Natural abundance nitrogen-15 NMR by enhanced heteronuclear spectroscopy

The detectton of NMR spectra of less sertstttve nuclei coupled to protons may be s@ficantly unproved by a two-dimez- sional Founer transform technique tnvolvmg a double transfer of pokuzatton. The method IS adequate to obtam natwaI abundance “N spectra m small sample volumes wth a commerctal spectrometer


Introduction
In spite of the obvious bIologica relevance, mtrogen-15 NMR has been hitherto rather hmited m its apphcatlons, rec_Jlrmg large, concentrated samples and lengthy accumulations [l ] . Nitrogen-1 5 chemical shifts can provide msight mto the sequencmg of peptldes because the slufts are not merely characterlstlc of the mdlvldual ammo acids but also depend on the nelghbormg residues [2]. Furthermore, the three-bond scalar coupling 3JNH to the (Y proton of the next residue may prowde mformation about the torslonal angle of the peptlde bond [3,4] -The sensitiwty IS hrmted by the 0 36% isotopic abundance and the gyromagnetrc ratio (TN/TH = -0.101) which entads a relative slgnal mtenslty of 0 001 compared to an equal number of protons, lsregardmg the unfavorable longltudmal relaxation tunes [5] In addltlon, the negative Overhauser effect (+I > 1 + Q > -3 9) IS often mcomplete and may lead to accIdenta signal cancellations for some molecular correlation times [6,7] A vanety of techruques have been proposed to enhance the sensltivlty of nuclei hke mtrogen-15. An attractive approach consists in observmg the proton spectrum of a I5 N ennched sample as a function of the offset of a cw nitrogen decoupler, to momtor the collapse of the heteronuclear scalar couplmg JNH when the decoupler has the precise frequency of the mtrogen chemical shft [8] -The method is less attractlve for samples with natural isotopic abundance, and tends to be rather tme consuming, since the changes rn the proton spectrum must be observed while the second rf field is stepped in a pomt-by-point manner througi~ the spectral range of the nucleus of interest. Maudsley and Ernst [9] have recently introduced heteronuclear twodimenslonal spectroscopy to extend the Fourrer advantage not only to the observatlon of the proton resonances, but to the spectrum of the msensitwe nucleus as well. In tlus experiment, the nitrogen-15 magnetrzatlon is generated initkdiy by a non-selective 90" pulse, and hence the sensitivity suffers from the inherently modest Boltzmann polarization, from the unfavorable Overhauser effect, and from saturation if the euperunent IS repeated before the longitudmal magnetization IS fully recovered. On the other hand, there is a considerable gain in sensitivity by observmg at the proton frequency after the transfer of the magnetization From the nitrogen to the proton transitions. Morris and Freeman [lo] for the detectron of msensitwe nuclei enhanced by polarization transfer (INEPT) follows an opposite strategy, by transferrmg magnetizatron from protons to the less sensitive nucleus. The resulting signal amplitude in the tsN spectra is proportional to the population difference that normally occurs across proton transltlons. In N-acetyl valine, the experimental enhancement achieved with the INEPT sequence was Found to be seventeen-fold (ii comparison with a normal undecoupled i5N spectrum, obtained with a flip angIe of 30" found to be optrmum for a pulse repetitron rate of 0 7 s common to both expenments).

A method proposed recently by
A ten-fold enhancement factor arises from the ratio rH/rR wmch governs the relatrve Boltzmann populatron drfferences, the addrtronal factor 1.7 IS due to the more favorable proton relaxation [ 111 As m conventional t5N NMR, the inherent drsadvantage of the detectron at a lower frequency has to be taken mto account Thrs letter describes a twodrmenslonal evperunent whtch nutrally generates proton magnetrzat:on, transfers the coherence to the nitrogen transrtions and. after an interval designed to probe the nitrogen chemical shrft. transfers the coherence back to the proton transrtrons to provide the advantage of the detectron at mgh frequencres The pulse sequence shown m fig. 1 indicates the double transfer of coherence symbohcally by the smusordal pattern representmg the precessron of the transverse magnetrzatron.
The 7 delays are approumately adJusted to (4JNH j-l and the mtrogen precessron 1s monitored by mcrementmg the evolutron per-rod rt m regular increments The proton decouplmg of the mtrcgen spectrum. whrch IS achreved by the 180" proton pulse m the middle of the evohrtron penod, may be omrtted. the mtrogen decouplmg of the proton spectrum durmg the acqursrtron perrod is also optronal The detads of the pulse sequence and phase alternatrons wrll be drscussed below. suffice rt to say here that the proton spectrum observed experimentally wrth thts technique vanrshes rf the mtrogen pulses are omltted or set too far off resonance_ The apphcation of thts pulse sequence to a 1 hl solution of 99% enriched N-acetyl valme m perdeuterated drmethylsulpho\rde (DMSOd6) produces the proton spectra shown m fig. 2 The amide proton gtves rrse  fig 2). the smaller sphttmg IS due to the cy proton of the ammo acid and IS not essential to the experiment To optumze the various delays and flip angles m the pulse sequence, rt IS sufficrent to maxrmrze the srgnal amplitude of the first spectrum obtained for tl =O ideally, the stgnal should be comparable to that obtamed after an ordmary 90° proton observation pulse, although the transverse decay. expressed by a factor exp (--4r/T2). should be taken mto account. As the evolution period cl IS incremented. the proton srgnals experrence an amphtude modulation which reflects the offset of the mtrogen transitions from the low-frequency transmrtter. avadable. The techmque is suitable both for studies ~II deuterated organic solvents as well as for non-exchanging NH groups of proteins dissolved in D20.

