The Use of NMR Spectroscopy in the Structure

8/6/2019 The Use of NMR Spectroscopy in the Structure

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102 NATURAL PRODUCT REPORTS, 1988of the pulse sequences that are presented achieve their results,it is useful to be able to follow how some of the simplestsequences work, particularly since these are the building blocksfrom which many apparently very complex sequences arecomposed. In many cases the operation of the sequences can beexplained in terms of a simple vector model. This will bedescribed first for the standard single-excitation-pulse n.m.r.experiment and then extended to show how two of the mostbasic pulse sequences produce their results. This section of thediscussion is restricted to nuclei of spin one-half.

2.1 The Single-Excitation-Pulse ExperimentAny particular line in an n.m.r. spectrum arises from thedifference between the populations of two energy levels. Foreach line, the equilibrium excess of nuclei (with spins orienteda) n the lower energy level is represented by a vector M,, whichis aligned in the direction of the external field Bo. The radio-frequency radiation behaves as another magnetic vector B,, ofconstant size, which rotates in a plane perpendicular to thedirection of B,. To make it easier to visualize how signals aredistinguished by the spectrometer and how sequences do theirjob it has become customary to view the system as if one werean observer sitting on the vector B ,, which represents theapplied radiofrequency radiation. More formally, a set of threemutually perpendicular axes is defined such that the x and yaxes rotate, at the frequency of the pulse, about the z axis,which coincides with the direction of the external field B,[Figure l(a)]. This set of axes is frequently referred to as therotating fram e of r e f e r e n ~ e . ~he radiofrequency vector B, istherefore stationary in this frame, and its application along,say, the x axis causes all of the magnetization vectors M (onefor each line in the spectrum) to be tipped through an angle 8(known as the flip angle) towards the y axis until the pulseceases [Figure 1 (b)]. It is easier at this point to think of each ofthese shifted vectors as being composed of two components.These are (i) a longitudinal component M , (= M cos0), n thedirection of the field, which is less than M , and which reflectsthe reduced population between the two levels, and (ii) atransverse component M y (= M sin 0),which is detected as then.m.r. signal. The size of Mg,and thus of the n.m.r signal, isdetermined by the pulse strength B , (usually several gauss) andits duration, which is usually the experimental parameter thatis adjusted. Clearly, a pulse length corresponding to O = 360"would leave the system unchanged. Pulse lengths correspondingto O = 90" and 180" are particularly significant and arecommonly used in multi-pulse experiments. A 90" (or n/2)pulse results in a maximum value for M y [Figure l(e)] andhence a maximum signal. There is no residual longitudinalmagnetization M y , indicating that the levels are equallypopulated. (This is not the same as saturation, in which thereis no magnetization in any direction.) A 180"(or n) ulse resultsin no transverse magnetization [Figure I (c)], and therefore non.m.r. signal, but inverts the populations of the energy levels,i.e. it changes the orientation of every nucleus.After a pulse has been applied, the system returns towardsthe equilibrium state : the magnetization vectors eventuallyresume their positions along the z axis and the n.m.r. signalsare obtained by recording the decay of the transversecomponents of M that have been created by applying thepulse.For a 180"pulse the return path is directly along the z axis[Figure l(d)]. This is relatively slow and may take severalseconds, depending upon the longitudinal (spin-lattice) relaxa-tion times (T ,) of the nuclei. For all other pulse angles thereturn path that is taken by M is dependent on the differencebetween the frequency of the pulse and the resonance frequencyof the nucleus. It is this difference in behaviour that enablesresonances at different frequencies to be distinguished, If theresonance frequency of the nucleus is the same as the pulsefrequency then, after a 90" pulse, the magnetization vector Mmoves back across the y z plane from the y axis to the z axis.

( k ) A3-Ir f Vr

Figure 1 The single-excitation-pulse n.m.r. experiment. (a) The magnet-ization vector M a t equilibrium (= M ,) in the rotating frame; (b) theposition of vector M immediately after a 0" pulse has been appliedalong the x axis of the rotating frame; (c) the position of vector Mimmediately after a 180' pulse; (d) the relaxation path taken byvector M after a 180" pulse; (e) the position of vector M immediatelyafter a 90" pulse; (f and g) the relaxation paths taken by the vectorM after a 90" pulse where the corresponding spin-resonancefrequencies either (0 equal or (8) differ from the pulse frequency; (hand i) the decay of the signal along the y axis of the rotating frame,corresponding to situations (0 and (g); (i and k) the positions ofspectral lines after Fourier transformation of (h) and (i).

Thus M , grows from zero to the original value M , and thetransverse component Mu decays to zero. If both relaxationprocesses occur at the same rate, the tip of the M vector movesin a circular path [Figure 1 (f)]. A detector monitoring changesin magnetization along the y axis would observe M y decay-ing exponentially with time [Figure l(h)]. If the resonance

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NATURAL PRODUCT REPORTS, 1988-1. H. SADLER 103frequency of the nucleus is greater than the pulse frequency,then, after a 90"pulse, the vector M also moves clockwise as itreturns to the z axis. Therefore, as M , grows to M,, thetransverse component (M,J rotates in the xy plane whilstdecaying to zero. This rotation is called free precession, and thefree-precession frequency of M ,, is equal to the differencebetween the frequencies of resonance and the pulse. If bothrelaxation rates are equal, the tip of the vector M moves in aspiral path on the surface of a hemisphere [Figure l(g)]. Adetector monitoring changes in magnetization along the y axis(My)observes a signal in the form of an exponentially decayingcosine wave [Figure 1(i)]. Since relaxation is relatively slow,magnetization vectors corresponding to signals more than afew hertz away from the pulse frequency precess many timesbefore they return to the z axis. Where several nuclear spinsof different resonance frequencies, are present they aredistinguished by their precession frequencies, and the FIDsignal that is obtained consists of a set of exponentiallydecaying cosine waves, one for each line in the spectrum.Provided the acquisition of signal is started immediately afterthe pulse, the spectrum that is obtainable by Fourier trans-formation can be 'phased' so that all of the peaks appear inthe pure absorption mode.

2.2 Th e Multi-pulse ExperimentIf there is a short delay ( 7 ) of a few milliseconds betweenapplying the pulse and acquiring the signal it is unlikely that itwill be possible to phase the spectrum correctly. Free precessionoccurs during the delay, so that the transverse magnetizationvectors have moved relative to each other by the time thatacquisition is started. The result in the final spectrum will bethat, if a resonance line at the same frequency as the pulse is apure absorption signal, any line whose magnetization vectorhas moved to the -y axis will appear inverted and lines whosemagnetization vectors are at other positions in the xy plane willappear badly phased (Figure 2). This apparently inconvenienteffect is turned to advantage in multi-pulse experiments.

The precession frequency of a vector corresponding to oneline in a multiplet is the sum of a contribution from thechemical shift (8) nd a contribution from the coupling constant(4.By applying an appropriate sequence of pulses, separatedby delays which are frequently inversely related to the couplingconstants, it is possible to remove the precession due to thechemical shift (&precession) whilst using that due to thecoupling (J-precession) to enhance, to remove, or to invertcertain resonances and to obtain properly phased spectra. Toavoid loss of signal intensity, the total duration of the pulsesequence must be kept short compared with the nuclearrelaxation times. Therefore, where possible, couplings in excessof 50 Hz ( i . e . principally one-bond couplings) are employed.In all except the simplest sequences the spectrometer mustnormally be capable of being programmed to apply bothobserving and decoupling pulses along any of the x, y , - , and- y directions and also of detecting signals in these directions.The direction along which the pulse is applied (or the signal isdetected) is often referred to as the phase of the pulse (or of thedetector). Most currently produced spectrometers have thisfacility to program pulse and detector phases, and on the most

recent spectrometers it is possible to specify phases in incrementsof 5" in the xy plane. On older spectrometers, however, it is notpossible to specify the decoupler phase or independently tospecify the observe transmitter and detector phases. This limits(rather than excludes) pulse-sequence work. In many exp-eriments the phases of the pulses and of the detector aresystematically changed through a set pattern from one scan tothe next; this process is known as phase ~ycling.~his maybe a refinement to remove artifacts arising from imperfect ormis-set pulses or from non-orthogonal directions of phases. Insome sequences, particularly those involving multiple-quantumtransitions, phase cycling is a necessary part of the experimentto eliminate strong unwanted signals. A simple form of phasecycling, the CYCLOPS6procedure, (in single-pulse-excitationexperiments) is commonly employed automatically on manyspectrometers to reduce the residual images that are caused bythe quadrature detection system.In the remainder of this section an attempt is made to showhow two simple one-dimensional pulse sequences achieve theirresults. The discussion is restricted to the observation of nucleithat are present in low abundance and where the situation isnot complicated by the presence of homonuclear coupling.2.3 The Spin-Echo SequenceWhere there is a delay between the excitation pulse and theacquisition of the signal, a properly phaseable spectrum isobtainable by applying a 180" pulse, in the middle of the delayperiod, to the nucleus that is being observed. This is illustrated(Figure 3) for the carbon- 13 magnetization vectors thatcorrespond to the two lines of a 13C-H doublet where bothlines are at frequencies greater than the pulse frequency. Thevector Ma orresponding to the higher-frequency line arisesfrom those carbon- 13 nuclei whose attached protons areorientated a and the vector M 8 corresponding to the lower-frequency line from carbon- I3 nuclei whose attached protonsare orientated p.During the first delay period 7 following the 90"pulse, thevectors Ma nd M s move clockwise and separate in the xyplane, as represented by instant (ii) in Figure 3. Application ofa carbon-13 180" pulse rotates each vector through 180", overthe surface of a cone, to new positions in the xy plane[Figure 3(iii)]. The vectors continue to move clockwise, at theirown frequencies, and since the slower-moving ( i . e . M B ) s nowahead, the two vectors converge and coincide along the - y axisat the end of the second delay period 7 . This sequence is knownas the spin-echo sequence' and the 180" pulse is referred to as arefocussing pulse. Acquisition of the decay signal at the pointwhere the vectors have re-aligned leads to a spectrum which canbe properly phased. Additionally, any line-broadening that hasbeen introduced during the period before the 180" pulse wasapplied as a result of inhomogeneity of the magnetic field isremoved during the period between applying the 180" pulse andacquiring data. Provided that the total time (27) betweenapplying the first pulse and acquiring the signal is sufficientlyshort, this sequence gives a spectrum identical with that from asimple experiment in which a single 90" pulse is applied.

