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 ratio between compound X and reference compound ref must also be considered to avoid that the integral ratios of a given NMR sample are outside a 10/1 to 1/10 range. For example, if there is a difference of &#60;2 logP units between compound X and ref, optimal mass ratio can assure that integration ratios in water and 1-octanol NMR samples are within a 10/1 to 1/10 range. In contrast, if an integration ratio of 50/1 in one layer is obtained, there will be more likely relatively larger errors in the integration for the peak with lower concentration. The equation below can be used to predict optimal compound mass ratio: 
	mX / mref = {(cPX / Pref)-0.5 * (MX/ Mref) * [(1 + cPX) / (1 + Pref)]} / (NX / Nref) 
	m, mass; M, molecular mass; N, number of F atoms; P, partition coefficients; cP, calculated partition coefficients.</li>
	<li>Place the flasks inside a temperature-controlled receptacle above a stirplate, and connect to a recirculating chiller. Stir the biphasic mixture at 25 &#176;C for 2 h, with stirring speed set at 600 rpm.</li>
	<li>Equilibrate the mixture at 25 &#176;C overnight (ca. 16 h), to allow for complete phase separation. 
	Note: In some cases, the formation of a foam between the n-octanol and water boundary can be observed. In this case, the mixture was transferred into a 4 mL glass vial and centrifuged till the disappearance of the foam. The biphasic mixture was then left to equilibrate again at 25 &#176;C overnight.</li>
</ol>
2. NMR Sample Preparation
<ol>
	<li>Fix the flask to a retort stand with a clamp.</li>
	<li>Take an aliquot of ca. 0.70-0.85 mL from both water and n-octanol layers, by using 1 mL disposable plastic syringes with long needles.
	<ol>
		<li>For taking the water aliquot, draw ca. 0.02 mL of air into the syringe before putting the needle into the mixture. While moving the needle through the upper n-octanol layer into water layer, gently push out the air to prevent n-octanol solution from entering the needle.</li>
		<li>Remove the long needle from the mixture. Discard a small amount of water sample, leaving ca. 0.6 mL of sample left in the syringe. Carefully wipe the needle with dry tissue, and inject ca. 0.5 mL of water sample into a clean NMR tube. Quickly close the NMR tube with a cap.</li>
		<li>For the n-octanol sample, remove the long needle from the n-octanol layer. Discard a small amount of n-octanol sample, leaving ca. 0.6 mL of sample left in the syringe. Carefully wipe the needle with dry tissue and inject ca. 0.5 mL of n-octanol sample into a clean NMR tube. Quickly close the NMR tube with a cap.</li>
	</ol>
	</li>
	<li>Visually inspect both n-octanol and water samples for any contamination (e.g., small droplets of n-octanol in water sample or small droplets of water in n-octanol sample). 
	Note: If there is any contamination, the aliquot sample needs to be re-prepared from the biphasic mixture. As the measurement is done in triplicate, six NMR tubes are obtained.</li>
	<li>To each NMR tube, add 0.1 mL of a deuterated NMR solvent that is miscible with both n-octanol and water (e.g., acetone-d6) to enable signal lock during NMR acquisition.</li>
	<li>For compounds with low boiling points (e.g., &#60;120 &#176;C), seal the NMR tubes using a blow torch, 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 19F NMR experiments.</li>
</ol>
3. NMR Experiments
<ol>
	<li>Run, using standard NMR parameter settings (NS 64, D1 1 s, SW 300 ppm, O1P -100 ppm), 19F{1H} NMR experiments to identify chemical shifts of 4,4,4-trifluorobutan-1-ol (compound X) and 2,2,2-trifluoroethanol (reference compound) in both n-octanol and water NMR samples.</li>
	<li>Measure the spin-lattice relaxation time (T1) for diagnostic fluorine nuclei by using an inversion-recovery sequence22. Gauge the level of appropriate pulse delay time (D1, set as &#8805; 5*T1) from the obtained T1 values for accurate quantitative NMR integration. 
	Note: This is very time-consuming, but a D1 of 60 s for the water phase sample, and of 30 s for the octanol phase sample, are conservative settings which will safely fulfill the D1 &#8805; 5*T1 criterium.</li>
	<li>Run 19F{1H} NMR experiments again with adjusted parameter settings as follows: a) use D1 &#8805; 5*T1; b) center the frequency offset point (O1P) between the two diagnostic fluorine signals so both nuclei can be equally excited; c) Set the spectral width (SW) as 300 ppm, but reduce if a better SNR ratio if needed; d) Set the number of transients (NS) as 64 but increase if higher SNR is required. 
	Note: Non-decoupled 19F NMR experiments can be also used for NMR data acquisition. However, proton-decoupled 19F NMR experiments are preferred here as it simplifies the fluorine signals by removing proton-fluorine couplings which also increases signal-to-noise ratio. We use inverse-gated decoupling to obtain a decoupled spectrum without nOe (nuclear Overhauser effect) enhancements23. For quantitative integration, a signal-to-noise ratio (&#8805;300) is desired.24</li>
</ol>
4. Data Processing
<ol>
	<li>Process the obtained data using ACD/NMR Processor Academic Edition or other custom NMR processing software.
	<ol>
		<li>Open the NMR data file, then open the pdata folder, followed by folder 1. Delete the 1r file.</li>
		<li>Return to the NMR data file and drag the fid file into the ACD/NMR Processor window.</li>
		<li>Click the WFunctions button, select Exponential, set LB value as 2, and click the OK button.</li>
		<li>Click the Zero Filling button, increase the Points Count to 4 times of its Original Points Count by clicking a small button next to the number, and click the OK button.</li>
		<li>Click the Fourier Tr. button.</li>
		<li>Click the Phase button, then click the Mouse Ph. button, click and hold the left mouse button, move the mouse forward or backward till the major peak of the spectrum is properly phased.
		<ol>
			<li>Click and hold the right mouse button, move the mouse forward or backward until the other peak(s) of the spectrum is properly phased. Then unclick the Mouse Ph. button, zoom into the spectral area with the fluorine peaks, click Fine Tuning, perform the phase correction if needed as described earlier till all peaks are correctly phased, and then click the Tick button.</li>
		</ol>
		</li>
		<li>Click the Baseline button, then the Options button. Select Spectrum Averaging for Automatic Models, adjust the number of points for Box Half Width if needed (in particular for spectrum with low S/R ratio), click OK | Auto, and then click the Tick button.</li>
		<li>Click Integration, integrate the diagnostic fluorine peaks, and click the Tick button. 
		Note: If the integral curve is not parallel to the baseline, click the Bias Corr. button, and adjust the tilt and slope until the curve is parallel to the baseline.</li>
	</ol>
	</li>
	<li>Obtain the integration ratios from n-octanol and water NMR samples and use in the logP calculation equation (Figure 1, eq. 4) to obtain the logP value of of 4,4,4-trifluorobutan-1-ol (compound X).</li>
</ol>

