Measurement of the sign and the magnitude of heteronuclear coupling constants from spin-state-edited...

MAGNETIC RESONANCE IN CHEMISTRYMagn. Reson. Chem. 2004; 42: 852–862Published online 19 July 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/mrc.1416

Measurement of the sign and the magnitudeof heteronuclear coupling constants fromspin-state-edited J-cross-polarization NMRexperiments

Teodor Parella,1∗ Jordi Belloc2 and Francesc Sanchez-Ferrando3

1 Servei de Ressonancia Magnetica Nuclear, Universitat Autonoma de Barcelona, E-08193 Bellaterra, Barcelona, Spain2 Research Center, Grupo Uriach, Camı Reial 51-57, E-08184 Palau-Solita i Plegamans, Spain3 Departament de Quımica, Universitat Autonoma de Barcelona, E-08193 Bellaterra, Barcelona, Spain

Received 30 December 2003; Revised 2 April 2004; Accepted 13 April 2004

New spin-state-selective (S3) NMR pulse sequences exclusively applying cross-polarization schemesto achieve optimum homonuclear and heteronuclear 1H–X coherence transfer are reported for thesimple and accurate measurement of the magnitude and sign of heteronuclear coupling constants forsamples at natural abundance. The proposed spin-edited HCP-TOCSY experiments are based on cleanheteronuclear S3 excitation, generated by simultaneous co-addition of two independent in-phase andanti-phase components created during the mixing heteronuclear J-cross-polarization (HCP) step, which isfinally transferred to other protons by a conventional homonuclear TOCSY mechanism. Selective 1D andnon-selective 2D approaches for the easy determination of long-range proton–carbon and proton–nitrogencoupling constants on any protonated and non-protonated heteronuclei are presented and discussed forseveral organic molecules. Copyright 2004 John Wiley & Sons, Ltd.

KEYWORDS: NMR; spin-state selective excitation; heteronuclear cross-polarization; long-range proton–carboncoupling constants

INTRODUCTION

Besides chemical shift information, homonuclear 1H,1Hcoupling constants and 1H–1H NOE values are the mostimportant NMR parameters used for structure elucidationof organic compounds. However, local lack of protons or theambiguity of the available NMR data can sometimes hinderthe complete structure determination. Alternative NMRrestraints such as heteronuclear long-range proton–carboncoupling constants, nJ(C,H) �n > 1�, can become usefultools when more data are required. However, the practicaluse of these coupling constants, usually extracted fromHMBC spectra, have generally been limited to qualitativepurposes because of the absence of a general, simple andwidely accepted experimental method for their quantitativemeasurement in samples at natural abundance. Recently,an excellent review has appeared on the more relevant2D proton-detected NMR experiments for measuring thesenJ(C,H) constants at natural abundance.1

ŁCorrespondence to: Teodor Parella, Servei de RessonanciaMagnetica Nuclear, Universitat Autonoma de Barcelona, E-08193Bellaterra, Barcelona, Spain. E-mail: [email protected]/grant sponsor: MCYT; Contract/grant number:BQU2003-01677.

Basically, long-range proton–carbon coupling constantsare measured using 1D or 2D versions of NMR experi-ments based on 13C-edited TOCSY2 – 4 or HMBC5 – 7 pulseschemes. All 13C-edited TOCSY experiments consist of twoindependent and consecutive transfer steps based on one-bond proton–carbon and proton–proton coupling constants,respectively. In practice, these experiments can only be suc-cessfully applied on protonated heteronuclei, and the overallsensitivity of the experiment generally depends on the trans-fer efficiency in the last homonuclear TOCSY process. nJ(C,H)values can be extracted from the spectra in the form of pure-absorption multiplets in 2D HSQC-TOCSY experiments,2

from E.COSY-type patterns in w1-filtered/edited 2D TOCSYexperiments3 or from spin-state-selective �S3� signal displace-ments in S3-TOCSY experiments.4

On the other hand, HMBC-like experiments5 – 7 involve adirect transfer between protons and carbons via a singleinterpulse delay optimized to a small value of nJ(C,H),typically in the range 5–10 Hz. The HMBC approach ishighly recommended when nJ(C,H) on both protonated andquaternary carbons must be simultaneously measured, butsome practical difficulties can appear for the measurementof small J values. Another major inconvenience is thatdetected signals in 2D HMBC spectra show an undesirableanti-phase/mixed multiplet pattern in which extra post-processing analysis using proper fitting algorithms is

Copyright 2004 John Wiley & Sons, Ltd.