Although, so far, there has been no gain over a single-pulseexperiment, this spin-echo sequence is probably the most

Figure 2 The effect on the spectrum of introducing a delay ( 7 ) of a few milliseconds between a 90" excitation pulse and the star t of the acquisitionof signal. Signals (b), (c), and (d) are, respectively, 45", 90", and 180" out of phase with signal (a).

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104 NATURAL PRODUCT REPORTS,

-1 8 0'H

I

B r o a d- BandD e c o u p l e

zI

i

I

-

( c 1 I

X

ii iii

i i I i I i v

1988

Figure3 The spin-echo pulse sequence. (a)A diagrammatic representation of the sequence; (b) the positions of magnetizationvectors in the rotatingframe at instants (it-(iv) of the sequence; (c) views of the xy plane corresponding to (b).

Figure 4 A sequence to distinguish carbon-I3 resonances of CH and CH, groups from those from C and CH, groups. The behaviour of thecarbon magnetization vectors is shown for CH and CH, groups.

important sub-sequence and is found as a component of nearlyall of the more complicated sequences. A slightly modifiedversion* provides a method in which resonances fromcarbon-13 nuclei of methyl and methine groups are invertedwith respect to those from methylene groups and fromquaternary carbon-13 nuclei. This is effected by applying aproton 180" pulse simultaneous with the carbon 180" pulse, byusing a delay 7 of (24- ' seconds, and by employing broad-band proton decoupling whilst acquiring the FID signal. Thissequence relies on the similarity in magnitude of most one-bond C-H coupling constants, which are assumed (for this

discussion) to be identical. The behaviours of CH and CH,groups are considered separately (Figure 4). As previously, thetwo carbon- 13 magnetization vectors for a CH group separateafter the 90" excitation pulse [Figure 4(a)] until, after a periodof (24- ' seconds, they lie at 180" to each other [Figure 4(b)]and also at 90" to a vector representing the chemical-shiftfrequency (i.e. the position of the magnetization vector, hadproton decoupling been employed). At this point the carbon180" pulse rotates the vectors to new positions [Figure 4(c)] inthe xy plane. If no further pulses were applied the vectorswould continue towards the - y axis and coincide there.

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NATURAL PRODUCT REPORTS, 1988-1. H. SADLER 105However, the application of a proton 180"pulse immediatelyfollowing or simultaneous with the carbon 180" pulse changesthe orientation of every proton. Thus carbon nuclei that hadformerly been bonded to protons that were orientated a arenow bonded to protons that are orientated p, and theircorresponding magnetization vectors become the slower-moving ones. Similarly, the magnetization vectors of carbonnuclei that are bonded to protons that had formerly beenorientated p but which are now orientated a become the faster-moving ones [Figure 4(d)]. Thus the direction of J-precessionof these vectors is reversed by the proton 180" pulse, and thevectors now return towards the + y axis, ultimately coincidingthere [Figure 4(e)] after a further period of (24-' seconds. Itcan similarly be deduced that the same behaviour is expectedfor the four carbon- I3 magnetization vectors for a CH, group.However, this is not so for CH, groups and quaternary carbonatoms.The centre line of a CH, triplet arises from carbon- I3 nucleiwhose attached protons are orientated in opposite directions,the high-frequency line arises where both attached protons areorientated a, and the low-frequency line arises where bothattached protons are orientated p. At the end [Figure 4(b)] ofthe first period of (24-l seconds following the application ofthe carbon 90" pulse the magnetization vectors M, and M aa(corresponding to the outer lines) have moved by 180" withrespect to the vector M , (corresponding to the central line),which shows only &-precession. As before, the carbon 180"pulse moves the vectors to new positions (c) and the proton180" pulse changes the orientation of every proton (d). However,this last pulse results in no net change for the Mapvector andso this continues to the --y direction; although the direction ofJ-precession of the other two vectors is reversed, their coincidentposition at this time also results in no net effect, and these alsocontinue towards the --y axis, where all three vectors coincide[Figure 4(e)] at the end of the second period of ( 24 - ' seconds.The signals will be inverted compared with those from CH andCH, groups. The precession of the single vector from aquaternary carbon atom is similarly not affected by the proton180" pulse and lies along the - y axis at the start of signalacquisition. The application of broad-band proton decouplingduring acquisition of the FID signal causes the multiplets tocollapse, as would be expected since J-precession is removed. Atypical spectrum that was obtained in this way (using anaverage value for lJCHf 135 Hz) is shown in Figure 5.Spectra showing resonances from only CH, groups andquaternary carbon nuclei may be obtained by combining bothsequences described above, i .e. by applying the proton 180"pulse on alternate scans.* In this case the signals from CH andCH, groups are alternately positive and negative, and cancelone another. If alternate FID signals are also subtracted ratherthan added, a spectrum is obtained in which only CH, and CHgroups can be seen. In all of these experiments there must be apre-excitation delay, whose magnitude is governed by therelaxation times of the carbon nuclei, between scans to ensurethat M , can adequately recover.2.4 A Polarization-Transfer Sequence :SensitivityEnhancementAnother process that is employed in many pulse sequences isincreasing the differences between populations of the energylevels that are responsible for the transitions, to improve thesignal intensities. At normal temperatures the populationdifference is proportional to the gyromagnetic ratio (y) of thenucleus. If a nucleus that has a lower value of y, e.g. carbon-13or nitrogen- 15, is spin-spin-coupled to a nucleus with a high y(frequently protons) it is possible to alter the average differencein populations for the low-y nucleus to that of the high-ynucleus. This is known as polarization transfer and will resultin an intensity gain of yhigh/ylowor the low-y nucleus. Thusproton coupling may be used to increase intensities of carbonsignals by a factor of four and those of nitrogen by a factor of

1 1 I 1 I 1 1 I 1160 140 120 100 80 60 40 20 0p.p.m.

Figure 5 The carbon-13 n.m.r. spectrum of linalool, obtained by thesequence of Figure 4. Signals from C and CH, groups appearinverted.

1 3

d d R = + 5 A w 2

C H

( C 1

l3C I II

Figure 6 (a) The energy-level populations of a CH system atequilibrium; (b) the corresponding proton magnetization vectors,where the subscripts refer to the orientation of the coupled carbon-13 nuclei; c) the normal carbon- 13 excitation sequence and spectrum.

ten. This is only a small improvement for carbon-13 since theintensity increase due to the nuclear Overhauser effect(NOE)9 hat can be obtained during proton decoupling is three-fold. However, the negative gyromagnetic ratio for nitrogen- 15can result in capricious results, including zero signal, whereNOE's are involved. Polarization transfer is therefore a farmore reliable and effective way of improving the sensitivitywhen observing this nucleus. A further advantage is that, wherepolarization transfer is utilized, the pre-excitation delay isgoverned by the relaxation rates of the protons and not of theobserved carbon- 13 or nitrogen-15 nuclei. This allows anadditional saving in time for both nuclei.In the simple situation of a sample such as 13CHC1,, where asingle carbon-13 nucleus is coupled to one proton, an increasein the sensitivity in the carbon-13 spectrum is obtainable byinverting the populations of either pair of energy levelscorresponding to one of the proton signals. The four energylevels, together with their equilibrium deviations from theaverage population, are shown in Figure 6(a). The proton

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106 NATURAL PRODUCT REPORTS, 1988

( b ) z zn s - 5 , !2

3

n = + 3 ~7 +1 O AlH rnon 3 l-66Figure 7 (a) The energy-level populations of a CH system after inversion of populations of the aa and ap levels; (b) the corresponding proton

magnetization vectors ; (c) a sequence for selective proton inversion followed by carbon- 13 excitation, and the resulting spectrum.

X ti

Figure 8 The INEPT pulse sequence, to invert the populations, for example, of all of the higher-frequency carbon-13 satellites in the protonspectrum. The subscripts f ndicate that the final 90" proton pulse alternates between the y and --y directions, and the signals that are detectedare alternately added to and subtracted from the accumulated FID, on successive scans. The vector diagrams represent the behaviour of theproton magnetization vectors, where the subscripts refer to the orientation of the coupled carbon- 13 nuclei.

magnetization is represented in Figure 6(b) by two vectorsM , and M a, lying on the + z axis. The vector M, correspondsto the excess a-orientated protons that are coupled to a-orientated carbon-13 nuclei, and gives rise to transition 3;Ma corresponds to the excess or-orientated protons coupled toP-orientated carbon-13 nuclei, and gives rise to transition 4.In this situation a carbon 90"pulse gives a normal spectrum, asseen in Figure 6(c).If this carbon excitation pulse is preceded by a selectiveproton 180" pulse, applied on the high-frequency line in theproton spectrum, the populations of the aa and a/3 levels areinverted [Figure 7(a)]. A selective pulse, sometimes called a'soft' pulse, is one that is of relatively low power and which issufficiently long (several milliseconds) to affect only a region ofa few hertz. The proton magnetization vectors M , and M a nowlie along the - z and + z directions [see Figure 7(b)]; in other

words, those excess a protons whose coupled carbon-13 nucleiwere also orientated a re now orientated p. This redistributionof populations is reflected in the carbon-13 spectrum, in whichthe high-frequency line is inverted and the intensities of linesincrease, on average, by a factor of four. This selective-population-inversion (SPI) techniquelo relies on a knowledge ofthe precise position of the carbon-13 satellites in the normalproton spectrum, and may be used satisfactorily in simplecases. It is used particularly for the observation of isolatednitrogen- 15 nuclei.

