INTRODUCTORY NMR BCMB 4190
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Date Created: 09/12/15
CHEM BCMB 419061908189 Introductory NMR Lecture 11 CHEM 41906190 The INEPT Experiment Sensitivit roblem in NMR 8 electromagnetic induction force in detection coil 3 2 2 s cc Ny h BO II 1 31ltBT Small SN in spectra of insensitive nuclei with low natural abundance eg 13C 15N is a main problem in NMR spectroscopy of organic molecules Example 8 13C 11 1 1 s CH 100 43 5818 One would need to record 33 million 58182 more scans in a 1D 13C spectrum to get equal signal intensity than in a 1D 1H spectrum Solutions to this problem are 1 Get more sample 2 Isotope labeling may be expensive and not practical 3 Record spectrum at higher field Bo 4 Record spectrum at lower temperature not significant effect 5 Special NMR experiments Selective Population Inversion SPIQ Experiment 39 Advantage of SPI Very useful to explain the principle of Selective Population Transfer that provides a means to quotrecoverquot one of the y factor 39 Disadvantage of SPI Not very practical because selective pulses are used Lets consider the twospin AX system 13CHCl3 with A1H sensitive nuclei and X13C insensitive nuclei A At equilibrium N4 X1 N4N oz N3NAC A1 Y BN3 N2NAH N1NACAH GB N2 A2 N2N4zN1N3 AH N3N4zN1N2AC X2 0101 AH4AC N1 For 13C spectrum X1 transition N3 N4 AC X2 transition N1 N2 AC X1 X2 B After a selective 180 pulse exciting the A2 transition are inverted N4 N A1 Ba N3 N3NDCDH N2NDH N1NAC ocB A2 The populations of N1 and N3 N4 Xl N N1 2 X1 transition N3 N4 AC AH SAC X2 transition N1 N2 AC AH 3AC 0101 X1 X2 C After a selective 180 pulse exciting the A1 transition The populations of N2 and N4 N4 X1 are inverted N4 N AH A1 Ba N3 N3 N AC N2 N B N1 N AC AH 0 N2 A2 X1 transition N3 N4 AC AH 3AC X2 transition N1 N2 AC AH 5AC X2 0 01 N1 X1 X2 After selective inversion of the A1 or A2 transition the signal ampli cation factors for the spectra of X are given by 139yA39YX and 1 39yA yX 4 The INEPT experiment INEPT Insensitive Nuclei Enhanced by Polarization Transfer Polarization transfer achieved using nonselective pulses Example 13CHCl3 A Pulse sequence in the 1H and 13C channels Note Without carbon pulses this is a spinecho experiment on 1H 90x 180x 90y B Vector diagrams showing the 1H magnetization vectors Z 2 MHCquotI and MHC395 are of approximately equal populations 0 de f g 180x 90x 13 C H MM ggvv b v 3CuHCl3 39VH JCHZ and v 13CpHC13 39VH JCHZ c 1 until then just like beginning of a spinecho experiment on 1H e Effect of 13C 180 phase of 180 doesn t matter X or y MC from z to z inverts population between N1 and N2 and between N3 and N4 M C becomes M C and M C becomes M C H H H H 6 f J CH continue to evolve instead of being refocused during the nextI delay g 1H 90 pulse rotates MHCu to 2 and MHCquot to z Same effect as the SP1 experiment but Without selective excitation The populations of N2 and N4 N4 X1 are inverted N4 N AH A1 N3 N3 N AC N2 N N1 N AC AH 1 6 N2 A2 X2 W N1 X1 transition N3 N4 AC AH 3AC X2 transition N1 N2 AC AH SAC C Vector diagrams showing the 13C magnetization vectors g h z SAC MCH or 4AC 3AC 01 x MCH g Note that MCHquotI is in its original position but that MCHquot is inverted h The 90 X pulse on 13C create transverse magnetization components which are observable The natural I spin magnetization in the INEPT experiment In many applications of polarization