Name: a new straightforward method for lipophilicity (logp) measurement using 19f nmr spectroscopy
Uploaded: 2019-01-30T13:30:00
Description: Watch this Scientific Journal Video about A New Straightforward Method for Lipophilicity logP Measurement using 19F NMR Spectroscopy at JoVE.com

This research is funded as part of EPSRC grants EP/K016938/1 and EP/P019943/1 (ZW, HRF) and of an EPSRC/AstraZeneca CASE conversion award (BFJ). The University of Southampton is thanked for additional support. The EPSRC is further thanked for a core capability grant EP/K039466/1.

1. Partitioning
<ol>
	<li>Add 4,4,4-trifluorobutan-1-ol (compound X, ca. 6.0 mg) and 2,2,2-trifluoroethanol (reference compound, ca. 3.0 mg) to a 10 mL pear-shaped flask, dissolve in n-octanol (HPLC grade, ca. 2 mL), and add water (HPLC grade, ca. 2 mL). 
	Note: This experiment is run in triplicate. Compound solubility in water and n-octanol must be checked. The amount of the compound used for partition must be carefully considered to avoid oversaturation of the compound in any layer. The mass ra...

<ol>
	<li>Arnott, J. A., Planey, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+lipophilicity+in+drug+discovery+and+design.">The influence of lipophilicity in drug discovery and design.</a> Expert Opinion on Drug Discovery. 7 (10), 863-875 (2012).</li><li>Lipinski, C. A., Lombardo, F., Dominy, B. W., Feeney, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+and+computational+approaches+to+estimate+solubility+and+permeability+in+drug+discovery+and+development+settings.">Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings.</a> Advanced Drug Delivery Reviews. 23 (1), 3-25 (1997).</li><li>Leeson, P. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipophilicity+in+drug+discovery.">Lipophilicity in drug discovery.</a> Expert Opinion on Drug Discovery. 5 (3), 235-248 (2010).</li><li>Linclau, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+the+Influence+of+(Deoxy)fluorination+on+the+Lipophilicity+of+Non-UV-Active+Fluorinated+Alkanols+and+Carbohydrates+by+a+New+log+P+Determination+Method.">Investigating the Influence of (Deoxy)fluorination on the Lipophilicity of Non-UV-Active Fluorinated Alkanols and Carbohydrates by a New log P Determination Method.</a> Angewandte Chemie International Edition. 55 (2), 674-678 (2016).</li><li>Derome, A. 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The protocol described in the paper is a straightforward method for logP measurement of fluorinated compounds. This method is applicable to fluorinated compounds with a logP value from -3 to 3. For more hydrophilic (logP &#60; -3) or lipophilic compounds (logP &#62; 3), this method can still be used but will require much longer NMR experiment time as extended number of transients are needed to obtain a good signal-to-noise ratio. Hence, this is a limitation of the method. There is no r...

