Name: line shape analysis of dynamic nmr spectra for characterizing coordination sphere rearrangements at a chiral rhenium polyhydride complex
Uploaded: 2022-07-27T14:25:00
Description: Watch this Scientific Journal Video about Line Shape Analysis of Dynamic NMR Spectra for Characterizing Coordination Sphere Rearrangements at a Chiral Rhenium Polyhydride Complex at JoVE.com

The authors thank the Department of Chemistry and Physics and the Creativity and Research Grant Program (Naik, Moehring) at Monmouth University for financial support of this work.

1. Sample preparation
<ol>
	<li>Preparation of ReH7(PPh3)235
	<ol>
		<li>Combine 0.15 g of sodium borohydride and 0.41 g of ReOCl3(PPh3)2 in a two- or three-necked 100 mL round-bottomed flask fitted with a rubber septum and gas port, or a 100 mL Kjeldahl flask (with a side arm gas port) fitted with a rubber septum (Supplementary Figure 1).</li>
		<li>Add a spin bar to the reaction vessel.</li>
		<...

There are four items in the preparation of ReH7(PPh3)2 that can impact the quantity and purity of the material that is produced. First, the use of an ice bath during the first 15 min of the reaction is important to remove heat from the reaction that occurs between sodium borohydride and water. Higher initial temperatures lead to a decreased yield of the ReH7(PPh3)2 product due to formation of the thermal decomposition product Re2H8(PP...

