Name: hot biological catalysis: isothermal titration calorimetry to characterize enzymatic reactions
Uploaded: 2014-04-04T13:45:00
Description: Watch this Scientific Journal Video about Hot Biological Catalysis: Isothermal Titration Calorimetry to Characterize Enzymatic Reactions at JoVE.com

The Specialty Fertilizer Products Company (SFP) is acknowledged for providing the funds necessary for this study.

1. Preparing Samples
<ol>
	<li>Prepare 2 ml of enzyme solution and 0.5 ml of substrate solution for each experimental run. Dilute concentrated stock solutions of enzyme and substrate in buffer solutions having identical composition to minimize the heat of dilution and mixing during the substrate addition.
	<ol>
		<li>Choose the buffer conditions that are adequate to prevent pH change during experiment. For example, 20 mM HEPES pH 7 is adequate for measurements at neutral pH. 
		NOTE: If proton exchange is...

<ol>
	<li>Leavitt, S., Freire, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+protein+binding+energetics+by+isothermal+titration+calorimetry.">Direct measurement of protein binding energetics by isothermal titration calorimetry.</a> Curr. Opin. Struct. Biol. 11, 560-566 (2001).</li><li>Ladbury, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+isothermal+titration+calorimetry+in+the+biological+sciences:+things+are+heating+up!+BioTechniques.">Application of isothermal titration calorimetry in the biological sciences: things are heating up! BioTechniques.</a> 37, 885-887 (2004).</li><li>Zambelli, B., Bellucci, M., Danielli, A., Scarlato, V., Ciurli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ni2++binding+properties+of+Helicobacter+pylori+NikR.">The Ni2+ binding properties of Helicobacter pylori NikR.</a> Chem. Commun. , 3649-3651 (2007).</li><li>Zambelli, 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=High-affinity+Ni2++binding+selectively+promotes+binding+of+Helicobacter+pylori+NikR+to+its+target+urease+promoter.">High-affinity Ni2+ binding selectively promotes binding of Helicobacter pylori NikR to its target urease promoter.</a> J. Mol. Biol. 383, 1129-1143 (2008).</li><li>Duff, M. R., Grubbs, J., Howell, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isothermal+titration+calorimetry+for+measuring+macromolecule-ligand+affinity.">Isothermal titration calorimetry for measuring macromolecule-ligand affinity.</a> J. Vis. Exp. , (2011).</li><li>Ghai, R., Falconer, R. J., Collins, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+isothermal+titration+calorimetry+in+pure+and+applied+research--survey+of+the+literature+from+2010.">Applications of isothermal titration calorimetry in pure and applied research--survey of the literature from 2010.</a> J. Mol. Recognit. 25, 32-52 (2012).</li><li>Todd, M. J., Gomez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme+kinetics+determined+using+calorimetry:+a+general+assay+for+enzyme+activity.">Enzyme kinetics determined using calorimetry: a general assay for enzyme activity.</a> Anal. Biochem. 296, 179-187 (2001).</li><li>Bianconi, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calorimetry+of+enzyme-catalyzed+reactions.">Calorimetry of enzyme-catalyzed reactions.</a> Biophys. Chem. 126, 59-64 (2007).</li><li>Demarse, N. A., Killian, M. C., Hansen, L. D., Quinn, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+enzyme+kinetics+via+isothermal+titration+calorimetry.">Determining enzyme kinetics via isothermal titration calorimetry.</a> Methods Mol. Biol. 978, 21-30 (2013).</li><li>Michaelis, L., Menten, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Die+kinetik+der+invertinwirkung.">Die kinetik der invertinwirkung.</a> Biochem. Z. 49, 333-369 (1913).</li><li>Walker, J., Wilson, K., Walker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principle and techniques of practical biochemistry. , 312-356 (2000).</li><li>Ciurli, S., Sigel, A., Sigel, H., Sigel, R. K. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nickel and its surprising impact in nature. 2, 241-278 (2007).