The 4D HMQC-NOESY-HMQC experiment is specifically designed to observe and unambiguously assign  ¹H-¹H NOEs even for protons with degenerate chemical shifts in X-labeled biomolecules. The mechanism involves an initial heteronuclear transfer from ¹H(A) to X via ¹J(XH) , a conventional homonuclear ¹H NOESY step from ¹H(A) to ¹H(B) and a final heteronuclear transfer from the ¹H(B) to X via ¹J(XH).

Implementation on AVANCE spectrometers is feasible. Improved versions using pulsed field gradients (PFGs) are also available and, therefore, in such cases gradient technology is required. The ¹³C/¹³C HMQC-NOESY-HMQC experiment is applied on ¹³C-labeled proteins in D₂O, the ¹⁵N/¹⁵N HMQC-NOESY-HMQC experiment is applied on ¹⁵N-labeled molecules in H₂O and the ¹³C/¹⁵N HMQC-NOESY-HMQC experiment is applied on ¹³C,¹⁵N-labeled molecules in H₂O.

The 4D HMQC-NOESY-HMQC pulse sequence ( 90SCI411 ) basically consists of the following steps:

• An 2D ¹H-X HMQC pulse train.
• A 2D NOESY pulse train.
• A second 2D ¹H-X HMQC pulse train.
• ¹H acquisition is performed under X-decoupling.

As a function of the X-nuclei, we can have three different experiments:

• 4D ¹³C/¹³C HMQC-NOESY-HMQC experiment ( 91BIO12 , 91JACS370 , 92JACS1082 , and 93JMRB210-101 ).

• 4D ¹⁵N/¹⁵N HMQC-NOESY-HMQC experiment ( 95JACS9592 and 95JACS9594 ).

• 4D ¹³C/¹⁵N HMQC-NOESY-HMQC experiment ( 90SCI411 and 93JB463 ).

Related sequences can be derived from the basic pulse scheme:

• Any HMQC block can be changed by an analog HSQC or TROSY block:
  4D HMQC-NOESY-HSQC ( 93JMRB210-101 , 93JB463 , and 99JMR232-139 ) or
  4D HSQC-NOESY-HSQC ( 95JACS9594 ) experiment.
  4D HSQC-NOESY-TROSY ( 00JB261-18 ) experiment.
  4D TROSY-NOESY-TROSY ( 00JB261-18 ) experiment.
• Use of PFGs ( 93JMRB210-101 and 95JACS9594 ) and PEP methodology ( 93JB463 ).
• Optimizing sensitivity and resolution using multidimensional decomposition ( 05JB1-33 ).
• Related 3D HMQC-NOESY-HMQC or similar experiments can be obtained by removing the variable evolution period in the NOESY part .

The 4D HMQC-NOESY-HMQC experiment can be recorded in automation mode. Each building block is usually optimized as their 2D counterparts.

The HMQC-NOESY-HMQC  experiment affords a 4D spectrum in which X, ¹H, X and ¹H chemical shifts are displayed in four independent dimensions. The F₂(¹H)-F₄(¹H) plane of a X/X HMQC-NOESY-HMQC spectrum will show a typical 2D F₁(¹H)-F₂(¹H) NOESY map in which the chemical shift of the directly-bonded X nuclei of the ¹H-¹H NOE contacts will be known.