 10.1002/mrc.2639

Problems, artifacts and solutions in theINADEQUATE NMR experiment†

Alex D. Bain,a∗ Donald W. Hughes,a Christopher K. Anand,b Zhenghua Niec

and Valerie J. Robertsond

The INADEQUATE experiment can provide unequalled, detailed information about the carbon skeleton of an organic molecule.However, it also has the reputation of requiring unreasonable amounts of sample. Modern spectrometers and probes havemitigated this problem, and it is now possible to get good structural data on a few milligrams of a typical organic smallmolecule. In this paper, we analyze the experiment step by step in some detail, to show how each part of the sequence can bothcontribute to maximum overall sensitivity and can lead to artifacts. We illustrate these methods on three molecules: 1-octanol,the steroid 17α-ethynylestradiol and the isoquinoline alkaloid β-hydrastine. In particular, we show that not only is the standardexperiment powerful, but also a version tuned to small couplings can contribute vital structural information on long-rangeconnectivities. If the delay in the spin echo is long, pairs of carbons with small couplings can create significant double-quantumcoherence and show correlations in the spectrum. These are two- and three-bond correlations in a carbon chain or through aheteroatom in the molecule. All these mean that INADEQUATE can play a viable and important role in routine organic structuredetermination. Copyright c© 2010 John Wiley & Sons, Ltd.

Keywords: NMR; 13C; INADEQUATE; steroid; alkaloid; pulse sequence; artifacts

Introduction

The INADEQUATE experiment[1 – 4] is potentially a very powerfulexperiment for structure determination in organic molecules.[5 – 13]

It can explicitly map out the carbon–carbon connectivity of amolecule, using the scalar coupling between two 13C nuclei. Thisgreat power is also its main drawback as it requires two suchnuclei to occur in a single molecule. At the natural abundance of13C, this is roughly one molecule in 104, so the sensitivity of theexperiment (even by NMR standards) is dreadful. Until recently,practically a neat sample was required to perform an experimentin a reasonable time.[14]

The situation has improved with modern high-field NMRspectrometers equipped with cryogenic probes.[6,15 – 17] It isnow feasible to do INADEQUATE-type experiments on tens ofmilligrams of a typical small molecule in an overnight experiment.The experiment is usually done with a refocusing delay of 1/(41JCC), but longer delays [often odd multiples of 1/(4 1JCC)] canbring up strongly coupled carbons and long-range correlations.However, there are drawbacks here as well. High fields mean largespectral widths, and cryogenic probes are quite limited in theRF power they will tolerate. These can combine to create seriousoffset effects and other artifacts in a realistic experiment. Becausesensitivity is an important issue, it is important to set all theparameters, so that signal/noise is maximized and all necessarycorrelations are visible. It is the purpose of this paper to analyzethe INADEQUATE experiment step by step, to delineate some ofthe problems and add to the list of suggested solutions.[10,18 – 22]

There are two INADEQUATE experiments: a one-dimensional(1D) version[1,2,4,20,23] and a 2D version.[3,24] Figure 1 shows thecoherence pathway for both these experiments. In the 1D exper-iment, the time between the last two pulses is very short, justallowing time to shift the phase in order to make this double-quantum (DQ) filter work. However, the resulting spectrum gets

very crowded and difficult to interpret for typically sized molecules.In the more common 2D version, this delay is the regularly incre-mented t1 delay that creates the modulation at the DQ frequencyand spreads the spectrum out in the f1 dimension. The basic mech-anism for the experiment is well known but details rely on a numberof assumptions. If these break down, it is important to recognizethe symptoms of problems, so that the solution can be applied. Inthis paper, we investigate some of the consequences of relaxation,imperfect pulses, values of the delays and off-resonance effects.

Experimental Procedures

All NMR spectra were acquired on a Bruker Avance 700-MHzspectrometer using a 5-mm triple resonance inverse cryoprobewith z-axis gradient capability. The 1D 13C NMR spectra wereacquired at 176.09 MHz using either the standard single-pulseexperiment or the J-modulated spin sort pulse sequence.

∗ Correspondence to: Alex D. Bain, Department of Chemistry and ChemicalBiology, McMaster University, 1280 Main Street West, Hamilton, Ontario,Canada L8S 4M1. E-mail: [email protected]

† Soon after the submission of this paper, Donald W. Hughes died for no knownreason. This paper is dedicated to his memory.

a Department of Chemistry and Chemical Biology, McMaster University, 1280Main Street West, Hamilton, Ontario, Canada L8S 4M1

b Department of Computing and Software, McMaster University, 1280 Main St.West, Hamilton, Ontario, Canada L8S 4K1

c School of Computational Engineering and Science, McMaster University, 1280Main Street West, Hamilton, Ontario, Canada L8S 4K1

d University of Guelph NMR Centre, Guelph, Ontario, Canada N1G 2 W1

Magn. Reson. Chem. 2010, 48, 630–641 Copyright c© 2010 John Wiley & Sons, Ltd.