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The authors have nothing to disclose.

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 requirement for the frequency of NMR spectrometer, as long as the conditions (NMR parameter settings and sufficient SNR) for quantitative integration are met. As for any shake flask method, it is critical to avoid oversaturation and contamination during the layer sampling.
Compared with previous shake-flask method and its variations, there are several advantages in our method with respect to existing methods. 1) Measurements of solute mass, volume of partition solvents and aliquots for NMR sample are not necessary. 2) The compound for measurement can be impure provided that the fluorine chemical shifts of the impurities are different from that of the measured compound. 3) Because of the intrinsic compensation effect when working with the ratio of a ratio, systematic errors are eliminated. 4) This method is applicable to non-UV-active fluorinated compounds. 5) This method is easy to use with open-access NMR facilities as no special NMR settings are needed (such as solvent suppression, applying a small excitation angle, etc.).
Currently, we are using this method to measure the lipophilicities of fluorinated carbohydrates, fluorohydrins and fluorinated amides, in order to investigate the influence of fluorination on lipophilicity and to identify fluorinated moieties with lipophilicity-lowering effect. Method development for logP measurement of more lipophilic compounds (logP &#62;3) and for fluorinated amines is ongoing in our group.
It can be pointed out that 19F NMR can also be used for critical micelle concentration (CMC) determination30.