NMR spectroscopy is commonly used to characterize dynamic processes that occur within or between molecules. For many simple intramolecular rearrangements, estimation of &#916;G&#8225; is as straight-forward as measuring the frequency difference, &#916;&#957;, between two resonances at the slow exchange limit and determining the coalescence temperature for those same resonances (Figure 1)1. The relationship,
&#916;G&#8225; = 4.575 x 10-3 kcal/mol x Tc [9.972 + log (Tc/&#916;&#957;)]
where Tc is the coalescence temperature for a pair of resonances that represent the slow exchange form of a dynamic sample, can be used to solve for the free energy of activation for such a dynamic rearrangement. More complex dynamic systems require line shape fitting of dynamic NMR spectra or another NMR technique such as two-dimensional exchange spectroscopy (2D-EXSY) or two-dimensional rotating-frame Overhauser effect spectroscopy (2D-ROESY) to estimate activation parameters.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64160/64160fig01.jpg" /> 
Figure 1: NMR spectra for a d8-toluene solution of ReH5(PPh3)2(sec-butyl amine) at two temperatures. The frequency difference between the two slow exchange doublets (lower trace, 117.8 Hz) and a coalescence temperature of 250 K (upper trace) correspond to an energy barrier (&#916;G&#8225;) of 11.8 kcal/mol. <a href="https://www.jove.com/files/ftp_upload/64160/64160fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Line shape fitting of dynamic NMR spectra is a common technique that has long been used for the estimation of activation parameters that describe dynamic rearrangements for substances with an activation energy of approximately 5 to 25 kcal/mol2,3,4,5. Determination of the energy barriers to proton exchange between water and amine molecules6, the energy barrier to rotation about the C-N bond in dimethylformamide7, or the general size of organic moieties8 are only a few examples of the many properties that have been assessed through line shape fitting of dynamic NMR spectra. This manuscript demonstrates the use of line shape fitting to characterize the intermolecular and intramolecular dynamic processes that occur for the complex ReH5(PPh3)2(sec-butyl amine). The goals of this and similar line shape fitting NMR experiments are to: 1) characterize all NMR observable intramolecular dynamic atom exchange processes if present, 2) identify and characterize NMR observable intramolecular dynamic atom exchange processes if present, 3) identify correlated intramolecular atom exchanges that occur for, in this example, both hydrogen and phosphorus atoms, and 4) for the example presented here, compare two published models for the dynamic processes that occur in the complex ReH5(PPh3)2(sec-butyl amine).
Eight-coordinate rhenium(V) polyhydride systems are complex dynamic systems in which the ligands participate in multiple dynamic processes and the phosphorus atoms can participate in a single dynamic process that is a second aspect of a hydride ligand exchange process9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26, 
27,28,29. Eight-coordinate, pseudododecahedral, rhenium(V) polyhydride complexes adopt a molecular geometry (Figure 2), which can be described as a pair of orthogonal trapezoids of ligands17,26. The vertices on the long edges of the trapezoids are commonly labelled as B sites and, in rhenium polyhydride complexes, are usually the sites occupied by neutral two-electron donor ligands such as tertiary phosphines or amine ligands. The vertices on the short edges of the trapezoids are commonly labelled as A sites and are typically occupied by anionic, two-electron donor, hydride ligands. The room temperature NMR spectra of rhenium(V) polyhydride complexes are, typically, deceptively simple due to the several dynamic processes that occur in room temperature solutions.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64160/64160fig02.jpg" /> 
Figure 2: A dodecahedral coordination set (left) and the complex ReH5(PPh3)2(sec-butyl amine) from the same perspective (right). The red-colored sites represent coordination sites that form a vertical trapezoid, and the blue-colored sites represent coordination sites that form a horizontal trapezoid. <a href="https://www.jove.com/files/ftp_upload/64160/64160fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Complexes of the form ReH5(PPh3)2(amine) are the most thoroughly studied class of rhenium polyhydride complexes with respect to dynamic processes9,10,12,13,16,30,31. Three dynamic processes (Figure 3) have been identified for ReH5(PPh3)2(amine) complexes: 1) a proton exchange between the sole B site hydride ligand and a proton from a water molecule (adventitious or intentional)9,13, 2) a turnstile exchange of a pair of A site hydride ligands with an adjacent B site hydride ligand9,11,13,30,31, and 3) a steric inversion (or pseudorotation) that manifests itself as a pairwise exchange of the A site hydride ligands and a pairwise movement of the B site atoms to the opposite side of the rhenium center (as depicted in Figure 4)4,5,6,8,26,27. The movement of B site atoms to the opposite side of rhenium is observable by dynamic NMR spectroscopy as: 1) a process that makes the inequivalent 3 and 5 protons of N = pyridine equivalent at room temperature10,30,31, 2) a process that causes the E and Z isomers of N = unsymmetrically substituted aromatic amine ligands to undergo fast exchange at room temperature9,10,13,30,31, or 3) a process that causes a fast exchange of the steric perspectives of a diastereotopic pair of phosphorus atoms with respect to a chiral center located on the amine ligand9,30,31. The previously unreported chiral complex ReH5(PPh3)2(sec-butyl amine) provides an opportunity to generally describe the methods that can be used to identify and characterize the dynamic rearrangements of rhenium polyhydride complexes.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64160/64160fig03.jpg" /> 
Figure 3: Representations of the dynamic processes that are observed by NMR spectroscopy for solutions of ReH5(PPh3)2(sec-butyl amine). Representation A depicts the exchange of a single proton of adventitious water for the unique B-site hydride ligand. Representation B depicts the turnstile exchange of three adjacent hydride ligands, two of which reside in A site while the third is the unique B site hydride ligand. Representation C depicts both the pairwise exchange of A site hydride ligands as well as the steric inversion of the phosphorus atoms with respect to the chiral amine ligand (N*). It should be noted that the A site hydride ligand pairwise exchange does not require a shift of the A site hydride ligands to the opposite side of the rhenium center. <a href="https://www.jove.com/files/ftp_upload/64160/64160fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
For chemical systems such as rhenium polyhydride complexes, which exhibit a complex set of dynamic processes, line shape fitting of dynamic NMR spectra is the most used NMR technique to characterize the processes9,11,13,16,21,29. Two-dimensional EXSY9,32 or 2D-ROESY11 are alternative dynamic NMR techniques that can also be used to quantitatively characterize the dynamic processes. Two-dimensional EXSY spectra are typically measured in the slow exchange temperature domain; two-dimensional ROESY spectra are typically measured in the fast exchange temperature domain. Both two-dimensional techniques may require considerable time in the spectrometer for data acquisition, in that each of the techniques is acquiring a much larger data set, at a given temperature, than the one-dimensional data sets needed for line shape fitting analysis. Simple dynamic processes that are well understood, such as the dynamic exchange of the two methyl groups of dimethylformamide, can be readily characterized by any of the three NMR techniques. More complex systems, such as ReH5(PPh3)2(sec-butyl amine), in which individual hydride ligands participate in multiple dynamic processes, or systems that are not necessarily well understood, such as a novel transition metal polyhydride complex which may or may not exchange protons between a hydride ligand and adventitious water, are more easily quantitatively characterized by the line shape fitting NMR method than by the two-dimensional NMR methods. Unlike the two-dimensional NMR methods, the line shape fitting method provides an easily interpretable visualization of the match between a tested model and the experimental data as well as visual evidence of an exchange that moves a hydride ligand beyond the inner coordination sphere of rhenium. Based upon peak heights and peak shapes in slow exchange spectra, even a complex dynamic system such as ReH5(PPh3)2(sec-butyl amine) can lead to an easily tested initial set of exchange models. Additionally, when multiple theoretical models have been reported for a molecular transformation, line shape fitting of dynamic NMR spectra can allow for a visual comparison of each model versus observed spectra.
Beyond the three NMR techniques mentioned above, isotopic substitution NMR experiments involving D2O or HD have been used to qualitatively demonstrate intermolecular exchange of atoms for complex rhenium polyhydride systems, but have not been used for quantitative characterizations9,33,34,35. Theoretical calculations present an additional method for characterizing the dynamic processes of complex dynamic systems30,31,36. Theoretical calculations have the advantage over line shape fitting in that they can be used to differentiate between possibilities that cannot be distinguished by line shape fitting analysis. For example, theoretical calculations have been used to describe an exchange that involves three adjacent hydride ligands on certain rhenium(V) complexes as a turnstile exchange of all three hydride ligands, rather than an alternating pair of pairwise exchanges with each pairwise exchange including a unique hydride ligand and one of two chemically equivalent hydride ligands30,31. The results of theoretical calculations are typically compared to experimentally observed quantitative characterizations from one of the three NMR techniques mentioned above as a check on the validity of the calculated results.
Line shape fitting of dynamic NMR spectra takes advantage of the change in the appearance of NMR spectra that occurs when NMR-active nuclei move between different chemical environments during an NMR measurement. Slow exchange NMR spectra (spectra with independent Lorentzian resonances for each set of exchanging nuclei) occur at temperatures where the frequency difference between resonances for nuclei that exchange is large compared with the rate of exchange of the nuclei37. Fast exchange NMR spectra (spectra with a single Lorentzian resonance for exchanging nuclei) occur at temperatures where the rate of exchange of the nuclei is much greater than the frequency difference between the slow exchange resonances37. Intermediate exchange rates occur for temperatures between the slow exchange temperature domain and the fast exchange temperature domain37. If the fundamental parameters of Larmor frequency, chemical shift of the exchanging nuclei, coupling constants (if any) for the exchanging nuclei, and relative populations of each nucleus type are known, rate constants for putative exchanges between nuclei can be determined by comparing simulated spectra to observed spectra at several intermediate temperatures. Good fits for simulations at several temperatures result in temperature and rate constant data that can be used with the Eyring equation to estimate activation parameters for the putative exchange(s). Results from the method have been found to be both accurate and reproducible.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bruker Avance II 400 MHz NMR spectrometer</td>
			<td>Bruker Biospin</td>
			<td></td>
			<td>The instrument includes a two channel probe (1H and X) with the X channel tunable from 162 MHz to 10 Mhz. The instrument is also VT capable with a dewar and heat exchanger for VT work.</td>
		</tr>
		<tr>
			<td>d8-toluene</td>
			<td>MilliporeSigma</td>
			<td>434388</td>
			<td></td>
		</tr>
		<tr>
			<td>Powerstat variable transformer</td>
			<td>Powerstat</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sec-butyl amine</td>
			<td>MilliporeSigma</td>
			<td>B89000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium borohydride</td>
			<td>MilliporeSigma</td>
			<td>452882</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrahydrofuran</td>
			<td>MilliporeSigma</td>
			<td>186562</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermowell C3AM 100 mL</td>
			<td>Thermowell</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Topspin 3.0 or 4.1.4 with dNMR</td>
			<td>Bruker Biospin</td>
			<td></td>
			<td>Data was acquired with Topspin version 3.0 and data handling was performed on a second computer that was running Topspin version 4.1.4..</td>
		</tr>
		<tr>
			<td>Trichlorooxobis(triphenylphosphine) rhenium(V)</td>
			<td>MilliporeSigma</td>
			<td>370193</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuubrand PC3000 vacuum pump with a CVC 3000 controller</td>
			<td>Vacuubrand</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