transfer the contribution from the natural 13C magnetization AC is unwanted There are multiple ways to remove it 1 Presaturate 13C at the start of the pulse sequence 2 Apply a 90 13C pulse followed by a gradient pulse at the start of the pulse sequence In cases 1 and 2 the populations at point a are N4 N AC2 N3 N AC2 N2 N AC2 AH N1 N AC2 AH The populations at point g are N2 and N4 inverted N4 N AC2 AH N3 N AC2 N2 N AC2 N1 N AC2 AH X1 transition N3 N4 AH 4AC X2 transition N1 N2 AH 4AC 3 By phase cycling Collect 2 experiments the phase of the last 90 pulse on 1H changes between y and y Lets analyze the effect of the 90 y pulse fUH 9 1H 30 h 30 Mom MHC31H90 y Z MHCOL Z MCHO Z SAC l gt V V MchS McHa y y y39 y39 MHCOL 3AC Xvl X MHCE X MchS X f1H MchS 1H 130 h 130 Q McH Z MHCfS 1H 903 Z MHCfS Z McHB Z SAC l gt i V McHa MchS M c y y y y H on 3AC X X4MHCQ X4MCHG X McHa At point g 1H 90 y pulse rotates MHCquot to z and MHCquot to 2 Same effect as the SP1 experiment but Without selective excitation The populations of N1 and N3 are inverted N4N N3NACAH N2NAH N1NAC X1 transition N3 N4 AC AH SAC X2 transition N1 N2 AC AH 3AC We have seen the effect of the 90 y pulse already X1 transition N3 N4 AC AH 3AC X2 transition N1 N2 AC AH SAC The first FID with 90 y and the second FID with 90 y will be recorded with phases of 0 and 180 for the receiver The net effect is substraction of the first spectrum to the second spectrum For X1 transition 3AC SAC SAC For X2 transition SAC 3AC SAC The trick here is that in these two experiments the natural 13C magnetization gives rise to a signal with a constant phase and the change in receiver phase will eliminate it 90y McHa Natural Transfered Magnetisation Magnetisation 5A0 1A0 1A0 4AC I I 3AC 4AC Mch 90 y Natural Transfered MCH Magnetisation Magnetisation 5A0 1A0 1A0 4A0 I I 393AC 4AC MCHOL 8AC Transfered 90y 90y Magnetisation Only 8AC INEPT pulse sequence applied to CHLand CH3 groups Use average value for 17 Jan 125150 Hz 1D 13C spectrum INEPT spectrum CH2 121 2y1H y 1300 2y1H y 0 CH3 1331 approx 311Hy 13C 371H 1 13C 393Y1H 1 13C 3Y1H Y 13C w AL W gure 1H7 Mulxiplclxohserwd in he quotCNMR spcclrum for H zmd 39H groups schcmalicl Len C spcclm wilhmxl decoup ling Rxgm lNEl Tspchru 11 Examplgs gt INEPT gngn39mm Note Experiments recorded for different times SN should not be compared H OH Refocused INEPT spectrum H W with 1H BB decoupling 0 H Ho oocu mm H OH H a I JH i Refocused INEPT spectrum Ii l I mi 1 i I l 39T i I i INEPT spectrum 0 uni i l quot1 H i i 13C spectrum with gated BB lH decoupling l3C spectrum with BB 1H decoupling The refocused INEPT experiment Provides additional delay 2A to refocus JCH coupling The additional 180 pulse refocuses chemical shift evolution during that delay Allows application of 1H BB decoupling during acquisition Optimal delays For CH groups the optimal delay A is 1 4JCH 179 ms For CH2 groups the optimal delay A is 1 8JCH 089 ms For CH3 groups only two of the four vectors can be refocused the optimal delay A is around 18JCH Need to find a compromise In practice a value of 3 8JCH 268 ms is usually chosen 70 mag 9 van 39N w 739 an u 9 h quotH n Ml Mr 4 0 VA J Mtquot x r k l m NIHquot 1 A Mn 399 J W 1 r X n glue rm I lu mmcu ca INEPT experiman Pulse Sequence in mu m and c clmnnc Vucmnlingmm for A mypn AX y m lh A In quotllzmdquotCmugnclizuuunvccmn upmlhcimlunlg39 mn Figumxrl lhcpwviuusg 39 39 m w 