</li><li>Zambelli, B., Musiani, F., Benini, S., Ciurli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry+of+Ni2++in+urease:+sensing,+trafficking,+and+catalysis.">Chemistry of Ni2+ in urease: sensing, trafficking, and catalysis.</a> Acc. Chem. Res. 44, 520-530 (2011).</li><li>Zonia, L. E., Stebbins, N. E., Polacco, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+of+urease+in+germination+of+nitrogen-limited+Arabidopsis+thaliana+seeds.">Essential role of urease in germination of nitrogen-limited Arabidopsis thaliana seeds.</a> Plant Physiol. 107, 1097-1103 (1995).</li><li>Follmer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+role+and+structure+of+plant+ureases.">Insights into the role and structure of plant ureases.</a> Phytochemistry. 69, 18-28 (2008).</li><li>Sumner, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+and+crystallization+of+the+enzyme+urease.">The isolation and crystallization of the enzyme urease.</a> J. Biol. Chem. 69, 435-441 (1926).</li><li>Krajewska, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ureases+I.+Functional,+catalytic+and+kinetic+properties:+A+review.">Ureases I. Functional, catalytic and kinetic properties: A review.</a> J. Mol. Cat. B. 59, 9-21 (2009).</li><li>Callahan, B. P., Yuan, Y., Wolfenden, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+burden+borne+by+urease.">The burden borne by urease.</a> J. Am. Chem. Soc. 127, 10828-10829 (2005).</li><li>Krajewska, B., van Eldik, R., Brindell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-+and+pressure-dependent+stopped-flow+kinetic+studies+of+jack+bean+urease.+Implications+for+the+catalytic+mechanism.">Temperature- and pressure-dependent stopped-flow kinetic studies of jack bean urease. Implications for the catalytic mechanism.</a> J. Biol. Inorg. Chem. 17, 1123-1134 (2012).</li><li>Hausinger, R. P., Karplus, P. A., Huber, R., Poulos, T., Wieghardt, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Metalloproteins. , 867-879 (2001).</li><li>Goldberg, R., Kishore, N., Lennen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamic+quantities+for+the+ionization+reactions+of+buffers.">Thermodynamic quantities for the ionization reactions of buffers.</a> J. Phys. Chem. Ref. Data. 31, 231-370 (2002).</li><li>Baumann, M. J., 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=Advantages+of+isothermal+titration+calorimetry+for+xylanase+kinetics+in+comparison+to+chemical-reducing-end+assays.">Advantages of isothermal titration calorimetry for xylanase kinetics in comparison to chemical-reducing-end assays.</a> Anal. Biochem. 410, 19-26 (2011).</li><li>Noske, R., Cornelius, F., Clarke, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+enzymatic+activity+of+the+Na+,K+-ATPase+via+isothermal+titration+microcalorimetry.">Investigation of the enzymatic activity of the Na+,K+-ATPase via isothermal titration microcalorimetry.</a> Biochim. Biophys. Acta. 1797, 1540-1545 (2010).</li><li>Harmon, K. M., Niemann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+competitive+inhibition+of+the+urease-catalyzed+hydrolysis+of+urea+by+phosphate.">The competitive inhibition of the urease-catalyzed hydrolysis of urea by phosphate.</a> J. Biol. Chem. 177, 601-605 (1949).</li><li>Benini, S., Rypniewski, W. R., Wilson, K. S., Ciurli, S., Mangani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-based+rationalization+of+urease+inhibition+by+phosphate:+novel+insights+into+the+enzyme+mechanism.">Structure-based rationalization of urease inhibition by phosphate: novel insights into the enzyme mechanism.</a> J. Biol. Inorg. Chem. 6, 778-790 (2001).</li><li>Segel, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Enzyme kinetics: behavior and analysis of rapid equilibrium and steady-state enzyme systems. , (1993).</li></ol>

Significance of ITC to study enzymatic activity with respect to existing methods
In addition to its classical applications to study binding equilibria, isothermal titration calorimetry provides a reliable and fast method to characterize enzymatic reactions in solution using the heat of reaction as a probe, without requiring system modification or labeling. The kinetic parameters kcat and KM are usually obtained through a set of time cour...