line shape analysis of dynamic nmr spectra for characterizing coordination sphere rearrangements at a chiral rhenium polyhydride complex

Dynamic solution nuclear magnetic resonance (NMR) spectroscopy is the typical method of characterizing the dynamic rearrangements of atoms within the coordination sphere for transition metal polyhydride complexes. Line shape fitting of the dynamic NMR spectra can lead to estimates for the activation parameters of the dynamic rearrangement processes. A combination of dynamic 31P-{1H} NMR spectroscopy of metal-bound phosphorus atoms with dynamic 1H-{31P} NMR spectroscopy of hydride ligands may identify hydride ligand rearrangements that occur in conjunction with a phosphorus atom rearrangement. For molecules that exhibit such a coupled pair of rearrangements, dynamic NMR spectroscopy can be used to test theoretical models for the ligand rearrangements. Dynamic 1H-{31P} NMR spectroscopy and line shape fitting can also identify the presence of an exchange process that moves a specific hydride ligand beyond the metal&#39;s inner coordination sphere through a proton exchange with a solvent molecule such as adventitious water. The preparation of a new compound, ReH5(PPh3)2(sec-butyl amine), that exemplifies multiple dynamic rearrangement processes is presented along with line shape fitting of dynamic NMR spectra of the complex. Line shape fitting results can be analyzed by the Eyring equation to estimate the activation parameters for the identified dynamic processes.