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INADEQUATE: problems and solutions

sin(α)cos2(β/2)

sin(α)sin2(β/2)

DETECTOR

0

+1

-1

+2

-2

D1 τ τ

p1 p2 p3 p4

t1

Figure 1. Pulse sequence and coherence pathway for the INADEQUATEpulse sequence. D1 is the relaxation delay between acquisitions, and t1 isthe incremented delay for a 2D experiment. The second line shows thegradient program, with the two gradients in a 1 : 2 ratio. The equationsin the coherence pathway show the dependence of the transfer on theflip angle of the final pulse, p4. Normally, α = β = p4, but if there aresignificant offset effects, the effective flip angles, α and β , felt by thetwo nuclei that make up the DQ coherence may be different. The phasecycles are p1: 0 135 90 45 180 315 70 225; p2: 0 135 90 45 180 315 70 225;p3 0 135 90 45 180 315 70 225; p4: 0 90 180 270 0 90 180 270; receiver:0 0 0 0 0 0 0 0.

The 2D INADEQUATE spectra were acquired in the absolutevalue mode. A pulse sequence specifically designed to suppresssingle-quantum artifacts[19] was not needed as these artifactswere usually small. Acquisition parameters for the 1-octanol, 17α-ethynylestradiol and β-hydrastine samples are as follows. For1-octanol, the standard INADEQUATE spectrum was recorded overa 10.000 kHz spectral width in 8K data points (0.410 s acquisitiontime). Each of the 256 FIDs in the t1 dimension was acquired with32 scans. The 1-octanol INADEQUATE spectra that demonstratedthe offset effects were recorded over a 42.372-kHz spectral widthin 8K data points (0.097 s acquisition time). Each of the 256 FIDs inthe t1 dimension was acquired with 16 scans. The relaxation delayfor the 1-octanol experiments was 2.0 s. The 17α-ethynylestradiolINADEQUATE spectrum was recorded over a 26.881-kHz spectralwidth in 4K data points (0.076 s acquisition time). Each of the 256FIDs in the t1 dimension was acquired with 96 scans. The relaxationdelay was 2.0 s. The β-hydrastine INADEQUATE spectrum wasrecorded with a spectral width of 27.100 kHz in 4K data points(0.076 s acquisition time). The number of scans for each FID in thet1 dimension was 96. In this case, the relaxation delay was 4.0 s.

The fixed delay time, τ , was set to 0.005 s for the standardINADEQUATE spectra, or 0.015 or 0.050 s for the long-rangecorrelation experiments. A 2.0-s relaxation delay was used forall long-range correlation experiments. The 13C 90◦ pulse widthwas 14.59 µs. The 180◦ adiabatic refocusing pulse was in theform of a composite smoothed chirp pulse that was designatedas Crp60comp.4 in the Bruker software.[25] The chirp pulse wasdefined by 4000 points and covered a spectral width of 60.0 kHzwith 20% smoothing. The pulse width was 2.0 ms. Two pulsed fieldgradients were used with one before and one after the final 120◦13C pulse (19.45 µs). The strength of the gradients was in the ratio1 : 2 at +10 and +20% of the maximum value.[26] The gradientpulse width was 1.0 ms. Broadband 1H decoupling was performedby the standard waltz-16 procedure. The data were processedusing exponential multiplication with a line broadening of 1.0 Hzin both dimensions. A power spectrum calculation was applied

in the f1 dimension. No significant difference was observed witha magnitude calculation (square root of the power spectrum),probably because the signals are close to the noise level. Powerspectra make the large peaks inordinately large but treat all points(spectra or noise) near the baseline in the same way. Forward linearprediction to 512 data points followed by zero-filling to 1024 datapoints was also performed in the f1 dimension.

The deuterated solvents used in this study were suppliedby Cambridge Isotope Laboratories Inc. The 1-octanol samplewas prepared as a 20% by volume solution in C6D6. The 17α-ethynylestradiol sample was prepared by dissolving 37.0 mg in250 µl of DMSO-d6, while 25.7 mg of β-hydrastine were dissolvedin 250 µl of CDCl3. The spectra of these samples were acquiredin Shigemi NMR tubes. Chemical shifts were referenced relativeto the residual solvent peaks: C6D6 at 128.06 ppm, DMSO-d6 at39.52 ppm and CDCl3 at 77.16 ppm.

Mechanism of Inadequate

The common assumptions are that we re-establish equilibriumbefore each acquisition and that the pulses and delays areperfect and appropriate. This means not only are the pulseswell calibrated but also they are infinitely short high-power hardpulses. A pulse is hard if the RF magnetic field associated withit dominates all the other terms in the Hamiltonian, includingoffsets (due to chemical shifts) from the transmitter frequency.This approximation is often violated, particularly for nuclei otherthan protons.[27,28] For example, if the 13C π/2 pulse is 15 µs, thenthis corresponds to an RF field (in frequency terms, this is γ B1,where γ is the magnetogyric ratio and B1 is the RF magneticfield) of 16.7 kHz. At a magnetic field of 14 T, 100 ppm of 13Ccorresponds to 20 kHz, so the hard-pulse assumption is certainlynot fully justified. For excitation, this effect is relatively harmlessbecause it merely generates an almost-linear phase distortionwhich is easily corrected. However, the refocusing pulse is not soforgiving.[10,18,19,29] It is important to look at the experiment indetail and see what happens if the assumptions are violated.