Quantitative determination of biochemical reactions provides insights into the biological processes at the basis of life. Calorimetry offers a label-free methodology to quantitatively characterize virtually every chemical reaction in solution. This technique measures the heat released or absorbed over time, and is therefore a universal detection system and a very convenient methodology to quantify the amount of reacting molecules (i.e. binding thermodynamics), as well as to measure the reaction rate (i.e. kinetics). In particular, isothermal titration calorimetry (ITC) has been adopted as method of choice to characterize the thermodynamics of biomolecular equilibria, involving protein-ligand, protein-protein, protein-metal ions and protein-DNA interactions1-6. In addition, the ability of ITC to provide kinetic information makes it a very powerful system to measure enzyme catalysis, although the potential of this application is still underestimated7-9.
The Michaelis-Menten equation10 is a quantitative description of enzymatic reactions, as it provides a relationship between the reaction rate and the substrate concentration, depending on two kinetic parameters: the Michaelis constant (KM) and the catalytic rate constant (kcat). The kcat/KM ratio is referred as the catalytic efficiency of an enzyme. In practice, determination of KM and kcat for a specific reaction provides a complete description of the catalysis.
In a typical enzymatic reaction (Figure 1), a substrate (S) interacts with the enzyme (E) forming the enzyme-substrate (ES) complex, which is subsequently activated into the transition state (ES*). The latter is converted into the enzyme-product (EP) complex that eventually dissociates. These steps are described by the following reaction.
<img fo:content-width="2in" src="/files/ftp_upload/51487/51487eq1.jpg" width="197" /> (1)
where k1 is the rate constant for the formation of the ES complex, k-1 is the rate constant for the dissociation of the ES complex, while kcat is the catalytic rate constant or turnover number.
According to the Michaelis-Menten equation10, the rate of the reaction can be calculated as:
<img fo:content-width="1.5in" src="/files/ftp_upload/51487/51487eq2.jpg" width="144" /> (2)
in which KM = (k-1 + kcat)/k1 and kcat = vmax/[E], with vmax being the maximal velocity reached when all enzyme is bound to the substrate.
The isothermal titration calorimeter is the instrument used in this study to characterize the enzymatic hydrolysis of urea. This instrument is made of an adiabatic shield containing two coined-shaped cells (Figure 1). These are connected to the outside with narrow access tubes. The sample cell (ca. 1.4 ml) is loaded with the enzyme solution, while the reference cell is generally filled with water or with the solvent used for the analysis. A rotating syringe with a long needle and a stir paddle attached, usually containing ca. 0.3 ml of substrate solution, is mounted on the sample cell. A thermoelectric device measures the difference of temperature between the sample and the reference cell and, using a &#8220;cell feedback network&#8221;, it maintains this difference at zero by adding or subtracting heat. During the experiment, the substrate is injected into the enzyme solution at a constant chosen temperature. When the enzymatic reaction takes place, the amount of heat released or absorbed is proportional to the number of substrate molecules that are converted into product molecules. In addition, the rate of heat flow is directly related to the rate of the reaction. The measured data, appearing as a deviation of the heat trace from initial baseline (Figure 1), represent the thermal power (&#956;cal/sec) supplied by the instrument to the sample cell, which is proportional to the heat flow occurring in the sample cell over time.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51487/51487fig1highres.jpg" width="500" /> 
Figure 1. Schematic representation of isothermal titration calorimeter to study enzymatic reactions. An enzymatic reaction occurring upon titration of the substrate (in the injection syringe) into the enzyme solution (in the sample cell) results in a change of the thermal power released by the calorimeter, needed to keep the difference of temperature between the sample cell and the reference cell constant. <a href="https://www.jove.com/files/ftp_upload/51487/51487fig1highres.jpg" target="_blank">Click here to view larger image.</a>
Overall, the heat change (Q) is proportional to the molar enthalpy of the reaction (&#916;H) and the number of moles of product generated (n), which in turn is given by the total volume times the concentration:
<img fo:content-width="2in" src="/files/ftp_upload/51487/51487eq3.jpg" width="200" /> (3)
The product formation over time (dP/dt), which corresponds to the reaction rate, can thus be related to the amount of heat generated over the same time (dQ/dt) through the relation:
<img fo:content-width="2in" src="/files/ftp_upload/51487/51487eq4.jpg" width="150" /> (4)
According to this equation, in order to obtain a Michaelis-Menten plot it is necessary to measure i) the total molar enthalpy &#916;H, and ii) the heat flow dQ/dt at different substrate concentrations. Usually, this is performed in two different experiments: in the first experiment (Method 1, M1), the substrate is injected into the enzyme solution and the heat for complete substrate conversion is measured; in the second experiment (Method 2, M2), multiple injections of substrate are performed and the rate of heat production is measured at different substrate concentrations. These two sets of data are sufficient to derive the kinetic parameters KM and kcat.
In the present article, a general protocol to determine the kinetic parameters for enzymatic reactions performed using ITC is described. We applied the method to urea hydrolysis by Canavalia ensiformis urease, as a reference system. The good agreement between the results obtained using this method and the data reported in literature demonstrates the reliability of this approach.

