Patent Application: US-61776403-A

Abstract:
a solid - state nuclear magnetic resonance method for investigating a sample material , which contains protons h and also spin - 1 / 2 hetero nuclei x , uses a sample material rotated at the magic angle . the method comprises the steps of increasing the equilibrium polarization of x ; eliminating proton magnetization ; transferring polarization from x to h ; and recording the proton signals under a condition of line narrowing . a radio frequency pulse sequence effects polarization transfer between x and the closest protons h , via dipole coupling with coupling constant d xh . a parameter , which is associated with the transfer process , is varied to determine the dipole coupling constant d xh and thereby permits precise determination of the binding separation x — h , even when the concentration of x is small compared to h .

Description:
the invention was tested by experiments to determine 15 n — 1 h binding lengths in natural abundance samples by means of inverse detection in solid - state nmr spectroscopy with fast mas . a 15 n — 1 h solid state correlation nmr experiment is provided which considerably increases the signal sensitivity by inverse 1 h detection and fast mas to permit precise determination of nh binding lengths via hetero nuclear 1 h — 15 n dipole - dipole couplings for samples having 15 n in natural abundance . pulsed field gradients or , alternatively , radio frequency pulses provide adequate suppression of undesired 1 h signals . this method permits routine application of 15 n — 1 h correlation nmr spectroscopy for solid state samples having natural isotope abundances in an experiment lasting a few hours . the dipolar coupling constants are determined from rotation sideband patterns , which are generated in the spectra by recently developed recoupling techniques . the information about 15 n / 1 h chemical shifts and quantitative 15 n — 1 h couplings can be directly combined in a “ split - t 1 ” approach . sensitivity increase by 1 h inverse detection ( 1 ) is almost universally used today in hetero nuclear multi - dimensional nmr experiments to determine the structure of molecules in solution . for solid state samples , the increase in 1 h resolution under fast magic angle spinning ( mas ) has recently proven to be sufficient to overcome the main obstacle of inverse detection in solid state samples , namely the width of the 1 h lines ( 2 ) . substantial sensitivity increases were reported for a series of 15 n and 13 c systems ( 2 )-( 4 ) . certain pulse sequences and detection variants guarantee high 1 h resolution and also permit high sensitivity increases in inversely detected static 2 h and 15 n powder spectra ( 5 )-( 6 ) . inverse detection techniques are subject to particular challenges if small isotopic concentrations are to be observed , since incomplete suppression of the large excess magnetization of uncoupled 1 h nuclei can produce serious signal artifacts . pulsed field gradients ( pfgs ) are a conventional nmr technique to eliminate undesired coherences in solutions ( 7 ) . for solid state samples , it has been shown that radio frequency ( rf ) pulses , which utilize strong dipole - dipole couplings , can achieve the same results without requiring special spectrometer equipment ( 4 ) . this invention presents two - dimensional 15 n — 1 h correlation nmr experiments , which are carried out with inverse 1 h detection and fast mas on samples having 15 n in natural abundance . pfgs or alternatively rf pulses suppress the undesired 1 h signal and 2d spectra can be recorded experimentally within a few hours . we show for the first time that hetero nuclear 1 h — 15 n dipole - dipole couplings and therefore reliable binding lengths for 15 n of natural occurrence can be determined from rotary sideband patterns which are generated by a recently developed dipolar recoupling technique ( 8 ) . the great importance of high - precision determinations of n — h binding lengths through solid state nmr in view of characterization of hydrogen bridge compounds was recently pointed out ( 9 ) . in our 15 n — 1 h spectra , the information about ( i ) chemical shift and ( i ) dipole - dipole couplings / binding lengths can be obtained either independently of each other or in combination ( by a “ split - t 1 ” approach ). the inventive method is based on the connection between two solid state nmr techniques which initially effect an incoherent and then a coherent polarization transfer between 1 h and 15 n ( fig1 ). initial 1 h polarization is transferred incoherently to 15 n through conventional cross - polarization ( cp ). subsequently , the final ( inverse ) detection of the weak 15 n signal on 1 h is facilitated by effectively removing strong 1 h residual polarization using a field gradient pulse ( with a length of 100 μs ) or with two rf pulses ( having a length of 400 μs each and under rotary resonance conditions ) ( 4 ) while the 15 n polarization remains stored in a longitudinal state . the performance of both dephasing techniques was comparable in practice . due to this pre - selection , the desired coherence path ( i . e . 