110 The cmlminnd mm 11 hch ludcnmdla m x quotc cxumplv I B dnli CHEM BCMB 419061908189 Introductory NMR Lecture 16 CHEM 41906190 TwoDimensional Correlated NMR Spectroscopy 1 TwoDimensional Heteronuclear H orrelated MR S ectrosco HET Ror H SY HETCOR HETeronuclear g 1 erelation CHCOSY gorrelated SpectroscopX Observed nuclei is rst A Pulse sequence and vector diagram 90 90 Lets consider a twospin AX system with A 1H and X 13C 13CHCl3 During t1 V MHCa 39VH 12JCH 39V MHCB 39VH 12JCH VH Larmor frequency in the absence of coupling Here 39VH gt frequency of the rotating frame Ignore effect of relaxation and eld inhomogeneity pa 2n VH 12JCH t1 qJB 2n 39VH 12JCH t1 6 pa 136 2IJCH t1 After the second 1H 90 X39 pulse 1H magnetization is transferred to the X39z plane The zmagnetization components are proportional to the population differences N1 and N3 for MHCa N2 and N4 for MHC In Figure 910 the populations of N1 and N3 are partially inverted and the population difference between N2 and N4 is modified In general the population differences depend on t1 39VH and J CH Population transfer from 1H to 13C as in SP1 and INEPT although here the transfer depends on t1 After the 13C 90 X39 pulse Turns the two longitudinal 13C vectors 2 and z into the y39 and y39 directions respectively Two frequencies are detected by the receiver VC 12JCH 39VC 12JCH B Spectrum 39 FT with respect to t2 gives two 13C signals vc 12JCH and 39vc 12JCH along the F2 axis one positive and one negativethese signals are modulated in t1 by W and JCH FT with respect to t1 gives two 1H signals VH 12JCH and 39VH 12JCH for each 13C signal along the F1 axis 39 2D NMR spectrum has therefore 4 signals 2 with negative amplitude 1H decoupling during acquisition would remove the signal Figure 911 Schematic two dimensional CH correlated NMR spectrum of a two spin AX system for pulse sequence see Fig 910 The two signals along the Fzdirection correspond to the onedimensional 13C NMR spectrum without decoupling except that the signals have oppo site signs Along the F 1directi0n is F I H Ar x X seen the doublet of the 1H NMR 2 spectrum with the CH coupling F2 130 the 13C satellites also with oppo site signal amplitudes 2 Modified HETCOR Pulse Sequence to Remove Splitting in F2 A Pulse sequence 39 Insertion of a delay 12J CH between the 13C 90quot pulse and the acquisition of the FD which allows refocusing of MCHu and 39 BB decoupling during acquisition cause MCHu and MCH to precess at the same rate during that time 90 I H II I I I I I I I I 1 130 tdzl A2 12J CH B Spectrum After FT with respect to t2 only one signal is observed in F2 with a frequency vc and this signal is modulated in t1 by VH and JCH FT with respect to t1 gives two 1H signals 39VH 12JCH and 39VH 12JCH along the F1 axis FII39H 39 Figure 913 AI P Schematic two dimensional CH I correlated NMR spectrum of a two spin AX system pulse sequence as Il in Fig 912 The 2D spectrum is reduced to two signals with opposite signs their separation along the F1 frequency axis is equal to 1 CH A2 x FII SEI 3 Modified HETCOR Pulse Sequence to Remove Splitting in F1 and F2 A Pulse sequence 1H I I HEW mm in Ho 130 I w lbw W I M r d a f a b I you an B Vector Diagram Insertion of a 13C 180 pulse in the middle of t1 which allows refocusing of MHCa and MHC Insertion of a delay 12J CH after t1 and before the second 1H 90quot pulse This constant delay is needed for