Figure 1 shows the pulse sequence and defines the nomen-clature for its elements. There is a delay, D1, which allows forrelaxation between acquisitions and serves to re-establish the zmagnetization. The first pulse, p1, is a 90◦ excitation pulse whichtakes that magnetization into the xy plane. The coherences evolveat frequencies determined by the chemical shifts and the 13C–13Ccouplings. The 180◦ pulse, p2, refocuses the chemical shifts butallows the homonuclear coupling to continue to evolve. At p3, the90◦ DQ creation pulse, we need the coherences for the carbons tobe in a suitable antiphase state in order to create the maximumDQ coherence. This means that the echo delay, τ , usually shouldbe 1/4JCC, but since there is a range of J’s, some average valueis used. Also, any odd multiple of this delay will serve the samepurpose. Both carbons of a coupled pair will contribute to a singleDQ coherence. This DQ coherence evolves during t1 at the DQfrequency for the pair of carbons, and then is brought back into thesingle-quantum manifold for acquisition by p4, the read pulse. Thisfinal pulse should have a flip angle of 60◦, 90◦ or 120◦, dependingon the experiment. The normally used 2D version works with a120◦ read pulse. Finally, the pulse sequence and the phase cyclingmust prevent any single-quantum coherence from reaching thedetector, because that will give spurious diagonal-type peaks atthe single-quantum frequency in f1. We now examine each ofthese parts of the pulse sequence in turn.

Magn. Reson. Chem. 2010, 48, 630–641 Copyright c© 2010 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/mrc

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Relaxation delay D1

It would be nice if we could wait 5 T1’s between scans, so that the zmagnetization is fully restored for each acquisition. However, 13CT1’s are long, particularly for quaternary carbons, and sensitivity isparamount. A shorter value of D1 partially saturates the steady-state z magnetization but allows more scans. For a spin witha relaxation time T1, the steady-state magnetization following aseries of 90◦ pulses separated by D1 is given by Eqn (1).[30 – 33]

Signal ∝ 1 − e−D1/T1 (1)

The number of acquisitions we can fit into a fixed time isproportional to 1/D1, and the signal/noise is proportional to thesquare root of the number of scans, as in Eqn (2).

Signal per unit time ∝ 1 − e−D1/T1

√D1

(2)

These countervailing effects mean that some compromise valueof D1 will give a maximum intensity for a given experiment time.The maximum of this function occurs when Eqn (3) is satisfied.

(1 − 2

D1

T1

)e

−D1

/T1 = 1 (3)

Although there is no analytical solution to this transcendentalequation, it has a maximum at approximately D1 = 1.26 T1.[33]

Two carbons (with different T1s) are required to create the singleDQ coherence, so the question arises as to which T1 to use. It isour opinion that the shorter T1 is appropriate because that spinwill not be saturated, although the other will be. This will createhalf the amount of DQ coherence that would have been created ifthe relaxation times were equal, but more scans will be collected.The true situation will depend on the actual values of the T1s, butusually they can be roughly sorted into short (protonated carbons)and long (quaternaries and methyls). If the correlation between aquaternary and a methine carbon is important, it is probably bestto use the T1 of the methine.

Excitation pulse p1

All pulse sequences need an initial pulse to start the evolutionof the spin system. A 90◦ pulse will give maximum signal, butthis probably does not give the maximum per unit experimentaltime. The z magnetization is fully saturated and needs a longtime to recover, whereas Ernst angle methods provide the mostefficient acquisition of 1D spectra. The SOFAST HMQC[34,35] hasonly one proton pulse, so the Ernst angle is applicable, but this isan unusual case for pulse sequences. Normally, there are enoughpulses in the sequence that the z magnetization is essentiallysaturated at the end. We compared an acquisition using a 90◦

excitation pulse with a 30◦ value for p1. The latter does notgive a good signal/noise, and the single-quantum artifact peaksare much stronger. The initial pulse actually plays two roles: bothexciting the spectrum and helping to suppress the single-quantumcontributions. Changing the excitation pulse from a standard 90◦

pulse is counter-productive.

Delay τ

This is the delay during which the spin system evolves in a spinecho in order to optimize the creation of DQ coherence. It is

usually set equal to 1/4J, where J is an average carbon–carbonone-bond coupling,[23,36 – 39] but longer values can be used topick up long-range correlations. Accordion methods[12,13,40] canbe used to cover a range of coupling constants, but since thisis a compromise, sensitivity is lost. From the beginning, it wasshown[4] that strongly coupled systems show a more complicateddependence on the delay in the spin echo,[41] as shown in Eqn (4).

I = 2 cos2 2θ sin[Jτ ] − sin 2θ (1 − sin 2θ )

sin[(C + J)τ ] − sin 2θ (1 + sin 2θ ) sin[(C − J)τ ] (4)

In this equation, J is the coupling constant, � is the chemicalshift difference, tan 2θ = J/� and C = √

�2 + J2.[42]

The second term, in (C + J), turns out to be essentially negligiblein most cases, and the third term, in (C − J), is zero in the weakcoupling limit. However, small amounts of strong coupling causethis term to contribute, and when J > �, it becomes the dominantterm in the creation of DQ coherence. It corresponds roughly toan antiphase state of the two central lines of the AB quartet.[4]

However, we have to wait a time proportional to 1/(C − J) for thisto happen and this tends to infinity in the limit of � going tozero. This is the limit when magnetically equivalent spins cannotgenerate DQ coherence.