Lipophilicity is a key physicochemical parameter of drug molecules which influences the properties of drug candidates in many aspects, including drug solubility, bioavailability, and toxicity1. Lipophilicity is measured as the logarithm (logP) of the ratio of compound concentrations after partitioning between n-octanol and water. Optimal lipophilicity ranges have been proposed based on statistical data of orally administered drugs, of which the Lipinski&#39;s &#34;rule of 5&#39;&#39; is the most famous example2,3. Indeed, controlling lipophilicity has shown to be essential for improving the prospect of drug candidates. Increasing drug binding affinity by elevated lipophilicity has been identified as one of the main problems in drug discovery projects during the past few decades, leading to increased attrition rates3. Therefore, it has been suggested that successful drug development is associated with keeping the molecular lipophilicity of the drug candidates within optimal boundaries during the affinity optimization process3,4. In that regard, new concepts (such as lipophilic efficiency indices) have been introduced5,6.
It is thus of great importance to accurately measure lipophilicity during the drug development process. Besides, the availability of straightforward methods for lipophilicity measurement is in demand as fundamental research aims to identify solutions for logP modulation. Currently, numerous established methods are accessible for lipophilicity determination1. The standard &#39;shake-flask (SF)&#39; method7and its variations are commonly employed to measure logP values directly, which in most cases depend on UV-Vis spectroscopy for quantification. The main disadvantage of this classic SF method is its labor-intensive nature. In addition, the formation of emulsions may occur, especially for highly lipophilic compounds8,9.Several methods were developed to circumvent such issues, such as by using flow injection analysis, dialysis tubing, etc.9,10.However, none of those methods are straightforward or easily applicable in non-specialized laboratories.
There are also many indirect methods available for use, such as potentiometric titration11, electrophoretic methods12,13, RP-HPLC-based chromatographic methods, mass-spectrometry-based methods14, etc. These are indirect methods, as the logP values are obtained by calibration curves. Among these methods, the RP-HPLC method has been widely used because it is user-friendly and time-saving. Nevertheless, its accuracy depends on the training set used to establish the calibration curve, and the estimated lipophilicity depends on the partition system used13,15.
There are a number of 1H NMR-based methods reported in the literature for lipophilicity determination. Mo et al. developed a method for logP measurement using 1H NMR without deuterated solvents. Water and octanol, as partition solvents, were used as references for the quantification of solute concentration in each phase16. Herth and co-workers also reported an approach, by which the partition experiment occurred directly in an NMR tube, where the NMR data of the bottom D2O aqueous layer were collected before and after the extraction with 1-octanol, to obtain the distribution coefficient17. In addition, Soulsby et al. exploited 1H NMR as an analysis tool, determining the amplitude of signals by using complete reduction to amplitude-frequency table software. The ratio of the amplitudes in both layers led to the measured partition coefficient18. These methods are relatively simple to use but often require the calibration of selective pulses and power levels or the use of shaped gradient pulses to ensure appropriate solvent suppression and signal selectivity.
Calculated logP (clogP) values for compounds can also be obtained. Several calculation methods and commercially available software are available. Such clogP values are commonly used in the pharmaceutical industry when evaluating large numbers of drug molecules. However, large errors from clogP values are not uncommon19,20.
The requirements of UV-activity for concentration analysis and the establishment of calibration curves for logP calculation impede research progress in this field. In particular, this is the case for non-UV-active aliphatic compounds. Fluorinated aliphatic moieties have become increasingly attractive for drug design in recent years, and their influence on overall lipophilicity of the compound is a research topic in our group21. In addition, 19F is a highly sensitive NMR-active nucleus, making 19F NMR a useful tool for analyzing fluorinated compounds. It also has a larger chemical shift range compared to that of 1H. Therefore, it is worthwhile to develop a straightforward method for logP determination of non-UV-active fluorinated compounds by 19F NMR spectroscopy. Hence, the overall goal of this method is to achieve convenient lipophilicity determination of fluorinated compounds.
The key principle of our 19F NMR-based method is to add a fluorinated reference compound in the partition experiment (Figure 1)21. Compound X and reference compound (ref) are partitioned between water and n-octanol. After equilibrating, an aliquot from each phase is taken into an NMR tube, and 19F NMR experiments are run on both NMR samples. The intensity of the fluorine peaks is proportional to compound concentration (C) and the number of fluorine atoms (n) of the compounds. Between compound X and ref, integral ratios can be obtained for both phases. The ratio in n-octanol layer is defined as &#961;oct, and &#961;aq for water layer (eq. 1). The ratio of &#961; values equals the ratio of partition coefficients (P) of compound X and ref (eq. 2). This leads to the final equation (eq. 4) for logP measurement of compound X. Therefore, in order to determine the logP value of an unknown compound X, only integration ratios (&#961;oct and &#961;aq) in both layers are needed to be measured by 19F NMR.