optimal population transfer After a delay of 12JCH MHCu and MHCp have a 180 phase difference The magnitude of the polarization transfer depends only on q which is independent of JCH pa p6 2n 39vH t1 a b A Pulse sequence for n we dimensmnal CHmrrnlalcd NMR cxperimenl which mince m 211 s mum of a twspin AX syswm pc lo only one a The vector diagrams n no 1 show Ihc positions 0 me 39H mncgnuiuljnn mom Mfu and X f I 45 v An um nN 3 m immnu indlcaud n A in I mg MSW fllsgramsamdonlylhcx y plnne x x s snown 6 C Spectra 39 FT with respect to t2 gives one 13C signal along the F2 axis with a frequency 39vc and this signal is modulated in t1 by VH only 39 FT with respect to t1 gives one 1H signal along the F1 axis with a frequency 39VH For 13CHCl3 F 39Hl V Vt 51 30 For more complex molecules 5 2 Nood niur ur oon C C 3 m mus leolweuo H1 130 Chemical Shift NOTE 1 Easy to assign 13C signals if 1H signals are assigned or vice versa 2 Little overlap of the correlation peak 4 T 39 39 39 quot 39 H EllCorrelated NMR Spectroscopy H HCOSY A Pulse sequence 90 E FlD Figure 917 Pulse sequence for the twodimen sional homonuclear HHcorrelated NMR experiment COSY The variable is t1 The pulse angle 9 is I r39 A 2 usually 90quot or 45 or occasionally H Lets consider the case where 9x39 90 X39 and lets consider a homonuclear twospin AX system First 90 X39 pulse Tilt both vectors MA and MX along y39 Due to JAX MA has two components MAXu and MAX and MX has two components MXAu and A9 During t1 VA Xu VA l2JAX VA X5 VA l2JAX VX An vx l2JAX vx A5 vx l2JAX qlVIAXa2n VA l2JAX t1 ipMAXp2m VA l2JAX t1 qJMXAa2n 39VX 12J AX t1 qMXAp2n 39VX l2JAX t1 At the end of t1 the vectors have components along X39 and y39 After the second 90 x39 pulse The y and y vector components are tilted along z and z which results in polarization transfer The transfer depends on t1 V and JAX Four frequencies are detected by the receiver A2 39VA Xa A1 39VA X3 X2 VX Au X1 vx Ap B Magnitude Spectrum FT with respect to t2 yields four signals at A1 A2 X1 and X2 These signals are modulated in t1 with these same four frequencies FT with respect to t1 gives a 2D NMR spectrum with four groups of signals each containing four signals Groups centered at VA VA and VX VX are diagonal peaks Groups centered at VA VX and VX VA are cross peaks Within each group separation in F1 and F2 is JAX 9 gure ms Schematic representation of a COSY experiment on a two spin eaks of a pair of mutually coupled nuclei and their cross peaks form the corners of a square C PhaseSensitive Spectrum Figun 541nm phwwmuiw msY rur unpmmmpm AX rn Dmgunulyx39 lmvt mom auuhlmmp Mvc m pt hmennmm dmbltvulnnmrimlmnhnpn u lt 1mm Illusumml In um um 4A lmmmcsmdmm p mw ca 0 9 co m 10 5 T 39 39 quot H Hl nrrelated NMR Spectroscopy COSY45 Same as COSY90 but second 1H pulse is 45 x39 instead of 90 x39 Reduces the intensity of the signal but simplifies the spectrum 39bbs39Y Qf 39 CHEM BCMB 419061908189 Introductory NMR Lecture 17 CHEM 41906190 Last Time HETCOR or C HCOSY 90 90 35 1 1 I 4 S C39Z HOOC IZH CHz CHz COOH C4 C 3 NH2 m H 4 11241 39 If I I I I r I 130 Chemical Shift mus leoweuo HL H The HSQC Experiment HETCOR or CHCOSY Versus HSQC Twodimensional Correlated NMR spectroscopy We have seen the CHCOSY experiment Where 1H is detected in t1 and 13C is detected in t2 Although there is population transfer from 1H to 13C the sensitivity of this experiment is poor