Summary of "Week 5 - Lecture 21"

Summary of Week 5 - Lecture 21

This lecture focuses on advanced NMR spectroscopy techniques, particularly heteronuclear correlation experiments such as HSQC (Heteronuclear Single Quantum Coherence), and extends to multidimensional NMR experiments combining different types of correlations to enhance spectral resolution and structural information.

Main Ideas and Concepts

1. Heteronuclear Single Quantum Coherence (HSQC) Experiment

HSQC is a common 2D NMR experiment that correlates proton (^1H) and heteronuclei (e.g., ^13C or ^15N) chemical shifts.
Requires two separate radiofrequency channels and coils: one for protons and one for heteronuclei (carbon or nitrogen).
The pulse sequence involves magnetization transfer from proton to heteronucleus and back, using the INEPT (Insensitive Nuclei Enhanced by Polarization Transfer) sequence.
Key steps in magnetization flow:
- Start with proton z-magnetization (Hz).
- Apply INEPT transfer (90°–τ–180°–τ–90° pulses) to transfer magnetization to heteronucleus (e.g., carbon-13).
- Heteronucleus magnetization evolves during t₁ with proton-carbon decoupling (spin echo with 180° pulse on proton channel).
- Reverse INEPT transfers magnetization back to proton for detection during t₂ with proton-carbon decoupling.
Result:
- t₁ dimension contains only heteronucleus frequencies.
- t₂ dimension contains only proton frequencies.
- Spectrum shows only cross-peaks (no diagonal), correlating protons with directly bonded heteronuclei.

2. Interpretation of HSQC Spectra

Carbon chemical shifts span a wide range (~0–130 ppm).
Proton chemical shifts range from 0 to 10 ppm.
Provides a “fingerprint” for molecular structure, especially useful in proteins.
In proteins, ^1H–^15N HSQC gives one peak per amino acid residue (except proline, which lacks amide proton), enabling counting and identification of residues.

3. Combining HSQC with TOCSY or NOESY

TOCSY (Total Correlation Spectroscopy) and NOESY (Nuclear Overhauser Effect Spectroscopy) can be combined with HSQC to relay magnetization from amide protons to other protons within the same residue or to neighboring residues.
This combination enhances assignment of spin systems and provides sequential and long-range correlations.
TOCSY-HSQC transfers magnetization among protons within the same residue.
NOESY-HSQC provides through-space correlations, including sequential connections.
Pulse sequences are modified to include TOCSY or NOESY mixing periods after the HSQC transfer steps.
Broadband decoupling is applied during acquisition to simplify spectra.

4. Multidimensional NMR: 3D and 4D Experiments

3D NMR experiments add a third frequency dimension by incrementing three time periods (t₁, t₂, t₃) and incorporating two mixing periods (m₁, m₂).
Example: Combining proton-proton correlation with ^15N chemical shift dimension to separate overlapping peaks.
This separation resolves ambiguities arising from overlapping proton resonances by spreading peaks along the nitrogen axis.
4D experiments add yet another dimension (e.g., carbon chemical shifts), further resolving spectral congestion.
Each additional dimension improves resolution but increases experimental time significantly.
Visualization changes from 2D planes to 3D “boxes” of peaks, with projections providing different correlation views.

Detailed Methodologies and Instructions

HSQC Pulse Sequence Steps

Start with proton magnetization (Hz).
INEPT transfer:
- 90° proton pulse → τ delay → 180° proton + 180° carbon pulse → τ delay → 90° carbon pulse → magnetization on carbon.
t₁ evolution with 180° proton pulse in the middle (spin echo) to decouple proton-carbon coupling.
Reverse INEPT transfer to proton magnetization.
t₂ acquisition with proton-carbon decoupling to detect proton signals.

Timing Parameters

τ delays are optimized based on coupling constants (e.g., τ ≈ 1/(4J_CH), typically ~1.7 ms for 150 Hz coupling).

Combining HSQC with TOCSY/NOESY

After HSQC magnetization transfer, introduce TOCSY or NOESY mixing period (τ_m).
TOCSY mixing uses spin-lock pulses to transfer magnetization among protons within the same residue.
NOESY mixing uses cross-relaxation for through-space correlations.
Adjust pulse sequences to include appropriate mixing pulses and delays.
Collect free induction decay (FID) with broadband decoupling.

3D NMR Experiment Design

Increment three time periods (t₁, t₂, t₃) to obtain three frequency dimensions (f₁, f₂, f₃).
Include two mixing periods (m₁, m₂) for magnetization transfer steps.
Example: Proton-proton correlation (2D) combined with ^15N chemical shift (3rd dimension).
Use projections to analyze individual 2D planes within the 3D data set.

Considerations

Longer experimental times with increasing dimensions.
Trade-off between resolution and experimental duration.
Practical limit often 3D; 4D used for very complex systems.

Speakers/Sources Featured

Primary Speaker: Lecturer presenting the NMR spectroscopy concepts and pulse sequences (name not provided).
Experimental Data: Protein HSQC and 3D NMR spectra shown as examples (specific proteins not named).
General References: Standard NMR methodologies (INEPT, HSQC, TOCSY, NOESY) as commonly used in biomolecular NMR.

This lecture provides a comprehensive overview of heteronuclear correlation NMR experiments, their pulse sequences, and how they can be combined and extended into multidimensional experiments to obtain detailed structural information, especially in biomolecular studies such as protein NMR.