Lecture 11: INEPT, HSQC Simultaneous spin echo INEPT 1 H 1 3C or15N 1 1 H 3C or15N 1 4J 1 y I ii i INEPT 1 H 1 3C or15N 1 1 H 3C or15N 1 4J 1 y I ii i z INEPT 1 H 1 3C or15N 1 1 H 3C or15N 1 4J i\y /5(e) = + cos^j^y - i«x sin (2j^z) - |«2^ INEPT 1 H 1 3C or15N 1 1 H 3C or15N 1 4J i\y I ii i p(e) = -j^t - -«i (2j^^) - -«2-^ INEPT 11 1 WL - X lx -2/1/2 lx lz y-2,ylzI2z INEPT P(g) = -*t - -«1 (2^y) + -«2^y INEPT P(g) = -*t - -«1 (2^y) + -K2^J/ INEPT y -C2 ISzSTy -> +s2 2jxyz —> +c2y y -s2yx —> | -c2cj 2fzyy { +c2sj yx | +s2cj 2j^x { +s2sj ^/ | +c2cj \ -c2sj 2yxjz -s2cj yx -s2sj 2yyyz Relaxation with J-coupling =>- = j?ix + \j?iy —>■ —\2j?i+j?2z different R2 • relaxation of depends on 2^1+^ relaxation of 2s±+S2z depends on cross-correlated cross-relaxation (ingnored here) cf. cross-relaxation of A{Mlz) and A(M22> (NOE) INEPT )} = A/"722^2^o 16kBT 1 Ä2 + _ #2 \ 72/7J « 16 for 13C 7i/72 ~ 100 for 15N Insensitive Nuclei Enhanced by Polarization Transfer HSQC Spectroscopy (Heteronuclear Single-Quantum Coherence) 1 3C or15N 1 4J 1 y t1 x/y f g i i h T 1 1 4J ill t2 1 COMPLEX EXPERIMENT ANALYSIS FACILITATED BY SIMPLIFICATIONS Using results of already analyzed building blocks (echoes) Ignoring components of 3 that cannot produce signal HSQC Spectroscopy Measured quantity: Mi_|_ (M2+ does not pass the frequency filters) Only jxM^-\. and JvM^-\. have non-zero traces: Tr{sy(Slx + \Sly)} = i Directly measurable: JXl Jy (in-phase single-quantum of nucleus 1) Evolve to measurable due to J coupling: IJx&z, l^y&z (anti-phase single-quantum of nucleus 1) Need 90° pulse + J coupling: Jz (90° pulse), IJz^z (populations, longitudinal polarization) .yx, yy, 2j?zy?x, 2Jz^y (single-quantum of nucleus 2) 1j?xyx, ISySfy, 2j?xyy, 2j?yyx (multiple-quantum) Never measurable: Jt (unit matrix) HSQC Spectroscopy 1 1 y 13C or15N I e BLOCK 1: INEPT p(a) = ^ + \k\ (.yz) + |«2^ p(e) = ^ - (2yzyy) + ^«2^?/ HSQC Spectroscopy 13C or15N BLOCK 2: DECOUPLING ECHO, INCREMENTED tx p(e) = \jt - \k\ (2j?zyy) + ^k2yy p(f) = §j«Jt + 5«l (C2l2^^y - S212^^x)+5«2 (C21^/ ~ «21^) HSQC Spectroscopy — Real 1 3C or15N I I ti I I g h 1 ■ ■ 1 y ■ — ■ 1 ■ 1 t2 l ill 4J |4J x BLOCK 3: TWO 90° PULSES, PHASE x (13C or 15N) p(f) = \st + \*>i (c212szyy - s212szyx) + \k2 (c21yy - s^i^x) p(g) = (c212syyz - s2i2syyx)+^k2 {c21yz - s21yx) HSQC Spectroscopy — Real 1 3C or15N I I ti I I g h 1 ■ ■ 1 y ■ — ■ 1 ■ 1 t2 l ill 4J |4J x BLOCK 3: TWO 90° PULSES, PHASE x (13C or 15N) p(f) = \st + \*>i (C2i2^zyy - s2i2yzyx) + \k2 (c2i^/ - s2iyx) ß(g) = —i«iC2i2^v^z+ unmeasurable (no more 90° pulses) HSQC Spectroscopy — Real i iy ± I 4J | 4J | | | 4J | x 13C or15N I { t1 I I e f g BLOCK 4: SIMULTANEOUS ECHO p(g) = —Akic2i2j^z+ unmeasurable —>• p(h) = ^/«iC2i^x+ unmeasurable HSQC Spectroscopy — Real 13C or15N KiC2lSx —>• 1 2«lC2iCi2 Sx \niC2lS\2 Sy +i«ic2ic12cj +2^ic2ici2sj 2j^^ r +2ttic21s12cj j^y -2^ic2isi2sj HSQC Spectroscopy — Imaginary 13C or15N BLOCK 3: TWO 90° PULSES, PHASE y (13C or 15N) p(f) = \st + \*>i {c2\2^zyy - s2i2yzyx) + \k2 (c2i^y - S2l^x) HSQC Spectroscopy — Imaginary 13C or15N BLOCK 3: TWO 90° PULSES, PHASE y (13C or 15N) p(f) = l^t + ^i (s212yzyy - s212yzyx) + ^k2 (c21yy - s21yx) ß(g) = —\K\s2\2yyyz+ unmeasurable (no more 90° pulses) HSQC Spectroscopy — Imaginary 13C or15N BLOCK 4: SIMULTANEOUS ECHO ^(g) = —^KiS2i2^yyz-\- unmeasurable —>• ^(h) = ^K\S2i^x-\- unmeasurable HSQC Spectroscopy — Imaginary -i -i X | 4J | 4J | 13C or15N i I i ti 1 f g 4J J t2 Jill iff h —«lS2i^x -> < 2K1S21C12 ->• +|K1S21C12CJ ^a; + 2«1S21C12SJ 2^y^ +o^ls21s12Cj -2«lS21s12sJ 2j^xj^ HSQC Spectroscopy — Hypercomplex 13C or15N ±«ie,Si2*l^ +4«ie,fí2íiCl2Cj Jx +2«ie'^2iiCl2Sj 2^ +§/íielQ2íiSl2Cj ^ -5«ie,n2il«i2*j HSQC Spectroscopy — Hypercomplex 1 3C or15N j_ 4J i y x/y fi \ g JUJ h T 1 4J ill t2 lllílk/W- |l| If y V WJ^^fPf" 16kBT í X i?2 2 + O - ^2)2 1^2 1 + O - Ql + ^J)2 ' Ä2 1 + - Qi - TT J) ß2,l ^2 ß2,l V Decoupling in direct dimension C22 2 ttJ Decoupling in direct dimension 1 3C or15N j_ j_ y 4J | 4J | ti 1 4J x/y A/712ft2£0 8kbt r12 + (uj- q2)2 r£ x + _ qx) -^2.2 ^2,1 Decoupling in direct dimension 1 3C or15N 1 1 y |4J|4J| 1 1 1 1 I 4J | 4J 1 t2 > 1 1 1 ti | x/y 1 1 ' GARP A/712 ft2 £0 8kbt r12 + (uj- q2)2 1 + _ m): #2.2 #2,1 Decoupling in direct dimension [ 2 ttJ ' wow 1 ^2 1 ^2 Benefits of HSQC • High sensitivity for 1JC or 1DN (higher by (7i/72)5//2 than by the direct detection • High resolution Second dimension and less peaks in spectrum (only 13C/15N-bonded protons and protonated 13C/15N visible) • Important structural information 1H-13C and 1H-15N correlation (it tells us which proton is attached to which 13C or 15N). HOMEWORK: COSY Section 11.3