because 13C not 1H is detected in t2 Higher sensitivity can be achieved by doing the quotreversequot experiment ie by detecting 1H in t2 and 13C in t1 The HSQC and HMQC are two experiments that achieve this heteronuclear HCcorrelation Here we will consider in more details the HSQC experiments I 1H PREPARATION 90 x 180 x 90 y The HSS 2C Pulse Seguence EVOLUTION MIXING t1 180 y 90 X 180 X DETECTION t2 180 x 90 x quotCIT 90 x 180 x quotCIT ml t1 5 n u WALTZ16 114J 39 In the 1H C HSQC X 13C 1 The PREPARATION period is an INEPT sequence 1H to 13C 2 The t1 EVOLUTION period allows for indirect 13C chemical shift detection 3 The MIXING period is a REVERSE INEPT sequence 13C to 1H 4 The t2 EVOLUTION period allows for direct 1H chemical shift detection 3 Review of the INEPT experiment A Pulse sequence in the 1H and 13C channels Q n x hm um a z Null At point g 1H 90 pulse rotates MHC to z and MHCa to z 5 The populations of N2 and N4 are inverted before INEPT after INEPT N4N NAH N4 X1 N3NAC NAC 1N3 N2NAH N N1NACAH NACAH A2 0 N2 X2 WNI X1 transition N3 N4 AC AH 3AC X2 transition N1 N2 AC AH SAC 39 In the case of the HSQC the contribution from the natural 13C magnetization AC is unwanted and is removed using one of the selected methods that we have seen previously The resulting populations and population differences are N4 N AC2 AH N3 N AC2 N2 N AC2 N1 N AC2 AH X1 transition N3 N4 AH 4AC X2 transition N1 N2 AH 4AC 39 At point g MCHquotI is in its original position but MCHquot is inverted I g z SAC MCH or 4AC 3AC or 4AC x M CH C Vector diagrams showing the 13C magnetization vectors g z 5 AC MCH or 4AC y 3AC or 4AC x M CH At point In lne 90 X pulse on 13C creates transverse magnetization components that evolve during t1 Note that these 13C magnetization components are 180 out of phase with each others at the beginning of t1 lk The t1 Evolution Period PREPARATION EVOLUTION MIXING DETECTION t1 12 90 x 180 x 90y 180 y 90 x 180 x n 1H I I anvnun 180 x 90 x t1 t1 90 x 180 x X 7 IT 7 397 WALTZ16 114J 1 The 13C chemical shift evolves to different points depending on the value of t1 2 There is no net 1H13C coupling evolution Note that the 13C magnetization components are 180 out of phase with each others at the beginning and the end of t1 t1 t1 E 180 x39 E gt I gt gt McH5 139 V V39 V MCH5 McHoc 2 2 J McHoc McH5 X McH5 McHoc 7 McHoc 5 The ReverseINEPT experiment A Pulse sequence in the 1H and 13C channels 90 gtlt 180 gtlt 1H t2 90 gtlt 180 gtlt X I T quotC 14J B Vector diagrams showing the effect of the first 13C 90 X39 pulse on the 13C magnetization vectors EX 13CHCl3 for various t1 values Four cases are shown below 2 2 Mom 90 gtlt39 90 gtlt39 Mom DC I Mow Mama y y y y McH Mama McHoc X McH3 X X X z 2 90 gtlt39 90 gtlt39 gt McH McH gt McH3 McHoc 5 i y y y y X 4M0Hm V rMtHoc X X39 X We will only consider the following case where after the first 13C 90 X pulse MCHquot is in its original position but MCHquotI is inverted Z 90 x39 M H C cc McHB y39 y39 4 McHB X MCHcc l The populations are described as followed same as after first 13C 90 X N4NAC2AH N4 X1 3333 A1 N1 N AC2 AH a N2 A2 A1 transition N2 N4 AH X2 WM A2 transition N1 N3 AH X1 transition N3 N4 AH 4AC X2 transition N1 N2 AH 4AC 39 The antiphase 1H magnetization is refocused during the 21 period The two 180 X39 pulse on 1H and 13C in the middle of the 21 period allows J coupling evolution but refocuses the 1H chemical