<table border="0" cellpadding="0" cellspacing="0" width="796">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">HEPES</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:119px;">H3375</td>
			<td style="width:323px;">dissolving in water and adjusting pH with NaOH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">TRIZMA-Base</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:119px;">T1503</td>
			<td>dissolving in water and adjusting pH with HCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Sodium dihydrogen phosphate</td>
			<td style="width:111px;">Riedel-de-Haen</td>
			<td style="width:119px;">4270</td>
			<td>dissolving in water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Sodium phosphate dibasic</td>
			<td style="width:111px;">Riedel-de-Haen</td>
			<td style="width:119px;">30427</td>
			<td>dissolving in water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Urea</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:119px;">U4128</td>
			<td>dissolving in water at 40 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">Canavalia ensiformis urease (type C-3)</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:119px;">U0251</td>
			<td>dissolving in 20 mM HEPES pH 7 and stored at -80 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;"></td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">VP-ITC on Origin 7.0</td>
			<td style="width:111px;">MicroCal (GE Healthcare)</td>
			<td style="width:119px;">SYS13901</td>
			<td>instrument&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">VPViewer2000 1.30.00 on Origin 7.0</td>
			<td style="width:111px;">MicroCal (GE Healthcare)</td>
			<td style="width:119px;"></td>
			<td>data acquisition software supplied with the instrument</td>
		</tr>
	</tbody>
</table>

hot biological catalysis: isothermal titration calorimetry to characterize enzymatic reactions

Isothermal titration calorimetry (ITC) is a well-described technique that measures the heat released or absorbed during a chemical reaction, using it as an intrinsic probe to characterize virtually every chemical process. Nowadays, this technique is extensively applied to determine thermodynamic parameters of biomolecular binding equilibria. In addition, ITC has been demonstrated to be able of directly measuring kinetics and thermodynamic parameters (kcat, KM, &#916;H) of enzymatic reactions, even though this application is still underexploited. As heat changes spontaneously occur during enzymatic catalysis, ITC does not require any modification or labeling of the system under analysis and can be performed in solution. Moreover, the method needs little amount of material. These properties make ITC an invaluable, powerful and unique tool to study enzyme kinetics in several applications, such as, for example, drug discovery.
In this work an experimental ITC-based method to quantify kinetics and thermodynamics of enzymatic reactions is thoroughly described. This method is applied to determine kcat and KM of the enzymatic hydrolysis of urea by Canavalia ensiformis (jack bean) urease. Calculation of intrinsic molar enthalpy (&#916;Hint) of the reaction is performed. The values thus obtained are consistent with previous data reported in literature, demonstrating the reliability of the methodology.