In the general situation of carbon-13 where there are manyresonances, the simultaneous selective pulsing of all high-frequency carbon-13 satellites in the proton spectrum would beimpossible. However, this result may be effected by the INEPT(Insensitive Nuclei Enhancement by Polarization Transfer)technique.l' This again relies on the similarity of magnitude of

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NATURAL PRODUCT REPORTS, 1988-1. H . SADLER 107

1 - 1 1 I I I140 120 100 8 0 G O 40 20

p.p.m.Figure 9 The fully proton-cou pled carbon- I 3 n.m.r . spectra of linalool at 90 MHz . (a) by the INE PT sequence; resonances for quaternary carbonsare absent, centre lines of triplets are missing, and the line intensities of the quartet are equal. The sensitivity is much greater than is obtained

(b) in the single-pulse-excitation method (without nuclear Overhauser enhancem ent) for the same experimental time.

all one-bond C-H couplings but uses non-selective ( i .e.'normal') pulses to invert all of the M u vectors, regardless oftheir frequencies. In this sequence (Figure 8) the directions(phases) in which the pulses are applied are important and aregiven as subscripts. The proton magnetization vectors Ma ndMpare first transferred [Figure 8 (a)] to the xy plane by the firstproton 90; pulse. After a period of (44-' seconds they haveprecessed to positions that are separated by 90", as shown inFigure 8(b). The proton 180," pulse then rotates these aroundthe x axis and the carbon- 13 180" pulse reverses their directionof travel [Figure 8(c)]. In this respect this sequence resemblesthe modified spin-echo sequence. After a further period of(44-' seconds the vectors have moved to positions along the -and + x directions. At this point [Figure 8(d)] a proton 90;pulse, applied along the y axis, rotates the vector M a to the zdirection and the vector M u to the - 2 direction. This will havebeen achieved for all such vectors, regardless of their frequen-cies. Excitation of the carbon- 13 nuclei with a 90" pulse at thispoint leads to a spectrum in which the high-frequency line ofevery CH doublet is inverted, and with the same gain in inten-sity. Had the final proton 90" pulse been applied along the - ydirection, the vector M 8 would have been inverted, resulting ina carbon spectrum in which the low-frequency lines of each CHdoublet had been inverted. Subtraction of this spectrum fromthe former results in equal intensities for each line. In practicethis effect is obtained by alternating the phase ( + y ) of the finalproton 90" pulse and alternately adding and subtractingsuccessive FID signals. It can be shown that CH, tripletsappear with relative intensities -1, 0, and + 1 and CH,quartets with relative intensities - , - 1, + 1, and + 1. A

typical result is shown in Figure 9. As it stands, this sequencedoes not allow proton decoupling while the signal is beingacquired since the multiplet components are opposite in phase,and would cancel each other, yielding no signals. The resolutionof this problem is considered in Section 3.2 and the methodsthat are described there may be used to obtain sensitivity-enhanced broad-band proton-decoupled spectra ofheteronucleithat are coupled to protons.

3 Methods for Distinguishing betweenCarbon-13 Resonances in Methyl, Methylene,and Methine Groups and QuaternaryCarbon-13 ResonancesSince the advent of routine pulse Fourier-Transform n.m.r.spectroscopy, the classification of carbon- 13 resonances hasbeen made by the single-frequency off-resonance proton-decoupling technique12 in which long-range proton


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108 NATURAL PRODUCT REPORTS, 1988Broad-Band'H Decouple

l3C t

Sequence 3A

I 1H B B

* & - -0. . . . . . . . . . . . . . . .

/"nib C // CH,//0/180

1J

0 1-

Sequence 3B APT; GASPE.-1 0 J

1 1 BB 1 Figure 10 Variation of intensities of carbon-I3 signals of CH,, CH,,and CH groups and of quaternary carbon with the delay 7 in

t t sequences 3A to 3C and with the angle 0 in Sequence 3D.Variations in intensity ( I ) are given by Z, = I, ; ZCH = Z, cos 0;I C H p I,, cos28 ; and Zca, = 10c0s38,where 8 = 180J7".

BBL Jl3C

Sequence 3C APT; GASPE.

3.1 Methods Excluding Polarization TransferSequence 3A, with the specific value of (24-' seconds for thedelay 7 as discussed above (Section 2.4), goes some waytowards classifying carbon resonances. 3The same result, i.e.resonances from CH, and CH groups appearing inverted withrespect to those from CH, groups and quaternary carbons, isobtained by using Sequence 3B or Sequence 3C, which areknown as GASPE14- 5(GAtedSpin-Echo) or APT16 (AttachedProton Test) sequences. In these the proton 180" pulse ofSequence 3A is omitted and replaced by broad-band protondecoupling, which is applied during one of the two delayperiods 7 . This has the advantage that the spectrometer doesnot require the capability to apply pulses on the decouplerchannel, although it must be able to switch the decoupler bothon and off independent of data acquisition. A theoreticaldisadvantage is that the duration of these sequences is twicethat of Sequence 3A, but in practice this is not a problem inthese applications. Indeed, it is likely that Sequence 3B andSequence 3C will give significantly better spectra than Sequence3A under comparable conditions since Sequence 3A isdependent on the homogeneity of the proton 180" pulse. In allof Sequences 3A to 3C the maximum peak intensities are onlyobtained for proton-bearing carbon atoms if the delay 7 isequal to J-l. The variation of signal intensities with 7 s shownin Figure 10, and arises because the individual magnetizationvectors of any particular proton- bearing carbon, althoughsymmetrically disposed about the y axis, do not coincide unless7 is an integral number of periods J-l. This variation canbe used to distinguish between signals from C and fromCH, groups but less easily between signals from CH andCH, groups. Problems arise, however, from the significantdifferences in lJCHor protons that are coupled to sp 3 (ca 125Hz), to sp 2 (ca 160 Hz), and to sp (ca 250 Hz) carbon. Thus adelay 7 of 8 ms, corresponding to 'JqH or non-strainedsaturated carbon atoms, yields negative signals at about 60 %of full intensity for aromatic, alkene, and cyclopropyl CHgroups but positive signals for acetylenic CH groups, not to beconfused with signals from C or from CH, groups. A delay of5 4 m s shows signals from CH, and saturated CH, groupswith markedly lower intensities than those from saturated CHgroups and from quaternary carbon. A delay T of 3 mseffectively nulls aromatic CH signals, leaves small residualsignals for CH, and saturated CH, groups, and shows aliphaticCH signals at about 50% of full intensity.

I 1 I I 1200 150 100 50 0p . p . m .Figure 11 Carbon-I3 n.m.r. spectra of carvone, obtained at 90 MHz bythe APT sequence (Sequence 3C) with different values for the delay

7 . (a) 7 = 0 ms, all resonances are positive; (b) T = 3.0 ms; (c) 7 = 3.5ms, principally quaternary resonances; (d) 7 = 5.5 ms, CH andalkene CH, resonances are largest, and CH and CH, signals areinverted; (e) 7 = 8.0 ms, sp2CH and CH, resonances are smaller, CHand CH, signals are inverted. The intensitiesofquaternary resonancesremained the same for all values of 7 but are low in absolute terms,due to a relatively short (3 s) pre-excitation delay.

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NATURAL PRODUCT REPORTS. 1988-1. H. SADLER 109A fair spectrum of resonances from quaternary carbon

atoms is obtained by using a mean value for J of 143 Hz ( i . e . ,7 = 3.5 ms). Signals from CH , and CH, groups are negligible,quaternary carbons give signals of full intensity, and most C Hgroups give significant ( < 20%) signals, these changing frompositive to negative as 'JcHncreases. Acetylenic CH signalshave full negative intensities. Typical spectra are shown inFigure 11.

To some extent it is possible to present subspectra, showingresonances of only one category of carbon, by adding and/orsubtracting the spectra that have been obtained at differentvalues of 7 n appropriate proportions. This process is frequentlyreferred to as 'subspectral editing'. For example, a subspectrumof CH signals (of similar lJCH), ontaining only small signals( < 10Yo)from other carbon nuclei, is ~b ta i n ed ' ~y subtractinga spectrum that has been obtained with 7 = 0.4J-' from onethat has been obtained with 7 = 0.6J-l, although the intensityof CH signals is only ca one-third of the full value. A variety"of other editing procedures have been described but these willnot be discussed, since a more satisfactory and faster method isprovided by the DEPT sequence (Sequence 3G; see Section3.2).

In an alternative sequence (Sequence 3D), known18 asSEMUT (Subspectral Editing by a Multiple-quantum Trap,variations in signal intensity are obtained by fixing the inter-pulse delays at (24- ' and selecting different values for the angle6 of the proton pulse. This pulse transfers part of the signalintensity into unobservable multi-quantum transitions (usuallycalled multiple-quantum coherences), and the variation ofsignal intensities with 8 parallels the variation of intensityversus 7 hat is observed for Sequences 3A to 3C (as shown inFigure 10) such that 6 is equivalent to 1 80 J~ ". his sequencerequires the spectrometer to be capable of applying pulses tothe decoupler channel, but a phase-shifting network (althoughalways an advantage) is not necessary. This sequence isreported to be less sensitive to variations in J than Sequence 3Bor Sequence 3C, and it is suggested that spectral editing is bestperformed by obtaining four spectra, with 6 = 0" (I), 6 = 60"(II), 6 = 120" (111), and 6 = 180" (IV), and collecting data fortwice as many scans for spectra (11) and (111) as for spectra (I)and (IV). Combining these, according to the Table below,yields acceptable subspectra provided the spread of values of Jdoes not exceed 20 YO

CH, subspectrum = (I)- IV)- II) ++(HI)CH, subspectrum = (I)+ IV)- 11)- 111)CH subspectrum = (11)- 111)-81)+ (IV)

C subspectrum = (11)+ 111)-51)- IV )A modification,19 known as SEMUT G L (Sequence 3E),markedly reduces signals of the wrong category in subspectra.These frequently arise with signals whose values of J differ

86' Hl3 C

Sequence 3D SEMUT.

