The 2D Constant-Time HSQC (CT-HSQC) experiment is a version of the conventional 2D HSQC experiment in which the typical variable ¹³C evolution period is replaced by a constant-time evolution period in which homonuclear ¹³C-¹³C coupling constants are refocused and, therefore, cross-peaks appear as singlets in the F₁ dimension. In addition, editing features can be included as a function of the constant-time period length. The use of CT periods has largely been used to enhance sensitivity and resolution in 3D and 4D NMR experiments of labeled biomolecules.

Implementation on any AVANCE spectrometer equipped with an inverse probehead.

In the 2D CT-HSQC pulse sequence ( 92JMR202-97 ), the evolution of ¹³C chemical shift takes place during a constant time period (T) consisting of the t₁/2-180º(¹H)-T/2-180º(¹³C)-(T-t₁)/2 block. Optimization of the T period has been discussed ( 92JMR637-97 ). A similar sequence including ¹³CO and ¹³N decoupling has also been reported ( 92JMR428-98 ).

Several modified experiments have been reported:

• To measure ²J(CO,HA) coupling constants from E.COSY cross peaks in ¹³C-labeled proteins ( 92JB401 ).
• To measure ³J(NC) coupling constants in spin-echo difference CT-HSQC experiments ( 93JACS5334 ).
• Assignments of carbons directly coupled to either an aromatic or to a carbonyl carbon, allowing the residue type assignment fro aromatic and Asn/Asp residues ( 93JB185 ) .
• Use of an hyperbolic secant 180º ¹³C pulse to achieve uniform ecitation over the complete ¹³C chemical shift range ( 95JB59-5 ).

The 2D CT-HSQC experiment can be recorded in routine/automation modes. Minor changes are required if a predefined parameter set is available. The length of the constant-time period (T) must be optimized as a function of J(CC):

• If T=2/J(CC) (for instance, 54 ms) , all cross peaks have the same sign.
• If T=1/J(CC) (for instance, 27 ms), the carbon signal intensity will depen of the number of directly-bonded carbon nuclei. Thus, ¹³C nuclei coupled to zero or two other carbons and those coupled to one or three other carbons appear with opposite sign.

Tutorials: 2D inverse experiments

Tutorials: 2D gradient-based inverse experiments

The CT-HSQC experiment affords a conventional 2D HSQC map in which J(CC) has been removed in the F1 dimension.

Related experiments:

2D Inverse experiments

2D Inverse gradient-enhanced experiments