Urease (EC 3.5.1.5; urea amidohydrolase) is a multisubunit nickel-containing enzyme found in archea, bacteria, unicellular eukaryotes and plants. This protein acts in the last step of organic nitrogen mineralization, catalyzing the hydrolysis of urea to ammonia and carbamate, which spontaneously decomposes to give a second molecule of ammonia and bicarbonate (Equation 6)12.
<img fo:content-width="4in" src="/files/ftp_upload/51487/51487eq6.jpg" width="450" /> (6)...

Watch this Scientific Journal Video about Hot Biological Catalysis: Isothermal Titration Calorimetry to Characterize Enzymatic Reactions at JoVE.com

Hot Biological Catalysis: Isothermal Titration Calorimetry to Characterize Enzymatic Reactions

Isothermal titration calorimetry measures heat flow released or absorbed in chemical reactions. This method can be used to quantify enzyme-catalysis. In this paper, the protocol for instrumental setup, experiment running, and data analysis is generally described, and applied to the characterization of enzymatic urea hydrolysis by jack bean urease.

Isothermal titration calorimetry (ITC) is a well-described technique that measures the heat released or absorbed during a chemical reaction, using it as ...

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Dichloro-bis(aminophosphine) complexes of palladium with the general formula of [(P{(NC5H10)3-n(C6H11)n})2Pd(Cl)2] (where n = 0-2), belong to a new family ...

Light-driven Enzymatic Decarboxylation

Oxidoreductases belong to the most-applied industrial enzymes. Nevertheless, they need external electrons whose supply is often costly and challenging. Recycling of the electron donors NADH or NADPH requires the use of additional enzymes and sacrificial substrates. Interestingly, several oxidoreductases accept hydrogen peroxide as electron donor. While being inexpensive, this reagent often reduces the stability of enzymes. A solution to this problem is the in situ generation of the cofactor. The continuous supply of the cofactor at low concentration drives the reaction without impairing enzyme stability. This paper demonstrates a method for the light-catalyzed in situ generation of hydrogen peroxide with the example of the heme-dependent fatty acid decarboxylase OleTJE. The fatty acid decarboxylase OleTJE was discovered due to its unique ability to produce long-chain 1-alkenes from fatty acids, a hitherto unknown enzymatic reaction. 1-alkenes are widely used additives for plasticizers and lubricants. OleTJE has been shown to accept electrons from hydrogen peroxide for the oxidative decarboxylation. While addition of hydrogen peroxide damages the enzyme and results in low yields, in situ generation of the cofactor circumvents this problem. The photobiocatalytic system shows clear advantages regarding enzyme activity and yield, resulting in a simple and efficient system for fatty acid decarboxylation.

We describe a protocol for the light-catalyzed generation of hydrogen peroxide &#8212; a cofactor for oxidative transformations.

Oxidoreductases belong to the most-applied industrial enzymes. Nevertheless, they need external electrons whose supply is often costly and challenging. ...

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 ...

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. ...

Employing Pressurized Hot Water Extraction (PHWE) to Explore Natural Products Chemistry in the Undergraduate Laboratory

A recently developed pressurized hot water extraction (PHWE) method which utilizes an unmodified household espresso machine to facilitate natural products research has also found applications as an effective teaching tool. Specifically, this technique has been used to introduce second- and third-year undergraduates to aspects of natural products chemistry in the laboratory. In this report, two experiments are presented: the PHWE of eugenol and acetyleugenol from cloves and the PHWE of seselin and (+)-epoxysuberosin from the endemic Australian plant species Correa reflexa. By employing PHWE in these experiments, the crude clove extract, enriched in eugenol and acetyleugenol, was obtained in 4-9% w/w from cloves by second-year undergraduates and seselin and (+)-epoxysuberosin were isolated in yields of up to 1.1% w/w and 0.9% w/w from C. reflexa by third-year students. The former exercise was developed as a replacement for the traditional steam distillation experiment providing an introduction to extraction and separation techniques, while the latter activity featured guided-inquiry teaching methods in an effort to simulate natural products bioprospecting. This primarily derives from the rapid nature of this PHWE technique relative to traditional extraction methods that are often incompatible with the time constraints associated with undergraduate laboratory experiments. This rapid and practical PHWE method can be used to efficiently isolate various classes of organic molecules from a range of plant species. The complementary nature of this technique relative to more traditional methods has also been demonstrated previously.