Spin-state selective HCP-TOCSY experiments 853

required to extract nJ(C,H) values.8 In analogy with thewidely used HMBC schemes, long-range heteronuclearconnectivities can also be traced out using the HSQC pulsetrain optimized to small J coupling values, the so-calledHSQMBC experiment.9

Another question to be answered when trying to measurethese nJ(C,H) coupling constants is the election for 1D or2D versions of these NMR experiments. 1D experimentsgenerally present better resolution and shorter acquisitiontimes, can be finely adjusted in terms of sensitivity andoptimized delays and can be specifically executed toextract specific coupling constant values. However, selectivepulses on specific carbon resonances are required andtherefore some drawbacks appear if signal overlappingoccurs or short transverse relaxation times are involved. 2Dexperiments represent a more general approach, allowingthe simultaneous measurement of more coupling constantvalues from a single experiment, but usually with longeracquisition times. Some important drawbacks associatedwith 2D experiments are the poor digital resolution attainableand the fact that interpulse delays are optimized to anaveraged J value. All proposed NMR experiments areusually based on the resolution of the coupling constantin some dimension but new approaches based on J-scaling methods,6 J-quantitative spectroscopy7 or J-resolvedspectroscopy10 have also been suggested.

One class of experiments that present very interestingfeatures are those based on the generation of S3 states.4 Forthe measurement of coupling constants, the basic idea of S3

relies on the generation of two subspectra, each with onlyone peak corresponding to a single ˛ or ˇ spin componentof a doublet. Comparison of the relative sense and thedisplacement between the two corresponding peaks canafford the sign and the magnitude of the active couplingconstant. Thus, it was shown that by inserting a non-selective90°�� 13C pulse just prior to acquisition in S3 HMQC andHSQC pulse sequences,4c a second anti-phase magnetizationcomponent (usually not detected) can be superimposed onthe original in-phase magnetization, affording sensitivity-enhanced clean selection of the ˛ or ˇ spin state only as afunction of the phase �� of this non-selective r.f. pulse.

On the other hand, it has also been demonstrated thatclean S3 excitation can be accomplished using heteronuclearJ-cross-polarization (HCP)11 as an alternative to traditionalHSQC and HMQC experiments to perform effective het-eronuclear coherence transfer in isotropic solutions betweena high-sensitivity nucleus (typically 1H and denoted I) and alow-sensitivity X nucleus (e.g. 13C or 15N and denoted S).12

Here we combine these two concepts into the sameNMR experiment. Thus, several gradient-enhanced S3 HCP-TOCSY pulse schemes are proposed for the measurement ofthe magnitude and sign of heteronuclear long-range couplingconstants from the corresponding ˛/ˇ spin state X-editedproton spectra with maximum sensitivity and high accuracy.As a main feature, all involved homonuclear 1H–1H and het-eronuclear 1H–X coherence transfer mechanisms are exclu-sively performed using cross-polarization pulse schemes. Inthe first step, heteronuclear transfer is accomplished by anout-and-back heteronuclear J-cross-polarization HCP–HCP

building block based on the application of simultaneous r.f.fields in both I and S channels. Clean S3 states can be gener-ated by appropriate manipulation of the second HCP mixingprocess taking into account its anisotropic behavior.13 Thepropagation of this particular S3 states to other protons viaconventional 1H–1H TOCSY transfer is fully preserved andsuccessfully used to determine the sign and the magnitudeof nJ(X,H) for samples at natural abundance. Additionally,the feasibility of the S3 HCP process is also presented fordirect long-range heteronuclear 1H–X coherence transfer,and its use is demonstrated for the measurement of specificnJ(X,H) coupling constants from in-phase multiplet patterns,irrespectively of carbon multiplicities.

The same experiments can be also applied to otherheteronuclei, such as in the measurement of long-rangeproton–nitrogen coupling constants, nJ(N,H) �n > 1�. Inaddition, different coupling pattern topologies can begenerated with minimal changes in the mixing HCP processof several non-selective spin-edited 2D HCP-TOCSY pulsesequences aimed at the same purpose. Examples are alsoprovided on single S3 editing in a F2-coupled correlation 2DHCP experiment (2D ˛,ˇ-HCP-TOCSY) and double S3 editingin F1,F2-coupled 2D correlation experiments in E.COSY-likeor TROSY-like manners.13

NMR EXPERIMENTS

Figure 1 shows two different ways to perform selective1D HCP-TOCSY experiments. The first proposed gradient-selected 1D ˛,ˇ-HCP-TOCSY pulse scheme [Fig. 1(A)]consists of a two-way doubly-selective cross-polarizationscheme14 with S3 editing by means of the phase of the last90° X pulse. All advantages on the use of weak r.f. fields foreffective heteronuclear transfer and selective excitation insolution-state HCP NMR experiments have been extensivelyreported14 and they will not be discussed here. After theinitial 90° �1H� pulse applied from the x-axis, coherence trans-fer takes place during the doubly-selective HCP(y) period,optimized to � D 1/1J�1H,X�, where cross-polarization andselective excitation are performed simultaneously by apply-ing weak r.f. continuous-wave fields to both on-resonance 1Hand X spins from the y-axis (with intensities ω1I D � IB1