Refocusing pulse p2

This pulse is normally the weakest link in the pulse sequence.Refocusing pulses are more difficult to improve than excitationor inversion pulses as the magnetization does not have a well-defined starting position. However, the solution is well known.Many composite or shaped pulses have been designed[43 – 48] andmake a big improvement.[10,18] (Fig. 2).

Creation pulse p3

The purpose of this pulse is to turn the antiphase xy magnetizationinto DQ coherence. As with the excitation pulse, there is nothingto be gained by using anything other than a 90◦ pulse.

Read pulse p4

The role of this pulse is to convert the shared DQ coherenceassociated with a coupled carbon spin pair back into single-quantum coherences for each spin. Although some sources (checkyour pulse sequence) always use a π/2 pulse here, which is correctfor the 1D experiment, the proper value for the usual coherencepathway in the 2D version is 2π/3[5,49 – 51] because that gives themaximum signal. Regardless of the flip angle, a hard pulse here willgive the same coherence to both the spins, so the carbon–carboncorrelation will be symmetrical in intensity.

For a flip angle α of the read pulse, the transfer from DQ tosingle quantum is given by Eqn (5) (Fig. 1).

2D ∝ sin(α) sin2(α/2)√2

(5)

Note that this function has a maximum at α = 2π/3 (120◦).[49,50]

Furthermore, some early 2D works used 135◦, which kept most ofthe sensitivity advantage, but significantly suppressed the othercoherence pathway (Fig. 1), getting around the need for 45◦ phaseshifts. However, on a modern spectrometer, 45◦ phase shifts areavailable, so the 120◦ flip angle for the final pulse gives the best

www.interscience.wiley.com/journal/mrc Copyright c© 2010 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2010, 48, 630–641

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(a) (b)

15202530354045505560 ppm 15202530354045505560 ppm

Figure 2. Comparison of INADEQUATE spectra of 20% 1-octanol in C6D6 acquired with (a) a standard 180◦ refocusing pulse and (b) an adiabatic 180◦

refocusing pulse. The spectral width in each case was 42.37 kHz and the transmitter offset was set at 110.5 ppm. The data set was recorded with 16 scansfor each of the 256 increments in t1 and a relaxation delay of 2.0 s.

ppm30405060

Figure 3. Rows from a 2D INADEQUATE spectrum of octanol whichdemonstrate offset effects. All the pulses are at high power, except for theread pulse, p4, whose power was varied but whose flip angle was always120◦ on resonance. The horizontal red arrow indicates the strength of theRF field, in frequency terms. In both cases, the high-frequency signal onthe left is on resonance.

intensity and eliminates the other pathway, provided the pulsesare hard. On the other hand, the 1D version of INADEQUATE usessignals from both coherence pathways (Fig. 1), and the signal thereis given by Eqn (6).

1D ∝ sin(α) sin2(α/2) + sin(α) cos2(α/2)√2

= sin(α)√2

(6)

This function does have a maximum at α = π/2.When RF offset effects are important,[27,28] asymmetric corre-

lations may be observed (Fig. 3). When the signal is weak, this

can lead to half of a correlation missing because it is in the noise.Because of offset effects, the effective RF fields that are felt by the Aspin and the X spin will be different. Although they are both draw-ing from the same reservoir, the A and the X spin single-quantumcoherence will have different values. For detection of A spin co-herence, the X coherence level will go from −1 to 0, but A will gofrom −1 to +1. The complete transfer from DQ to single-quantumcoherence will be the product of these two transfer efficiencies,giving the intensity of the line at the A single-quantum frequencyin f2. Conversely, the signal at the X single-quantum frequency willinvolve X going from −1 to +1, and A going from −1 to 0. Becausethe effective field angle will be different for the two offsets, thetwo correlations at A and at X will have different intensities (Fig. 3).

Acquisition

Disk space is cheap, so many acquisition points leading to relativelylong acquisitions can be done in t2. Longer acquisition times canjust be subtracted from the interscan delay. Good digitization inf2 allows us to resolve the genuine carbon–carbon doublets andclearly distinguish them from residual single-quantum artifacts,which appear as singlets.

Applications of the Experiment

With the theoretical background to the INADEQUATE experimentin place, some applications of the technique were then examined.The principal focus of these applications dealt with how thevariation in the delay in the spin echo, τ , provided additionalinformation that could be useful for the structure elucidationand/or confirmation of natural products. Initially, the alteration ofthe τ delay was used to examine the issue of the considerable lossof cross-peak intensity when dealing with adjacent 13C nuclei thatwere strongly coupled or second order.[4,11,33] This is an importantconsideration, as nuclei with close chemical shifts can lead to someof the biggest ambiguities. This effect can occur even in moleculesthat are considered to have a relatively simple structure. If careis not taken when dealing with molecules of unknown structureand/or in cases where the standard 2D methods fail to establish the


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direct connection between the second-order carbons, the reducedcross-peak intensity may result in the wrong interpretation of theINADEQUATE spectrum.

1-Octanol

The INADEQUATE spectrum of a 20% solution of 1-octanol[8] inC6D6 recorded at 176 MHz with a τ delay of 0.005 s (τ = 1/41JCC

with 1JCC = 50 Hz) showed correlations in the 34–22 ppm regionwith good intensity (Fig. 4(a)). However, at the contour level usedto plot this spectrum, the correlations between carbons 4 and 5were not observed. An examination of the C-4 and C-5 chemicalshifts indicated that these carbons have a frequency separationof 24.6 Hz which was almost 10 Hz smaller than the average 1JCC

of 34.8 Hz measured from the 1-octanol INADEQUATE spectrum.This small frequency separation relative to the size of 1JCC resultedin the C-4 and C-5 spin system being second order.