I38 1iH Tl

widely from that selected for 7, nd this is referred to as ' Jcrosstalk'. In this sequence an additional group of protonpulses (90~-r-1 80~-~- 90~,nown as a 'purging sandwich ) isintroduced before the 6 pulse. The three delays T ~ ,,, and 7,take different values, to accommodate a range of values of J.These delays are conveniently selected from the maximum andminimum values of J as follows: 7y 1 = 2(Jm,,+O.O7AJ), 7i1=(Jmin J,,,), and 7i1 = 2(Jmi,- . 0 7 A 4 , where A J =

By using delays of T~ = 3.86 ms, 7, = 3.17 ms, and 7,=2.7 ms, coupling ranges of 120-160 Hz for CH, groups, of120 -18 0Hz for CH, groups, and of 125-190Hz for CHgroups can be tolerated with less than 2 % crosstalk. Thissequence, however, does require a phase-shifting network forthe proton decoupler and extensive phase cycling.For the direct generation of a 'quaternary carbon atomsonly' spectrum, the use20v21 f Sequence 3D, with 6 = 90, isprobably the most satisfactory. Errors from imperfect carbon180" pulses can be removed by cycling the phase of the 19pulsein steps of 90", accompanied by simultaneous shifts by 180" inthe phase of the detector. When used across the normal spectralregion of carbon (i .e.0-220 p.p.m.), a compromise value of Jofca 135 Hz may lead to substantial positive signals ( d 8 "O) foraromatic and alkene CH groups. It is frequently useful to usea delay 7 hat is based on a value of J that is appropriate to theregion of the spectrum of greatest interest and to set 0 slightlygreater than 90, to ensure that any residual CH o r CH, signalsare negative. A typical spectrum is shown in Figure 12.

It has been proposed that a reduction in intensity of theunwanted signals might be achieved by setting the periodbefore the proton 90" pulse to a value of (24- ' that correspondsto a compromise value of the delay 7 for aliphatic groups andthe following period to a value corresponding to a compromise7 for aromatic groups. In this modification the carbon 180"pulse must be set at the centre of the total delay period, and no tcoincident with the proton 90" pulse. A somewhat involvedsequence that has been proposed22 n the hope that it wouldeliminate signals from proton-bearing carbon atoms and showonly the signals of quaternary carbon atoms has been shownz3to give signals of reduced intensity for some CH, groupsalso.In this discussion it has been assumed that sufficient timeelapses between one acquisition of data and the next so that thenormal equilibrium population distribution of the energy levelsis re-established. This will be governed by the longest carbonrelaxation time in the sample. However, a delay of at least 5 sis needed between the scans for most medium-sized moleculesotherwise signals from quaternary carbon atoms will be severelyreduced. In a single-pulse experiment a reduction in the pre-excitation delay is coupled with a smaller pulse angle. In thesemulti-pulse experiments the pre-excitation delay can be reducedby using a greater value than 90" ( e . g . 155") for the first carbonpulse.

The distinction between CH, CH,, CH,, CHD, CH,D,CHD,, CD, CD,, and CD, groups by spin-echo sequences hasbeen However, since these methods are moreappropriate to analyses in isotopic labelling studies, they willnot be examined here.*

Jmax - min.

3.2 Methods Utilizing Polarization TransferThe sequences that are described in this section rely on one-bond proton-carbon couplings not only to distinguish betweensignals from CH,, CH,, and CH groups but also to produce thesignals; herefore quaternary carbon atoms, since they have nodirectly bonded protons, are not observable. In principle, long-range couplings could be used to observe quaternary carbonatoms; however, these couplings are relatively small and show

Sequence 3E SEMUT GL. * See J. C. Vederas, Nat. Prod. Rep., 1987, 4, 277 for a discussion ofapplications of n.m.r. to isotopic labelling studies.

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110 NATURAL PRODUCT REPORTS, 1988

(a

r I I I 1 1 11 4 0 12 0 100 80 60 4 0 20

p . p . m .Figure 12 Carbon-13 n.m.r. spectra of lanosterol at 90 MHz. (a) A normal proton-decoupied spectrum; (b) a spectrum of quaternary carbons (a),obtained by the SEMUT sequence (Sequence 3D), with 8 = 90"and using a pre-excitation delay of 10 s.

Sequence 3F Refocussed INEPT (7 = 1/2J).

such wide variations that in practice their use is severelylimited.The INEPT sequence'' (see Section 2) alone will result in nosignals at all if broad-band proton decoupling is applied duringthe acquisition period since the positive and negative parts ofthe multiplets cancel each other. This can be overcome byadding a further delay, A , to the INEPT sequence; 180" pulsesare applied to both nuclei, at the centre of this delay before theacquisition of the FID, with broad- band proton decoupling.This allows the components of multiplets to regain the samephase. The sequence (Sequence 3F) is known25 s the refocussedINEPT sequence. The optimum delay 7 is (24-1 for all CH,groups since each proton is coupled to only one carbonnucleus. However, signal intensities for CH, CH,, and CH,groups vary differently with the delay d since the rates ofJ-precession are different. This variation is shown in Figure13. Approximately equal intensities are obtained for CH,CH,, and CH, signals if d is ca 0.3.J-'; a value of d of ca0.7J-' will show positive signals for CH and CH, groupsand negative signals for CH, groups. Only signals from CHgroups should be obtained if d is equal to (24-', but, since theintensities of signals from CH, groups show their maximumrate of change at this point, slight variations in values of J fromthat selected for d lead to significant signals from CH, groups,and Sequence 3F (like Sequences 3A to 3C) is very susceptibleto intensity errors due to differences in values of J .A more accurate CH spectrum is provided26 by the EPT

Figure 13 Variation of intensities of carbon-13 signals of CH,, CH,,and CH groups with the delay d in the refocussed INEPT sequence(Sequence 3F) and with the pulse 8 in the DEPT sequence (Sequence3G). Variations in intensity (I) are given by I C H= I, sinf?; I CH =I , sin2 8 ; and I C H ,= 31, (sin 38+ sin 8)/4, where 8= 180JAO.

(Exclusive Polarization Transfer) sequence [Sequence 3G ( 0 =90")], which is a special case of the more general DEPTZ7(Distortionless Enhancement by Polarization Transfer)sequence [Sequence 3G (8 is variable)]. This sequence has fewerpulses than the refocussed INEPT sequence and effectspolarization transfer in a different way. Except for the specialcase of 8 = 90", this cannot be described by using the simplevector pictures that have been presented earlier since multiple-quantum effects are involved. In this sequence the separation

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NATURAL PRODUCT REPORTS, 1988-1. H. SADLER 111

Sequence 3G DEPT ; EPT (0 = 90").

I

Ac 0 I

rN 4 1

I I8 0 6 0 I40 120p.p.m.Figure 14 Carbon- 13 n.m.r. spectra of a triterpene derivative (40 mg in0.4ml CDCl,) at 90 MHz. Spectrum (a) is a normal proton-decoupled spectrum (2000 scans), showing all resonances for sp 3carbon; spectra (b)+d) were obtained by using the DEPT sequence(Sequence3G) (320 scans each; pre-excitation delay of 3 s) and showthe proton-bearing carbons. In spectrum (b) 0 = 45" and allresonances are positive; in (c) 8 = 90, and only CH resonances arevisible; in (d) 0 = 135", and whereas CH and CH, resonances arepositive, the CH, resonances are negative.

between the pulses remains fixed at (24-l and signal intensitiesare varied by altering the value of the third (0 ) proton pulse.This variation of intensity (see Figure 13) with 0 parallels thatobtained by changing d in Sequence 3F, and there is a generalcorrespondence28between 0 in DEPT and 18OJd" in Sequence3F. A comparison of the DEPT and refocussed INEPTtechniques forms a significant part of a review,' of pulsedmethods for polarization transfer in carbon- 13 n.m.r. spec-troscopy. Although the total duration of the DEPT sequenceis greater, this is of little significance in practice for carbon-13spectra, and the DEPT sequence is preferred since, amongother reasons, it tolerates greater differences in values of J.Residual signals from CH, and CH, groups that might beobtained by using a compromise value for J of 135 Hz aresignificantly less in an EPT spectrum than for a refocussedINEPT spectrum with the equivalent value of A . In most casesthe CH,, CH,, and CH groups are readily distinguished byvisual examination of two DEPT spectra. The spectrum that isobtained by using a compromise value for J o f 135 Hz, a pre-excitation delay of 5 seconds, and with a value of 0 equal to 90"

I I I I80 60 40 20P.P m

Figure 15 The carbon-13 n.m.r. spectra of Figure 14, edited to showseparate subspectra for CH, CH,, and CH, resonances.

shows CH resonances only; if 0 is ca 130" the spectrum showsCH and CH, groups as positive signals and CH, groups asnegative signals. Typical spectra are shown in Figure 14.Features arising from values of J that differ widely from thatchosen for the calculation of a suitable inter-pulse delay includethe following. Acetylenic CH groups may give very small orzero signals. Alkenemethylene and methylenedioxy groupsappear with up to 50 % of full intensity in a 'CH group only'(0 = 90") spectrum but are readily identified (as negativesignals, of reduced intensity) in the other spectrum (0 = 130").These signals also lie in the relatively empty region between thealiphatic and the aromatic resonances.Should editing be considered necessary, the DEPT sequenceprovides more accurate subspectra than the refocussed INEPTsequence. Three spectra are obtained21 using values of 0 of 45"(I), 90" (11), and 135" (111), with twice as many scans beingacquired fo r spectrum (11)] and these are combined as follows:

CH, subspectrum = (I) + 111)- II)/d2CH, subspectrum = (I )- 111)CH subspectrum = (11)

In practice these proportions may-need to be adjusted slightlyto give the best results, due to imperfections in both the timingand the homogeneity of pulses. Such imperfections may alsoresult in small CH, signals in a CH subspectrum. Unwantedsignals arising from a variation of 10% in J constitute less than7 % . In particular, CH, subspectra show no residual CH orCH, signals from this source of error; CH, subspectra show noresidual CH signals and CH subspectra show less than 1YOofresidual CH, signals. The largest errors arise from CH, signalsin a CH subspectrum and for CH, signals in a CH, subspectrum.Negative CH, signals (arising from methoxy-groups or acetylgroups) in a CH subspectrum are usually indicative of too shorta pre-excitation delay, but this is a diagnostically useful error.The variations assume that the 90" pulse and the 180" pulse forboth nuclei are accurately set. Edited DEPT spectra are shownin Figure 15.

