生物大分子波谱学原理 吴季辉 7.1异核相关谱 蛋白质研究中用的异核相关谱均利用单键偶合，分为两类： HSQC (Heteronuclear Single-Quantum Coherence) Simple HSQC Decoupled HSQC Constant-time HSQC HMOC (Heteronuclear Multiple-Quantum Coherence)
7.1 异核相关谱 蛋白质研究中用的异核相关谱均利用单键偶合，分为两类： HSQC (Heteronuclear Single-Quantum Coherence) Simple HSQC Decoupled HSQC Constant-time HSQC HMQC (Heteronuclear Multiple-Quantum Coherence) 生物大分子波谱学原理 吴季辉
Modification of a refocused I-S INEPT into a 2D HSQC sequence,and use of States-TPPI quad detection INEPT 2-spin INEPT order 31 φ12 φ1=6-x φ2=x,x,-x,-x φ31=x-七-6x G: 2LS±21S2±2lS2 /4s φ31 φ2 φ1=6x +States-TPPI =1(0)+(ni-1)/sw1 2=xx,-x,-x G φ31=北，-6-x,x +States-TPPl 21S.21-S.2S,21Scos(os1)21S.215 ×cos(元J11) -21-Sx sin(os n) ×cos(πJt)
生物大分子波谱学原理 吴季辉 间接维正负频率区别 Experiment Increment Pulse phase Receiver phase TPPI (4+1) t1(0)+(4k)△ X X (4k+2) t1(0)+(4k+1)△ y x (4k+3) t1(0)十(4k+2)△ x x (4k+4) t1(0)+(4k+3)△ y X States (4k+1) t1(0)+(4k)2△ X (4+2) t1(0)+(4k)2△ y X (4k+3) t(0)+(4k+1)2△ X X (4+4) t1(0)计(4k+1)2△ TPPI-States (4k+1) t1(0)+(4k)2△ X (4k+2) t1(O)t(4)2△ y x (4k+3) t1(0)+(4k+1)2△ -X -X (4k+4) t1(0)+(4k+1)2△ y -X
间接维正负频率区别 生物大分子波谱学原理 吴季辉
Important Features of HSQC Spectra Start with 1H magnetization:gain YH/YN A factor of 10 in sensitivity Detect 1H magnetization:gain (YH /YN)2 Another factor of 100 Even though you must acquire 32-64 t1 points,far more sensitive than direct detection. 相干阶路线？
相干阶路线?
States quadrature:change the phase by 900 for each t increment to select cosine and sine components of S-spin magnetization 1.For each value of t, 14 4 4 31 acquire two FIDs which phase =1 are stored separately,cor- 1=6x 1=x,-术 responding to phase=1 2■x,-水，-x and phase=2. 31=,-x-， G 2.For the FID with phase=1,set 01=x,-x.This transfers 215.21.s 21.S es 21S es 21,S.cs ly cos(osh) IS cos(@st)to proton 21Sx ss -25x ss 2fSx ss (at the end of the se- 2I-S cs=cos(oos 11) quence ss =sin(os 1) 2.For the FID with phase=2,set 4 phase =2 4 31 1=y-y.This transfers IS sin(@st to proton 01=5-y =5少 (at the end of the se- 2=x,x-x,-x quence. φ31=x,--x,x 2S21S 21.S,cs -21Sxes 2hSxes 21-Sy ss 21-S:ss 2lxSzss ly sin(os f1)
Basic HSQC sequence with States-TPPI quadrature detection 1H coupled HSQC-13C peaks are split by C-H couplings in /4.Jrs 中31 01=x,-=x States-TPPI 2=x,x,-6,x 1H decoupled HSQC-13C peaks are singlets in 01 at natural abundance, 031=K,一x,-x,x split by 13C-13C couplings in 13C labeled systems +States-TPP 12 4 1/4US Φ31 1/2 gradients must be of opposite signs to continue to dephase I spins not coupled to S
The duration (A)of S-spin chemical shift evolution during the first t1 increment(Ist two FIDs) dictates the extent of phase correction required in the o dimension. 1H coupled HSQC t红 pwx G △=2×(2pwx/π)+4(0) off-resonance effects 1 st order(lp1)=-360×sw1×△ zero order(rp1)=-phc1/2 1H decoupled HSOC 2xpw 1/4Js t1/2 2 G △=2×(2DwX/π)+2×Dw+t.(0)
If△=1/(2×sw1)or half the dwell time, 1 st order(lp1)=-360×sw1×1/(2×sw1)=-180 zero order(rp1)=90 there is no need to calculate the phase constants 1H decoupled HSQC 2xpw 1/4Us 1/4Js 1 1/2 t/2 G 4(0)= -2x(2×pwx/π)-2×pW (2×swl) △=2×(2×pwx/π)+2×pw+4(O) 1 (2×swI)
Another way to avoid phase corrections in is to introduce a 180s pulse at the end of the t period, followed by a delay 8,to refocus chemical shift precession during t(0) 1st order(Ip1)=zero order (rp1)=0 if t1(0)~0.0 1st order (Ip1)=-180,zero order(rp1)=90 if t1(0)=1/(2sw1) 1H decoupled HSQC 2xpw 02 1/2 t/2 δ=2×pw+t(0) the off-resonance effects of the two 90S pulses are refocused by the 180S pulse, so they do not have to be included in the phase constant calculation. The Jis coupling evolves for the period 8,but since 8~us,the effect is negligible
HMOC (904 (a→b)1z→-y 2hsiSz g T=1/2J (90)Sx 21xSz +2lxSz-2lxSy 12 (b→c) 21Sycos hcosh Decoupling (24)Sz 2lxSxcos nitisin st -2lySysin itcos st The 180 pulse of I-spin in the middle of t refocuses +2lySxsin itisin st the /-spin chemical shift.As a result,magnetization (9075x transfer from points a to d is as follows: (c→d) 2lxSxcos nitisin st (1801x 2lySxsin it sin st -2lxSzcos nt cos nst (b→c) -2lx(Sycos st -Sxsin sh) (Qsh)Sz -2lySzsin niticos sti (90S (c →d) >-2lxSzcos nst 2lxSxsin Dst 21glt →2 IxSxcos2t1sin2st1 2JilSz (r=1/21s) -lycos st 2lxSxsin sti [6b] (r=1/2Js 2lySxsin itisin st-lycos ifcos st Ixsin ificos st [6a] HMQC和HSQC谱图 看起来非常类似
HMQC HMQC和HSQC谱图 看起来非常类似