As described above (Eqn (4)), the intensity of INADEQUATE cross-peaks from second-order 13C–13C spin systems can be recoveredby acquiring spectra with longer delays such as τ = 3/41JCC. This

modified value of τ was then used to record an INADEQUATEof 1-octanol (Fig. 4(b)) which showed a strong correlation forcarbons 4 and 5. In fact, the cross-peaks between C-4 and C-5 werethe most intense peaks in the spectrum. The significant intensityvariation of the C-4/C-5 cross-peaks as a function of the τ delaybecomes evident by comparing the f2 rows through the C-4/C-5correlations (Fig. 5). Figure 5(a) shows the considerable loss ofcross-peak intensity in the standard INADEQUATE spectrum forthe second-order spin system relative to neighboring correlations.When the τ delay was set to 3/41JCC (Fig. 5(b)), the C-4/C-5cross-peaks were very intense and in fact appeared as an ABspin system. Normal intensities for the C-4/C-5 cross-peaks wereobtained by increasing the frequency separation of these carbonsthrough the combined addition of a shift reagent and recordingthe standard INADEQUATE spectrum at a higher field strength.Second-order effects must be considered when applying theINADEQUATE experiment to a molecule with carbon signals ofsmall frequency separation.

A further benefit of the use of longer τ delay times inthe 1-octanol experiments was the detection of INADEQUATE

(a) (b)

232425262728293031323334 ppm

2 6 4 5 3 7

232425262728293031323334 ppm

2 6 4 5 3 7

Figure 4. (a) Standard INADEQUATE spectrum of 20% 1-octanol in C6D6 recorded with τ = 1/41JCC with 1JCC = 50 Hz. The C-4 to C-5 cross-peaks werenot detected at this contour level. (b) The INADEQUATE spectrum acquired with τ = 3/41JCC which shows strong C-4/C-5 cross-peaks (circled).

(a) (b)

27282930313233 ppm 27282930313233 ppm

29.830.030.2 ppm

Figure 5. F2 rows showing the difference in intensity of the C-4/C-5 cross-peaks as a function of the delay, τ . (a) Spectrum acquired with τ = 1/41JCC.(b) Spectrum acquired with τ = 3/41JCC.


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16171819202122232425262728293031323334 ppm

2 6 4 5 3 7

5 8

74

36

52

8

Figure 6. Expansion of the INADEQUATE spectrum of 20% 1-octanol in C6D6 acquired with τ = 0.05 s (τ = 1/4nJCC where nJCC = 5.0 Hz). The dashedlines connect carbons that have a three-bond 13C–13C coupling interaction.

correlations from long-range 13C–13C coupling interactions(Fig. 6). When the INADEQUATE spectrum of 1-octanol wasacquired with τ = 1/4nJCC = 0.05 s (nJCC = 5.0 Hz), all possiblethree-bond 13C–13C coupling interactions were detected (Fig. 6).The one-bond cross-peaks and C-4/C-5 correlation were alsopresent. Since the two- and three-bond 13C–13C couplingstend to be relatively small (≤5.0 Hz), the long-range cross-peaksappeared as a single contour and could be readily differentiatedfrom the one-bond cross-peaks (Fig. 6). This modification ofthe INADEQUATE experiment has been mentioned in the NMRliterature.[33,52,53] However, its application has been limited tovery specific classes of compounds and/or the measurementof the long-range 13C–13C coupling constants where modifiedINADEQUATE pulse sequences were used to help improvesensitivity. With the sensitivity gains provided by cryogenicprobes in combination with high-field magnets, we have takena more general approach of simply modifying the τ delay inthe standard INADEQUATE pulse sequence and focusing on theconnectivity information much like the information in a 1H–13CHMBC experiment.

17α-Ethynylestradiol

In order to demonstrate the information that can be obtainedwhen this type of INADEQUATE experiment is applied tonatural products, a series of experiments with different τ delayswere performed on the steroid 17α-ethynylestradiol and theisoquinoline alkaloid β-hydrastine (Fig. 7). The 13C chemical shift

assignments are presented in Table 1. This study began withthe 17α-ethynylestradiol because it contained a full range ofcarbon types: aliphatic, aromatic and acetylenic. Figure 8 showsan expansion of the standard INADEQUATE spectrum of 17α-ethynylestradiol that was acquired with τ = 0.005 s. This contourplot focused on the correlations observed for the aliphatic carbonsand provided all the required connectivities to identify thecarbons of the steroid framework. The exception was carbons19 (88.98 ppm) and 20 (74.96 ppm) of the acetylene group. NoINADEQUATE cross-peaks were observed between the acetylenecarbons and carbons 17 (78.18 ppm) and 19. This resulted from thelarge difference in the magnitude of the one-bond 13C couplingsof acetylene versus aliphatic carbons. The τ delay used for thespectrum in Fig. 8 was determined from an average 13C–13Ccoupling of 50 Hz, whereas the coupling between acetylenecarbons is usually in the range of 160–220 Hz. The one-bondcoupling between sp and sp[3] carbons would generally be in the70 Hz range which would account for the lack of a correlationbetween carbons 17 and 19. Under the conditions used to acquirethe spectrum in Fig. 8, the INADEQUATE experiment was unableto show the presence of the acetylene and also could not locatethe site of substitution of the acetylene on the steroid molecule.