Details
The mecharusm of the ten-pulse sequence shown in fig 1 is best dlscussed for a heteronuclear AX system consisting of one proton coupled to one 15N nucleus. Such a urut occurs in all peptide bonds with the exceptlon of prohne. with scalar coupling corlstants& typIcally around 92 Hz [ 121. The proton magnetization, conslstmg of two vectors precessmg with frequencles 6, f $JNH, is mitlally rotated into the +,' axis OF the rotatmg fr&qe, and refocused by sunultaneous 180" pulses applied to both nuclei, to form an ech?i at time 2~ = (2J)-I.
The heteronuclear coupling &uses the two vectors to refocus along opposite +X and --x axes of the rotating frame. As m INEPT, a 10; proton pulse, apphed at the top of the echo, flips one of these vectors back into the eqtuhbnum positton, wMe the other is inverted and ends up along the -z auis. it can be ready shown that the two t5N transitions have become associated with dramatically enhanced population differences, of +2A+ 26 and -2A f 26 respectively (m this conventional notation, the equlbrium populatlon tiferences are defined to be 2A for protons and Volume 69. number 1 CHEMICAL PHYSICS LETTERS 1 January 1980 26 for mtrogen transrtrons, wrth A/s = 10). An alterna-trr2 expenment uses a 900,. proton pulse at the top of the echo to mvert the other proton magnetizatron v2ctor instead, and yields populatron drfferences across the mtrogrn transrttons of-2A + 26 and +?A + 26 respectrv2Iy. The subtraction of the signals obtamed from two such expertments cancitls the 26 terms and therefore ehminates the nuclear Overhausser effect (the latter stems from a net redrstrrbutron of populations across the rutrogen transrtrons, and IS reflected by a change rn S only). Now the 15N magnetrzatron IS brought into the transrzrse plane of a frame rotating in synchronism with the nitrogen earner frequency.
Because of the populatrons prevathng just before the 90: nitrogen pulse, the two doublet components pomt along opposite +I' and -J' axes. A proton I SO0 pulse at i tl mterchanges the identrty of the two nnrogsn vectors, a vector nutrally rotatmg wnh the frequency fi + Lf N 2 I$H resuming its pr2cesslon with the frequency 6, -iJNH in the second half of the evolution period. At the end of the tl Interval. each vector has accumulated a phase 2ns~t~ The second 901 mtrogen pulse rotates they components back mto the 2 axis, thus generatmg populatlon &fferenczs whch are -'read" by a 90: proton pulstt At ~hls pomt, the two proton magnetization vectors are m opposite phase. but after an mterval2r = (2JNH)-1 both \ectors acquire the earne phase [13]. The strnultaneous apphcation of 180' pulses to both nucler m the mrddle of #IIS mterval remows the frequency dependent phase shift and ehmmates srgnal losses due to ~omogen2ous decay m this mterval. Once the proton doublet 1s in phase, a contmuous nitrogen decoupler may be applied.
To cancel the proton signals arlsmg from molecules contammg I4N, the phase of the first 90" rutrogen pulse IS alternated, which reverses the afgebraic sign of the lnfo~atlon transferred to the protons [9]_ The proton signals are added or subtracted accordmg to the scheme m table 1, which also has the \qrtue of ehmmatmg the nuclear Overhauser effect from the lsN spectra Any spurious transverse magnetization generated by the first mtrogen "180a'-pulse IS also cancelled.
The expenments were performed with a Bruker 270 MHz spectrometer.
The 5 mm probe, orlgmaily deslgned for proton decoupled fluorine-19 NMR, was tuned for proton observation, th2 decoupler cod berg r2tuned to resonate for l5 N. A frequency synthesrzer Table 1 In a tmrne rotatmg in synchromsm with the proton earner frequency, the phases of alI proton pulses m f&-1 are along the x a.\rs except for the phase q+ of the fhrrd proton puise, which alternates between +y and --,' as shown. In the mtrogen frame, ah phases are along the x axrs except for the phase &N of the second 1sN pulse Addrng and subtractmg the free inductron decays as shown suppresses aII stgmds except those arrsing from a double transfer of m~net~atlon from protons to mttogen and back, and ehmmates the nuctear Overhauser effect