Employing Pressurized Hot Water Extraction PHWE to Explore Natural Products Chemistry in the Undergraduate Laboratory

Here, we employ a pressurized hot water extraction (PHWE) method, which utilizes an unmodified household espresso machine to introduce undergraduates to natural products chemistry in the laboratory. Two experiments are presented: PHWE of eugenol and acetyleugenol from cloves and PHWE of seselin and (+)-epoxysuberosin from the Australian plant Correa reflexa.

A recently developed pressurized hot water extraction (PHWE) method which utilizes an unmodified household espresso machine to facilitate natural products ...

Imine Metathesis by Silica-Supported Catalysts Using the Methodology of Surface Organometallic Chemistry

With this protocol, a well-defined singlesite silica-supported heterogeneous catalyst [(&#8801;Si-O-)Hf(=NMe)(&#951;1-NMe2)] is designed and prepared according to the methodology developed by surface organometallic chemistry (SOMC). In this framework, catalytic cycles can be determined by isolating crucial intermediates. All air-sensitive materials are handled under inert atmosphere (using gloveboxes or a Schlenk line) or high vacuum lines (HVLs, &#60;10-5 mbar). The preparation of SiO2-700 (silica dehydroxylated at 700 &#176;C) and subsequent applications (the grafting of complexes and catalytic runs) requires the use of HVLs and double-Schlenk techniques. Several well-known characterization methods are used, such as Fourier-transform infrared spectroscopy (FTIR), elemental microanalysis, solid-state nuclear magnetic resonance spectroscopy (SSNMR), and state-of-the-art dynamic nuclear polarization surface enhanced NMR spectroscopy (DNP-SENS). FTIR and elemental microanalysis permit scientists to establish the grafting and its stoichiometry. 1H and 13C SSNMR allows the structural determination of the hydrocarbon ligands coordination sphere. DNP SENS is an emerging powerful technique in solid characterization for the detection of poorly sensitive nuclei (15N, in our case). SiO2-700 is treated with about one equivalent of the metal precursor compared to the amount of surface silanol (0.30 mmol&#183;g-1) in pentane at room temperature. Then, volatiles are removed, and the powder samples are dried under dynamic high vacuum to afford the desired materials [(&#8801;Si-O-)Hf(&#951;2&#960;-MeNCH2)(&#951;1-NMe2)(&#951;1-HNMe2)]. After a thermal treatment under high vacuum, the grafted complex is converted into metal imido silica complex [(&#8801;Si-O-)Hf(=NMe)(&#951;1-NMe2)]. [(&#8801;Si-O-)Hf(=NMe)(&#951;1-NMe2)] effectively promotes the metathesis of imines, using the combination of two imine substrates, N-(4-phenylbenzylidene)benzylamine, or N-(4-fluorobenzylidene)-4-fluoroaniline, with N-benzylidenetert-butylamine as substrates. A significantly lower conversion is observed with the blank runs; thus, the presence of the imido group in [(&#8801;Si-O-)Hf(=NMe)(&#951;1-NMe2)] is correlated to the catalytic performance.

A new group IV metal catalyst for imine metathesis is prepared by grafting amine metal complex onto dehydroxylated silica. Surface metal fragments are characterized using FT-IR, elemental microanalysis, and solid-state NMR spectroscopy. Further dynamic nuclear polarization surface enhanced NMR spectroscopy experiments complement the determination of the coordination sphere.