1 h → 15 n → 1 h ) can be selected by a four - stage phase cycle and increased to a total of 16 or 32 stages for complete purification of the signal . while rf pulses for dephasing can be directly applied using standard nmr equipment , pfgs require installation of gradient coils at the top and bottom of the mas stator . the 15 n signal is finally returned to 1 h for detection via a coherent tedor recoupling step ( 8 ),( 10 ) thereby permitting measurement of the 15 n — 1 h coupling . two independent spectral dimensions can be incorporated in this cp - tedor transfer scheme , one dipolar decoupled ( dd ) 15 n dimension ( t 1 ) between the two transfer blocks and a t 1 ′ dimension in the center of the tedor sequence . while the first provides access to chemical shift information via 15 n , the latter produces modulation of a dipolar arranged state through recoupling . this method of “ rotor encoding ” has already been suggested in another homo ( 11 ) and hetero nuclear ( 8 ) correlation experiment . the coupling information can be obtained from the t 1 ′ modulation of the detected signal which is converted into a side band pattern by means of fourier transformation . these patterns greatly depend on the product of recoupling time and coupling strength . ( 8 ) fig2 a shows a 15 n — 1 h correlation nmr spectrum of l - histidin . hcl . h 2 o , which was recorded in a 2 . 5 mm rotor system at 30 khz mas . unfortunately , the use of small rotors is absolutely necessary and this limits the technique , in samples having natural isotope abundance , to smaller molecules due to the small sample amounts . fast mas is essential for an efficient and quantifiable tedor transfer . ( 8 ) an alternative possibility is the use of a lee - goldburg cross - polarization instead of the tedor / rept transfer in connection with multi - pulse supported 1 h acquisition in t 2 ( step 8 ) thereby also permitting determination of binding lengths with systems using larger rotors with slower rotation . nevertheless , only 8192 runs of the pulse sequence of fig1 were required for recording the full 2d spectrum in fig2 a . to obtain a pure correlation spectrum of the chemical shifts , t 1 was incremented in steps of full rotor periods ( τ r ) without rotor encoding ( t 1 ′= 0 ). in this fashion , 15 n — 1 h correlation spectra can be recorded in their natural concentrations within 4 to 10 hours , which also permits routine use of the technology for solid state samples . the inversely detected cp - tedor technique provides a sensitivity gain ( 2 ) of a factor between 6 and 8 compared to the analog 15 n detected conventional 2d tedor experiment . the spectrum of fig2 a contains all three expected nh correlation signals when the 15 n — 1 h couplings are recoupled for a duration of 2 × 6τ r in the tedor step . for shorter recoupling periods (& lt ; 2 × 4τ r ) the signal of the fast rotating nh 3 + groups vanishes due to the weaker coupling . the rotary sideband pattern , which is introduced into the spectra by incrementing t 1 ′ in steps of τ r / n simultaneously with t 1 (“ split - t 1 ” approach ), permits precise measurement of the couplings . the total number n determines the splitting width δν of the ( uneven ) sideband configurations in the spectra in accordance with δν = 2ν r / n , wherein ν r is the mas frequency . the spectrum of fig2 b was recorded with n = 30 . dipolar recoupling was applied in the tedor step for a duration of 2 × 6τ r . 15 n — 1 h couplings in the region of d nh / 2π & gt ; 2 khz can be determined which corresponds to n — h separations of up to 180 pm . since the signal is distributed over a pattern in the “ split - t 1 ” experiment , 5 to 10 times more signal accumulations are required compared to the pure shift correlation experiment . if the 15 n shift information is not required , the t 1 dimension can be omitted and a purely rotor - encoded signal in the t 1 ′ dimension can be recorded . fig2 d shows the sideband patterns which are observed for the δ 1 - nh and the ε 2 - nh for ˜ 18 ppm or ˜ 13 ppm in the 1 h spectrum . a total of 20480 experiment runs ( 2 . 