Two INADEQUATE experiments were then performed withdifferent τ delay times. The first experiment was acquired withτ = 3/41JCC (0.015 s) where 1JCC was set to 50 Hz and the resultingspectrum is shown in Fig. 9. The correlations indicated in Fig. 9arose from two- and three-bond 13C–13C coupling interactions.Table 2 provides a listing of the two- and three-bond correlations


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A. D. Bain et al.

13

14

8

9

10

17

5

19

12

11

15

7

CH20

1

6

16

4

3

OH

2

CH318

HO

HH

H

3'a 3'

7'a1'

O2'

8a

1

4'

N2

4a

87

6

7'

5

O

O

O

5'

3

4

6'

OO

CH3

CH3

H3C

H H

Figure 7. Structure and numbering of 17α-ethynylestradiol (C20H24O2) and the isoquinoline alkaloid β-hydrastine (C21H21NO6).

Table 1. 13C chemical shifts for 17α-ethynylestradiol andβ-hydrastine

17α-Ethynylestradiola β-Hydrastineb

Carbon Carbon

1 126.07 1 66.07

2 112.75 NCH3 44.95

3 154.95 3 49.14

4 114.94 4 26.74

5 137.13 4a 130.41

6 29.19 5 108.52

7 27.05 6 146.71

8 39.16 7 145.79

9 43.33 8 107.80

10 130.31 8a 124.78

11 26.17 1′ 83.02

12 32.60 3′ 167.78

13 46.71 3′a 119.67

14 49.01 4′ 147.90

15 22.50 5′ 152.51

16 38.84 6′ 118.58

17 78.18 7′ 117.80

18-CH3 12.75 7′a 140.73

19 88.98 6,7-OCH2O 100.88

20 74.96 4′-OCH3 62.31

5′-OCH3 56.80

a Solvent: DMSO-d6.b Solvent: CDCl3.

that were detected in the 17α-ethynylestradiol experiment.Figure 10 shows some selected long-range correlations on thesteroid structure. These correlations were able to show theconnectivity between carbons through the various ring junctions(C-10 and C-14, C-4 and C-9, C-8 and C-17) or between carbonsaround the outer edge of the molecule (C-11 and C-17, C-4 and C-7).The important result is that these two- and three-bond correlationsare defining larger fragments of the molecular structure (Fig. 10).

A closer examination (Fig. 11) of this spectrum revealed cross-peaks from the acetylene carbons that were absent in the standardINADEQUATE spectrum. The longer τ delay has allowed thecreation of antiphase magnetization arising from the larger one-bond 13C couplings between the acetylene carbons 19 and 20(1JCC = 163.5 Hz) and also carbons 17 and 19 (1JCC = 72.1 Hz)

Table 2. The two- and three-bond 13C–13C coupling interactions de-tected in the long-range INADEQUATE spectra of 17α-ethynylestradioland β-hydrastine

17α-Ethynylestradiol β-Hydrastine

3JCC3JCC

1–4 1–4

1–6 1–5

1–11 4–8

2–9 1′ –4′

2–5 3′ –5′

3–6 3′ –7′

3–10 4′ –7′

4–9 6′ –3′a4–7 5′ –7′a6–14 1′ –NCH3

7–11 4–NCH3

7–13 5′OCH3 –6′

8–17 5′OCH3 –4′

9–15 6,7OCH2O–5

10–12 6,7OCH2O–8

10–14

11–17

18-CH3 –192JCC

2JCC

1–9 1–8

2–10 4–5

2–4 5–7

4–6 6–8

8–13 3′ –7′a14–17 4′ –6′

17–20

prior to the formation of DQ coherence by the second 90◦ pulse.The observation of these correlations allowed the identificationof the acetylene carbons based on the measurement of thelarge one-bond carbon coupling constant. These correlations alsoconfirmed the site of substitution of the acetylene group at C-17by the presence of both one-bond and two-bond INADEQUATEcross-peaks.

The second long-range INADEQUATE experiment involved theuse of a longer τ delay of 0.05 s that was determined by usingan average long-range 13C–13C coupling constant of 5.0 Hz in the


63

7


2030405060708090100110120130140150 ppm

6 712 1113 18

15168 711914 15

121389

14 8

14 13

17 16

1317

6510 9

181511

7

61216

8

913

14

201719

241

1053

Figure 8. Expansion of the standard INADEQUATE spectrum of 17α-ethynylestradiol recorded with τ = 0.005 s (τ = 1/41JCC where 1JCC = 50 Hz).