The characterizations of both rhenium polyhydride products described in this manuscript are best accomplished by 1H-{31P} and 31P-{1H} NMR spectroscopy. In a room temperature d6-benzene solution, the hydride ligand resonance of ReH7(PPh3)2 appears as a binomial triplet at &#948; = -4.2 ppm with 2JPH = 18 Hz by 1H NMR spectroscopy (Supplementary Figure 2). The same d6-benzene...

Watch this Scientific Journal Video about Line Shape Analysis of Dynamic NMR Spectra for Characterizing Coordination Sphere Rearrangements at a Chiral Rhenium Polyhydride Complex at JoVE.com

Line Shape Analysis of Dynamic NMR Spectra for Characterizing Coordination Sphere Rearrangements at a Chiral Rhenium Polyhydride Complex

Line shape analysis of NMR spectra collected over a range of temperatures serves as a guide for the rearrangement of inner coordination-sphere atoms at a chiral, eight-coordinate, rhenium(V) polyhydride complex, ReH5(PPh3)2(sec-butyl amine). Line shape analysis is also used to determine the activation parameters &#916;H&#8225;, &#916;S&#8225;, and &#916;G&#8225; for those atom rearrangements.

Dynamic solution nuclear magnetic resonance (NMR) spectroscopy is the typical method of characterizing the dynamic rearrangements of atoms within the ...