I/2�and ω1X D �XB1

X/2� about J/2 Hz, respectively). Under theseconditions, inhomogeneous r.f. fields do not significally affectthe transfer efficiency.14 The resulting transverse in-phase Sy

magnetization is allowed to evolve during a gradient spin-echo period in which heteronuclear coupling constants andchemical shifts are fully refocused by the effect of the cen-tral 180° X pulse. After this dephasing period, a secondHCP(y) block, identical with the first HCP block, transfersback the selected X magnetization to the directly-attachedI nucleus also via 1J�1H,X�, whose magnetization is refo-cused with the last G3 gradient echo and, finally, the signalis detected without heteronuclear decoupling. The secondpart of the sequence is a conventional homonuclear 1H–1HTOCSY transfer exclusively applied on the 1H channel totransfer from the original S3 state to other protons. By apply-ing the refocusing condition, a ��I/�S� : � ��I/�S� : 1 gradientratio selects the p1 D C1 and p2 D �1 �SC ! S� ! I��

Copyright 2004 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2004; 42: 852–862

854 T. Parella, J. Belloc and F. Sanchez-Ferrando

Figure 1. Two different pulse sequences of the gradient-selected 1D 1H–X ˛,ˇ-HCP-TOCSY experiment to measurelong-range heteronuclear coupling constants, nJ�1H,X� �n > 1�.A minimum four-step EXORXYCLE phase cycle is applied onthe 180° X pulse �x, y, �x, �y� and the receiver �x, �x�. TheHCP mixing transfer is executed as (A) selective CW r.f. fieldsor (B) non-selective DIPSI-2 pulse trains simultaneouslyapplied from the y-axis in both channels during the period. In(B), selectivity is achieved by the selective 180° X pulseindicated by a shaded shape placed between the twodephasing G1 and G2 gradients. Homonuclear TOCSY transfercan be accomplished in the traditional way by means of aDIPSI-2 or MLEV-16 pulse train during the 0 period. Gradientsof duration υ are indicated by shaded shapes on the line Gz. 1Hand X offsets are set on resonance of the selected resonancesthrough out the experiment. The gradient ratio should be set to��I/�S� : � ��I/�S� : 1. See text for discussion about the

phase of the last 90° S pulse for ˛/ˇ spin-state selection.

coherence-order transfer pathway, where I represents thehigh abundant nucleus �1H� and S represents the low-abundant nucleus (13C or 15N). The available X magnetizationafter the first HCP(y) block is better described as the sum oftwo orthogonal in-phase cartesian operators:

SC D Sx C iSy

180°x �S�

��!S� D Sx � iSy �1�

The behaviour of these different coherences during theHCP mixing sequence depends strongly on the phase ofthe HCP r.f. fields.12,13 Thus, maximum in-phase to in-phaseheteronuclear transfer is achieved when � D 1/JIS for theparallel Sy magnetization:

Sy

HCPy�� D 1/J��!Iy �2a�

but the Sx or Sz magnetization components are convertedto anti-phase and multiple-quantum magnetization, respec-tively, as follows:

Sx

HCPy�� D 1/J��!SyIz �2b�

Sz

HCPy�� D 1/J��! � SyIx �2c�

As described above, the effect of the second mixingHCP(y) process for each one of the two components definedin Eqn (1) will depend on their relative phase:

S� D Sx � iSy

HCP�y��!SyIz � iIy �3�

The first term stands for anti-phase X magnetization whichneeds to be reconverted whereas the second term stands forin-phase 1H magnetization which is ready to be detected.

The second approach of the selective HCP-TOCSY exper-iment displayed in Fig. 1(B) presents two major differences.HCP is performed in a non-selective way by means of con-ventional DIPSI-2 pulse schemes also applied during a period from the y-axis simultaneously from the two chan-nels. On the other hand, selectivity is introduced only in thecarbon frequency domain by the selective spin-echo period.This approach is less selective than the sequence of Fig. 1(A)and the long selective 13C pulse can be inconvenient whenhigh selectivity is required. However, the major advantageof this approach is that it can be applied to any carbonmultiplicity and becomes a very interesting general pulsescheme to measure the magnitude and the sign of nJ(C,H) onmethylene CH2 and methyl CH3 spin systems.

