13C-13C Connectivity via 1H-13C 1,1-ADEQUATE

One of the most valuable pieces of information one could obtain in elucidating the structure of a small organic molecule is carbon-carbon connectivity information.  This information can sometimes be indirectly deduced from HMBC and/or H2BC data with reasonable sensitivity.  The same information can be determined directly, albeit with dramatically less sensitivity, using the ¹³C INADEQUATE technique.  Another option for obtaining carbon-carbon connectivity information is the 1,1-ADEQUATE technique (Adequate sensitivity DoublE QUAnTum spEctroscopy).  This method is proton detected and relies on a 1-bond INEPT transfer between ¹H and ¹³C.  One-bond ¹³C-¹³C double quantum coherence between the carbon bound to the proton used for the initial INEPT transfer and adjacent carbons is allowed to evolve in much the same way as in the INADEQUATE technique.  Magnetization is transferred back to single quantum coherence for proton detection.  The 2D NMR data show correlations between the proton resonances and the double quantum frequencies between the carbon attached to the proton and those carbons bound to that carbon.  The carbon-carbon connectivity information is provided in the double quantum carbon frequencies.  One drawback to the 1,1-ADEQUATE technique is that connectivity cannot be established between two quaternary carbon atoms not attached to protonated carbons.  Connectivity information between a quaternary carbon bound to a protonated carbon can however be established.  The sensitivity advantage of the 1,1-ADEQUATE technique compared to the ¹³C INADEQUATE technique arises from ¹H rather than ¹³C detection and that the recycle delay depends on the proton T₁'s rather than the ¹³C T₁'s.  Here is an example of how one could use the 1,1-ADEQUATE technique with other methods to unambiguously assign the structure of a small organic molecule.  The edited HSQC spectrum of the unknown molecule with separately acquired ¹H and ¹³C NMR spectra as projections is shown in the figure below.

 The ¹³C spectrum provides all of the ¹³C frequencies, while the edited HSQC signals provide the ¹H-¹³C one-bond connectivity and multiplicities for each protonated carbon.  Note that the carbon frequencies could also be determined from a high resolution HMBC spectrum if insufficient material is available for a direct ¹³C measurement.  From the carbon frequencies, one can determine all of the double quantum frequencies as shown in the table below, taking into account the ¹³C offset frequency expressed in ppm, 'o1p'.

Those  highlighted in pink are those that are present in the 1,1-ADEQUATE spectrum which is shown below.

The spectrum was acquired on a concentrated sample at 600 MHz with a cryoprobe using the standard 'adeq11etgpsp' Bruker pulse program .  The total data collection time was less than 1 hour.  The carbon-carbon connectivity is labelled on the spectrum based on the double quantum frequencies using the numbering scheme from the ¹³C spectrum presented as the projection on the edited HSQC spectrum above.  From these connectivities, the structure of the compound can unambiguously be assigned to limonene.

2D 13C INADEQUATE

The 2D ¹³C INADEQUATE (Incredibe Natural Abundance DoublE QUAmtum Transfer Experiment) is undoubtedly one of the most definitive, yet under-used NMR techniques able to assign chemical structures of small organic molecules.  It gives a connectivity map for all carbon atoms in the molecule.  The reason that it is so under-used is that it relies on one bond ¹³C - ¹³C coupling therefore, adjacent carbon atoms must both be of the ¹³C isotope.  Since ¹³C is only 1.1% naturally abundant, the chance of having two adjacent ¹³C atoms is approximately 1 in 8300, reducing the sensitivity of the measurement drastically.  As a result, 2D INADEQUATE spectra can only be run on very concentrated samples.  The sensitivity afforded by high magnetic field strengths and cryogenically cooled probes has certainly made these measurements more accessible than they have been in the past and they may be within reach when sample quantity and solubility are not a problem.  The 2D ¹³C INADEQUATE spectrum of ~450 mg of limonene in benzene-d6 was acquired on a Bruker TCI H/C/N cryoprobe at 600 MHz and is shown in the figure below.

The spectrum was acquired with the gradient version of the INADEQUATE pulse sequence using a shaped refocusing pulse (Bruker pulse program inadgpqfsp).  It was acquired in 11.8 hours with 64 scans for each of 128 increments using a 5 second recycle delay. The proton decoupled ¹³C spectrum is in the horizontal F2 domain.  The spectrum is interpreted by locating the vertical cross peaks for each ¹³C resonance.  Each cross peak has a partner peak along the horizontal axis.  The partner peak lies on the same vertical axis as the carbon atom bonded to the initial carbon.  This is shown in the figure for C1 which is bonded to C2, C3 and C4.  The entire carbon skeleton of the molecule can be traced unambiguously in this manner to provide a complete assignment.  The same sample was run under the same conditions in 45 minutes with only 4 scans.  A comparison of the signal-to-noise ratio for both data sets is shown in the figure below.

It is clear that usable 2D INADEQUATE data can be acquired in less than an hour for extremely concentrated samples at high field with a cryoprobe.