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I B B80-180

A full discussion2' of sources of errors in DEPT spectra hasbeen given, along with full experimental details for measuringthe pulse times and for checking the accuracy of the phase shiftsof the proton decoupler pulses.

Both Sequence 3F and Sequence 3G require that spectro-meters should be used that are capable of providing phase shiftsof 90" on the proton-decoupler channel, and therefore it maynot be possible to use these sequences on some olderspectrometers. It is, however, possible30 to carry out polar-ization-transfer experiments with spectrometers which do nothave this facility, but in which pulses may be applied from theproton decoupler, by using Sequence 3H. This is a modifiedrefocussed INEPT sequence in which the first pair of 180"pulses on both nuclei is omitted from the centre of the delay 7.This results in an extent of polarization transfer (and thereforesignal intensity) which, assuming 7 = (24-l and a fixed value ofA , varies with sin{180vo/J), where v is the separation betweenthe proton decoupler frequency and the individual protonresonance frequencies. This sine dependence has led to theacronym SINEPT for Sequence 3H. Signal intensities aremaximum where the relevant proton resonance lies such thatv = (2n+ 1)J/2 and zero when v = nJ. This dependence ofsignal intensity is markedly reduced by alternating the protondecoupler frequency between two values, differing by O S J , onsuccessive scans. Alternatively, half of the scans may beacquired at one proton transmitter frequency and the accumu-lated FID be combined with that from the remainder, which isobtained with the shifted frequency. In this way proton-bearingcarbon atoms are observed with at least 70% of their 'INEPT'intensities.The DEPT sequence may be similarly modified30 by omittingthe second proton 180" pulse to give the MODEPT sequence(Sequence 31). In this sequence, in which 7 = (24- ', signals areat a maximum when v = (2n+ 1)J/4 and zero when v = nJ/2 forany specified value of 8. n this instance the variations in signalintensity are reduced by choosing proton decoupler frequencieswhich differ by 0.25J.

A new sequence (Sequence 3J), known as POMMIE (PhaseOscillations to MaxiMIze Editing), has been described,' as analternative to the DEPT technique. This differs from thepreviously discussed sequences in that, in addition to the inter-pulse delays being fixed at (24-', the pulses are all 90" and 180"pulses and intensity variations are achieved by cycling thephase of one additional decoupler pulse. Such experiments canonly be carried out on the most recent instruments, whichemploy digital phase-shifting networks that allow incremental

YSequence 3H SINEPT (7 = 1 /24 .

BB'ti1-c 2 J

Sequence 31 MODEPT.

'H BB1 1-J-3C 2J

Sequence 35 P O MMI E ; the last proton 90"pulse uses a variable phaseangle 9.

variations in phase ( e . g .by 5") rather than the standard 90"and180" shifts that are employed in most spectrometers. Thisallows more accurate setting of phases and more extensivephase cycling, for which reason POMMIE is expected to be lessprone to errors than DEPT. However, this option is notavailable to most workers at present. investigations intothe use of polarization transfer from deuterium to carbon- 13 todistinguish between CD, CD,, and CD, groups have beencarried out. Selective enhancement of CD groups is straight-forward but the distinction between CD, and CD, is moredifficult. The possibility of obtaining separate subspectra ofCD, CD,, CD,, CDH, CDH,, and CD,H groups by combiningpolarization transfer with gated spin-echo sequences has beenexplored34 but in practice these methods are unlikely to beerror-free. All of these methods require a spectrometer that iscapable of pulsing and decoupling at the deuterium frequencyas well as the proton frequency, and a fourth nucleus would benecessary for locked operation.

Some e~perimen tal~~'3 and

4 Methods for the S implification of FullyProton-Coupled Carbon-I3 SpectraProton-coupled carbon- 13 spectra of natural products arefrequently very difficult, if not impossible, to analyse, due tooverlap of multiplets from carbon resonances whose chemicalshifts lie in the same region of the spectrum. Completeseparation is achievable by two-dimensional methods35 butlarge amounts of data storage and plenty of material are neededfor high resolution, and distortions in multiplet spectra mayoccur if the proton spectrum is not first-order. One-dimensionalsimplification methods involve obtaining individual spectra foreach multiplet or the careful use of editing techniques, based onthe number of protons bonded to the carbon- 13 nuclei. In somecases the positive/negative presentation of the simple INEPTsequence with the absence of the centre section of triplets canprovide sufficient separation of multiplets. The additionalintensity in the outer sections of quartets can also be valuablewhere sensitivity is low.4.1 Separate-MultipletSpectraTwo methods have been proposed which allow individualproton-coupled carbon- 13 multiplets to be observed separately.One method (Sequence 4A) relies on selective excitation of acarbon resonance at the pulse frequency whilst employingbroad-band proton decoupling and switching off the decouplerduring the acquisition of the FID, to restore the proton

Selective excitation is achieved by using a train of nshort pulses, each of flip angle 8 = 90"/n, spaced 7 secondsapart. This alternately tips the magnetization vectors throughan angle 8 and allows them to precess, during the delays,through an angle q5 = 180~7,where v is the separation of thevector from the pulse frequency. Vectors from signals locatedat the pulse do not precess during the delays, and at the end ofthe pulse train they have in effect received a 90" pulse. Vectorsat intervals of 1/7 Hz from the pulse are also tipped through90" since they precess by an integral number of revolutionsduring each delay. Provided enough pulses are employed,magnetization vectors from resonances more than a few hertzaway from the pulse never stray far from the z axis of therotating frame, and significant signals are only obtained fromresonances within a narrow band f /n7 Hz around the pulsefrequency and at intervals of 1/7 Hz from it . The behaviour of

B B'H Q I

W u e n c e 4A DANTE; selective excitation.

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NATURAL PRODUCT REPORTS, 1988-1. H. SADLER 113these vectors has led to this sequence acquiring the nameDANTE (Delays Alternating with Nutations for TailoredExcitation). A typical experiment may employ a train of thirtypulses of 3", at 2 ms intervals, and to be able to define suchsmall pulses it is frequently necessary to attenuate the power tothe carbon- 13 signal transmitter. Suitable selection of theinterval 7 allows two carbon-13 multiplets that are far enoughapart not to overlap to be excited simultaneously, with aconsequent saving in time. If excitation employs the equivalentof a 90" pulse, relatively long pre-excitation delays (of 1&20seconds) may be needed.An alternative to the DANTE sequence relies on selectiveacquisition3' of the desired signals rather than selectiveexcitation, and similar spectra are obtained. A single, hardexcitation pulse is applied at the chemical-shift frequency of thecarbon resonance whose multiplet is to be observed and then,after a short delay, t, the FID is acquired (Sequence 4B). As inDANTE, broad-band proton decoupling is applied at all timesexcept during the acquisition of data. During the total numberof scans the delay t is regularly incremented by an amount 6tfrom an initial value of 6t to a final value t , . Selectivity isobtained by making use of the variations in phase that areundergone during the delay periods by signals that are notcoincident with the pulse. Every time the delay is extended byone increment the phase of any off-resonance signal, v Hz fromthe pulse, alters by an amount 4 = 180v6to,whereas the phaseof a signal that is located at the pulse and at multiples of1/6t Hz from it remains unchanged. Provided enough shortincrements are employed, the alterations in phase of the signalthroughout the experiment result in a reduction of the final off-resonance signals to an insignificant level, and only signalsfrom a resonance that is located at the pulse frequency and atintegral units of 1/6t Hz from it are observed.The application of this method to part of the carbon- 13 n.m.r.spectrum of P-pinene is shown in Figure 16. Similar results areobtained with DANTE. Severe overlap in the fully proton-coupled spectrum (a) prevents satisfactory analysis of themultiplets. Selective-acquisition spectra [(bt-(f)] show themultiplets clearly, and the power of these methods is illustratedby the multiplets that can be obtained for the two methylenecarbons whose centres lie only 10 Hz apart . As a guide forgeneral useage, good selectivity can be obtained if not less than25 increments are used and if the size of the increments is setsuch that the final delay is not less than 0.8/Af, where Af is theseparation between the closest pair of carbon resonances in theproton-decoupled spectrum. The final delay should be made asshort as possible, consistent with the desired selectivity, since ifit is too long the final signal intensity will be reduced byrelaxation. The excitation pulse may be set at the flip angle thatis normally used to obtain fully proton-coupled spectra with anappropriate pre-excitation delay, which should be at least onequarter of the acquisition time to retain the nuclear Overhauserenhancement. It is not necessary to reduce the power of thecarbon- 13 transmitter.