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 experimentally measured logP value +1.20). 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-difluoro-1,4-butanediol, the difference in measured logP values by using 2-fluoroethanol and its trans isomer is also very small (0.01 logP units, Figure 2B). This demonstrated that the selection of reference compound does not have impact on the logP measurement. In addition, a rather small standard deviation (&#60;0.01) indicated good reproducibility of our method.
Using our method, a series of compounds with known logP values was measured as shown in Table 1. The difference between literature data and the values measured using our method is shown in the last column of the Table. Overall, the experimentally obtained logP values (at 25 &#176;C) have good to excellent accordance with the literature values, which further validated our method.
Additional selected examples21 were shown in Figure 3. All these non-UV-active aliphatic compounds (from fluorinated carbohydrates to fluorohydrins) can be easily measured with our method.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58567/58567fig1.jpg" /> 
Figure 1: Principle of the logP determination method. This figure has been reproduced with permission from Wiley-VCH Verlag GmbH &#38; Co. KGaA.21. This shake-flask method is based on 19F NMR spectroscopy. A reference compound is used for partition experiment. Aliquots for both n-octanol and water phase were taken for NMR experiment. Integration ratios between reference compound and the compound to be measured are obtained for the determination of logP value. Detailed mathematical deduction of equations, which leads to the final equation for measurement, are also given. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58567/58567fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58567/58567fig2.jpg" /> 
Figure 2: Examples for internal validation21. Two sets of control experiments, using two different reference compounds to measure logP value of one compound, were conducted. The logP difference between those experiments is negligible. Standard deviation (&#60;0.01) from experiments run in triplicate shows good reproducibility of the method. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58567/58567fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58567/58567fig3.jpg" /> 
Figure 3: Further selected examples of logP measurement using our method. Applying this method, logP values for 8 fluorinated compounds (such as fluorinated carbohydrates, acyclic alkanols and conformationally restricted fluorohydrins) were obtained. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58567/58567fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Comparison between literature data and the experimental logP values using our method21. logP values for 14 fluorinated compounds (with known logP data) were measured using this new method. The reference compounds used for each measurement were also tabulated. Comparision (logP) between literature values and logP results from our method demonstrated good accuracy of this method. a2,2,2-Trifluoroethanol (TFE), 2-Fluoroethanol (FE); bAveraged logP value from at least three experiments; cExperimentally measured logP value by our method (-0.75) was used as reference. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58567/Table1.xlsx">Please click here to download this file.</a>

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 ...

a-new-straightforward-method-for-lipophilicity-logp-measurement-using

Nanyang Technological University

Research

JoVE Journal

Chemistry

14.2K Views.  University of Southampton. A novel and straightforward variation of the shake-flask method was developed for accurate lipophilicity measurement of fluorinated compounds by 19F NMR spectroscopy.

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

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 reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

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

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

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 samples. These samples were used as simulants for soil and contained minor and trace elements commonly found in soil at a wide range of concentrations. For this study, over 100 calibration curves were prepared using different pulse energies and timing parameters; detection limits and sensitivities were determined from the calibration curves. Plasma temperatures were also measured using Boltzmann plots for the various energies and the timing parameters tested. The electron density of the plasma was calculated using the full-width half maximum (FWHM) of the hydrogen line at 656.5 nm over the energies tested. Overall, the results indicate that the use of lower pulse energies and non-gated detection do not seriously compromise the analytical results. These results are very relevant to the design of field- and person-portable LIBS instruments.

LIBS detection capabilities on soil simulants were tested using a range of pulse energies and timing parameters. Calibration curves were used to determine detection limits and sensitivities for different parameters. Generally, the results showed that there was not a significant reduction in detection capabilities using lower pulse energies and non-gated detection.