shift evolution For simplicity one can ignore the effect of chemical shift Which is refocused during the reverseINEPT period 2 z B a T 39 1 4J y y X a B B a 180 X39 180 X39 39 E 1 gt gt H 13C 14J y y y X39 X39 X39 39 At the end of the reverseINEPT a and 5 are in phase therefore we can turn on BB 13C decoupling during acquisition 6 HS 11 Spectrum Example 1H C HSQC of Glutamic Acid H l H0061 J 39H CH1CHZ C00H H4 2 2 39 Signals not obtained for 1H that are not bound to 13C 39 Comparison with CH COSY 1 Axes are interchanged in HSQC 1H is detected in HETCOR 13C is detected 2 HSQC is more sensitive and a good spectrum can be recorded quickly Example for 1 mM uniformly 13C or 15N isotopically labeled samples typical recording times are 1530 minutes for HSQC 24 hours for C H COSY 11 Proton H Nitrogen N Correlations C c I t of III c rl I R5 Lysine K Ligand Binding in Protein 7 Imnnrtant m Biased 1HlSN HSQC for Analysis of CASE 1 Protein Folding Upon Ligand Binding Domains of the A N Protein 1 22 34 47 73 107 N 122 boxB RNA 1H chemical shift ppm 0 ASN and GLN side chains 3 ARC side chains folded niuogens j LYS side chains folded nitrogens CASE 2 Mapping Ligand Binding Sites in Folded Proteins 1164 1158 1152 N ppm IITiO 1176 TOCSY htal Qorrelation SpectroscopX Also known as HOHAHA HOmonuclear HArtmannmhn The TOCSY Experiment 39 Very useful experiment for determining the structures of oligosaccharides and peptides as well as many other organic compounds 1 Pulse seguence The pulse sequence is similar to that of the COSY experiment except that the second 1H 90 x39 pulse is replaced by a spinlock pulse train 90 t1 spinlocktm FID The spinlock allows polarization transfer from one selected proton to all protons Within a coupled system aka spin system preparation evolution mixing detection t1 02 90 90 COSY t1 90 90 90 NOESY t1 Em 90 TOCSY t1 90x 180x 90y 180 90 180 1H H I H I H I H I HSQC 180x 90x i 90 180 13C t12 t12 decouple 2 Spin sytems Lets take u methyl3Omethylcellobioside for example 39 In sugar I H139 is coupled to H239 ie J u 0 H239 is coupled to H339 H339 is coupled to H439 H439 is coupled to H539 H539 is coupled to H639a and H639b JH5 H6 a gt JH5 H6 b H639a is coupled to H639b H139 H239 H339 H439 H539 H639a and H639b form a spin system 39 In sugar 11 H1 is coupled to H2 ie J u 0 H2 is coupled to H3 H3 is coupled to H4 H4 is coupled to H5 H5 is coupled to H6a and H6b JHsH a gt JHsH b H6a is coupled to H6b H1 H2 H3 H4 H5 H6a and H6b form a spin system 39 Because there is no 1HlH J coupling between sugar I and sugar II they both form independent spin systems Differences between SY and T SY In a COSY spectrum we observe one crosspeak for H139 F2 H139F2H239F1 We also observed the symmetrical peak H239F2 H139 F1 Simulated COSY spectrum of u methyl3Omethylcellobi0side O sugarl O sugarll LA mus 90UJ9HO HL o o o 000 39 c98 o o o o 09 00 no 8 0 o o o I 3 39 In a TOCSY spectrum we observe multiple crosspeaks for H139 H139 H139 H139 H139 H139 H139 39 We also observed the symmetrical peaks across the diagonal H239 H339 H439 H539 H639a H639b 39 TOCSY spectrum of amethyl3 Omethylcellobioside H39DO coocu ocu In H n all 39I39 a V 40 I10 I II W lo a I quot39 I r II39 I In In I u 39 539s 639b 339 539 439 239 u H II M l 5 6h 4 2 3 l m I In I I I I quot 39 l 1 5 4 n 35 5 1H Chemical Shift F2 17 I l La mus 90UJ9HO HL
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