4.2 Multiplet Spectra by EditingIn Section 3 it was described how spin-echo and polarization-transfer sequences could be used to give separate proton-decoupled subspectra for CH, CH,, and CH, groups. In manycases a similar simplification of fully proton-coupled spectrareduces the overlap of signals to an acceptable level. Clearly,obtaining several carbon- 13 multiplets in a single spectrumsaves time compared with the individual observation methods

l3C -JLbar iedSe


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Sequence 4C DEPT GL+; he final pro ton pulse (----) is applied on Sequence 5A SHECOR; the selective proton 180" pulse (---) isalternate scans only; extensive phase cycling is required. applied on alternate scans only.

The DEPT sequence without proton decoup1ing2'. is farless prone to these spectral distortions but is not entirely freefrom them. In all of these sequences, however, it is frequentlyimpossible to achieve completely clear edited subsp ectra du e tovariations in the magnitude of the one-bond proton-carboncouplings. To overcome these problems, several modificationsto these sequences have been proposed. The addition, forexample, of a single proton 90" pulse to the APT and to therefocussed INEPT sequences immediately before data areacquired, t o give the AP T+ and INEP T+ sequence^,^^.^^ resultsin distortionless coupled spectra [Figure 17 (b)]. How ever, thesesequences often lead to poorly edited subspectra. Probably themost satisfactory modification for proton-bearing carbons isthat39 described as the DE PT GL' sequence (Sequence 4C), inwhich the delays 71,,' and 73 re set according to the range ofJ,, values that is required (as described under SEMUT G L;see Section 3.1).In Sequence 4C the final proton 180" pulse is applied onalternate scans only. Satisfactory editing is obtained by usingvalues of 38", 90", and 142" for the 0 pulse and by combiningthese spectra as described previously (Section 2). Fully coupledspectra in which only quaternary carbons are seen are bestobtained by using the analogous SEMUT GL+sequence.

5 Heteronuclear Chemical-Shift-CorrelationMethods5.1 One-Bond CorrelationsTh e correlation of carbon-1 3 resonances with those of theirbonded protons has formerly been obtaine dg0 rom a series ofsingle-frequency proton-decou pled carb on- 13 n.m.r. spectra.Assignment may, however, be ambiguous or impossible in thisway in crowded regions of the carbon-13 spectrum and alsowhere the pro ton spectrum shows strong homonuclear coupl-ing. Elegant proton-carb on correlations ca n be obtained witha very high degree of certainty by using two-dimensionalmethods,41 but these frequently need large data m atrices andare very time-consuming if only small quantities of material areavailable or if high resolution is necessary. In many instances arelatively small num ber of one-dim ensional spectra suffice, andrepresent a considerable saving in time. A variety of one-dimension al sequences for C-H correlation are available. Allinvolve selective polarization transfer from specific proton(s),so that the signal from the corresponding coupled ca rbon nucleusapp ears with increased intensity. These methods fall into one oftwo classes, according to whether or not they employ selective(soft) pulses to initiate the polarization transfer process. Clearly,such methods a re applicable to the correlation of proto ns withnuclei other than carbon, but the problems are rarely socomplex and can frequently be solved by conventionaldecoupling experiments. It is important in all experiments thatinvolve selective irradiation of protons whilst other nuclei arebeing observed that the same probe is used to determine theproton spectrum as will be used for the main experiment. If aswitchable prob e is not available, satisfactory proto n spectracan usually be obtai ned by using the proton-decoup ler coils. Inthe case of long accumulations it is also necessary to preventevaporation of solvent since this may result in a shift of thefrequencies of the proton resonances.

5.1.I Sequences Employing a Selective Proton PulseAll of these sequences use a selective pro ton 180" pulse, appliedat one of the carbon-13 satellites of a particular proton, toidentify the corresponding carbon resonance. In the simplestsequence (Sequence 5A), know n as SH EC OR g2 (SelectiveHE tero nuc lear COR relation), the initial selective prot on pulseis followed by a gated spin-echo sequence; the F ID is acquiredwith broad-ban d decoupling. No phase cycling is necessary andFourier trans formation yields a spectrum in which there is anenhanced fully proton-decoupled signal for the correspondingcarbon resonance. Typical durations for the selective proton180" pulse are 25-50 ms and pre-ex citation delays of 2-3 sare used. In practice, non-enhanced signals are removed byapplying the selective pulse on altern ate scans and alternatelyadding and subtracting successive FID signals. This has thesame effect as subtracting a norm al spectrum from a selectivelyenhanced one. Enhancements for CH, CH,, and CH, signalsvary with the delay 7 . In particular, signals are obtained onlyfrom C H groups if 7 = (24-'. Nearly even enhancements aregiven for all proton-bearing carbon s if 7 = (4 4- I; alternatively,if 7 = 3/4J, CH, signals are inverted with respect to CH andCH , signals. Use of this value for the delay 7 s not recommendedsince signals are inverted (for all values of 7 ) f a low-frequencyrather than a high-frequency satellite is irradiated. Where thepro ton spectrum is well resolved an d clearly assignable, a fewcarefully chosen irradiation positions should suffice to givea correlation of the two spectra. However, in more complexcases the high-frequency carbon-13 satellite of one protonresonance may be very close to the low-frequency satellite ofano ther. In such cases it is preferable to increase the frequencyof the selective pulse in steps of 10 Hz across the proton regionand to present the da ta as a stack of one-dimensional spectra.Spectra of the methylene and methyl carbons in linalool thathave been obtained in this way are shown in Figure 18. Eachcarbon resonance shows a maximum positive intensity at thefrequency of the high-frequency carbon satellite of thecorresponding proton resonance and a maximum negativeintensity at the frequency of the corresponding low-frequencycarb on satellite. The corresponding prot on resonance lies at theaverage of these two frequencies. Thus the carbon resonancesof 6 = 17.7 and S = 25.7 correlate with the alkene methyl-proton resonances a t S = 1.54 and 6= I .62 respectively and thecarbon resonance at S= 42.6 correlates with the methylene-proto n multiplet tha t is centred aro und S = 1.5. Although theproton region that is examined covers only one satellite fromeach of the extreme pro ton resonances, there is no ambig uity inthe correlation of the remaining two carbon resonances, that atS = 23.1 correlating with the methylene-proton multipletcentred aroun d S= 2.0 and that a t S = 27.7 correlating with themethyl resonance at 6 = 1.21. Additional low-intensity peaksmay occasionally arise from long-range couplings. Severalvariants of Sequence 5A have been proposed which result inroughly a tw o-fold increase in signal- o-noise ratio .43 Th eseleaive pulse is applied on every scan and consecutive FIDsignals are all added. Additionally, a carbon 180" pulse isapplied a t (i) on alternate scans or a carbon 90" pulse is appliedat (i) or at (ii), alternating the phase of this pulse between + xan d - x on successive scans. The nulling of un-enhancedresonances is more sensitive to imperfections in the pulses herethan in the unmodified sequence.

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45 40 3 5 30 2 5 20 15p.p.m.13c

Figure 18 A stacked plot of carbon-I3 n.m.r. spectra of CH, and CH, resonances of linalool, obtained at 90 MHz by the SHECOR technique withselective irradiation of protons at intervals of 10 Hz over the range 6= 1.2 to 6 = 2.0. The power of the proton pulse was 45 db below 0.2 W , itsduration was 59 ms, and the pre-excitation delay was 3 s. Frequencies of correlated protons and carbon nuclei lie ( 0 ) t the average of the protonfrequencies corresponding to the maximum positive and the maximum negative intensities of the carbon resonances.

l3C

Sequence 5B SEPT; the first hard proton pulse (----) is applied onalternate scans only.

Sequence 5C SDEPT.

BB

vSequence 5D Selective suppression.

A related sequence (Sequence 5B), known as SEPT44(Selective inEPT), is used in a similar fashion to SHECOR.Un-enhanced signals are removed by applying a normal ( i . e .non-selective) proton 180" pulse, simultaneously with thecarbon 90"pulse, on alternate scans; successive FID signals arealternately added and subtracted. If a carbon 90"purging pulseis applied at (ii), to remove out-of-phase signals, then phasecycling must be employed. The variation of signal enhancementswith 7 ollows that described for SHECOR, and the same careis required for complex proton spectra. No direct comparisonof the two sequences has been reported. However, SEPT (andtherefore presumably SHECOR) has been reported to suffersome of the disadvantages of INEPT, namely error signalsarising from variations in J . In an attempt to overcome this,Sequence 5C, known as SDEPT (Selective DEPT), has been45 although it requires an extensive phase-cycling

procedure if optimum results are to be attained. Signalintensities display the same dependence on 8 as in the DEPT

sequence and show maxima where the frequencies of theselective pulses coincide with carbon- 13 satellite. However, thesignal is not inverted if the selective pulse is transferred fromone satellite to the other, and a value of 0 of 135" will invertCH, signals with respect to CH and to CH, signals in a seriesof experiments in which the frequency of the selective pulse isincreased in uniform increments over a region of the protonspectrum. One possible experimental drawback to Sequences5Band 5C is the need for very rapid switching of the power levelof the decoupler from the selective pulse to the first non-selective pulse, and the spectrometer may need to be modifiedto enable this.In situations where the positions of the proton resonancesand corresponding satellites can be clearly identified it ispossible to use a technique (Sequence 5D) in which thecorresponding carbon signals can be s u p p r e ~ s e d . ~ ~refocussedINEPT sequence is preceded by a modified SPT sequence,made up of a selective proton 180" pulse sandwiched betweentwo normal carbon- 13 90"pulses. This effectively equalizes thepopulations of the four levels of the selected CH system andthus no signals are possible for this carbon. The magnetizationremains at equilibrium for all other protons and thus resonancesfor all other carbons are observed. In this way it has beenpossible to correlate the six resonances of methyl protons andthe corresponding carbon resonances in the cyclic depsipeptidevalin~mycin,~~nd the method has also been successfullyapplied to the proton and nitrogen resonances of the NHgroups in the same m01ecule.~~lthough no DEPT version ofthis method has been reported, there appears to be no reasonwhy this should not be a viable method.5.1.2 Sequences Employing Only Non-selective Proton PulsesSince the use of non-selective proton pulses in a standardDEPT or refocussed INEPT sequence leads to equivalentenhancements for all carbon resonances, regardless of thefrequency of the proton pulse, these sequences must be modifiedin some way, ideally to provide an enhancement only for thecarbon resonance corresponding to the proton resonance thatcoincides with the frequency of the proton pulse. If this ideal isnot attainable, and more than one carbon resonance is obtained,then hopefully the intensity of the carbon might be related insome simple way to the separation of the corresponding protonresonance frequencies and the proton pulse frequencies. Thislatter situation is considered first since the method does notrequire phase cycling of the decoupler. Variation of the

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NATURAL PRODUCT REPORTS, 1988

EB 1W u e n c e 5E SINEPT-2 (T + 7 = I / 2 4 .