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 with the synthesis of a hydrophilic poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) homopolymer using reversible addition-fragmentation chain-transfer (RAFT) polymerization. Under mild visible light irradiation (&#955; = 460 nm, 0.7 mW/cm2), this macro-chain transfer agent (macro-CTA) in the presence of a ruthenium based photoredox catalyst, Ru(bpy)3Cl2 can be chain extended with a second monomer to form a well-defined block copolymer in a process known as Photoinduced Electron Transfer RAFT (PET-RAFT). When PET-RAFT is used to chain extend POEGMA with benzyl methacrylate (BzMA) in ethanol (EtOH), polymeric nanoparticles with different morphologies are formed in situ according to a polymerization-induced self-assembly (PISA) mechanism. Self-assembly into nanoparticles presenting POEGMA chains at the corona and poly(benzyl methacrylate) (PBzMA) chains in the core occurs in situ due to the growing insolubility of the PBzMA block in ethanol. Interestingly, the formation of highly pure worm-like micelles can be readily monitored by observing the onset of a highly viscous gel in situ due to nanoparticle entanglements occurring during the polymerization. This process thereby allows for a more reproducible synthesis of worm-like micelles simply by monitoring the solution viscosity during the course of the polymerization. In addition, the light stimulus can be intermittently applied in an ON/OFF manner demonstrating temporal control over the nanoparticle morphology.

This article describes a process for producing polymeric self-assembled nanoparticles using visible light mediated dispersion polymerization. Using low energy visible light to control the polymerization allows for the reproducible formation of self-assembled worm-like micelles at high solids content.

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

This detailed protocol describes the new Spin Saturation Transfer Difference Nuclear Magnetic Resonance protocol (SSTD NMR), recently developed in our group to study processes of mutual-site chemical exchange that are difficult to analyze by traditional methods. As the name suggests, this method combines the Spin Saturation Transfer method used for small molecules, with the Saturation Transfer Difference (STD) NMR method employed for the study of protein-ligand interactions, by measuring transient spin saturation transfer along increasing saturation times (build-up curves) in small organic and organometallic molecules undergoing chemical exchange.
Advantages of this method over existing ones are: there is no need to reach coalescence of the exchanging signals; the method can be applied as long as one signal of the exchanging sites is isolated; there is no need to measure T1 or reach steady state saturation; rate constant values are measured directly, and T1 values are obtained in the same experiment, using only one set of experiments.
To test the method, we have studied the dynamics of the hindered rotation of N,N-dimethylamides, for which much data is available for comparison. The thermodynamic parameters obtained using SSTD are very similar to the reported ones (spin-saturation transfer techniques and line-shape analysis). The method can be applied to more challenging substrates that cannot be studied by previous methods.
We envisage that the simple experimental set up and the wide applicability of the method to a great variety of substrates will make this a common technique amongst organic and organometallic chemists without extensive expertise in NMR.

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

A detailed protocol describing the SSTD NMR method is presented here to help new users apply this new method to obtain the kinetic parameters of their own systems undergoing chemical exchange.

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)

Glucosinolates are a well-studied and highly diverse class of natural plant compounds. They play important roles in plant resistance, rapeseed oil quality, food flavoring, and human health. The biological activity of glucosinolates is released upon tissue damage, when they are mixed with the enzyme myrosinase. This results in the formation of pungent and toxic breakdown products, such as isothiocyanates and nitriles. Currently, more than 130 structurally different glucosinolates have been identified. The chemical structure of the glucosinolate is an important determinant of the product that is formed, which in turn determines its biological activity. The latter may range from detrimental (e.g., progoitrin) to beneficial (e.g., glucoraphanin). Each glucosinolate-containing plant species has its own specific glucosinolate profile. For this reason, it is important to correctly identify and reliably quantify the different glucosinolates present in brassicaceous leaf, seed, and root crops or, for ecological studies, in their wild relatives. Here, we present a well-validated, targeted, and robust method to analyze glucosinolate profiles in a wide range of plant species and plant organs. Intact glucosinolates are extracted from ground plant materials with a methanol-water mixture at high temperatures to disable myrosinase activity. Thereafter, the resulting extract is brought onto an ion-exchange column for purification. After sulfatase treatment, the desulfoglucosinolates are eluted with water and the eluate is freeze-dried. The residue is taken up in an exact volume of water, which is analyzed by high-pressure liquid chromatography (HPLC) with a photodiode array (PDA) or ultraviolet (UV) detector. Detection and quantification are achieved by conducting comparisons of the retention times and UV spectra of commercial reference standards. The concentrations are calculated based on a sinigrin reference curve and well-established response factors. The advantages and disadvantages of this straightforward method, when compared to faster and more technologically advanced methods, are discussed here.