This method examines the dynamic behavior of the eight atoms that are bound to a central metal atom through line shape fitting of dynamic NMR spectrum. The visual nature of the line shape fitting technique allows for ready development of dynamic exchange models in comparisons of the model results with the observed spectra. Line shape fitting of NMR spectra is a method that is used to investigate a variety of dynamic molecular processes with activation energies in the range of 5 to 25 kilocalories per mole.I would expect novice users to have questions regarding how to complete the inputs for the line shape fitting application. Practicing on the application, especially with an experienced user, is helpful. To begin, combine 0.15 grams of sodium borohydride and 0.41 grams of oxotrichlorobis triphenylphosphine rhenium-V in a two-or three-necked 100-milliliter round-bottom flask fitted with the rubber septum and gas port, or a 100-milliliter Kjeldahl flask fitted with a rubber septum.In a fume hood, use a piece of rubber pressure tubing to connect the gas port of the reaction vessel with one of the stopcocks of a dual glass manifold for vacuum and nitrogen gas. Connect the glass vacuum manifold to a vacuum pump with rubber pressure tubing, glass nitrogen manifold to a regulated nitrogen gas cylinder, and the exit gas from the nitrogen gas manifold to a stopcock that can be used to direct the vented gas through either a two-centimeter column of mineral oil or mercury. Then, open the tap on the nitrogen cylinder and adjust the pressure on the flowing gas to 34 pounds per square inch and vent the nitrogen gas flow through the mercury bubbler.Next, evacuate the gas inside the reaction vessel by adjusting the stopcock on the glass manifold to connect the vessel to the vacuum manifold, and fill the reaction vessel with nitrogen gas by changing the glass manifold stopcock that it connects the gas manifold with the reaction vessel. Then, add eight milliliters of deoxygenated water and eight milliliters of deoxygenated tetrahydrofuran to the solids in the reaction vessel via a syringe. Upon achieving an orange to tan color for the reaction mixture, filter the mixture through a 30-milliliters medium-centered glass funnel and wash the recovered solid three times each with 15-milliliters portions of water, methanol, and ethyl ether.Next, fit the flask to a condenser equipped with a gas port and add a volume of eight milliliters of deoxygenated tetrahydrofuran to the reaction vessel via a syringe by cracking the joint between the round-bottomed flask and the condenser. Then, pour the reaction mixture into 25 milliliters of methanol in a 125-milliliter Erlenmeyer flask and add five milliliters of water to induce the formation of a flocculent yellow precipitate. To prepare the spectrometer, enter a flow rate of 200 liters per hour for the cooling gas and a target temperature of 290 kelvin for the probe, while allowing the spectrometer to stabilize at the target temperature for two minutes.After shimming the sample at 290 kelvin, change the file name for each of the previously measured spectra by adding the temperature to the end of the file name and acquire a set of three spectra at 290 kelvin. Then increase the cooling gas flow rate by more than or equal to 30 liters per hour as needed to stabilize at the next temperature and decrease the target temperature by 10 kelvin. For line shape analysis of the measured spectra, click on the Edit Range button to enter the upper and lower chemical shifts for line shape fitting, and click the OK button to accept those limits.Then, start a model for line shape fitting by clicking on the SpinSystem tab in the line shape fitting window and click on the Add button to allow for the building of a model spin system. Next, unselect LB and enter the value for line broadening manually with the mouse and the LB button on the line shape fitting toolbar. Add the first nucleus into the model by clicking on the Nucleus tab, followed by clicking on the Add button, and a set of default values will appear for nucleus one.Then, adjust the chemical shift for nucleus one by entering a value for chemical shift in the new NuISO box or with the chemical shift tool on the line shape fitting toolbar. For nucleus one, input the number of equivalent nuclei for nucleus one with each spin half nucleus equivalent to 0.5 in counting, and enter the sum of the spins into the Pseudo Spin box in order to account for all equivalent nuclei. Using the In Molecule box, assign resonances that arise from different molecules to separate molecules using designations such as 1, 2, et cetera for different molecules, and for resonances that arise from a single molecule, assign 1 for all In Molecule values.Next, add the second and all subsequent nuclei to the model by clicking on the Nucleus tab, followed by clicking on the Add button. Then, include spin-spin coupling between nuclei by either entering the coupling in the appropriate JM box or by adjusting the scaler coupling button on the line shape fitting toolbar. Begin the process of describing the atom exchanges by clicking on the Reaction tab and click on the checkbox.if the rate constant for the exchange is to be varied in line shape fitting, then enter the number of nuclei to be exchanged into the Exchanges box for the first exchange in the model. Next, define the exchanges between nucleus tabs in the boxes below the Exchanges box, ensuring that exchanges are cyclic, in that if a nucleus is moved from nucleus one, another nucleus has to be moved into nucleus one. Use the exchange speed button on the line shape fitting toolbar to change the initial value of K in order to iteratively adjust the value of K, even if the checkbox is selected for the rate constant.Add more exchanges to the model by clicking on the Reaction tab, followed by clicking on the Add button. Use the tools on the line shape fitting toolbar to adjust the starting variables and begin iterative line shape fitting by clicking on the Start the spectrum fit button on the line shape fitting toolbar. Continue iterative fitting until no change is found in the best overlap between spectrum and model or until 1, 000 iterations are reached.If fitting stops at 1, 000 iterations, continue further iterations with the Start the spectrum fit button, and the model spectrum is displayed with the actual spectrum for comparison. The dynamic proton-decoupled phosphorus-31 NMR spectra of the rhenium complex were measured at several temperatures. The spectra show the coalescence of the two resonances that arise from the diastereotopic phosphorous atoms into a single resonance at higher temperatures.The temperature dependence of the difference in chemical shifts between the two proton-decoupled phosphorous-31 resonances was determined. Extrapolation allows for estimation of the chemical shifts of the individual resonances at higher temperatures. The temperature dependence for the hydride resonance chemical shifts was determined.The chemical shifts calculated from the best linear fits were used for the line shape fitting of the observed spectra. The results of line shape fitting for pairwise exchange of A site hydride ligands, turnstile exchange of three adjacent hydride ligands, and proton exchange between one proton of water and the unique hydride ligand were compared with the observed hydride region of a series of proton-decoupled phosphorous-31 NMR spectra from 225 kelvin to 240 kelvin. A comparison of the models for rearrangement of hydride ligands, with and without proton exchange, versus the proton-decoupled phosphorous-31 NMR spectrum measured at 225 kelvin.Rate constants arising from the line shape fitting of proton-decoupled phosphorous-31 NMR spectra show a good fit for the Eyring equation. Temperature changes for the sample should not exceed 10 kelvin, and the target temperature should be maintained for at least two minutes to protect the probe of the instrument.

line-shape-analysis-dynamic-nmr-spectra-for-characterizing

Research

JoVE Journal

Chemistry

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 ...