2030405060708090100110120130140150 ppm

3 6

105 14 8

11

42

99 6

116 7

44

17 14

14 6

159

18

1511

7

61216

8

913

14

201719

241

1053

2030405060708090100110120130140150 ppm

3 5 10

1 4 2

19

17

20

14

13

9 8

16 12 6

7

1115

18

3 610 14

851 9

10 124 9

9211

611

44

67

17 14

17 8

17 111819

81314 6

13 79 15

7 11(a) (b)

Figure 9. Comparison of the long-range INADEQUATE spectra of 17α-ethynylestradiol recorded with (a) τ = 0.015 s (τ = 3/41JCC where 1JCC = 50 Hz)and (b) τ = 0.05 s (τ = 1/41JCC where 1JCC = 5 Hz). The dashed lines indicate correlations arising from two- and three-bond 13C–13C couplinginteractions.

formula τ = 1/4nJCC. The resulting spectrum in Fig. 9(b) showedmore two- and three-bond coupling interactions compared withthe spectrum in Fig. 9(a). The one-bond correlations betweenthe acetylene carbons 19 and 20 and 19 and 17 were stillpresent but the two-bond cross-peaks between 20 and 17 werenot detected. However, carbon-17 did show new three-bondcorrelations to carbons 8 (39.16 ppm) and 11 (26.17 ppm) in theadjacent six-member ring. These results, combined with the two-bond correlation between C-17 and C-14 (49.01 ppm), definedthe carbons at the junction between the five- and six-memberrings (Fig. 10). This is a particularly useful observation because

C-17 is usually a site of substitution in steroids and can displaya well-resolved and readily assigned signal. Therefore, C-17 canbe used as a starting point to derive chemical shift assignmentsof neighboring carbons. An unexpected observation was thedetection of weak cross-peaks between the acetylene carbon C-19and the 18-CH3 (12.75 ppm) that was attributed to a three-bondcoupling. An examination of a computer-generated molecularmodel indicated that these carbons have a torsion angle ofapproximately 150◦. Based on a standard Karplus analysis, thisangle is in the range where three-bond or vicinal couplingstend to increase in magnitude. Potentially, these long-range


63

8

A. D. Bain et al.

14

10

1711

CH

6

3

OHCH3

HO

HO HO

HO

HH

H

8

9

17 CH

4

OHCH3

HH

H

14

8

17

5

11CH

1

OHCH3

HH

H9

15

7

CH

64

OHCH3

HH

H

Figure 10. 17α-Ethynylestradiol structures showing some of the two- and three-bond 13C–13C coupling pathways between the steroid ring carbons thatwere detected from the long-range INADEQUATE spectra.

757677787980818283848586878889909192 ppm

1917

20

19 20

1719

17 20

17 C19

CH20

OHCH318

HO

HH

H 2JC17,C20

1JC19,C20 = 163.5 Hz

1JC17,C19 = 72.1 Hz

Figure 11. Expansion of the long-range INADEQUATE spectrum acquired with τ = 0.015 s (τ = 3/41JCC where 1JCC = 50 Hz) showing the one-bondcorrelations between the acetylene carbons 19 and 20 and 19 and 17 and the two-bond cross-peaks between carbons 17 and 20.

13C connectivities may provide stereochemical information aboutcarbon substituents especially in cases where there is a lack ofprotons for NOE experiments or the proton distances may be large.

β-Hydrastine

Another application of long-range 13C–13C coupling interactionsfrom the INADEQUATE experiment is the case where the molecule

contains 1H-depleted regions and the carbons will generally havelong T1 relaxation times. An example of this situation was theisoquinoline alkaloid β-hydrastine (Fig. 7) where there were fivequaternary carbons in the phthalide ring (C-3′ to C-5′ and C-7′a).The standard INADEQUATE experiment was acquired (Fig. 12) witha relaxation delay (D1) of 4.0 s and a τ delay of 0.005 s (τ = 1/41JCC

where 1JCC = 50 Hz). The majority of carbons in the isoquinolinepart of the molecule showed strong cross-peaks with the exception


63

9


90100110120130140150160 ppm

3'a 5'

6'

6 77'a 4a 8a

3'a

6' 7' 5 8 1'

7'a 1'

8a 86' 7'

4a 5

4a 8a7 8

7'7'a7'a 3'a

5' 6'

6 7

90100110120130140150160 ppm

3' 5' 4'

6

77'a 4a 8a 3'a

6' 7' 5 8 1'

3' 5'

3' 7'a

5' 7'a

3' 7'

4' 6'

4' 7'

3'a 6'4' 1'

6,7OCH2O

(a) (b)

Figure 12. Comparison of (a) the standard INADEQUATE spectrum of β-hydrastine with (b) the long-range INADEQUATE spectrum obtained withτ = 0.05 s (τ = 1/4nJCC where nJCC = 5.0 Hz) which shows the additional correlations from the phthalide quaternary carbons.

30405060708090100110120130 ppm

4

NCH3

3

11'857'6'

3'a8a4a

4

5'OCH3

6'OCH36,7OCH2O

NCH3

41

1' NCH3

35

5'OCH3

4a 3

6 8

5 1

1

30405060708090100110120130 ppm

4

NCH3

3

11'857'6'

3'a8a4a

3 4

1' 1

4a 4

8a 1

5'OCH3

6'OCH36,7OCH2O

(a) (b)

Figure 13. Comparison of (a) the standard INADEQUATE spectrum of β-hydrastine with (b) the long-range INADEQUATE spectrum obtained withτ = 0.05 s (τ = 1/4nJCC where nJCC = 5.0 Hz) which shows the long-range correlations in the aliphatic region. Of interest are the 13C–13C couplingsthrough the N and O atoms.

of the C-5 to C-6 cross-peaks which were very weak and the C-6 toC-7 cross-peaks which were not detected. The difficulty inobserving the C-6/C-7 correlation most likely resulted from longrelaxation times as the frequency separation of these of carbons(162 Hz) was larger than their expected mutual 13C spin coupling(55–70 Hz for aromatic carbons). The standard INADEQUATEspectrum did provide new information that resulted in a revisionof the original 13C chemical shift assignments of carbons 4a(130.41 ppm) and 8a (124.78 ppm)[54] and allowed a definitiveassignment of carbons 6 (146.71 ppm) and 7 (145.79 ppm)(Table 1).