With this protocol, a well-defined singlesite silica-supported heterogeneous catalyst [(&#8801;Si-O-)Hf(=NMe)(&#951;1-NMe2)] is designed and prepared ...

Enzymatic Synthesis of Epoxidized Metabolites of Docosahexaenoic, Eicosapentaenoic, and Arachidonic Acids

The epoxidized metabolites of various polyunsaturated fatty acids (PUFAs), termed epoxy fatty acids, have a wide range of roles in human physiology. These metabolites are produced endogenously by the cytochrome P450 class of enzymes. Because of their diverse and potent biological effects, there is considerable interest in studying these metabolites. Determining the unique roles of these metabolites in the body is a difficult task, as the epoxy fatty acids must first be obtained in significant amounts and with high purity. Obtaining compounds from natural sources is often labor intensive, and soluble epoxide hydrolases (sEH) rapidly hydrolyze the metabolites. On the other hand, obtaining these metabolites via chemical reactions is very inefficient, due to the difficulty of obtaining pure regioisomers and enantiomers, low yields, and extensive (and expensive) purification. Here, we present an efficient enzymatic synthesis of 19(S),20(R)- and 16(S),17(R)-epoxydocosapentaenoic acids (EDPs) from DHA via epoxidation with BM3, a bacterial CYP450 enzyme isolated originally from Bacillus megaterium (that is readily expressed in Escherichia coli). Characterization and determination of purity is performed with nuclear magnetic resonance spectroscopy (NMR), high-performance liquid chromatography (HPLC), and mass spectrometry (MS). This procedure illustrates the benefits of enzymatic synthesis of PUFA epoxy metabolites, and is applicable to the epoxidation of other fatty acids, including arachidonic acid (AA) and eicosapentaenoic acid (EPA) to produce the analogous epoxyeicosatrienoic acids (EETs) and epoxyeicosatetraenoic acids (EEQs), respectively.

We present a method useful for large-scale enzymatic synthesis and purification of specific enantiomers and regioisomers of epoxides of arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) with the use of a bacterial cytochrome P450 enzyme (BM3).

The epoxidized metabolites of various polyunsaturated fatty acids (PUFAs), termed epoxy fatty acids, have a wide range of roles in human physiology. These ...

A Microwave-Assisted Direct Heteroarylation of Ketones Using Transition Metal Catalysis

Heteroarylation introduces heteroaryl fragments to organic molecules. Despite the numerous available reactions reported for arylation via transition metal catalysis, the literature on direct heteroarylation is scarce. The presence of heteroatoms such as nitrogen, sulfur and oxygen often make heteroarylation a challenging research field due to catalyst poisoning, product decomposition and the rest. This protocol details a highly efficient direct &#945;-C(sp3) heteroarylation of ketones under microwave irradiation. Key factors for successful heteroarylation include the use of XPhos Palladacycle Gen. 4 Catalyst, excess base to suppress side reactions and the high temperature and pressure achieved in a sealed reaction vial under microwave irradiation. The heteroarylation compounds prepared by this method were fully characterized by proton nuclear magnetic resonance spectroscopy (1H NMR), carbon nuclear magnetic resonance spectroscopy (13C NMR) and high-resolution mass spectrometry (HRMS). This methodology has several advantages over literature precedents including broad substrate scope, rapid reaction time, greener procedure and operational simplicity by eliminating the preparation of intermediates such as silyl enol ether. Possible applications for this protocol include, but are not limited to, diversity-oriented synthesis for the discovery of biologically active small molecules, domino synthesis for the preparation of natural products and ligand development for new transition metal catalytic systems.

Heteroaryl compounds are important molecules utilized in organic synthesis, medicinal and biological chemistry. A microwave-assisted heteroarylation using palladium catalysis provides a rapid and efficient method to attach heteroaryl moieties directly to ketone substrates.

Heteroarylation introduces heteroaryl fragments to organic molecules. Despite the numerous available reactions reported for arylation via transition metal ...