Atomically Defined Templates for Epitaxial Growth of Complex Oxide Thin Films

Atomically defined substrate surfaces are prerequisite for the epitaxial growth of complex oxide thin films. In this protocol, two approaches to obtain such surfaces are described. The first approach is the preparation of single terminated perovskite SrTiO3 (001) and DyScO3 (110) substrates. Wet etching was used to selectively remove one of the two possible surface terminations, while an annealing step was used to increase the smoothness of the surface. The resulting single terminated surfaces allow for the heteroepitaxial growth of perovskite oxide thin films with high crystalline quality and well-defined interfaces between substrate and film. In the second approach, seed layers for epitaxial film growth on arbitrary substrates were created by Langmuir-Blodgett (LB) deposition of nanosheets. As model system Ca2Nb3O10- nanosheets were used, prepared by delamination of their layered parent compound HCa2Nb3O10. A key advantage of creating seed layers with nanosheets is that relatively expensive and size-limited single crystalline substrates can be replaced by virtually any substrate material.

Various procedures are outlined to prepare atomically defined templates for epitaxial growth of complex oxide thin films. Chemical treatments of single crystalline SrTiO3 (001) and DyScO3 (110) substrates were performed to obtain atomically smooth, single terminated surfaces. Ca2 Nb3 O10- nanosheets were used to create atomically defined templates on arbitrary substrates.

Atomically defined substrate surfaces are prerequisite for the epitaxial growth of complex oxide thin films. In this protocol, two approaches to obtain ...

Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of metabolic transformations. Hyperpolarization via dissolution DNP circumvents part of the sensitivity issues thanks to the large out-of-equilibrium nuclear magnetization stemming from the electron-to-nucleus spin polarization transfer. The high NMR signal obtained can be used to monitor chemical reactions in real time. The downside of hyperpolarized NMR resides in the limited time window available for signal acquisition, which is usually on the order of the nuclear spin longitudinal relaxation time constant, T1, or, in favorable cases, on the order of the relaxation time constant associated with the singlet-state of coupled nuclei, TLLS. Cellular uptake of endogenous molecules and metabolic rates can provide essential information on tumor development and drug response. Numerous previous hyperpolarized NMR studies have demonstrated the relevancy of pyruvate as a metabolic substrate for monitoring enzymatic activity in vivo. This work provides a detailed description of the experimental setup and methods required for the study of enzymatic reactions, in particular the pyruvate-to-lactate conversion rate in presence of lactate dehydrogenase (LDH), by hyperpolarized NMR.

The sensitivity enhancement provided by dissolution dynamic nuclear polarization (DNP) enables following metabolic processes in real time by NMR and MRI. The characteristics and performances of a dedicated dissolution DNP setup designed for study enzymatic reactions are discussed.

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of ...

Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, and that these chemical fingerprints can be used for plant species identification. The mass spectral data can be acquired rapidly and in a high throughput manner without the need for sample extraction, derivatization or pH adjustment steps. The use of this technique bypasses challenges presented by more conventional techniques including lengthy chromatography analysis times and resource intensive methods. The high throughput capabilities of the direct analysis in real time-high resolution mass spectrometry protocol, coupled with multivariate statistical analysis processing of the data, provide not only class characterization of plants, but also yield species and varietal information. Here, the technique is demonstrated with two psychoactive plant products, Mitragyna speciosa (Kratom) and Datura (Jimsonweed), which were subjected to direct analysis in real time-high resolution mass spectrometry followed by statistical analysis processing of the mass spectral data. The application of these tools in tandem enabled the plant materials to be rapidly identified at the level of variety and species.

A method for species identification of botanical material by direct analysis in real time-high resolution mass spectrometry and multivariate statistical analysis is presented.

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, ...

On-line Analysis of Nitrogen Containing Compounds in Complex Hydrocarbon Matrixes

The shift to heavy crude oils and the use of alternative fossil resources such as shale oil are a challenge for the petrochemical industry. The composition of heavy crude oils and shale oils varies substantially depending on the origin of the mixture. In particular they contain an increased amount of nitrogen containing compounds compared to the conventionally used sweet crude oils. As nitrogen compounds have an influence on the operation of thermal processes occurring in coker units and steam crackers, and as some species are considered as environmentally hazardous, a detailed analysis of the reactions involving nitrogen containing compounds under pyrolysis conditions provides valuable information. Therefore a novel method has been developed and validated with a feedstock containing a high nitrogen content, i.e., a shale oil. First, the feed was characterized offline by comprehensive two-dimensional gas chromatography (GC &#215; GC) coupled with a nitrogen chemiluminescence detector (NCD). In a second step the on-line analysis method was developed and tested on a steam cracking pilot plant by feeding pyridine dissolved in heptane. The former being a representative compound for one of the most abundant classes of compounds present in shale oil. The composition of the reactor effluent was determined via an in-house developed automated sampling system followed by immediate injection of the sample on a GC &#215; GC coupled with a time-of-flight mass spectrometer (TOF-MS), flame ionization detector (FID) and NCD. A novel method for quantitative analysis of nitrogen containing compounds using NCD and 2-chloropyridine as an internal standard has been developed and demonstrated.