The most important feature is that both proposed 1DHCP-TOCSY experiments include separate acquisition oftwo data sets employing two different but completelyequivalent alternatives: inversion of the phase or inversionof the refocusing G3 gradient. Thus, spin-edited 1D spectraare obtained by detecting simultaneously both in-phaseand anti-phase magnetization components (S3 excitation)by applying D Cx (addition) or D �x (subtraction),respectively:

[3] D SyIz � iIy

90y�I��!90x�S�

SzIx � iIyυ � 180�I� � υ��!SzIx

C iIy ) I�Sz C I� D I�S˛ �4a�

[3] D SyIz � iIy

90y�I��!90�x�S�

� SzIx � iIyυ � 180�I� � υ��! � SzIx

C iIy ) �I�Sz C I� D I�Sˇ �4b�

The resulting clean S3 selection is suitable for the accuratedetermination of the magnitude and the sign of nJ(C,H) bymeasurement of the relative displacement of relayed signalsin the corresponding ˛ and ˇ 1D subspectra. Couplingconstant values even smaller than the linewidth can bemeasured by comparison of the relative displacement of the1D TOCSY multiplets:

I�1 S˛/ˇ

TOCSY�I��!I�2 S˛/ˇ �5�

High-quality 1D spectra with maximum sensitivity areobtained by using gradient coherence selection because ofthe excellent suppression level achieved for the unwanted1H–12C magnetization (mandatory in natural abundancesamples) and preservation and simultaneous detection of



two independent orthogonal terms giving rise to in-phaseand anti-phase components just before the HCP process. Fora given IS spin system, the amount of cross-talk contributionshould be proportional to the ratio

Isuppressed

IselectedD 1 � sin��J��

1 C sin��J��6�

However, in 1D experiments the presence of such undesiredartefacts can be largely minimized by finely adjusting themost important acquisition parameters such as frequencycarriers, power levels and transfer durations from a pre-liminary and fast 1D spectrum without the last TOCSYtransfer.

RESULTS AND DISCUSSION

The compounds considered are shown in Scheme 1.The H1–C1 pair of menthol was chosen as a test model

to verify these predictions (Fig. 2) by recording several 1DHCP spectra without the last DIPSI-2 pulse train. The in-phase [Fig. 2(A)], the anti-phase [Fig. 2(B)] or the selectiveselection of the ˛ [Fig. 2(C)] or ˇ [Fig. 2(D)] spin statecomponents can be detected by a proper setting of thephase of the 90° 1H and 13C pulses applied just afterthe second HCP block. Figures 3(B) and (C) and 3(D) and(E) show the corresponding clean ge-1D ˛,ˇ-HCP-TOCSYspectra obtained after S3 excitation of the H1–C1 and H5–C5methine pairs, respectively, and followed by a homonuclearTOCSY transfer of 80 ms. Extraction of the correspondingnJ(C,H) can be performed directly from the 1D in-phasemultiplet without any post-processing method (Fig. 4). It isclearly evident that such measurements can be successfullyperformed in overlapped regions and independently of themultiplet pattern.

(1)

(2)

(3)

Scheme 1. Molecules studied.

Figure 2. Multiplet patterns of the H1C1 pair of menthol (1)after applying the ˛,ˇ-HCP experiment of Fig. 1(A) without thelast homonuclear TOCSY transfer. (A) In-phase patternobtained using D y; (B) anti-phase pattern when the last 90°1H and 13C pulses are applied from the x-axis; (C) and (D)spin-state selective ˛ and ˇ pattern obtained using D x and D �x, respectively. Thirty-two scans were recorded for each1D spectrum using an HCP period of D 7 ms.

Figure 3. 1D 1H–13C ˛,ˇ-HCP-TOCSY spectra recorded afterselective cross-polarization of the H1–C1 (B) and (C) andH5–C5 (D) and (E) spin pairs, respectively. 1D data wereacquired using 128 acquisitions and a TOCSY mixing time of0 D 80 ms. All other parameters as described in the captionof Fig. 2. The excited spin-selected multiplet is marked withan asterisk.



Figure 4. Several relayed multiplets taken from the 1D 1H–13C ˛,ˇ-HCP-TOCSY spectra shown in Fig. 3(B) and (C).

Figure 5. Several relayed multiplets taken from the 1D 1H–13C ˛,ˇ-HCP-TOCSY spectra shown in Fig. 3(D) and (E). In (B) theequivalent multiplets obtained from a non-spin-edited HCP experiment [recorded as described in Fig. 2(A)] are shown to comparemultiplet structures with the edited ones (C) or with the regular 1H multiplets (A).