Sequence 5F CHORTLE (T+7= 1 / 2 4 ; extensive phase cycling isrequired.

intensity of carbon resonances with the frequency of the protonpulse has been briefly considered above in the SINEPT sequence(Section 3.2), where signal intensities of zero for carbon areobtained for corresponding proton resonances which lie atintervals of 'JCHrom the frequencies of proton pulses. Sequence5E, which is known4' as SINEPT-2, is more flexible in thatpositions yielding zero or maximum intensity can be placed atany desired separation from the frequency of the proton pulseby adjusting the period 7 . Sequence 5E is a refocussed INEPTsequence in which the first pair of 180" pulses is placed off-centre in the (24-l period during which polarization transferoccurs. Assuming a fixed value for A and that all pulses have thesame phase, then the intensities of carbon signals vary accordingto I,, sin(360vT0), where I,, is the maximum INEPT intensityand v is the separation of the corresponding proton resonancefrom the pulse frequency. The delay d is selected as in therefocussed INEPT sequence. Carbon signals of zero intensityarise where corresponding proton resonances lie at thefrequency of the transmitter and at intervals of (27)-l Hz oneither side of that frequency. Other carbon resonances appearwith positive or negative intensities up to the maximum,according to the position of their corresponding protons. Bysuperimposing a sine curve of appropriate wavelength ( i . e . 1 7 )on the proton spectrum it is frequently possible to selectsuitable values for the frequency of the proton pulse and for thedelay 7 to allow correlation of proton and carbon- 13 resonancesby qualitative inspection of the spectra, provided the protonspectrum is well resolved and not too complex.

A qualitative approach is unlikely to be satisfactory formany proton-carbon correlations, and a more rigorousmethod has been Sequence 5F, known asCHORTLE (Carbon-Hydrogen correlation from One-dimen-sional polaRization-Transfer spectra by LEast-squares analy-sis), differs from SINEPT-2 primarily in the extensive cycling ofall of the transmitter and receiver phases, so that any errorsthat have been caused by phase or pulse imperfections areremoved and peak intensities can be used reliably. Additionally,the last proton 180" pulse is replaced by gating the decouplerand half of the accumulations are carried out with the period 7placed at the start of the initial (24-' period. Two spectra areobtained for each value of 7 . Where the two proton 90" pulseshave the same phase (or differ by 180") the intensities show thesine dependence as in SINEPT-2, and the spectrum is referredto as the 'sine spectrum'. If, however, the phases of the protonpulses differ by 90" (or by 270"), the intensities show a cosinedependence and the spectrum is referred to as a 'cosinespectrum'. Thus, in its simplest form, 'sine' and 'cosine'spectra are obtained for one value of 7 (e.g. the inverse of thespectral width) and the intensity ratio !JIG is determined foreach carbon resonance. The frequency Y of the correspondingproton resonance relative to the pulse frequency is given bylJ IC tan (360~7") r v = [tan-' (1,/1,)]/360~".If the carbon isbonded to two non-isochronous nuclei, the frequency v is the

5h0I r I S 7: = 1.8msv = 1.336

IC

ST = 1.2msv = 1.33 6

C

( f 1

Y I . I I S \ T = 1 . 8 m s

I I r I 1 I50 40 30 20 10 0P.P.m

Figure 19 Carbon- 13 n.m.r. spectra of camphor at 90 MHz. Spectrum(a) is the proton-broad-band-decoupled pectrum, with assignments:(b)-(g) were obtained by using the CHORTLE pulse sequence.Locations of proton pulses, delays 7, nd spectrum types ['sine' (s)or 'cosine' (c)] are as indicated.

Sequence 5G Direct correlation.

average of that for the two protons. It is clearly more desirableto obtain data with several different values of 7 and/orfrequencies of the proton pulse to provide a check. Additionally,non-linear least-squares analysis of the intensities will yieldchemical shifts of protons to within a few hertz. This degree ofprecision, however, requires that any systematic errors shouldbe minimized and entails holding the pre-excitation delay,(24-', and d constant within a series of spectra together withextensive phase cycling. This procedure allows the positionsof both protons in a non-equivalent pair to be located. Goodresults can be obtained from four pairs of spectra, such as thoseshown for camphor in Figure 19.

Spectra that show only a single carbon resonance, correlatingwith a proton resonance that is located at the frequency of theproton pulse, may be obtainedg9 with Sequence 5G. Thissequence also resembles SINEPT except that the first delayperiod t is varied during the course of the experiment and thephase of the second proton 90" pulse differs from that of thefirst by 90". The extent of polarization transfer depends on boththe value of 'JcHand cos(360vt0), where v is the separation

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NAT URAL PRODUCT REPORTS, 1988-1. H. SADLER

(g )

I1 7

4 - H

3-exo - H

( f ) 5-exo-H

1

(d 1 anti-Me - H

I I I I I 150 40 30 20 10 0P P.m

(C1

Figure 20 Carbon- 13 n.m.r. spectra of camphor at 90 MHz. Spectrum(a) is the proton-broad-band-decoupled pectrum; spectra (b)--(h)were obtained by using the variable-delay sequence 5G and eachshow one principal carbon resonance. Corresponding locations ofproton pulses are as indicated.

1-Me-H

between the resonance frequency of the proton that isresponsible for polarization transfer and the frequency of theproton pulse. The sequence requires that the delay t be variedfrom one scan to the next in such a way that, over a largenumber of scans, polarization transfer is maximum for protonsthat are located at the pulse frequency and zero for thoseelsewhere. It has been shown that this is effected by increasingthe value of t in regular increments or by randomly varying thevalue of t between K / J and ( K + l)/J, where K is an integer.The selectivity increases with increasing K , but more artefactsare generated and the experiment becomes more susceptible topulse and phase errors. In practice, these problems are overcomeby using equal numbers of scans for values of K equal to 0, 1,2, and 3 and subtracting the FIDs for the even values of K fromthose for the odd values of K . In each set of scans the value oft is incremented or chosen at random between the limits definedby K . This gives a proton selectivity of ca20 Hz. Proton-correlated carbon- 13 spectra of camphor that have beenobtained in this way are shown in Figure 20.Of these three sequences in which only non-selective pulsesare employed, SINEPT-2 is the only one which may be used onspectrometers where phase shifting in the decoupling (proton)channel is not possible. However, proton pulse frequenciesmust be selected with great care to obtain unambiguous results.The CHORTLE sequence has the advantages that relativelyfew proton irradiation positions are necessary for a completecorrelation and it does not require knowledge of the protonspectrum. However, extensive phase-shifting is necessary ifreliable quantitative intensity measurements are to be obtained.

( b )

The sequence in which the polarization- transfer period is variedprovides the clearest visual correlations, as a series of essentiallysingle-line spectra. However, a knowledge of the positions ofthe proton resonances is necessary.

sy n -Me -HA

5.2 Long-Range Correlations5.2.1 Correlation through CouplingTwo- or three-bond proton-carbon correlations can frequentlybe obtained from carbon-13 n.m.r. spectra by selectiveirradiation of specific proton resonances at low power, since thecoupling constants are small (3-1 5 Hz). This method is regularlyused for the assignment of resonances of quaternary carbonnuclei and is also valuable,50 hough less frequently used, forthe assignment of resonances of protonated carbons. The sametechnique can obviously be applied for nitrogen resonances inpolyaza-compounds and for silicon resonances in multisilylethers. However, in carbon spectra, the resonances of somenon-quaternary carbons may show marked changes in intensityand in multiplicity if one of the corresponding carbon-13satellites in the proton spectrum lies near a proton that is beingirradiated. This problem can be markedly attenuated byemploying51 proton broad-band irradiation during thepre-excitation delay and by switching to single-frequencydecoupling during excitation and acquisition of the FID signal.The principal drawback of the experiment for quaternarycarbon nuclei is its greater duration, necessitated by thecomparatively long relaxation times of carbon nuclei. Thesituation is even worse for nitrogen- 15 and silicon-29 nuclei,where incomplete nuclear Overhauser effects can result in therebeing no signals. A marked improvement should be obtained inmany cases by employing a modified refocussed INEPTsequence [Sequence SH, known as INAPT (Insensitive NucleiAssigned by Polarization Transfer)] in which all of the protonpulses are selective and are applied to a specific protonresonance, and thereby52 using long-range couplings forpolarization transfer. Since these couplings are much smallerthan one-bond couplings, inter-pulse delays are significantlylonger and some relaxation may occur while the pulse sequenceis being applied. Thus signal enhancements of two- or three-fold are observed for carbon- 13 resonances rather than theexpected four-fold. Since the lengths of proton pulses are nowrelatively long (10-15 ms), this must be taken into accountwhen calculating the delays A , and A, , for which values areotherwise set as discussed previously. Since the strength of theselective proton pulses (expressed in hertz) is much smaller thanthe one-bond coupling constant, no signal is obtained from thedirectly bonded carbon- 13 nucleus. If the chosen protonresonance coincides with a carbon-13 side-band of anotherthen a small additional signal may be observed. The spectrumthus only shows resonances from carbon- 13 nuclei which havelong-range couplings [of ca (2A)-'] with the resonance at thefrequency of the proton pulse. The wide variations in J meanthat the delay A cannot be optimized for all likely values of J .In practice this delay is rarely set longer than 50 ms, to avoidtoo much signal being lost by relaxation. The main restrictionis that proton resonances which are coupled to that selectedmust be greater than 30 Hz away, otherwise multiple-quantumeffects will result in further losses of signal intensity. Protonsthat are close to but which are not coupled to the chosen protonwill not affect the polarization transfer. The method has alsobeen used for the observation and assignment of nitrogen- 15resonances in small pep tide^.^^

Sequence5H INAPT; all proton pulses are selective.