A method combining comprehensive two-dimensional gas chromatography with nitrogen chemiluminescence detection has been developed and applied to on-line analysis of nitrogen containing compounds in a complex hydrocarbon matrix.

The shift to heavy crude oils and the use of alternative fossil resources such as shale oil are a challenge for the petrochemical industry. The ...

Synthesis of a Water-soluble Metal&#8211;Organic Complex Array

We demonstrate a method for the synthesis of a water-soluble multimetallic peptidic array containing a predetermined sequence of metal centers such as Ru(II), Pt(II), and Rh(III). The compound, named as a water-soluble metal-organic complex array (WSMOCA), is obtained through 1) the conventional solution-chemistry-based preparation of the corresponding metal complex monomers having a 9-fluorenylmethyloxycarbonyl (Fmoc)-protected amino acid moiety and 2) their sequential coupling together with other water-soluble organic building units on the surface-functionalized polymeric resin by following the procedures originally developed for the solid-phase synthesis of polypeptides, with proper modifications. Traces of reactions determined by mass spectrometric analysis at the representative coupling steps in stage 2 confirm the selective construction of a predetermined sequence of metal centers along with the peptide backbone. The WSMOCA cleaved from the resin at the end of stage 2 has a certain level of solubility in aqueous media dependent on the pH value and/or salt content, which is useful for the purification of the compound.

Synthesis of a Water-soluble Metal&amp;#8211;Organic Complex Array

A potential general method for the synthesis of water-soluble multimetallic peptidic arrays containing a predetermined sequence of metal centers is presented.

We demonstrate a method for the synthesis of a water-soluble multimetallic peptidic array containing a predetermined sequence of metal centers such as ...

Facet-to-facet Linking of Shape-anisotropic Colloidal Cadmium Chalcogenide Nanostructures

Here, we describe a protocol that allows for shape-anisotropic cadmium chalcogenide nanocrystals (NCs), such as nanorods (NRs) and tetrapods (TPs), to be covalently and site-specifically linked via their end facets, resulting in polymer-like linear or branched chains. The linking procedure begins with a cation-exchange process in which the end facets of the cadmium chalcogenide NCs are first converted to silver chalcogenide. This is followed by the selective removal of ligands at their surface. This results in cadmium chalcogenide NCs with highly reactive silver chalcogenide end facets that spontaneously fuse upon contact with each other, thereby establishing an interparticle facet-to-facet attachment. Through the judicious choice of precursor concentrations, an extensive network of linked NCs can be produced. Structural characterization of the linked NCs is carried out via low- and high-resolution transmission electron microscopy (TEM), as well as energy-dispersive X-ray spectroscopy, which confirm the presence of silver chalcogenide domains between chains of cadmium chalcogenide NCs.

A protocol detailing how shape-anisotropic colloidal cadmium chalcogenide nanocrystals can be covalently linked via their end facets is presented here.

Here, we describe a protocol that allows for shape-anisotropic cadmium chalcogenide nanocrystals (NCs), such as nanorods (NRs) and tetrapods (TPs), to be ...

Enzymatic Cascade Reactions for the Synthesis of Chiral Amino Alcohols from L-lysine

Amino alcohols are versatile compounds with a wide range of applications. For instance, they have been used as chiral scaffolds in organic synthesis. Their synthesis by conventional organic chemistry often requires tedious multi-step synthesis processes, with difficult control of the stereochemical outcome. We present a protocol to enzymatically synthetize amino alcohols starting from the readily available L-lysine in 48 h. This protocol combines two chemical reactions that are very difficult to conduct by conventional organic synthesis. In the first step, the regio- and diastereoselective oxidation of an unactivated C-H bond of the lysine side-chain is catalyzed by a dioxygenase; a second regio- and diastereoselective oxidation catalyzed by a regiodivergent dioxygenase can lead to the formation of the 1,2-diols. In the last step, the carboxylic group of the alpha amino acid is cleaved by a pyridoxal-phosphate (PLP) decarboxylase (DC). This decarboxylative step only affects the alpha carbon of the amino acid, retaining the hydroxy-substituted stereogenic center in a beta/gamma position. The resulting amino alcohols are therefore optically enriched. The protocol was successfully applied to the semipreparative-scale synthesis of four amino alcohols. Monitoring of the reactions was conducted by high performance liquid chromatography (HPLC) after derivatization by 1-fluoro-2,4-dinitrobenzene. Straightforward purification by solid-phase extraction (SPE) afforded the amino alcohols with excellent yields (93% to &#62;95%).