Alternatively, these nJ(C,H) coupling constants could beextracted by acquisition of multiplets with and without theheteronuclear splitting because the active heteronuclear cou-pling constants introduces an additional in-phase doubletsplitting with respect to the conventional proton multiplet[see Fig. 5(B)]. Thus, in the case of the simplest resonancepatterns, the J value could be measured from a direct mul-tiplet analysis. In more complex multiplets, this additionalcoupling usually complicates the analysis and a fitting pro-cedure can be highly recommended.15 The usefulness of theproposed S3 approach is clearly demonstrated comparing themultiplet patterns in Fig. 5(B) and (C). Even coupling valuessmaller than the natural linewidth can be determined, along

with their signs, as shown for the small three-bond couplingconstant between H1 and C5 �C0.85 Hz� corresponding tothe axial position of the H1 proton (dihedral angle about62°). When compared with the conventional 1D proton spec-trum, the non-spin-edited HCP-TOCSY multiplet shows anincreased line width [Fig. 5(B)], for which direct multipletanalysis is inappropriate. In fact, this complicated resonancecan be seen as the addition of the two more simple spin-edited multiplets shown together in Fig. 5(C), from whichthe coupling constant can be extracted.

An interesting advantage of the selective 1D ˛,ˇ-HCP-TOCSY sequence of Fig. 1(B) is that it can be also applied tomethylene CH2 and methyl CH3 spin systems. In principle,



Figure 6. 1D 1H–13C ˛,ˇ-HCP-TOCSY spectra recorded withthe pulse sequence of Fig. 1(B) for the H6C6 spin pairbelonging to a methylene CH2 spin system. Experimentalconditions as described in Fig. 3 except that the HCP mixingwas set to 5 ms. The excited spin-selected multiplet is markedwith an asterisk and the geminal proton (marked with a circle)also displays the corresponding ˛ or ˇ spin state.

the ˛,ˇ-HMQC and ˛,ˇ-HSQC experiments4c were limited toIS spin systems owing to the need for perfect adjustment ofthe refocusing/defocusing periods. In contrast to CH groups,the theoretical optimal transfer for I2S and I3S groups shouldbe expected to occur after 0.7/J and 0.625/J, respectively.16

Figure 6 shows the ˛,ˇ-HCP-TOCSY spectra after selectiveexcitation of the diastereotopic C6H6 methylene pair ofmenthol using an HCP mixing duration of 5 ms. In additionto the corresponding long-range peaks, it can be seen thatthe geminal H60 proton also displays the corresponding clean˛/ˇ spin state owing to its 1J(C,H) coupling constant. Finally,Fig. 7 shows the resulting 1D ˛,ˇ-HCP-TOCSY spectra afterselective HCP on the methyl H9–C9 spin pair from whichcoupling constants are also measured in the way described.

The idea of selective HCP transfer, usually adjusted tolarge 1J�IS� coupling constants, can be also extrapolated tolong-range optimized HCP (LR-HCP) experiments. The HCPsequence (Fig. 1), with omission of the final homonuclearTOCSY transfer, can be used for this purpose with improvedresults with respect to the previously reported 1D HCPsequence17 that allowed the direct extraction of long-rangeproton–carbon coupling constants from highly appropriatein-phase multiplets.17,18 Experimentally, the HCP process isoptimized to a small J value, typically about 10–15 Hz, and

Figure 7. 1D 1H–13C ˛,ˇ-HCP-TOCSY spectra recorded withthe pulse sequence of Fig. 1(B) for the H9C9 methyl spinsystem. The excited spin-selected multiplet is marked with anasterisk. In (D) note that the overlapping existing between thespin-state component (marked with an asterisk) and thecorresponding geminal methyl C8 signal does not hinder theprecise measurement of the coupling value. Experimentalconditions as described in Fig. 3 except that the HCP mixingperiod was set to 5 ms.

the effective bandwidth of on-resonance CW r.f. fields isstrongly attenuated and carefully calibrated to lower powerlevels.

Figure 8(C) shows the 1D LR-HCP multiplets obtainedfor the H3–C1 and H4–C6 spin pairs of 1. Comparison of theresulting multiplet patterns with one reference [for instance,the proton spectrum (Fig. 8(A)) or the decoupled LR-HCPspectrum (Fig. 8(B))] could allow the measurement of thisadditional J splitting value. In the case of large J values insimple multiplets, nJ(C,H) can be measured directly, evenfor quaternary carbons (see Fig. 9). It is also shown that itis possible to apply the same described S3 principles to LR-HCP experiments, affording ˛,ˇ-LR-HCP spectra [Fig. 8(D)and (E)], in which the original in-phase multiplet patternis fully retained and the presence of undesired cross-talkand transfer efficiency can be carefully controlled. In theseedited spectra, the measurement of the active coupling isgreatly simplified, thus allowing the direct coupling valuemeasurement.