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118 NATURAL PRODUCT REPORTS, 1988In principle, an analogous selective DEPT is also possible.

However, an additional pulse interval of (2.9- increases theopportunity for relaxation, and therefore signals of lowerintensity are likely to be obtained.

5.2.2 Correlation through Space :Difference SpectroscopyA method for correlating the resonances of protons andcarbon-13 nuclei that are not bonded to each other and whichis just beginning to receive the attention it deserves makes useof the heteronuclear Overhauser effjxt. Briefly, if a proton issufficiently close (usually within 3 A) to a nearby nucleus Athen irradiation and saturation of the proton resonance leadsto an increase in intensity of the resonance of A. The ratio (0 )of the intensities of the resonance of A with and withoutirradiation of the proton resonance is defined as the nuclearOverhauser enhancement, NOE. For molecules of molecularweight less than about 1000 this enhancement does not exceed1 +(-yH/2yA).Thus carbon intensities may be increased by anyfigure up to a factor of 3, and this is frequently found forproton-bearing carbons in a broad- band proton-decoupledcarbon- 13 n.m.r. spectrum. Selective low-power ( e . g .y B , / 2 n ofca 2-3 Hz) irradiation of a proton generates nuclear Overhauserenhancements on the resonances of only a few carbon nucleithat are two (or sometimes more) bonds away. Combining thistechnique with difference spectroscopy5* provides a powerfulalternative to selective proton decoupling or selective polar-ization transfer for identifying or for assigning resonances,particularly (but not only) of quaternary carbons. In differencespectroscopy a control spectrum, which has been obtainedwith the irradiation point located in a blank region of theproton spectrum, is subtracted from a spectrum where aspecific proton has been irradiated, so that only the effects ofirradiation are visible in the difference spectrum and unaffectedsignals are missing. Apart from the position of irradiation, allexperimental parameters must be identical for both spectra.The data-handling systems of most modern spectrometers arecapable of performing subtractions of this kind, and theincreases in intensity (which are usually quoted as a percentageof the intensities in the control spectrum) may be measuredaccurately. The earlier procedure of direct integration of theindividual spectra is too unreliable to give accurate values,whereas difference spectra can (in favourable cases) detectincreases in intensity of less than 1 YO. everal procedures havebeen used for these experiments; of t h e ~ e , ~ ~ , ~ ~hat describedbelow is probably the most satisfactory. A series of FIDs isobtained, one for each position of irradiation and one for thecontrol. Selective low-power irradiation of one proton iscarried out during a long (10-20 seconds) pre-excitation delay,as shown in Sequence 51, and the carbon-13 spectrum isobtained with broad-band proton decoupling at the usualpower levels. The long delay is to ensure a maximum selectiveNOE for the nucleus that is being studied and minimum NO Eat other sites which might otherwise arise from the broad-banddecoupling. To average out variations due to spectrometerinstability or to minor fluctuations in temperature during along accumulation, it is preferable to use a small number ofscans ( e . g . 32 or 64) for each FID and to cycle through all ofthe irradiation points several times, co-adding the corres-ponding decays. It is usually worthwhile setting up all likelyirradiation points in a single session, since observing a numberof no-result irradiations usually wastes less time than severalextra runs, each with its control spectrum. The occasional no-

H s e l e c t i v e c o n t i n u o u s i r r a d ia t io n J BB Ir

Sequence 51 Heteronuclear NOE measurement.

result irradiation is always comforting in that it confirms thereliability of the method. Difference spectra may be obtainedeither by subtracting the control FID from each of the o thersin turn and transforming the difference FIDs or bytransforming all of the FIDs and subtracting the controlspectrum from each of the other spectra. All transforms shouldbe carried out on an absolute scaling basis. Enhancements areobtained by direct integration of the difference spectra and ofthe control spectrum. Some spectra of this type are shown inFigure 21 that have been obtained for andibenin B by irradiatingthe previously assigned resonances of methyl protons. The levelof power that is selected for the irradiation ensures that directlybonded nuclei are not affected. In each case the quaternarycarbon is clearly identified in the difference spectrum. Smallerresponses are also seen from C-4 and C-5 if the 5- and4a-methyl protons, respectively, are irradiated, due to somepower leaking into one proton resonance whilst the other isbeing irradiated, since these resonances are only 12 Hz apart.(The proton spectrum is shown in Figure 28). Where anirradia tion position also corresponds with (or lies close to) oneof the one-bond carbon-I3 satellites of another protonresonance, a strong response of the corresponding carbon isalso obtained, due to selective-population- ransfer effects. Thusirradia tion of the 10-methyl protons [see Figure 21(c)] alsogives responses from the 3-, the 5 - , and the 4a-methyl carbons,and responses from the 10-methyl carbon are obtained whenthe 3- and 5-methyl protons are irradiated [Figure 21 (b) and(d)]. A response from the 4a-methyl carbon is also obtained (c)

0

L

5

10I 3 - M e r e

1 0 - M e( b )

I 980 60 40 20p . p . m .Figure 21 Carbon-13 n.m.r. spectra of andibenin B at 90 MHz .Spectrum (a) is the proton-broad-band-decoupled pectrum; spectra(b)--(f) are N O E difference spectra with irradiation at the proto n

resonances corresponding to (b) 3-methyl, (c) 10-methyl, (d) 5-methyl, (e) 4a-methyl, and ( f ) 4P-methyl groups. Conditions : 256pulses, 30 pulse, pre-excitation irradiation period 10 s, acquisitiontime 0.4 s.

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NATURAL PRODUCT REPORTS. 1988-1. H. SADLER 119as the 7P-proton multiplet lies partly under the 10-methylproton resonance. It is important that the irradiation point forthe control spectrum is selected carefully since, if this coincideswith a one-bond carbon-13 satellite, a negative signal is seen forthe corresponding carbon resonance in the difference spectrum.Negative signals may also arise57where a carbon nucleus andan irradiated proton are not sufficiently close to give a directenhancement of the carbon signal but are both sufficiently closeto a second proton for the effect of irradiation to be relayed.Where a particular carbon is thought to be relaxed by two (ormore) protons that have different chemical shifts, strongerenhancements may sometimes be by rapidly cyclingthe irradiating frequency through all of the line positions of allresonances during the pre-excitation delay. This also allowslower irradiation powers to be used, thereby improvingselectivity.The use of difference spectroscopy in NOE and other double-resonance experiments has been thoroughly and carefullyreviewed,56 nd the reader is referred to that source for a muchmore detailed consideration.

6 Methods for the Direct Correlation ofCarbon-13 ResonancesImportant information concerning the structure and theconfiguration and conformation of the carbon framework ofmolecules is provided by the carbon- 13 homonuclear spin-coupling Where one-bond couplings between pairsof adjacent carbon atoms are sufficiently different it should, inprinciple, be possible to deduce carbon-carbon connectivitiesand to map out the carbon skeleton by measuring andcomparing these couplings. The geometrical dependence oflonger-range couplings is useful in studies of molecularconformation.The experimental measurement of these couplings presents anumber of difficulties. The low natural abundance of carbon- 13nuclei means that relatively very few molecules possess a pairof carbon-13 nuclei. This is advantageous in that it gives arelatively simple AX or AB pair of doublets, but significantquantities of material are required if the experiment is not tolast an unrealistic time. Even if quantity is not a problem, afurther difficulty arises with natural-abundance samples. At thecentre of each doublet lies a signal that is two-hundred times asstrong, from molecules that contain only a single carbon-13nucleus. This signal may completely cover any long-rangecoupled doublets, and even one-bond doublets may not be easyto identify, due to the presence of small spinning side-bands orof odd lines that are due to incomplete or inhomogeneousproton decoupling or to the presence of minor impurities.Additionally, couplings to quaternary carbons are frequentlysimilar in magnitude, and the four overlapping doublet signalscannot be readily resolved. Fortunately, methods are nowavailable to alleviate or to overcome these problems and areexamined below.

6.1 The Suppression of Strong Signals from UncoupledCarbon-13 NucleiSignals from uncoupled carbon- 13nuclei may be suppressed bythe INADEQUATE60 (Incredible Natural-AbundanceDoublE-QUAnTum Excitation) sequence (Sequence 6A). Thismakes use of the special phase properties6' of signals that arederived by double-quantum excitation of an AX system such astwo carbon- 13 nuclei that are coupled only to each other. Theresults that are obtained by using the sequence cannot beexplained in terms of simple vector diagrams of the type thatwere used ea

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The Use of NMR Spectroscopy in the Structure

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