5 times more than for the spectrum of fig2 a ) were used for the recording . the n — h separations determined from these patterns agree with results of earlier investigations , ( 9 ), ( 12 ) ( see table 1 ). besides the usual over - estimation of the separations in nmr , ( 9 ) a further systematic deviation is to be expected due to the influence of couplings of 15 n to additional protons . the separations can nevertheless be reliably determined even when the disturbing coupling is ˜ 30 % of the main coupling ( 8 ) . it is to be noted that , in a system in which many protons couple with one single 15 n core , the technique only provides access to the dominant ( strongest ) coupling . two or more couplings of similar strength produce destructive interferences during recoupling and therefore signal losses . ( 8 ),( 13 ) in summary , one expects a wide range of applications for these and related ( 4 ),( 6 ) inverse 1 h detection techniques , since they provide information about dipolar couplings ( i . e . binding lengths ) together with chemical shift resolution in samples having 15 n in natural abundance . this technique is particularly important for investigations of n — h . . . n and n — h . . . o hydrogen bridges . with regard to biomolecules , the sensitivity increase for 15 n in combination with 15 n — 1 h coupling information will be substantially helpful to improve nmr experiments for determining the structure of solid state peptide main chains . further technical developments , in particular optimization of the 1 h rf oscillating circuit for detection are expected to further increase the signal sensitivity and therefore the practical importance of 1 h detected solid state nmr experiments . fig1 shows an inventive pulse sequence for 1 h detected 15 n — 1 h correlation nmr spectroscopy in fast solid state mas . the time is plotted on the right - hand side ; pulse sequences on h , x and gradient pulses in the z direction g z are indicated on three lines . black and white ( not filled in ) bars represent 90 ° and 180 ° pulses , respectively . the alternative possibilities to suppress excessive 1 h magnetization are shown with horizontal hatches ( pfgs ) and vertical hatches ( dephasing rf pulses ). in the time interval 1 , cross - polarization is carried out with corresponding sequences 1 a on h and x followed by dephasing in the time interval 2 . during the time interval t 1 , marked with reference numeral 3 , the dipolar decoupling 3 a and encoding of the 15 n chemical shift takes place followed by the time intervals 4 , 5 and 6 , which , together , form the tedor / rept sequence . a first recoupling takes place in the time interval 4 . the pulses in square brackets can thereby be repeated several times in time periods of half a rotor period . rotor encoding 5 a takes place in the time interval t 1 ′, marked with reference numeral 5 , followed by a second recoupling in the time interval 6 , wherein the pulses in square brackets may be repeated . the time interval 7 marks a dephasing step and the proton signals are recorded in the time interval 8 . fig2 a shows a 15 n — 1 h correlation spectrum of l - histidin . hcl . h 2 o recorded with 30 khz mas in a 16 . 4 t magnet ( 700 mhz 1 h larmor frequency ) with a recoupling time of 2 × 6τ r and signal accumulation of 8192 experiment runs of the pulse sequence of fig1 ( with rf dephasing pulses and t 1 ′= 0 ). the chemical shifts of 1 h and 15 n are plotted towards the right - hand and upper side . fig2 b shows the same spectrum as fig2 a but with pfg dephasing and dipolar rotary sideband patterns in the 15 n dimension recorded by simultaneous incrementation of t 1 and t 1 ′ in steps τ r and τ r / 30 , respectively . fig2 c shows cuts through fig2 b along the 15 n dimension with the corresponding 1 h positions . fig2 d shows a purely rotor - encoded sideband pattern recorded by incrementation of t 1 ′ with t 1 = 0 ( using rf dephasing pulses ). calculated patterns are shown in fig2 c and 2 d with finely dotted lines above the experimentally determined patterns . the n — h dipole - dipole couplings and n — h separations determined from the patterns are listed in table 1 . 1 . 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