Some important characteristics of the proposed LR-HCPmethod are as follows: (i) it can be applied to both protonatedand non-protonated carbons; (ii) accurate nJ(C,H) values can



Figure 8. Expansions of 1D 1H–13C LR-HCP spectra for theH3–C1 and H4–C6 spin pairs of menthol (1) acquired with thesequence in Fig. 1(A) without the last TOCSY transfer. A veryselective 15 Hz CW HCP mixing field was applied on-reson-ance on the selected signals for 120 ms. (B), (C) In-phasemultiplets with and without the optional 13C GARP decouplingduring acquisition, respectively. (D), (E) The two separate 1D1H–13C ˛,ˇ-LR-HCP spectra acquired with the specified pulsephases D x and �x, respectively. A total of 256 acquisitionswere accumulated for each 1D spectrum.

be extracted directly from multiplet analysis; (iii) it can beapplied to protons displaying highly complicated multipletpatterns or resonating in overlapped regions; (iv) easy set-up, only requiring one to specify the offsets of the selectednuclei. The main drawbacks are similar to those describedfor the one-bond optimized experiments. Problems can arisewhen there is degeneracy on both proton and carbon spectraand, on the other hand, two experiments are required for eachsingle CH pair. In addition, the signal intensity will show anexperimental dependence on cos��J�� and, sometimes, highsensitivity ratios can be difficult to obtain, for instance whenusing long mixing delays or when short transverse relaxationtimes are present.

The described HCP experiments can also be appliedto nuclei other than 13C, as shown by the LR-HCPspectra applied for the 15N-natural abundance compound

Figure 9. Expansions of 1D 1H–13C LR-HCP spectra appliedon selected proton and quaternary carbons of (2). In (B) and(C), two different multiplets resulting from the LR-HCP transfer(125 ms) between the simple H2 proton resonating at 8.61 ppmand the quaternary C4 and C9 carbons, respectively. A total of256 acquisitions were accumulated for each 1D spectrum.

albaconazole (2), a broad-spectrum oral azole antifungalagent developed in Grupo Uriach. The idea has alreadybeen used to measure specific relaxation rates in 15N-labeled proteins.14 More importantly, the sequence can besuccessfully applied on non-protonated nitrogen centerswhere other methods fail. When the J value is large, theadditional multiplet lines can be directly determined asshown in Fig. 10(B) and (C). In cases of small values orcomplicated multiplets, the S3 editing affords a robustapproach for this measurement, as shown by comparisonFig. 10(D) and expansions in Fig. 10(E).

In many cases, it can be preferable to record non-selective 2D experiments when, for instance, no selectivecross-polarization can be applied or when many differentcoupling values are to be measured at the same time.Figure 11 shows different 2D pulse sequences to record spin-edited HCP-TOCSY experiments exclusively using cross-polarization in both HCP and TOCSY building blocks. Alltheoretical details concerning to the heteronuclear transfermagnetization under HCP mixing conditions have beenextensively described13 and they are not discussed here. Inall 2D HCP-TOCSY approaches, the sign and the magnitudeof the corresponding coupling constant can be extracted fromthe coupling topologies displayed in Fig. 12 and the choicebetween them will depend on the complexity of the resulting2D heteronuclear correlation spectra. The first approach is anF2-coupled S3-edited 2D HCP-TOCSY experiment [Fig. 11(A)with the 180° 1H pulse in the center of t1] in which the ˛ andˇ spin components are extracted from two separate spectraas described for the 1D version. In fact, the sequence inFig. 11(A) is the 2D version of the selective 1D version ofFig. 1(B) and the results are equivalent to those known for˛,ˇ-edited 2D HSQC-TOCSY experiments.19



D)

C)

B)

A)

E)

Figure 10. Expansions of 1D 1H–15N LR-HCP spectra appliedon selected proton and some non-protonated nitrogen centersof 2. In (B) and (C), the direct LR-HCP transfer (75 ms) betweenthe H2 proton and the N3 and N1 nitrogens, respectively,affords two clean doublets. In (D) �0 D 160 ms�, the smallcoupling between the methyl H9 protons and the N3 nitrogenis not well resolved. The separate 1D ˛- and ˇ-spectra in(E) clearly demonstrate the usefulness of the spin-state editingapproach in direct LR-HCP transfers. A total of 256acquisitions were accumulated for each 1D spectrum.

