We have developed a straightforward method for lipophilicity measurement of frenetic compounds using fluorine NMR which is acrid enough to reproduce we measure small differences in lipophilicity. This method can be used to systematically study the impact of formation of organic compounds on the resolved changing lipophilicity changes, which is of interest in general medicinal chemistry. Advantages of this method include that compounds do not have to be reactive, and they do not have to be completely pure.
There is no need to measure exact mass or volumes, and there is no need to establish calibration curves. An appropriate Reference Compound should be chosen, and cross-contamination of the aliquot should be avoided during the sampling phase. In this video we will demonstrate appropriate practical skills for sampling, NMR tube sealing, and correct use of NMR software for data processing.
Add 6.0 milligrams of Compound X and 3.0 milligrams of the Reference Compound to a 10 milliliter pear-shaped flask. Dissolve the compounds in approximately two milliliters of HPLC-grade n-Octanol, and add two milliliters of HPLC-grade water. Place the flasks inside a temperature controlled receptacle above a stir plate, and connect to a recirculating chiller.
Stir the biphasic mixture at 25 degrees Celsius for two hours, with stirring speed set at 600 RPM. Equilibrate the mixture at 25 degrees Celsius overnight to allow for complete phase separation. Fix the flask to a retort stand with a clamp.
Take an aliquot of approximately 0.70 to 0.85 milliliters from both water and n-Octanol layers by using one milliliter disposable plastic syringes with long needles. For taking the water aliquot, draw approximately 0.02 milliliters of air into the syringe before putting the needle into the mixture. While moving the needle through the upper n-Octanol layer into the water layer, gently push out the air to prevent the n-Octanol solution from entering the needle.
Remove the long needle from the mixture. Discard a small amount of water sample, leaving approximately 0.6 milliliters of sample left in the syringe. Carefully wipe the needle with dry tissue, and inject approximately 0.5 milliliters of the water sample into a clean NMR tube.
Quickly close the NMR tube with a cap. For the n-Octanol sample, remove the long needle from the n-Octanol layer. Discard a small amount of n-Octanol sample, leaving approximately 0.6 milliliters of sample left in the syringe.
After carefully wiping the needle with dry tissue, inject approximately 0.5 milliliters of the n-Octanol sample into a clean NMR tube. Quickly close the NMR tube with a cap. Visually inspect both n-Octanol and water samples for any contamination from the other phase.
To each NMR tube, add 0.1 milliliters of a deuterated NMR solvent that is miscible with both n-Octanol and water, to enable signal lock during NMR acquisition. For compounds with low boiling points, seal the NMR tubes using a blowtorch, and after cooling invert the tube to check for any leaks. Carefully invert the sealed or non-sealed NMR tubes 20 times to obtain a homogenous solution for F19 NMR experiments.
Run proton decoupled fluorine NMR experiments to identify chemical shifts of Compound X and the Reference Compound in both n-Octanol and water NMR samples. Use standard NMR parameter settings as listed in the text protocol. Measure the spin-lattice relaxation time, or T1, for diagnostic fluorine nuclei by using an inversion recovery sequence.
Gauge the level of appropriate pulse delay time from the obtained T1 values for accurate quantitative NMR integration. Run a quick F19 H1 spectrum to set up the initial parameters. Measure the 90 degree pulse width, then set the parameter for the T1 measurement experiment, and run the T1 measurement experiment using an inversion recovery sequence.
Run proton decoupled fluorine NMR experiments again with adjusted parameter settings. Set D1 and center the frequency offset point between the two diagnostic fluorine signals so that both nuclei can be equally excited. Also, set the spectral width as 300 PPM, and set the number of transients to 64.
Adjust these values if a higher signal-to-noise ratio is required. Process the obtained data using ACD NMR Processor Academic Edition, or other custom NMR processing software. Open the NMR data file.
Then, open the pdata folder, followed by the folder 1. Delete the 1r file. Return to the NMR data file, and drag the FID file into the ACD NMR Processor window.
Click the window function button, select exponential, set line broadening value as two, and click the okay button. Now, click the zero filling button, increase the points count to four times of its original points count by clicking a small button next to the number, and click the okay button. Select the fourier transform button.
Next, click the phase button, and then click the mouse phasing button. Click and hold the left mouse button, and proceed to move the mouse forward or backward till the major peak of the spectrum is properly phased. Now, click and hold the right mouse button and move the mouse forward or backward until the other peaks of the spectrum are properly phased.
Then, un-click the mouse phasing button and zoom into the spectral area with the fluorine peaks. Click fine tuning, perform the phase correction if needed as described earlier, and then click the tick button. Click the baseline button, and then the options button.
Select the spectrum averaging for automatic models, adjust the number of points for box half width if needed. Click okay, auto, and then click the tick button. Next, click integration, integrate the diagnostic fluorine peaks, and click the tick button.
Finally, obtain the integration ratios from n-Octanol and water NMR samples and use them in the logP calculation equation to obtain the logP value of Compound X.Using 2, 2, 2-Trifluoroethanol as a Reference Compound, a logP value of 0.75 was obtained for two fluoroethanol, and a logP value of positive 1.20 was found for 3, 3, 3, 2, 2-pentafluoropropanol. Subsequently, the lipophilicity of two fluoroethanol was determined again, but with 3, 3, 3, 2, 2-pentafluoropropanol as the reference. The measured logP value was 0.76, which only had a difference of 0.01 logP units when compared with the value measured using 2-2-2-trifluoroethanol as reference.
Likewise, for cis-2, 3-difluro-1, 4-butane-diol, the difference in measured logP values by using two fluoroethanol and its trans-isomer are very small. This demonstrates good reproducibility, and that the selection of Reference Compound does not have impact on the logP measurement. Additional selected examples are shown here.
All of these non-UV active allophatic compounds, from fluorinated carbohydrates, to fluorohydrins, can be easily measured with this method. Other proto NMR-based methods can be used if your compound does not contain fluorine, and if the compound contains UV Chromofor, then the logP measurement can be carried out using UV quantification. The lipophilicity of a wide range of non-UV active fluorohydrins and deoxifluonated carbohydrates was measured.
The obtained data library was used to establish trends and rules for the impact of aliphatic fluorination on lipophilicity. Octanol and water pose minimal safety hazards. Of course, the hazards that are measured and the Reference Compound should always be taken into consideration.
