HSQC = Heteronuclear Single-Quantum Coherence. ) in order to maximize the coherence transfer from I to S

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1 HSQC = Heteronuclear Single-Quantum Coherence The pulse sequence: Consider: Ω I, Ω S and J IS τ = 1/(4J IS in order to maximize the coherence transfer from I to S The overal pulse sequence can be subdivided as follows: INEPT + echo + INEPT. The first INEPT is to improve the sensitivity of the low magnetogyric ratio nuclei ( 15 N, 13 C. During the defocused delay (echo,, only Ω S evolves (coupling is refocused. The final INEPT-type unit is to perform inverse detection (observing the frequency of the low magnetogyric ratio nuclei as 1 H magnetization.

2 σ[eq.] I z Ĥ = π/2 (Î x x σ[0] -I y this is a homo type echo: τ and τ πτ Ĥ = πs x τ and τ πτ the chemical shift is refocused but coupling evolves, thus the effective Hem(s are: π2τ, and Ĥ = πs x σ[0] π2τ Ĥ = πs x -I y -I y cos(πj IS 2τ +2I x sin(πj IS 2τ +I y cos(πj IS 2τ +2I x sin(πj IS 2τ +I y cos(πj IS 2τ -2I x sin(πj IS 2τ

3 +I y cos(πj IS 2τ -2I x sin(πj IS 2τ τ = 1/(4J IS in order to maximise the coherence transfer from spin I to S if τ = 1/(4J IS, then after 2τ sin(πj IS 2τ = 1 and cos(πj IS 2τ = 0 Ĥ = π/2î y Ĥ = π/2s x [1/2 ] π[1/2 ] [1/2 ] π[1/2 ] Hem = -2I x (anti-phased magnet. on I +2I z -2I z (anti-phased magnet. on S

4 Coupling is refocused during the hetero type echo and the chemical shift of I doesn t evolve. Hem = Ĥ = π/2î x Ĥ = π/2s x -2I z +2I z +2I z cos -2I z S x sin -2I y cos +2I y S x sin -2I y cos +2I y S x sin this is a homo type echo: τ and τ πτ Ĥ = πs x τ and τ πτ the chemical shift is refocused so the effective Hem(s areĥ= 2Î z π2τ, πi x and πs x

5 so: -2I y cos +2I y S x sin (multiple quantum term π2τ -2I y cos cos(πj IS 2τ +I x cos sin(πj IS 2τ Ĥ = πs x -2I z cos cos(πj IS 2τ +I x cos sin(πj IS 2τ +2I z cos cos(πj IS 2τ +I x cos sin(πj IS 2τ τ = 1/(4J IS to maximise the coherence transfer from I to S (if τ = 1/(4J IS then after 2τ sin(πj IS 2τ = 1 and cos(πj IS 2τ = 0 So before ACQ the following term remains: +I x cos ACQ [ ] and [ ] π[ ] +I x cos cos cos(πj IS +2I y cos cos sin(πj IS +I y cos sin cos(πj IS -2I x cos sin sin(πj IS

6 memo 1: put the receiver on x therefore only the single x term remains : +I x cos sin cos(πj IS memo 2: sin(acos(b = 1/2[sin(A+B+sin(A-B] therefore: -1/2I x cos [+sin{ +πj IS }+ sin{ πj IS }] the following term can be found - I x [ ] at Ω I, Ω S if one sets the phase that cos is absorptive (a in sin is absorptive (d in I x [ +a.. +a..] at Ω I, Ω S Singulet in F1 and the in phased doublet in F2. ( 1,1 J[H,N] Hz, 1,1 J[H,C] 150 Hz if we decouple during ( π[ ] is not active then the spectrum is I x [ +a ] at Ω I, Ω S

7 Heteronukleáris egyszeres-kvantum koherencia spektrum HSQC = Heteronuclear Single-Quantum Coherence 1,1 J~Hz H z Ω N -2H z N y +2H z N y cos(ω N H x cos(ω N H x cos(ω N cos(ω H Ω H

8 HSQC-SE = Heteronuclear Single-Quantum Coherence with sensitivity enhancement τ τ τ τ -/+H y cos(ω N +H x sin(ω N 1 H dec 13 C H z INEPT: Frequency labeling: H z N y cos(ω N H z N x cos(ω N Both anti-phase N from -H y coherence is transferred to the less sensitive nuclei resulting in anti-phase coherence on N: H z N y coherences are to be selected REVERSE INEPT: from H z N y to ±H x and form H z N x to H y N x Both coherence pathways are considered SE module: -/+H y cos(ω N cos(ω H -H x sin(ω N sin(ω H from +/-H x to -/+H y form H y N x to H x (phase modulated signal with the contribution of both coh. trans. pathways: gain a factor of 2. F.J.M van de Ven Palmer et al. J.Magn.Reson. 1991, 93, Cavanagh & Rance J.Magn.Reson. 19, 88, 72-85

9 HSQC = Heteronuclear Single-Quantum Coherence Monitoring H 2 O along the sequence: +z y y y x y y x z +z 1 H 15 N φ 2 dec G z What is happanining with the balk water during the HSQC pulses? +I z ( x I y ( x +I y ( y +I y ( x I y ( x I z ( x +I z Water before acquisition is aligned along +z. Thus, most of the water is not detected during. Unfortunately, that part of the water which is not parallel to z will be detected as residual water. The latter amount of water should be minimized, (dynamic range problem of the detectors.

10 1 H- 13 C HSQC-SE: Destroy all residual 13 C z : Put it into the transverse plane and defocused by grad. INEPT: from H z generates +2H z C y Labeling by 13 C cĥ shift: from 2H z C y generates 2H z C y cos(ω N REVERSE INEPT: from H z C y to ±H x and form H z C x to H y C x (in a no-se version the latter path is ignored SE module: from +/-H x to -/+H y form H y C x to H x (phase modulated signal with the contribution of both coh. trans. pathways: gain a factor of 2. τ τ τ τ 1 H dec 13 C G z Grad.7: before converting -2H z C z into -2H z C y defocuses any other transverse coherences present (kills everything not coupled to 13 C Coh. selection by grad.: 2H z C x and 2H z C y are selected (SE experiment only. ( refocuses evolution occurring during the long gradient 3: a hetero nuclear echo. Coh. selection by grad.: refocuses on proton (H y what was defocused on carbon (2H z C x.