The second experiment does not use the 180° protonpulse at the middle of the evolution t1 period, allowing theevolution of J(IS) and υ�S� during this period. In the resultingF1,F2-coupled 2D correlation spectra, a different couplingpattern would be obtained as a function of the 90° pulsesapplied after and before the HCP mixing process. It hasbeen reported that many different possibilities are feasiblefor these purposes.13 Applying the sequence in Fig. 11(B), aheteronuclear correlation map (2D HCP-TOCSY-E.COSY) isobtained in which the cross peaks show an E.COSY pattern[Fig. 12(C)] as found in analogous 2D HSQC-E.COSY orHECADE20 experiments and, therefore, the sign and thevalue of coupling constants can be extracted from this singleexperiment (see Fig. 13 as an example).

The major drawback of this approach is that the numberof peaks is doubled compared with the other approaches andoverlapping can become a problem for large molecules or

DIPSI-2(x) DIPSI-2(y) DIPSI-2(y)

DIPSI-2(y)

DIPSI-2(y)

DIPSI-2(y)

DIPSI-2(y)DIPSI-2(x)

DIPSI-2(x)

DIPSI-2(x)

Figure 11. Pulse schemes of (A) F2-˛/ˇ-edited (or TROSY) and(B) E.COSY-edited 2D HCP-TOCSY experiments. Non-selec-tive 90° and 180° pulses are indicated by thin and thick blackbars, respectively. All pulses are applied from the x-axis unlessotherwise specified. The optional white 180° 1H pulse placed inthe center of the t1 period in (A) refocuses heteronuclearcoupling evolution during this period and when applied, theresulting spectra shows the typical TROSY pattern (see text formore details). The echo–anti-echo procedure is applied byinverting the G2 gradient and the phase on alternate scans.The phase of the last 90° X pulse and other relevantexperimental details are set as described for 1D HCPexperiments.

Figure 12. Schematic representation of the differentmultiplet-pattern spin topology obtained for the different 2Dspin-state-edited HCP-TOCSY experiments displayedin Fig. 11.

in overlapped regions. In these cases, a possible alternativeshould be a TROSY-like pattern sequence (2D HCP-TOCSY-TROSY),13 in which the two ˛˛ and ˇˇ spin states are obtainedin two separate spectra.21 This experiment is obtained fromthe sequence in Fig. 11(A) but omitting the 180° 1H pulseplaced in the middle of the t1 period. As a comparisonof all these 2D methodologies, Fig. 14 shows an expansionof the corresponding 2D maps of menthol in which directcorrelation peaks clearly represent the type of S3 selectionachieved by the pulse sequence. In addition, it can be seenthat this spin-editing selection is irrespective of the carbonmultiplicity. The analysis of cross peaks is performed in theusual way as described for 1D or analogues 2D experiments.

An important aspect of the 1D and 2D HCP experimentsto consider is the precision obtained in the measurement ofthe J values. There are several experimental questions thatmust be addressed, such as the presence of cross-talk artefacts



Figure 13. Expansions of the methyl region of the 2D 1H–15N E.COSY-HCP-TOCSY spectrum of 3 acquired using the pulsesequence in Fig. 11(B). Data were recorded using 128 ð 2048 points in t1 and t2, respectively and with spectral widths of 2000 Hz inF1 and 2500 Hz in F2 32 scans per t1 increment and a total measurement time of 80 min. The size of the processed data was2048 ð 512 points. A TOCSY mixing time of D 80 ms was used.

due to unmatched J optimization, the preservation of thespin state of the passive nucleus during the mixing process,the effect of digital resolution, the lineshape distortionsdue to differential relaxation behaviour during the mixingprocess and the importance of the signal-to-noise ratiosand also the magnitude of the J value to be measured.In principle, all these requirements for the precise andaccurate measurement of coupling constants remain exactlythe same as in other equivalent spin-edited 1D4 and 2D19,20

HSQC- and HMQC-type experiments already described inthe literature. Therefore, although in many cases couplingconstant values can be directly extracted from the 1D or 2Dspectra, fitting procedures can be also applied to improve theaccuracy and precision of these measurements. For typicalsmall molecules, 1D spectra show excellent digital resolutionand allow precise set-up optimization. In these cases, directextraction of the J value can usually be performed frommultiplets showing good sensitivities and well-resolvedsubmultiplet components. However, it is recognized thatall spin-edited spectra are susceptible to other undesirablecontributions and, as a consequence, systematic errors canbe accumulated in the determination of J values. Thetheoretical cross-talk contribution dependence for each S3