Chiral amino alcohols are versatile molecules for use as scaffolds in organic synthesis. Starting from L-lysine, we synthesize amino alcohols by an enzymatic cascade reaction combining diastereoselective C-H oxidation catalyzed by dioxygenase followed by cleavage of the carboxylic acid moiety of the corresponding hydroxyl amino acid by a decarboxylase.

Amino alcohols are versatile compounds with a wide range of applications. For instance, they have been used as chiral scaffolds in organic synthesis. ...

Synthesis of Terpolymers at Mild Temperatures Using Dynamic Sulfur Bonds in Poly(S-Divinylbenzene)

Elemental sulfur (S8) is a byproduct of the petroleum industry with millions of tons produced annually. Such abundant production and limited applications lead to sulfur as a cost-efficient reagent for polymer synthesis. Inverse vulcanization combines elemental sulfur with a variety of monomers to form functional polysulfides without the need for solvents. Short reaction times and straight forward synthetic methods have led to rapid expansion of inverse vulcanization. However, high reaction temperatures (&#62;160 &#176;C) limit the types of monomers that can be used. Here, the dynamic sulfur bonds in poly(S-divinylbenzene) are used to initiate polymerization at much lower temperatures. The S-S bonds in the prepolymer are less stable than S-S bonds in S8, allowing radical formation at 90 &#176;C rather than 159 &#176;C. A variety of allyl and vinyl ethers have been incorporated to form terpolymers. The resulting materials were characterized by 1H NMR, gel permeation chromatography, and differential scanning calorimetry, as well as examining changes in solubility. This method expands on the solvent-free, thiyl radical chemistry utilized by inverse vulcanization to create polysulfides at mild temperatures. This development broadens the range of monomers that can be incorporated thus expanding the accessible material properties and possible applications.

Synthesis of Terpolymers at Mild Temperatures Using Dynamic Sulfur Bonds in PolyS-Divinylbenzene

The goal of this protocol is to initiate polymerization using dynamic sulfur bonds in poly(S-divinylbenzene) at mild temperatures (90 &#176;C) without using solvents. Terpolymers are characterized by GPC, DSC and 1H NMR, and tested for changes in solubility.

Elemental sulfur (S8) is a byproduct of the petroleum industry with millions of tons produced annually. Such abundant production and limited applications ...

Application of Voltage in Dynamic Light Scattering Particle Size Analysis

Dynamic light scattering (DLS) is a common method for characterizing the size distribution of polymers, proteins, and other nano- and microparticles. Modern instrumentation permits measurement of particle size as a function of time and/or temperature, but currently there is no simple method for performing DLS particle size distribution measurements in the presence of applied voltage. The ability to perform such measurements would be useful in the development of electroactive, stimuli-responsive polymers for applications such as sensing, soft robotics, and energy storage. Here, a technique using applied voltage coupled with DLS and a temperature ramp to observe changes in aggregation and particle size in thermoresponsive polymers with and without electroactive monomers is presented. The changes in aggregation behavior observed in these experiments were only possible through the combined application of voltage and temperature control. To obtain these results, a potentiostat was connected to a modified cuvette in order to apply voltage to a solution. Changes in polymer particle size were monitored using DLS in the presence of constant voltage. Simultaneously, current data were produced, which could be compared with particle size data, to understand the relationship between current and particle behavior. The polymer poly(N-isopropylacrylamide) (pNIPAM) served as a test polymer for this technique, as pNIPAM's response to temperature is well-studied. Changes in the lower-critical solution temperature (LCST) aggregation behavior of pNIPAM and poly(N-isopropylacrylamide)-block-poly(ferrocenylmethyl methacrylate), an electrochemically active block-copolymer, in the presence of applied voltage are observed. Understanding the mechanisms behind such changes will be important when trying to achieve reversible polymer structures in the presence of applied voltage.

Here, a protocol to apply voltage to solution during dynamic light scattering particle size measurements with the intent to explore the effect of voltage and temperature changes on polymer aggregation is presented.

Dynamic light scattering (DLS) is a common method for characterizing the size distribution of polymers, proteins, and other nano- and microparticles. ...