<table>
	<tbody>
		<tr>
			<td>NMR (400 MHz) with Bruker 5 mm SEF probe</td>
			<td>Bruker</td>
			<td>n/a</td>
			<td>AVIIIHD400</td>
		</tr>
		<tr>
			<td>NMR (400 MHz) with Bruker 5 mm SMART probe</td>
			<td>Bruker</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>DrySyn Snowstorm reactor</td>
			<td>Asynt</td>
			<td>ADS13-S</td>
			<td></td>
		</tr>
		<tr>
			<td>recirculating chiller</td>
			<td>Asynt</td>
			<td>n/a</td>
			<td>model:Grant-LTC2</td>
		</tr>
		<tr>
			<td>magnetic stirplate</td>
			<td>Asynt</td>
			<td>ADS-HP-NT</td>
			<td></td>
		</tr>
		<tr>
			<td>ACD/NMR processor software</td>
			<td>ACD/Labs</td>
			<td>n/a</td>
			<td>ACD/NMR processor academic edition or ACD/Spectrus processor 2015</td>
		</tr>
	</tbody>
</table>

a new straightforward method for lipophilicity (logp) measurement using 19f nmr spectroscopy

Fluorination has become an effective tool to optimize physicochemical properties of bioactive compounds. One of the applications of fluorine introduction is to modulate the lipophilicity of the compound. In our group, we are interested in the study of the impact of fluorination on lipophilicity of aliphatic fluorohydrins and fluorinated carbohydrates. These are not UV-active, resulting in a challenging lipophilicity determination. Here, we present a straightforward method for the measurement of lipophilicity of fluorinated compounds by 19F NMR spectroscopy. This method requires no UV-activity. Accurate solute mass, solvent and aliquot volume are also not required to be measured. Using this method, we measured the lipophilicities of a large number of fluorinated alkanols and carbohydrates.

Two sets of data as control experiments are shown in Figure 221. Using 2,2,2-trifluoroethanol as reference compound, logP values were obtained for 2-fluoroethanol and 3,3,3,2,2-pentafluoropropanol as -0.75 and +1.20, respectively (Figure 2A). Subsequently, the lipophilicity of 2-fluoroethanol was determined again but with 3,3,3,2,2-pentafluoropropanol as the reference (using its previous experimen...

Watch this Scientific Journal Video about A New Straightforward Method for Lipophilicity logP Measurement using 19F NMR Spectroscopy at JoVE.com

A New Straightforward Method for Lipophilicity logP Measurement using 19F NMR Spectroscopy

A novel and straightforward variation of the shake-flask method was developed for accurate lipophilicity measurement of fluorinated compounds by 19F NMR spectroscopy.

A New Straightforward Method for Lipophilicity (logP) Measurement using 19F NMR Spectroscopy

Fluorination has become an effective tool to optimize physicochemical properties of bioactive compounds. One of the applications of fluorine introduction ...

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.

Research

JoVE Journal

Chemistry

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

Dependence of Laser-induced Breakdown Spectroscopy Results on Pulse Energies and Timing Parameters Using Soil Simulants

The  dependence of some LIBS detection capabilities on lower pulse energies (&lt;100  mJ) and timing parameters were examined using synthetic silicate ...

Facile Synthesis of Worm-like Micelles by Visible Light Mediated Dispersion Polymerization Using Photoredox Catalyst

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins ...

Spin Saturation Transfer Difference NMR (SSTD NMR): A New Tool to Obtain Kinetic Parameters of Chemical Exchange Processes

Spin Saturation Transfer Difference NMR SSTD NMR: A New Tool to Obtain Kinetic Parameters of Chemical Exchange Processes

This detailed protocol describes the new Spin Saturation Transfer Difference Nuclear Magnetic Resonance protocol (SSTD NMR), recently developed in our ...

A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography (HPLC)

A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography HPLC

Glucosinolates are a well-studied and highly diverse class of natural plant compounds. They play important roles in plant resistance, rapeseed oil ...

NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential ...

Fabrication and Testing of Photonic Thermometers

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being ...

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the ...

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

Electrochemical Impedance Spectroscopy as a Tool for Electrochemical Rate Constant Estimation

Electrochemical impedance spectroscopy (EIS) was used for advanced characterization of organic electroactive compounds along with cyclic voltammetry (CV). ...