For the phthalide portion of β-hydrastine, no one-bondcorrelations could be detected between the carbonyl carbon

C-3′ and C-3′a, C-3′a and C-4′, and C-4′ and C-5′. This was alsothe case when the experiment was repeated with D1 delays of5.0 and 8.0 s. When the INADEQUATE experiment was performedwith τ = 0.05 s (τ = 1/4nJCC where nJCC = 5.0 Hz) and a D1delay of 2.0 s (Fig. 12(b)), the phthalide carbonyl carbon C-3′

showed three-bond correlations with C-5′ and C-7′ and two-bond cross-peaks with C-7′a. Additional three-bond couplingsobserved in the phthalide ring included C-3′a and C-6′, C-4′

and C-7′, and C-5′ and C-7′a. Similarly, quaternary carbons C-6and C-7 of the isoquinoline ring showed three-bond correlationswith C-4 and C-1, respectively, which confirmed the assignmentof these aromatic carbons. Weak one-bond correlations were infact detected between carbons 6 and 7 in this spectrum. This


64

0

A. D. Bain et al.

3'

1'

O

4'

N

8

7'

O

O

O4

OO

CH3

CH3

H3CH3C H3C

H H

1'

O

1

4'

N

8

5

O

O

O4

6'

OO

CH3

CH3

H H

3'

O

N

8

5

O

O

O

5'

4

OO

CH3

CH3

H H

Figure 14. β-Hydrastine structures showing a selection of two- and three-bond 13C–13C coupling pathways between them were detected from thelong-range INADEQUATE spectra.

long-range variation of the INADEQUATE experiment is providingcorrelation information into regions of the molecule where thelack of protons and long relaxation times make signal detectiondifficult in the standard experiment.

The long-range INADEQUATE experiment on β-hydrastine alsoshowed 13C–13C coupling interactions through the nitrogen andoxygen atoms. In the aliphatic region, three-bond couplingswere detected between the N-CH3 carbon (41.96 ppm) andC-4 (26.74 ppm) of the isoquinoline ring and the N-CH3 and C-1′ ofthe phthalide ring (Figs 13 and 14). These long-range correlationsare providing information about the structural relationship ofthe N-methyl carbon relative to neighboring carbons in both theisoquinoline and phthalide rings.

As far as the oxygen substituents were concerned, themethylenedioxy carbon at 100.88 ppm did not show any two-bond correlations with carbons 6 and 7 but did show three-bondcross-peaks with carbons 5 (108.52 ppm) and 8 (107.08 ppm).Similarly in the phthalide ring, the methoxyl carbons did notdisplay any two-bond cross-peaks with carbons 4′ and 5′. However,the 5′ methoxyl carbon (56.80 ppm) had three-bond cross-peakswith both C-6′ at 118.52 ppm and C-4′ at 147.90 ppm. No three-bond correlations were observed for the C-4′ methoxyl carbon at62.31 ppm. These results indicated that the C-5′ methoxyl carbonhas more conformational freedom and can adopt a co-planarstructure with the aromatic ring and allow transmission of thecoupling interaction to carbons 4′ and 6′. In contrast, the C-4′methoxyl carbon is being forced out of a co-planar structurebecause of steric interactions with the adjacent substituents.These arguments have also been used to account for the higherfrequency chemical shift of 62.31 ppm observed for the C-4′methoxyl group.[55]

These are excellent examples of the rich information that amodern application of INADEQUATE can do for realistic amountsof realistic molecules.

Conclusions

We have mapped out the best values of all the parameters in theINADEQUATE pulse sequence in Fig. 1. The total interscan delay,which is the sum of the relaxation delay and the acquisition time,should be about 1.3 times the T1 of the faster relaxing carbonin an important correlation. The first pulse, p1, should be a 90◦

pulse, both to get maximum signal and to help suppress single-quantum artifacts. The delay τ in the spin echo that creates the

antiphase coherence needed to create DQ coherence should bethe reciprocal of four times the average carbon–carbon coupling,either one-bond or multiple-bond, unless strong coupling effectsare important. The refocusing pulse, p2, is the weakest link andshould be some sort of composite or adiabatic pulse. The resultingantiphase magnetization is best transferred to DQ coherence by a90◦ pulse for p3, but the DQ coherence should be read back out forfinal detection with a p4 of 120◦. Finally, the FID can be generouslydigitized (4K or 8K points) to give relatively good digitizationand resolution in f2. Experiments on 1-octanol, the steroid17α-ethynylestradiol and the isoquinoline alkaloid β-hydrastineillustrated these effects on realistic molecules. Implementation ofall these recommendations means that INADEQUATE is a feasibleovernight experiment on roughly 10–25 mg of a typical organicsmall molecule in a relatively standard instrument.

Acknowledgements

We would like to thank the Natural Sciences and EngineeringResearch Council of Canada for funding.

References

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