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

Here, we describe in great detail an established and robust protocol for the extraction of glucosinolates from ground plant materials. After an on-column sulfatase treatment of the extracts, the desulfoglucosinolates are eluted and analyzed by high-pressure liquid chromatography.

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 nutrients. The objective of this work is to demonstrate the qualitative and quantitative analysis of encapsulated fish oil supplements using high-resolution 1H and 13C NMR spectroscopy utilizing two different NMR instruments; a 500 MHz and an 850 MHz instrument. Both proton (1H) and carbon (13C) NMR spectra can be used for the quantitative determination of the major constituents of fish oil supplements. Quantification of the lipids in fish oil supplements is achieved through integration of the appropriate NMR signals in the relevant 1D spectra. Results obtained by 1H and 13C NMR are in good agreement with each other, despite the difference in resolution and sensitivity between the two nuclei and the two instruments. 1H NMR offers a more rapid analysis compared to 13C NMR, as the spectrum can be recorded in less than 1 min, in contrast to 13C NMR analysis, which lasts from 10 min to one hour. The 13C NMR spectrum, however, is much more informative. It can provide quantitative data for a greater number of individual fatty acids and can be used for determining the positional distribution of fatty acids on the glycerol backbone. Both nuclei can provide quantitative information in just one experiment without the need of purification or separation steps. The strength of the magnetic field mostly affects the 1H NMR spectra due to its lower resolution with respect to 13C NMR, however, even lower cost NMR instruments can be efficiently applied as a standard method by the food industry and quality control laboratories.

Here, high-resolution 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy was used as a rapid and reliable tool for quantitative and qualitative analysis of encapsulated 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 leveraged for developing sophisticated photonic sensors. Silicon photonic sensors seek to exploit the strong confinement of light in nano-waveguides to transduce changes in physical state to changes in resonance frequency. In the case of thermometry, the thermo-optic coefficient, i.e., changes in refractive index due to temperature, causes the resonant frequency of the photonic device such as a Bragg grating to drift with temperature. We are developing a suite of photonic devices that leverage recent advances in telecom compatible light sources to fabricate cost-effective photonic temperature sensors, which can be deployed in a wide variety of settings ranging from controlled laboratory conditions, to the noisy environment of a factory floor or a residence. In this manuscript, we detail our protocol for the fabrication and testing of photonic thermometers.

We describe the process of 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

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 average content of fluorinated drugs in their formulated drug product forms: tablets or capsules. This method is specific to fluorinated drugs because it detects only the content of fluorine, avoiding interference from the excipients that lack fluorine. The advantages of measuring the active content of fluorinated drugs using low-field 19F TD-NMR versus high-field 19F solid-state (SS) NMR are the simplicity of the method; the low cost; and the non-destructive nature of the technique, with all samples recoverable in intact forms (e.g., powders, tablets, and capsules), making this technique affordable for any laboratory.
We have tested the method with three fluorinated drug products available on the market - cinacalcet, lansoprazole, and ciprofloxacin - with doses ranging from 15 to 500 mg. The results of the analyses, measured by low-field 19F TD-NMR, supported the reported label claims for the average drug content.
Based on the simplicity and reproducibility of the analysis, we envision this methodology being implemented in any laboratory, including manufacturing plants, as a process analytical technology (PAT) tool in the pharmaceutical industry.

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

A simple and non-destructive technique that measures the average content of drug substances in formulated drug products containing fluorine using low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) is presented here. The technique can be applied to the development and manufacturing of drugs in the pharmaceutical industry.

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 the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography &#8211; mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

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). In the case of fast reversible electrochemical processes, current is predominantly affected by the rate of diffusion, which is the slowest and limiting stage. EIS is a powerful technique that allows separate analysis of stages of charge transfer that have different AC frequency response. The capability of the method was used to extract the value of charge transfer resistance, which characterizes the rate of charge exchange on the electrode-solution interface. The application of this technique is broad, from biochemistry up to organic electronics. In this work, we are presenting the method of analysis of organic compounds for optoelectronic applications.

Electrochemical impedance spectroscopy (EIS) of species that undergo reversible oxidation or reduction in solution was used for determination of rate constants of oxidation or reduction.

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