HCP experiment will be described in detail elsewhere andanalyzed as a function of the InS multiplicities.22 For systemswith J�IS� coupling constants spanning a relatively narrowrange of values, as found in peptides and proteins, theunwanted cross-talk between different transitions can becontrolled and its undesirable effects largely minimized.However, in applications where a wide range of J�IS� values

exists, residual cross-talk can be present. In these cases thecontribution of the other spin state is characterized by amultiplet distortion and the corresponding decrease of theoverall sensitivity. For large molecules, relaxation effects alsoplay an important role in lineshape distortions and it has beenshown that, in these cases, post-processing protocols basedon peak picking using peak interpolation or point-to-pointtrace fitting in the frequency or time domain greatly improvethe accuracy of the coupling measurement.23

CONCLUSIONS

Useful 1D and 2D heteronuclear cross-polarization exper-iments have been proposed to measure the sign andmagnitude even of small heteronuclear coupling constants.Spin-state selective excitation is achieved in a very simpleway and without need of specific single-transition excita-tion. Maximum sensitivity ratios, excellent spectral quality,easy and minimum set-up and the simplicity of data anal-ysis and interpretation are sufficient reasons to use them.The long-range ˛,ˇ-HCP experiment also demonstrates thatit is possible to achieve spin selection in long-range opti-mized experiments taking profit from its in-phase nature.The principles outlined here can be applied to any type ofheteronucleus, irrespective of its multiplicity, and might alsobe applied for other purposes, such as the measurement ofrelaxation properties of the different multiplets componentsand the study of isotope effects.



Figure 14. 2D 1H–13C spin-edited HCP-TOCSY spectra ofmenthol: (A) fully-coupled; (B) E.COSY; (C) anti-TROSY;(D) TROSY; (E) ˛-F2-edited; (F) ˇ-F2-edited. DIPSI-2 pulsetrains were used for HCP (5 ms) and for homonuclear TOCSY(50 ms), which was applied after the HCP mixing process andbefore the last gradient echo. For clarity, one-bond correlationcross peaks are marked in each spectrum. The time domaindata matrices consist of 256 ð 2K data points, with acquisitiontimes of 14.5 ms �t1� and 455 ms �t2�, and 32 transients perFID. The total measuring time was 2 h for each 2D spectrum.

EXPERIMENTAL

All NMR experiments were recorded on a Bruker Avance500 MHz spectrometer equipped with a triple-resonanceinverse probehead incorporating a z-gradient coil. In selec-tive experiments, all 1H and 13C (or 15N) pulses wereapplied on-resonance to the corresponding 1H–X pair.The amplitudes of the selective CW r.f. fields in selec-tive 1D HCP experiments were approximately matched to� IB1

I/2� D �SB1S/2� D J�IS�/2 Hz and placed on resonance

to the desired signals. For instance, in HCP experimentsoptimized to 1J(C,H), effective r.f. fields of about 70 Hz wereused (about 47 dB attenuation). In the case of LR-HCP exper-iments, the power level was attenuated up to 62 dB (effectivefield 15 Hz). Calibrations of the power levels for CW pulsetrain were performed by maximizing the selected signalintensity in the 1D experiment. A 5.5 kHz DIPSI-2 pulse train

was applied for both homonuclear TOCSY and non-selectiveHCP mixing sequences.

The following general experimental conditions wereapplied: pre-scan delay of 1 s, HCP mixing delay of D 1/J(C,H) D 7 ms (for CH), 5 ms (for CH2), 4.5 ms(for CH3) and 11 ms (for NH). Gradient ratios in 1D HCPexperiments were set to 2 : �2 : 1 for 13C and 5 : �5 : 1 for 15N(see Fig. 1) and to 80 : 20.1 for 13C and 80 : 8.1 15N in 2D 1H–15NHCP-TOCSY experiments (see Fig. 11). Gradients of duration�υ� of 1 ms with a recovery time of 100 µs were alwaysapplied. The number of transients needed for the optimumsignal-to-noise ratio depends on the sample concentration.The samples used were (a) 50 mg of menthol (1) dissolvedin 0.6 ml of CDCl3 for 1D and 2D 1H–13C HCP experiments,(b) 40 mg of albaconazole (2) dissolved in 0.6 ml of CDCl3 for1D 1H–13C and 1H–15N HCP experiments and (c) 40 mg ofN-succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (3) dissolved in0.6 ml of DMSO-d6 for 2D 1H–15N HCP-TOCSY experiments.2D experiments were recorded in phase-sensitive mode usingthe echo/anti-echo gradients. See the figure captions for moreexperimental details.

AcknowledgmentsFinancial support for this research provided by MCYT (projectBQU2003-01677) is gratefully acknowledged. We thank the Serveide Ressonancia Magnetica Nuclear, UAB, and Grupo Uriach forallocating instrument time to this project.

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Measurement of the sign and the magnitude of heteronuclear coupling constants from spin-state-edited...

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